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Sample records for enzyme activity measured

  1. Measuring the Enzyme Activity of Arabidopsis Deubiquitylating Enzymes.

    Science.gov (United States)

    Kalinowska, Kamila; Nagel, Marie-Kristin; Isono, Erika

    2016-01-01

    Deubiquitylating enzymes, or DUBs, are important regulators of ubiquitin homeostasis and substrate stability, though the molecular mechanisms of most of the DUBs in plants are not yet understood. As different ubiquitin chain types are implicated in different biological pathways, it is important to analyze the enzyme characteristic for studying a DUB. Quantitative analysis of DUB activity is also important to determine enzyme kinetics and the influence of DUB binding proteins on the enzyme activity. Here, we show methods to analyze DUB activity using immunodetection, Coomassie Brilliant Blue staining, and fluorescence measurement that can be useful for understanding the basic characteristic of DUBs.

  2. Measurement of enzyme activity.

    Science.gov (United States)

    Harris, T K; Keshwani, M M

    2009-01-01

    To study and understand the nature of living cells, scientists have continually employed traditional biochemical techniques aimed to fractionate and characterize a designated network of macromolecular components required to carry out a particular cellular function. At the most rudimentary level, cellular functions ultimately entail rapid chemical transformations that otherwise would not occur in the physiological environment of the cell. The term enzyme is used to singularly designate a macromolecular gene product that specifically and greatly enhances the rate of a chemical transformation. Purification and characterization of individual and collective groups of enzymes has been and will remain essential toward advancement of the molecular biological sciences; and developing and utilizing enzyme reaction assays is central to this mission. First, basic kinetic principles are described for understanding chemical reaction rates and the catalytic effects of enzymes on such rates. Then, a number of methods are described for measuring enzyme-catalyzed reaction rates, which mainly differ with regard to techniques used to detect and quantify concentration changes of given reactants or products. Finally, short commentary is given toward formulation of reaction mixtures used to measure enzyme activity. Whereas a comprehensive treatment of enzymatic reaction assays is not within the scope of this chapter, the very core principles that are presented should enable new researchers to better understand the logic and utility of any given enzymatic assay that becomes of interest.

  3. A Simple and Accurate Method for Measuring Enzyme Activity.

    Science.gov (United States)

    Yip, Din-Yan

    1997-01-01

    Presents methods commonly used for investigating enzyme activity using catalase and presents a new method for measuring catalase activity that is more reliable and accurate. Provides results that are readily reproduced and quantified. Can also be used for investigations of enzyme properties such as the effects of temperature, pH, inhibitors,…

  4. Enzyme activity measurement via spectral evolution profiling and PARAFAC

    DEFF Research Database (Denmark)

    Baum, Andreas; Meyer, Anne S.; Garcia, Javier Lopez

    2013-01-01

    The recent advances in multi-way analysis provide new solutions to traditional enzyme activity assessment. In the present study enzyme activity has been determined by monitoring spectral changes of substrates and products in real time. The method relies on measurement of distinct spectral...... fingerprints of the reaction mixture at specific time points during the course of the whole enzyme catalyzed reaction and employs multi-way analysis to detect the spectral changes. The methodology is demonstrated by spectral evolution profiling of Fourier Transform Infrared (FTIR) spectral fingerprints using...

  5. Methodological Considerations and Comparisons of Measurement Results for Extracellular Proteolytic Enzyme Activities in Seawater

    Directory of Open Access Journals (Sweden)

    Yumiko Obayashi

    2017-10-01

    Full Text Available Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO and 2-methoxyethanol (MTXE. The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube, protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In

  6. Measurement of peroxisomal enzyme activities in the liver of brown trout (Salmo trutta, using spectrophotometric methods

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    Resende Albina D

    2003-03-01

    Full Text Available Abstract Background This study was aimed primarily at testing in the liver of brown trout (Salmo trutta spectrophotometric methods previously used to measure the activities of catalase and hydrogen peroxide producing oxidases in mammals. To evaluate the influence of temperature on the activities of those peroxisomal enzymes was the second objective. A third goal of this work was the study of enzyme distribution in crude cell fractions of brown trout liver. Results The assays revealed a linear increase in the activity of all peroxisomal enzymes as the temperature rose from 10° to 37°C. However, while the activities of hydrogen peroxide producing oxidases were strongly influenced by temperature, catalase activity was only slightly affected. A crude fraction enriched with peroxisomes was obtained by differential centrifugation of liver homogenates, and the contamination by other organelles was evaluated by the activities of marker enzymes for mitochondria (succinate dehydrogenase, lysosomes (aryl sulphatase and microsomes (NADPH cytochrome c reductase. For peroxisomal enzymes, the activities per mg of protein (specific activity in liver homogenates were strongly correlated with the activities per g of liver and with the total activities per liver. These correlations were not obtained with crude peroxisomal fractions. Conclusions The spectrophotometric protocols originally used to quantify the activity of mammalian peroxisomal enzymes can be successfully applied to the study of those enzymes in brown trout. Because the activity of all studied peroxisomal enzymes rose in a linear mode with temperature, their activities can be correctly measured between 10° and 37°C. Probably due to contamination by other organelles and losses of soluble matrix enzymes during homogenisation, enzyme activities in crude peroxisomal fractions do not correlate with the activities in liver homogenates. Thus, total homogenates will be used in future seasonal and

  7. Enzyme Activities in Waste Water and Activated Sludge

    DEFF Research Database (Denmark)

    Nybroe, Ole; Jørgensen, Per Elberg; Henze, Mogens

    1992-01-01

    The purpose of the present study was to evaluate the potential of selected enzyme activity assays to determine microbial abundance and heterotrophic activity in waste water and activated sludge. In waste water, esterase and dehydrogenase activities were found to correlate with microbial abundance...... measured as colony forming units of heterotrophic bacteria. A panel of four enzyme activity assays, α-glucosidase, alanine-aminopeptidase, esterase and dehydrogenase were used to characterize activated sludge and anaerobic hydrolysis sludge from a pilot scale plant. The enzymatic activity profiles were...... distinctly different, suggesting that microbial populations were different, or had different physiological properties, in the two types of sludge. Enzyme activity profiles in activated sludge from four full-scale plants seemed to be highly influenced by the composition of the inlet. Addition of hydrolysed...

  8. ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay.

    Science.gov (United States)

    Sehgal, Pankaj; Olesen, Claus; Møller, Jesper V

    2016-01-01

    Enzymatic coupled assays are usually based on the spectrophotometric registration of changes in NADH/NAD(+) or NADPH/NADP(+) absorption at 340 nm accompanying the oxidation/reduction of reactants that by dehydrogenases and other helper enzymes are linked to the activity of the enzymatic reaction under study. The present NADH-ATP-coupled assay for ATPase activity is a seemingly somewhat complicated procedure, but in practice adaptation to performance is easily acquired. It is a more safe and elegant method than colorimetric methods, but not suitable for handling large number of samples, and also presupposes that the activity of the helper enzymes is not severely affected by the chemical environment of the sample in which it is tested.

  9. Monovalent Cation Activation of the Radical SAM Enzyme Pyruvate Formate-Lyase Activating Enzyme.

    Science.gov (United States)

    Shisler, Krista A; Hutcheson, Rachel U; Horitani, Masaki; Duschene, Kaitlin S; Crain, Adam V; Byer, Amanda S; Shepard, Eric M; Rasmussen, Ashley; Yang, Jian; Broderick, William E; Vey, Jessica L; Drennan, Catherine L; Hoffman, Brian M; Broderick, Joan B

    2017-08-30

    Pyruvate formate-lyase activating enzyme (PFL-AE) is a radical S-adenosyl-l-methionine (SAM) enzyme that installs a catalytically essential glycyl radical on pyruvate formate-lyase. We show that PFL-AE binds a catalytically essential monovalent cation at its active site, yet another parallel with B 12 enzymes, and we characterize this cation site by a combination of structural, biochemical, and spectroscopic approaches. Refinement of the PFL-AE crystal structure reveals Na + as the most likely ion present in the solved structures, and pulsed electron nuclear double resonance (ENDOR) demonstrates that the same cation site is occupied by 23 Na in the solution state of the as-isolated enzyme. A SAM carboxylate-oxygen is an M + ligand, and EPR and circular dichroism spectroscopies reveal that both the site occupancy and the identity of the cation perturb the electronic properties of the SAM-chelated iron-sulfur cluster. ENDOR studies of the PFL-AE/[ 13 C-methyl]-SAM complex show that the target sulfonium positioning varies with the cation, while the observation of an isotropic hyperfine coupling to the cation by ENDOR measurements establishes its intimate, SAM-mediated interaction with the cluster. This monovalent cation site controls enzyme activity: (i) PFL-AE in the absence of any simple monovalent cations has little-no activity; and (ii) among monocations, going down Group 1 of the periodic table from Li + to Cs + , PFL-AE activity sharply maximizes at K + , with NH 4 + closely matching the efficacy of K + . PFL-AE is thus a type I M + -activated enzyme whose M + controls reactivity by interactions with the cosubstrate, SAM, which is bound to the catalytic iron-sulfur cluster.

  10. Measurement and purification of Alanine aminotransferase (ALT enzyme activity in patients with celiac disease

    Directory of Open Access Journals (Sweden)

    Taghreed U. Mohammed

    2017-09-01

    Full Text Available Celiac disease (CD is the most common genetically - based disease in correlation with food intolerance. The aim of this study is to measure the activity of ALT enzyme and purify enzyme from sera women with celiac disease. Alanine aminotransferase (ALT activity has been assayed in (30 women serum samples with celiac disease, age range between (20-40 year and (30 serum of healthy women as control group, age range between (22-38 year. In the present study, the mean value of ALT activity was significantly higher in patients with celiac disease than healthy group (p<0.01. The ALT enzyme was partial purified from sera women with celiac disease by dialysis, gel filtration using Sephadex G- 50 and ion exchange chromatography using DEAE- cellulose A-50 . The results showed a single peak by using gel filtration and the activity reached 31-15 U/L .Two isoenzymes were obtained by using ion exchange chromatography and the purity degree of isoenzymse (I, II were (5.7 and (5.53 fold respectively

  11. Lysosomal enzyme activation in irradiated mammary tumors

    International Nuclear Information System (INIS)

    Clarke, C.; Wills, E.D.

    1976-01-01

    Lysosomal enzyme activity of C3H mouse mammary tumors was measured quantitatively by a histochemical method. Following whole-body doses of 3600 rad or less no changes were observed in the lysosomal enzyme activity for 12 hr after the irradiation, but very large increases in acid phosphatase and β-naphthylamidase activity were, however, observed 24 hr after irradiation. Significant increases in enzyme activity were detected 72 hr after a dose of 300 rad and the increases of enzyme activity were dose dependent over the range 300 to 900 rad. Testosterone (80 mg/kg) injected into mice 2 hr before irradiation (850 rad) caused a significant increase of lysosomal enzyme activity over and above that of the same dose of irradiation alone. If the tumor-bearing mice were given 95 percent oxygen/5 percent carbon dioxide to breathe for 8 min before irradiation the effect of 850 rad on lysosomal acid phosphatase was increased to 160 percent/that of the irradiation given alone. Activitation of lysosomal enzymes in mammary tumors is an important primary or secondary consequence of radiation

  12. Effects of de-icing salt on soil enzyme activity

    Energy Technology Data Exchange (ETDEWEB)

    Guentner, M; Wilke, B M

    1983-01-01

    Effects of de-icing salt on dehydrogenase, urease, alkalinephosphatase and arylsulfatase activity of O/sub L/- and A/sub h/-horizons of a moder and a mull soil were investigated using a field experiment. Additions of 2.5 kg m/sup -2/ and 5.0 kg m/sup -2/ of de-icing salt reduced activities of most enzymes within four weeks. Eleven months after salt addition there was nearly no reduction of enzyme activity to be measured on salt treated soils. The percentage of reduced enzyme activity was generally higher in the moder soil. It was concluded that reductions of enzyme activity were due to decreases of microbial activity and not to inactivation of enzymes.

  13. [Interaction between CYP450 enzymes and metabolism of traditional Chinese medicine as well as enzyme activity assay].

    Science.gov (United States)

    Lu, Tu-lin; Su, Lian-lin; Ji, De; Gu, Wei; Mao, Chun-qin

    2015-09-01

    Drugs are exogenous compounds for human bodies, and will be metabolized by many enzymes after administration. CYP450 enzyme, as a major metabolic enzyme, is an important phase I drug metabolizing enzyme. In human bodies, about 75% of drug metabolism is conducted by CYP450 enzymes, and CYP450 enzymes is the key factor for drug interactions between traditional Chinese medicine( TCM) -TCM, TCM-medicine and other drug combination. In order to make clear the interaction between metabolic enzymes and TCM metabolism, we generally chose the enzymatic activity as an evaluation index. That is to say, the enhancement or reduction of CYP450 enzyme activity was used to infer the inducing or inhibitory effect of active ingredients and extracts of traditional Chinese medicine on enzymes. At present, the common method for measuring metabolic enzyme activity is Cocktail probe drugs, and it is the key to select the suitable probe substrates. This is of great significance for study drug's absorption, distribution, metabolism and excretion (ADME) process in organisms. The study focuses on the interaction between TCMs, active ingredients, herbal extracts, cocktail probe substrates as well as CYP450 enzymes, in order to guide future studies.

  14. Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity

    DEFF Research Database (Denmark)

    Hendriksen, Niels Bohse; Winding, Anne

    2012-01-01

    Extracellular enzyme activity assay as indicator of soil microbial functional diversity and activity Niels Bohse Hendriksen, Anne Winding. Department of Environmental Science, Aarhus University, 4000 Roskilde, Denmark Soils provide numerous essential ecosystem services such as carbon cycling...... of soil microbial functions is still needed. In soil, enzymes originate from a variety of organisms, notably fungi and bacteria and especially hydrolytic extracellular enzymes are of pivotal importance for decomposition of organic substrates and biogeochemical cycling. Their activity will reflect...... the functional diversity and activity of the microorganisms involved in decomposition processes. Their activity has been measured by the use of fluorogenic model substrates e.g. methylumbelliferyl (MUF) substrates for a number of enzymes involved in the degradation of polysacharides as cellulose, hemicellulose...

  15. Vitality Improvement of the Mediterranean Fruit Fly, Ceratitis capitata Wied 1- Measured by using dehydrogenase Enzyme Activities

    International Nuclear Information System (INIS)

    Salama, M.S.; Shoman, A.A.; Elbermawy, S.M.; Abul Yazid, I.

    2000-01-01

    The present study searches for the improvement vitality of the Mediterranean fruit fly, Ceratitis capitata Wied. Through the induction of a specific variance (mutation) in the genetic material. Several types of treatments that were thought to cause this mutation were used, as IGR's, temperature, formaldehyde, colchicine, alcohols, several types of larval rearing media and gamma-rays. Generally, the activities of the energy enzymes alpha-glycerophosphate dehydrogenase (alpha-GPDH) enzyme lactate dehydrogenase (LDH) enzyme and malate dehydrogenase (MDH) enzyme, when used as a direct measure for the fly vitality, increased due to treatments of the egg stage by the previously mentioned treatments specially by the usage of rice hulls in the larval rearing medium alone or followed by irradiation of the pupal stage with 90 Gy

  16. Descriptive and predictive assessment of enzyme activity and enzyme related processes in biorefinery using IR spectroscopy and chemometrics

    DEFF Research Database (Denmark)

    Baum, Andreas

    the understanding of the structural properties of the extracted pectin. Secondly, enzyme kinetics of biomass converting enzymes was examined in terms of measuring enzyme activity by spectral evolution profiling utilizing FTIR. Chemometric multiway methods were used to analyze the tensor datasets enabling the second......-order calibration advantage (reference Theory of Analytical chemistry). As PAPER 3 illustrates the method is universally applicable without the need of any external standards and was exemplified by performing quantitative enzyme activity determinations for glucose oxidase, pectin lyase and a cellolytic enzyme blend...... (Celluclast 1.5L). In PAPER 4, the concept is extended to quantify enzyme activity of two simultaneously acting enzymes, namely pectin lyase and pectin methyl esterase. By doing so the multiway methods PARAFAC, TUCKER3 and NPLS were compared and evaluated towards accuracy and precision....

  17. An evaluation on elastase enzyme activity in gingival crevicular fluid in periodontitis

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    Qujeq D

    2003-08-01

    Full Text Available Statement of Problem: Changes in protein levels, host calls enzymes and inflammatory mediators in gingival"ncrevicular Fluid (GCF are considered as diagnostic indicators of Periodontitis."nPurpose: he aim of the present study was to measure the elastase enzyme activity in gingival crevicular Fluid"namong patients with periodontitis."nMaterial and Methods: In this study, 52 periodontitis patients (experimental group and 51 healthy subjects"nwithout any gingival inflammatio (control group were participated. Subjects of the periodontitis group"nshowed pockets of 4-5 mm depth without gingival enlargement and recession or pockets of 1-2 mm depth"nwith gingival recession. For enzyme activity measurement, lOOu,! of gingival fluid of each sample was mixed"nwith lOOu! of enzyme substrate on the tube. The mixture was incubated at 34°c for lh with a buffer solution"nof 1ml volume and absorbance was read at 410nm with spectrophotometer. The enzyme activity differences"nbetween two groups were analyzed by student t test."nResults: The elastase enzyme activity in gingival crevicular fluid in subjects with periodontium destruction"nand control subjects was 153±11.3 and 52.7±10.4 enzyme unit in ml per minute, respectively. The difference"nbetween groups was statistically significant (PO.05."nConclusion: Based on the findings of this study, the measurement of elastae enzyme activity could be a useful"nindication of tissue changes that may ultimately manifest clinically as periodontitis.

  18. Enzyme hydration, activity and flexibility : A neutron scattering approach

    International Nuclear Information System (INIS)

    Kurkal-Siebert, V.; Finney, J.L.; Daniel, R.M.; Smith, Jeremy C.

    2006-01-01

    Recent measurements have demonstrated enzyme activity at hydrations as low as 3%. The question of whether the hydration-induced enzyme flexibility is important for activity is addressed by performing picosecond dynamic neutron scattering experiments on pig liver esterase powders at various temperatures as well as solutions. At all temperatures and hydrations investigated here, significant quasielastic scattering intensity is found in the protein, indicating the presence of anharmonic, diffusive motion. As the hydration increases a temperature-dependent dynamical transition appears and strengthens involving additional diffusive motion. At low temperature, increasing hydration resulted in lower flexibility of the enzyme. At higher temperatures, systems containing sufficient number of water molecules interacting with the protein exhibit increased flexibility. The implication of these results is that, although the additional hydration-induced diffusive motion and flexibility at high temperatures in the enzyme detected here may be related to increased activity, they are not required for the enzyme to function

  19. Development Of Enzyme Digestive Activity Of Blue Crab Portunus Pelagicus Larvae

    OpenAIRE

    Nikhlani, Andi; Sukarti, Komsanah

    2017-01-01

    Seed production continuity of Portunus pelagicus larvae had been conducted but the results were still un-consistent Digestive activity was known to be associated with the type of feed consumed by larvae. Amylase, lipase, and trypsin enzymes were used as a biological indicators to measure the digestion of feed. The aim of this study was to describe the activity of digestive enzymes in blue swimming crab larvae. Digestive enzyme activity data obtained was presented in graphical form and anal...

  20. Chaperone-Like Activity of ß-Casein and Its Effect on Residual in Vitro Activity of Food Enzymes

    DEFF Research Database (Denmark)

    Sulewska, Anna Maria

    ABSTRACT Activity of endogenous enzymes may cause browning of fruits and vegetables. These enzymes can be inactivated, for example by heat treatment, but the response of enzymes to heat treatment depends on many factors. Foods are very complex systems and the stability of enzymes......-casein on the enzymatic activity of three targets was tested by monitoring enzyme activity after heat treatment and by measuring the intensity of scattered light during and after heat treatment. β-Casein was shown to interact at elevated temperatures with three selected targets:horseradish peroxidase, tyrosinase from......, residual activity of horseradish peroxidase was lower in samples containing BSA than in samples without any addition. Horseradish peroxidase heated with BSA did not regain activity within one hour after treatment. BSA is often added to enzyme solutions to prevent enzyme adhesion to vial surfaces...

  1. Finding Biomass Degrading Enzymes Through an Activity-Correlated Quantitative Proteomics Platform (ACPP)

    Science.gov (United States)

    Ma, Hongyan; Delafield, Daniel G.; Wang, Zhe; You, Jianlan; Wu, Si

    2017-04-01

    The microbial secretome, known as a pool of biomass (i.e., plant-based materials) degrading enzymes, can be utilized to discover industrial enzyme candidates for biofuel production. Proteomics approaches have been applied to discover novel enzyme candidates through comparing protein expression profiles with enzyme activity of the whole secretome under different growth conditions. However, the activity measurement of each enzyme candidate is needed for confident "active" enzyme assignments, which remains to be elucidated. To address this challenge, we have developed an Activity-Correlated Quantitative Proteomics Platform (ACPP) that systematically correlates protein-level enzymatic activity patterns and protein elution profiles using a label-free quantitative proteomics approach. The ACPP optimized a high performance anion exchange separation for efficiently fractionating complex protein samples while preserving enzymatic activities. The detected enzymatic activity patterns in sequential fractions using microplate-based assays were cross-correlated with protein elution profiles using a customized pattern-matching algorithm with a correlation R-score. The ACPP has been successfully applied to the identification of two types of "active" biomass-degrading enzymes (i.e., starch hydrolysis enzymes and cellulose hydrolysis enzymes) from Aspergillus niger secretome in a multiplexed fashion. By determining protein elution profiles of 156 proteins in A. niger secretome, we confidently identified the 1,4-α-glucosidase as the major "active" starch hydrolysis enzyme (R = 0.96) and the endoglucanase as the major "active" cellulose hydrolysis enzyme (R = 0.97). The results demonstrated that the ACPP facilitated the discovery of bioactive enzymes from complex protein samples in a high-throughput, multiplexing, and untargeted fashion.

  2. Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid.

    NARCIS (Netherlands)

    Miners, J.S.; Verbeek, M.M.; Olde Rikkert, M.G.M.; Kehoe, P.G.; Love, S.

    2008-01-01

    Neprilysin, a zinc-metalloendopeptidase, has important roles in the physiology and pathology of many diseases such as hypertension, cancer and Alzheimer's disease. We have developed an immunocapture assay to measure the specific enzyme activity of neprilysin in brain tissue homogenates and

  3. Substrate-Competitive Activity-Based Profiling of Ester Prodrug Activating Enzymes.

    Science.gov (United States)

    Xu, Hao; Majmudar, Jaimeen D; Davda, Dahvid; Ghanakota, Phani; Kim, Ki H; Carlson, Heather A; Showalter, Hollis D; Martin, Brent R; Amidon, Gordon L

    2015-09-08

    Understanding the mechanistic basis of prodrug delivery and activation is critical for establishing species-specific prodrug sensitivities necessary for evaluating preclinical animal models and potential drug-drug interactions. Despite significant adoption of prodrug methodologies for enhanced pharmacokinetics, functional annotation of prodrug activating enzymes is laborious and often unaddressed. Activity-based protein profiling (ABPP) describes an emerging chemoproteomic approach to assay active site occupancy within a mechanistically similar enzyme class in native proteomes. The serine hydrolase enzyme family is broadly reactive with reporter-linked fluorophosphonates, which have shown to provide a mechanism-based covalent labeling strategy to assay the activation state and active site occupancy of cellular serine amidases, esterases, and thioesterases. Here we describe a modified ABPP approach using direct substrate competition to identify activating enzymes for an ethyl ester prodrug, the influenza neuraminidase inhibitor oseltamivir. Substrate-competitive ABPP analysis identified carboxylesterase 1 (CES1) as an oseltamivir-activating enzyme in intestinal cell homogenates. Saturating concentrations of oseltamivir lead to a four-fold reduction in the observed rate constant for CES1 inactivation by fluorophosphonates. WWL50, a reported carbamate inhibitor of mouse CES1, blocked oseltamivir hydrolysis activity in human cell homogenates, confirming CES1 is the primary prodrug activating enzyme for oseltamivir in human liver and intestinal cell lines. The related carbamate inhibitor WWL79 inhibited mouse but not human CES1, providing a series of probes for analyzing prodrug activation mechanisms in different preclinical models. Overall, we present a substrate-competitive activity-based profiling approach for broadly surveying candidate prodrug hydrolyzing enzymes and outline the kinetic parameters for activating enzyme discovery, ester prodrug design, and

  4. Microbial enzyme activities of peatland soils in south central Alaska lowlands

    Science.gov (United States)

    Microbial enzyme activities related to carbon and nutrient acquisition were measured on Alaskan peatland soils as indicators of nutrient limitation and biochemical sustainability. Peat decomposition is mediated by microorganisms and enzymes that in turn are limited by various ph...

  5. Thermodynamic activity-based intrinsic enzyme kinetic sheds light on enzyme-solvent interactions.

    Science.gov (United States)

    Grosch, Jan-Hendrik; Wagner, David; Nistelkas, Vasilios; Spieß, Antje C

    2017-01-01

    The reaction medium has major impact on biocatalytic reaction systems and on their economic significance. To allow for tailored medium engineering, thermodynamic phenomena, intrinsic enzyme kinetics, and enzyme-solvent interactions have to be discriminated. To this end, enzyme reaction kinetic modeling was coupled with thermodynamic calculations based on investigations of the alcohol dehydrogenase from Lactobacillus brevis (LbADH) in monophasic water/methyl tert-butyl ether (MTBE) mixtures as a model solvent. Substrate concentrations and substrate thermodynamic activities were varied separately to identify the individual thermodynamic and kinetic effects on the enzyme activity. Microkinetic parameters based on concentration and thermodynamic activity were derived to successfully identify a positive effect of MTBE on the availability of the substrate to the enzyme, but a negative effect on the enzyme performance. In conclusion, thermodynamic activity-based kinetic modeling might be a suitable tool to initially curtail the type of enzyme-solvent interactions and thus, a powerful first step to potentially understand the phenomena that occur in nonconventional media in more detail. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:96-103, 2017. © 2016 American Institute of Chemical Engineers.

  6. ENZYME ACTIVITIES OF PADDY SOILS AND RELATIONSHIPS WITH THE SOIL PROPERTIES

    Directory of Open Access Journals (Sweden)

    Rıdvan KIZILKAYA

    1998-03-01

    Full Text Available This study was carried out to determine the effect of soil properties on enzyme activities of paddy soils, the sample of which were taken from Üçpınar, Harız, Doğancı, Kaygusuz, Emenli, Sarıköy and Gelemenağarı villages where rice cultivation is an intensive agricultural system. In this study, soil properties having effects on urease, phosphatase, ß-glucosidase and catalase enzyme activities were setforth. Urease enzyme activities of soil samples varied from 24.12 to 39.03 mg N 100 g dry soil -1 . Significant correlations were determined between urease enzyme activities and organic matter (r = 0.89**, extractable Mn (r = 0.74**, exchangable K (r = 0.73** and total P content of soil (r = 0.81*. Acid phosphatase enzyme activity varied between 3.00-17.44 mg phenol 100 g dry soil -1 , alkaline phosphatase enzyme activity between 12.00-25.53 mg phenol 100 g dry soil-1 . Exchangable Mg (r = 0.71* and extractable Cu (r = 0.74* were found to have positive effect on acid phosphatase enzyme activity and pH (r = 0.73*, exchangable Ca (r = 0.74*, exchangable Mg (r = 0.71*, exchangable total basic cations (r = 0.79* and extractable Cu (r = 0.70* had positive effects on alkaline phosphatase enzyme activity, whereas total P (r = - 0.84** affected the activity negatively. ß-glucosidase enzyme activity was measured to vary between 1.12-3.64 mg salingen 100 g dry soil -1 . It was also observed that extractable Zn content of soil samples (r = - 0.97** had negative effect on ß-glucosidase activity, wheras total exchangable acidic cations (r = 0.70* affected the activity positively. Catalase enzyme activities of soils changed between 5.25 - 9.00 mg O2 5 g dry soil -1 . Significant correlations were found between catalase activities and fraction of soils and extractable Fe content. Positive correlations, however, were determined between catalase activities and clay fraction (r = 0.82* and salt content (r = 0.83** of samples.

  7. Visualization of enzyme activities inside earthworm pores

    Science.gov (United States)

    Hoang, Duyen; Razavi, Bahar S.

    2015-04-01

    In extremely dynamic microhabitats as bio-pores made by earthworm, the in situ enzyme activities are assumed as a footprint of complex biotic interactions. Our study focused on the effect of earthworm on the enzyme activities inside bio-pores and visualizing the differences between bio-pores and earthworm-free soil by zymography technique (Spohn and Kuzyakov, 2013). For the first time, we aimed at quantitative imaging of enzyme activities in bio-pores. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). After two weeks when bio-pore systems were formed by earthworms, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine-aminopeptidase, and phosphatase. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. However, the differences in activity of cellobiohydrolase and leucine aminopeptidase between bio-pore and bulk soil were less pronounced. This demonstrated an applicability of zymography approach to monitor and to distinguish the in situ activity of hydrolytic enzymes in soil biopores.

  8. Enzyme activity screening of thermophilic bacteria isolated from Dusun Tua Hot Spring, Malaysia

    Science.gov (United States)

    Msarah, Marwan; Ibrahim, Izyanti; Aqma, Wan Syaidatul

    2018-04-01

    Thermophilic bacteria have biotechnological importance due to the availability of unique enzymes which are stable in extreme circumstances. The aim of this study includes to isolate thermophilic bacteria from hot spring and screen for important enzyme activities. Water samples from the Dusun Tua Hot Spring were collected and the physiochemical characterisation of water was measured. Eight thermophilic bacteria were isolated and determined to have at least three strong enzyme activity including protease, lipase, amylase, cellulase, pectinase and xylanase. The results showed that HuluC2 displayed all the enzyme activities and can be further studied.

  9. County-Scale Spatial Distribution of Soil Enzyme Activities and Enzyme Activity Indices in Agricultural Land: Implications for Soil Quality Assessment

    Directory of Open Access Journals (Sweden)

    Xiangping Tan

    2014-01-01

    Full Text Available Here the spatial distribution of soil enzymatic properties in agricultural land was evaluated on a county-wide (567 km2 scale in Changwu, Shaanxi Province, China. The spatial variations in activities of five hydrolytic enzymes were examined using geostatistical methods. The relationships between soil enzyme activities and other soil properties were evaluated using both an integrated total enzyme activity index (TEI and the geometric mean of enzyme activities (GME. At the county scale, soil invertase, phosphatase, and catalase activities were moderately spatially correlated, whereas urease and dehydrogenase activities were weakly spatially correlated. Correlation analysis showed that both TEI and GME were better correlated with selected soil physicochemical properties than single enzyme activities. Multivariate regression analysis showed that soil OM content had the strongest positive effect while soil pH had a negative effect on the two enzyme activity indices. In addition, total phosphorous content had a positive effect on TEI and GME in orchard soils, whereas alkali-hydrolyzable nitrogen and available potassium contents, respectively, had negative and positive effects on these two enzyme indices in cropland soils. The results indicate that land use changes strongly affect soil enzyme activities in agricultural land, where TEI provides a sensitive biological indicator for soil quality.

  10. Stimulation of Escherichia coli DNA photoreactivating enzyme activity by adenosine 5'-triphosphate

    International Nuclear Information System (INIS)

    Koka, P.

    1984-01-01

    A purification procedure consisting of Biorex-70, single-stranded DNA-agarose, and ultraviolet (UV) light irradiated DNA-cellulose chromatography has been adopted for the Escherichia coli photoreactivating enzyme, to obtain enzyme preparations that are free of extraneous nucleic acid or nucleotides. The purification yields high specific activities (75 000 pmol h -1 mg -1 ) with a 50% recovery. Enzyme preparations have also been obtained from UV-irradiated DNA-cellulose by exposure to visible light. These enzyme preparations contain oligoribonucleotides, up to 26 nucleotides in length in relation to DNA size markers, but these are not essential for enzymatic activity. When the enzyme is preincubated with exogenous ATP a 10-fold stimulation in the enzyme activity has been observed. It has been determined by polyacrylamide gel electrophoresis and high-voltage diethylaminoethyl paper electrophoresis that the light-released enzyme samples from a preincubated and washed mixture of the enzyme, [γ- 32 P]ATP, and UV-irradiated DNA-cellulose contained exogenous [γ- 32 P], which eluted with the enzyme-containing fractions when subjected to Bio-Gel P-30 chromatography. GTP caused a slight enhancement of the enzyme activity while ADP strongly inhibited photoreactivation, at the same concentration and conditions. Higher (X5) concentrations of ADP and adenosine 5'-(β, γ-methylenetriphosphate) totally inhibited the enzyme activity. Dialysis of a photoreactivating enzyme preparation against a buffer solution containing 1 mM ATP caused a 9-fold stimulation of the enzyme activity. In addition, there is an apparent hydrolysis of ATP during photoreactivation as measured by the release of 32 P from [γ- 32 P]ATP

  11. Distribution of enzyme activity hotspots induced by earthworms in top- and subsoil

    Science.gov (United States)

    Hoang, D. T. T.

    2016-12-01

    Earthworms (Lumbricus terrestris L.) not only affect soil physics, but they also boost microbial activities and consequently create important hotspots of microbial mediated carbon and nutrient turnover through their burrowing activity. However, it is still unknown to which extend earthworms change the enzyme distribution and activity inside their burrows in top- and subsoil horizons. We hypothesized that earthworm burrows, which are enriched in available substrates, have higher percentage of enzyme activity hotspots than soil without earthworms, and that enzyme activities decreased with increasing depth because of the increasing recalcitrance of organic matter in subsoil. We visualized enzyme distribution inside and outside of worm burrows (biopores) by in situ soil zymography and measured enzyme kinetics of 6 enzymes - β-glucosidase (GLU), cellobiohydrolase (CBH), xylanase (XYL), chitinase (NAG), leucine aminopeptidase (LAP) and acid phosphatase (APT) - in pore and bulk soil material up to 105 cm. Zymography showed a heterogeneous distribution of hotspots in worm burrows. The hotspot areas was 2.4 to 14 times larger in the burrows than in soil without earthworms. However, the dispersion index of hotspot distribution showed more aggregated hotspots in soil without earthworms than in soil with earthworms and burrow wall. Enzyme activities decreased with depth, by a factor of 2 to 8 due to fresh C input from the soil surface. Compared to bulk soil, enzyme activities in topsoil biopores were up to 11 times higher for all enzymes, but in the subsoil activities of XYL, NAG and APT were lower in earthworm biopores than bulk soil. In conclusion, hotspots were twice as concentrated close to earthworm burrows as in surrounding soil. Earthworms exerted stronger effects on enzyme activities in biopores in the topsoil than in subsoil. Keywords: Earthworms, hotspots, enzyme activities, enzyme distribution, subsoil

  12. Dynamic relationships between microbial biomass, respiration, inorganic nutrients and enzyme activities: informing enzyme based decomposition models

    Directory of Open Access Journals (Sweden)

    Daryl L Moorhead

    2013-08-01

    Full Text Available We re-examined data from a recent litter decay study to determine if additional insights could be gained to inform decomposition modeling. Rinkes et al. (2013 conducted 14-day laboratory incubations of sugar maple (Acer saccharum or white oak (Quercus alba leaves, mixed with sand (0.4% organic C content or loam (4.1% organic C. They measured microbial biomass C, carbon dioxide efflux, soil ammonium, nitrate, and phosphate concentrations, and β-glucosidase (BG, β-N-acetyl-glucosaminidase (NAG, and acid phosphatase (AP activities on days 1, 3, and 14. Analyses of relationships among variables yielded different insights than original analyses of individual variables. For example, although respiration rates per g soil were higher for loam than sand, rates per g soil C were actually higher for sand than loam, and rates per g microbial C showed little difference between treatments. Microbial biomass C peaked on day 3 when biomass-specific activities of enzymes were lowest, suggesting uptake of litter C without extracellular hydrolysis. This result refuted a common model assumption that all enzyme production is constitutive and thus proportional to biomass, and/or indicated that part of litter decay is independent of enzyme activity. The length and angle of vectors defined by ratios of enzyme activities (BG/NAG versus BG/AP represent relative microbial investments in C (length, and N and P (angle acquiring enzymes. Shorter lengths on day 3 suggested low C limitation, whereas greater lengths on day 14 suggested an increase in C limitation with decay. The soils and litter in this study generally had stronger P limitation (angles > 45˚. Reductions in vector angles to < 45˚ for sand by day 14 suggested a shift to N limitation. These relational variables inform enzyme-based models, and are usually much less ambiguous when obtained from a single study in which measurements were made on the same samples than when extrapolated from separate studies.

  13. Epigenetics of dominance for enzyme activity

    Indian Academy of Sciences (India)

    Unknown

    dimer over a wide range of H+ concentrations accounts for the epigenetics of dominance for enzyme activity. [Trehan K S ... The present study has been carried on acid phosphatase .... enzyme activity over mid parent value (table 3, col. 13),.

  14. Activation of lysosomal enzymes and tumour regression caused by irradiation and steroid hormones

    International Nuclear Information System (INIS)

    Ball, A.; Barratt, G.M.; Wills, E.D.

    1982-01-01

    The lysosomal enzyme activity and membrane permeability of mouse C3H mammary tumours has been studied using quantitative cytochemical methods following irradiation of the tumours with doses of 1500, 3500 or 6000 rad ν rays. No change in the lysosomal enzyme activity was observed immediately after irradiation, but increased enzyme activity and increased membrane permeability were observed 24 hr after irradiation with doses of 3500 or 6000 rad. Twenty-four hours after injection of prednisolone there was a marked increase of lysosomal membrane permeability and enzyme activity, and injection of prednisolone soon after irradiation enhanced the effect of irradiation. After a dose of 6000 rad and prednisolone, the lysosomal membrane permeability increased to 191% of the control and the enzyme activity to 326% of the value of the control tumours. Measurement of tumour size after irradiation or after a combined treatment with irradiation and prednisolone showed that a close correlation exists between tumour regression and lysosomal enzyme activity. The experiments support the view that lysosomal enzymes play an important role in tumour regression following irradiation. (author)

  15. Development of a solid-phase assay for measurement of proteolytic enzyme activity

    International Nuclear Information System (INIS)

    Varani, J.; Johnson, K.; Kaplan, J.

    1980-01-01

    A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When 125 I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. 125 I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When [ 14 C]elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases

  16. Spatial distribution of enzyme activities in the rhizosphere

    Science.gov (United States)

    Razavi, Bahar S.; Zarebanadkouki, Mohsen; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    The rhizosphere, the tiny zone of soil surrounding roots, certainly represents one of the most dynamic habitat and interfaces on Earth. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. That is why there is an urgent need in spatially explicit methods for the determination of the rhizosphere extension and enzyme distribution. Recently, zymography as a new technique based on diffusion of enzymes through the 1 mm gel plate for analysis has been introduced (Spohn & Kuzyakov, 2013). We developed the zymography technique to visualize the enzyme activities with a higher spatial resolution. For the first time, we aimed at quantitative imaging of enzyme activities as a function of distance from the root tip and the root surface in the soil. We visualized the two dimensional distribution of the activity of three enzymes: β-glucosidase, phosphatase and leucine amino peptidase in the rhizosphere of maize using fluorogenically labelled substrates. Spatial-resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography visualized heterogeneity of enzyme activities along the roots. The activity of all enzymes was the highest at the apical parts of individual roots. Across the roots, the enzyme activities were higher at immediate vicinity of the roots (1.5 mm) and gradually decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify spatial distribution of enzyme activities in the rhizosphere hotspots. References Spohn, M., Kuzyakov, Y., 2013. Phosphorus mineralization can be driven by microbial need for carbon. Soil Biology & Biochemistry 61: 69-75

  17. Development of an enzyme-linked immunosorbent assay-based method for measuring galactosyltransferase activity using a synthetic glycopolymer acceptor substrate.

    Science.gov (United States)

    Oubihi, M; Kitajima, K; Kobayashi, K; Adachi, T; Aoki, N; Matsuda, T

    1998-03-15

    A lectin-assisted enzyme-linked immunosorbent assay (ELISA)-based method using a synthetic glycopolymer as an acceptor substrate was developed for measuring beta 1,4-galactosyltransferase (GalT) activity. A polyacrylamide derivative having a beta-linked N-acetylglucosamine (GlcNAc beta) moiety on each monomeric unit was synthesized chemically and immobilized on a polystyrene microtiter plate as an acceptor substrate for GalT. After the plate was incubated with bovine GalT, the enzyme reaction product, beta-linked Gal residue on the polyacrylamide-bound GlcNAc residue, was detected by using Ricinus communis agglutinin 1 (RCA1), rabbit anti-RCA1 antibody, and a peroxidase-labeled anti-rabbit IgG. The lowest GalT concentration detectable by this method was about 0.5 mU/ml, which is comparable to those by the previously reported ELISA-based assays. The unique property of the glycopolymer, PAP(GlcNAc beta), of binding noncovalently but tightly to the polystyrene microtiter plate allowed the use of this acceptor substrate for the GalT activity measurement even in the presence of 1% Triton CF-54 and X-100. Our system was successfully applied to assess GalT activity in milk of various mammals.

  18. Enzymes and Enzyme Activity Encoded by Nonenveloped Viruses.

    Science.gov (United States)

    Azad, Kimi; Banerjee, Manidipa; Johnson, John E

    2017-09-29

    Viruses are obligate intracellular parasites that rely on host cell machineries for their replication and survival. Although viruses tend to make optimal use of the host cell protein repertoire, they need to encode essential enzymatic or effector functions that may not be available or accessible in the host cellular milieu. The enzymes encoded by nonenveloped viruses-a group of viruses that lack any lipid coating or envelope-play vital roles in all the stages of the viral life cycle. This review summarizes the structural, biochemical, and mechanistic information available for several classes of enzymes and autocatalytic activity encoded by nonenveloped viruses. Advances in research and development of antiviral inhibitors targeting specific viral enzymes are also highlighted.

  19. Temperature and UV light affect the activity of marine cell-free enzymes

    Directory of Open Access Journals (Sweden)

    B. Thomson

    2017-09-01

    Full Text Available Microbial extracellular enzymatic activity (EEA is the rate-limiting step in the degradation of organic matter in the oceans. These extracellular enzymes exist in two forms: cell-bound, which are attached to the microbial cell wall, and cell-free, which are completely free of the cell. Contrary to previous understanding, cell-free extracellular enzymes make up a substantial proportion of the total marine EEA. Little is known about these abundant cell-free enzymes, including what factors control their activity once they are away from their sites (cells. Experiments were run to assess how cell-free enzymes (excluding microbes respond to ultraviolet radiation (UVR and temperature manipulations, previously suggested as potential control factors for these enzymes. The experiments were done with New Zealand coastal waters and the enzymes studied were alkaline phosphatase (APase, β-glucosidase, (BGase, and leucine aminopeptidase (LAPase. Environmentally relevant UVR (i.e. in situ UVR levels measured at our site reduced cell-free enzyme activities by up to 87 % when compared to controls, likely a consequence of photodegradation. This effect of UVR on cell-free enzymes differed depending on the UVR fraction. Ambient levels of UV radiation were shown to reduce the activity of cell-free enzymes for the first time. Elevated temperatures (15 °C increased the activity of cell-free enzymes by up to 53 % when compared to controls (10 °C, likely by enhancing the catalytic activity of the enzymes. Our results suggest the importance of both UVR and temperature as control mechanisms for cell-free enzymes. Given the projected warming ocean environment and the variable UVR light regime, it is possible that there could be major changes in the cell-free EEA and in the enzymes contribution to organic matter remineralization in the future.

  20. Patterns of functional enzyme activity in fungus farming ambrosia beetles.

    Science.gov (United States)

    De Fine Licht, Henrik H; Biedermann, Peter H W

    2012-06-06

    In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae), wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow) and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i) 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii) four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4)-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily accessible hemicellulose components of the ray

  1. Enzyme activity assay of glycoprotein enzymes based on a boronate affinity molecularly imprinted 96-well microplate.

    Science.gov (United States)

    Bi, Xiaodong; Liu, Zhen

    2014-12-16

    Enzyme activity assay is an important method in clinical diagnostics. However, conventional enzyme activity assay suffers from apparent interference from the sample matrix. Herein, we present a new format of enzyme activity assay that can effectively eliminate the effects of the sample matrix. The key is a 96-well microplate modified with molecularly imprinted polymer (MIP) prepared according to a newly proposed method called boronate affinity-based oriented surface imprinting. Alkaline phosphatase (ALP), a glycoprotein enzyme that has been routinely used as an indicator for several diseases in clinical tests, was taken as a representative target enzyme. The prepared MIP exhibited strong affinity toward the template enzyme (with a dissociation constant of 10(-10) M) as well as superb tolerance for interference. Thus, the enzyme molecules in a complicated sample matrix could be specifically captured and cleaned up for enzyme activity assay, which eliminated the interference from the sample matrix. On the other hand, because the boronate affinity MIP could well retain the enzymatic activity of glycoprotein enzymes, the enzyme captured by the MIP was directly used for activity assay. Thus, additional assay time and possible enzyme or activity loss due to an enzyme release step required by other methods were avoided. Assay of ALP in human serum was successfully demonstrated, suggesting a promising prospect of the proposed method in real-world applications.

  2. Phytobiotic Utilization as Feed Additive in Feed for Pancreatic Enzyme Activity of Broiler Chicken

    Directory of Open Access Journals (Sweden)

    Sri Purwanti

    2015-09-01

    Full Text Available This research was conducted to evaluate the effect of turmeric water extract, garlic and combination turmeric and garlic as a feed additive in the broiler diet on pancreatic enzyme activity of broiler chicken. Effectivity of treatments was assessed by addition of phytobiotic (control, 015% zinc bacitracin, 2.5% TE, 2.0% GE, 2.5% TGE which were arranged Completely Randomized Design with 4 replications. The variables measured were pancreatic enzyme activity(amylase enzyme activity, protease enzyme activity  and lipase enzyme activity.The results showed that enzyme protein activity content of 2.5% TE supplementation is also high at 82.02 U/ml, then supplemented 2.5% TGE, 2.0% GE, negative control and positive control respectively 75.98 ; 72.02; 68.74; and 66.57 U/ml. The lipase enzyme activity whereas the negative control and a positive control differ significantly higher (P<0.05 to treatment with the addition of 2.5% TE, 2.0% GE and 2.5% TGE phytobiotic. The research concluded that the incorporation of 2.5% TE, 2% GE and combined 2.5% TGE as feed additive enhanced pancreatic enzyme activity.

  3. The ultrasound technology for modifying enzyme activity

    Directory of Open Access Journals (Sweden)

    Meliza Lindsay

    2016-06-01

    Full Text Available Enzymes are protein complexes compounds widely studied and used due to their ability to catalyze reactions. The food processing mainly aims the inactivation of enzymes due to various undesirable effects. However, there are many processes that can be optimized by its catalytic activity. In this context, different technologies have been applied both to inactivate or to improve the enzymes efficiency. The Ultrasound technology emerges as an alternative mainly applied to achieve the enzyme inactivation. On the contrary, very few investigations show the ability of this technology under certain conditions to achieve the opposite effect (i.e. increase the catalytic activity of enzymes. The objective of this study was to correlate the ultrasonic energy delivered to the sample (J/mL with the residual enzymatic activity and explain the possible mechanisms which results in the enzymatic activation/inactivation complex behavior. The activity of POD in coconut water was evaluated as a model. The enzymatic activity initially increased, followed by reduction with a trend to enzyme inactivation. This complex behavior is directly related to the applied ultrasonic energy and their direct mechanical effects on the product, as well as the effect in the enzymatic infinite intermediate states and its structural conformation changes. The obtained results are useful for both academic and industrial perspectives.

  4. Lignin-degrading enzyme activities.

    Science.gov (United States)

    Chen, Yi-ru; Sarkanen, Simo; Wang, Yun-Yan

    2012-01-01

    Over the past three decades, the activities of four kinds of enzyme have been purported to furnish the mechanistic foundations for macromolecular lignin depolymerization in decaying plant cell walls. The pertinent fungal enzymes comprise lignin peroxidase (with a relatively high redox potential), manganese peroxidase, an alkyl aryl etherase, and laccase. The peroxidases and laccase, but not the etherase, are expressed extracellularly by white-rot fungi. A number of these microorganisms exhibit a marked preference toward lignin in their degradation of lignocellulose. Interestingly, some white-rot fungi secrete both kinds of peroxidase but no laccase, while others that are equally effective express extracellular laccase activity but no peroxidases. Actually, none of these enzymes has been reported to possess significant depolymerase activity toward macromolecular lignin substrates that are derived with little chemical modification from the native biopolymer. Here, the assays commonly employed for monitoring the traditional fungal peroxidases, alkyl aryl etherase, and laccase are described in their respective contexts. A soluble native polymeric substrate that can be isolated directly from a conventional milled-wood lignin preparation is characterized in relation to its utility in next-generation lignin-depolymerase assays.

  5. Study on the Correlation between Gene Expression and Enzyme Activity of Seven Key Enzymes and Ginsenoside Content in Ginseng in Over Time in Ji'an, China.

    Science.gov (United States)

    Yin, Juxin; Zhang, Daihui; Zhuang, Jianjian; Huang, Yi; Mu, Ying; Lv, Shaowu

    2017-12-11

    Panax ginseng is a traditional medicine. Fresh ginseng is one of the most important industries related to ginseng development, and fresh ginseng of varying ages has different medicinal properties. Previous research has not systematically reported the correlation between changes in key enzyme activity with changes in ginsenoside content in fresh ginseng over time. In this study, for the first time, we use ginseng samples of varying ages in Ji'an and systematically reported the changes in the activity of seven key enzymes (HMGR, FPS, SS, SE, DS, CYP450, and GT). We investigated the content of ginsenoside and gene expression of these key enzymes. Ginsenoside content was measured using HPLC. HPLC, GC-MS, and LC-MS were combined to measure the enzyme activity of the key enzymes. Quantitative PCR was used in the investigation of gene expression. By analyzing the correlation between the enzyme activity and the transcription level of the key enzymes with ginsenoside content, we found that DS and GT enzyme activities are significantly correlated with the ginsenoside content in different ages of ginseng. Our findings might provide a new strategy to discriminate between ginseng of different years. Meanwhile, this research provides important information for the in-depth study of ginsenoside biosynthesis.

  6. The ultrasound technology for modifying enzyme activity

    Directory of Open Access Journals (Sweden)

    Meliza Lindsay Rojas

    2016-01-01

    Full Text Available Enzymes are protein complexes compounds widely studied and used due to their ability to catalyze reactions. The food processing mainly a ims the inactivation of enzymes due to various undesirable effects. However, there are many processes that can be optimized by its catalytic activity. In this context, different technologies have been applied both to inactivate or to improve the enzymes ef ficiency. The Ultrasound technology emerges as an alternative mainly applied to achieve the enzyme inactivation. On the contrary, very few investigations show the ability of this technology under certain conditions to achieve the opposite effect (i.e. increase the catalytic activity of enzymes. The objective of this study was to correlate the ultrasonic energy delivered to the sample (J/mL with the residual enzymatic activity and explain the possible mechanisms which results in the enzymatic activation/in activation complex behavior. The activity of POD in coconut water was evaluated as a model. The enzymatic activity initially increased, followed by reduction with a trend to enzyme inactivation. This complex behavior is directly related to the applied ultr asonic energy and their direct mechanical effects on the product, as well as the effect in the enzymatic infinite intermediate states and its structural conformation changes. The obtained results are useful for both academic and industrial perspectives.

  7. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

    Directory of Open Access Journals (Sweden)

    Fiona Karen Harlan

    Full Text Available Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research

  8. Enzyme with rhamnogalacturonase activity.

    NARCIS (Netherlands)

    Kofod, L.V.; Andersen, L.N.; Dalboge, H.; Kauppinen, M.S.; Christgau, S.; Heldt-Hansen, H.P.; Christophersen, C.; Nielsen, P.M.; Voragen, A.G.J.; Schols, H.A.

    1998-01-01

    An enzyme exhibiting rhamnogalacturonase activity, capable of cleaving a rhamnogalacturonan backbone in such a manner that galacturonic acids are left as the non-reducing ends, and which exhibits activity on hairy regions from a soy bean material and/or on saponified hairy regions from a sugar beet

  9. Effects of Recurring Droughts on Extracellular Enzyme Activity in Mountain Grassland

    Science.gov (United States)

    Fuchslueger, L.; Bahn, M.; Kienzl, S.; Hofhansl, F.; Schnecker, J.; Richter, A.

    2015-12-01

    Water availability is a key factor for biogeochemical processes and determines microbial activity and functioning, and thereby organic matter decomposition in soils by affecting the osmotic potential, soil pore connectivity, substrate diffusion and nutrient availability. Low water availability during drought periods therefore directly affects microbial activity. Recurring drought periods likely induce shifts in microbial structure that might be reflected in altered responses of microbial turnover of organic matter by extracellular enzymes. To study this we measured a set of potential extracellular enzyme activity rates (cellobiohydrolase CBH; leucine-amino-peptidase LAP; phosphatase PHOS; phenoloxidase POX), in grassland soils that were exposed to extreme experimental droughts during the growing seasons of up to five subsequent years. During the first drought period after eight weeks of rain exclusion all measured potential enzyme activities were significantly decreased. In parallel, soil extractable organic carbon and nitrogen concentrations increased and microbial community structure, determined by phospholipid fatty acid analysis, changed. In soils that were exposed to two and three drought periods only PHOS decreased. After four years of drought again CBH, PHOS and POX decreased, while LAP was unaffected; after five years of drought PHOS and POX decreased and CBH and LAP remained stable. Thus, our results suggest that recurring extreme drought events can cause different responses of extracellular enzyme activities and that the responses change over time. We will discuss whether and to what degree these changes were related to shifts in microbial community composition. However, independent of whether a solitary or a recurrent drought was imposed, in cases when enzyme activity rates were altered during drought, they quickly recovered after rewetting. Overall, our data suggest that microbial functioning in mountain grassland is sensitive to drought, but highly

  10. Patterns of functional enzyme activity in fungus farming ambrosia beetles

    Directory of Open Access Journals (Sweden)

    De Fine Licht Henrik H

    2012-06-01

    Full Text Available Abstract Introduction In wood-dwelling fungus-farming weevils, the so-called ambrosia beetles (Curculionidae: Scolytinae and Platypodinae, wood in the excavated tunnels is used as a medium for cultivating fungi by the combined action of digging larvae (which create more space for the fungi to grow and of adults sowing and pruning the fungus. The beetles are obligately dependent on the fungus that provides essential vitamins, amino acids and sterols. However, to what extent microbial enzymes support fungus farming in ambrosia beetles is unknown. Here we measure (i 13 plant cell-wall degrading enzymes in the fungus garden microbial consortium of the ambrosia beetle Xyleborinus saxesenii, including its primary fungal symbionts, in three compartments of laboratory maintained nests, at different time points after gallery foundation and (ii four specific enzymes that may be either insect or microbially derived in X. saxesenii adult and larval individuals. Results We discovered that the activity of cellulases in ambrosia fungus gardens is relatively small compared to the activities of other cellulolytic enzymes. Enzyme activity in all compartments of the garden was mainly directed towards hemicellulose carbohydrates such as xylan, glucomannan and callose. Hemicellulolytic enzyme activity within the brood chamber increased with gallery age, whereas irrespective of the age of the gallery, the highest overall enzyme activity were detected in the gallery dump material expelled by the beetles. Interestingly endo-β-1,3(4-glucanase activity capable of callose degradation was identified in whole-body extracts of both larvae and adult X. saxesenii, whereas endo-β-1,4-xylanase activity was exclusively detected in larvae. Conclusion Similar to closely related fungi associated with bark beetles in phloem, the microbial symbionts of ambrosia beetles hardly degrade cellulose. Instead, their enzyme activity is directed mainly towards comparatively more easily

  11. Photoperiodism and Enzyme Activity

    Science.gov (United States)

    Queiroz, Orlando; Morel, Claudine

    1974-01-01

    Metabolic readjustments after a change from long days to short days appear, in Kalanchoe blossfeldiana, to be achieved through the operation of two main mechanisms: variation in enzyme capacity, and circadian rhythmicity. After a lag time, capacity in phosphoenolpyruvate carboxylase and capacity in aspartate aminotransferase increase exponentially and appear to be allometrically linked during 50 to 60 short days; then a sudden fall takes place in the activity of the former. Malic enzyme and alanine aminotransferase behave differently. Thus, the operation of the two sections of the pathway (before and after the malate step) give rise to a continuously changing functional compartmentation in the pathway. Circadian rhythmicity, on the other hand, produces time compartmentation through phase shifts and variation in amplitude, independently for each enzyme. These characteristics suggest that the operation of a so-called biological clock would be involved. We propose the hypothesis that feedback regulation would be more accurate and efficient when applied to an already oscillating, clock-controlled enzyme system. PMID:16658749

  12. Product-selective blot: a technique for measuring enzyme activities in large numbers of samples and in native electrophoresis gels

    International Nuclear Information System (INIS)

    Thompson, G.A.; Davies, H.M.; McDonald, N.

    1985-01-01

    A method termed product-selective blotting has been developed for screening large numbers of samples for enzyme activity. The technique is particularly well suited to detection of enzymes in native electrophoresis gels. The principle of the method was demonstrated by blotting samples from glutaminase or glutamate synthase reactions into an agarose gel embedded with ion-exchange resin under conditions favoring binding of product (glutamate) over substrates and other substances in the reaction mixture. After washes to remove these unbound substances, the product was measured using either fluorometric staining or radiometric techniques. Glutaminase activity in native electrophoresis gels was visualized by a related procedure in which substrates and products from reactions run in the electrophoresis gel were blotted directly into a resin-containing image gel. Considering the selective-binding materials available for use in the image gel, along with the possible detection systems, this method has potentially broad application

  13. Effects of misonidazole, irradiation and hyperthermia on lysosomal enzyme activity in mouse tumours

    International Nuclear Information System (INIS)

    Barratt, G.M.; Wills, E.D.

    1981-01-01

    Male C3H mice bearing transplanted tumours were treated with hyperthermia, gamma radiation and the radiosensitising drug misonidazole. The activity of tumour lysosomal acid phosphatase and β-glucuronidase was determined using quantitative cytochemical techniques which measure both lysosomal membrane permeability and enzyme activity. Misonidazole had no effect on the membrane permeability or enzyme activity of tumour lysosomes 1 hr after injection; but 25 hr after the drug treatment the permeability of the lysosomal membrane to the substrate was increased to 1.7 times control. Increases in the lysosomal enzyme activity and membrane permeability were observed 1 hr after combined treatment with misonidazole and irradiation, although neither the drug nor irradiation given alone affected the lysosomes 1 hr after treatment. Twenty-five hours after treatment of tumours with misonidazole given 25 minutes before irradiation of tumours, permeability of the lysosomal membrane had increased to 2.3 times the control. The effects of the irradiation and the radio-sensitisers were thus synergistic. Hyperthermic treatment of tumours increased and misonidazole decreased the lysosomal membrane permeability and enzyme activity measured immediately after exposure. Thus misonidazole and irradiation act synergistically to cause increased lysosomal activity but misonidazole depresses the effect of hyperthermia on lysosomes. (author)

  14. Compounds from silicones alter enzyme activity in curing barnacle glue and model enzymes.

    Science.gov (United States)

    Rittschof, Daniel; Orihuela, Beatriz; Harder, Tilmann; Stafslien, Shane; Chisholm, Bret; Dickinson, Gary H

    2011-02-17

    Attachment strength of fouling organisms on silicone coatings is low. We hypothesized that low attachment strength on silicones is, in part, due to the interaction of surface available components with natural glues. Components could alter curing of glues through bulk changes or specifically through altered enzyme activity. GC-MS analysis of silicone coatings showed surface-available siloxanes when the coatings were gently rubbed with a cotton swab for 15 seconds or given a 30 second rinse with methanol. Mixtures of compounds were found on 2 commercial and 8 model silicone coatings. The hypothesis that silicone components alter glue curing enzymes was tested with curing barnacle glue and with commercial enzymes. In our model, barnacle glue curing involves trypsin-like serine protease(s), which activate enzymes and structural proteins, and a transglutaminase which cross-links glue proteins. Transglutaminase activity was significantly altered upon exposure of curing glue from individual barnacles to silicone eluates. Activity of purified trypsin and, to a greater extent, transglutaminase was significantly altered by relevant concentrations of silicone polymer constituents. Surface-associated silicone compounds can disrupt glue curing and alter enzyme properties. Altered curing of natural glues has potential in fouling management.

  15. Simultaneous measurement of two enzyme activities using infrared spectroscopy: A comparative evaluation of PARAFAC, TUCKER and N-PLS modeling

    DEFF Research Database (Denmark)

    Baum, Andreas; Hansen, Per Waaben; Meyer, Anne S.

    2013-01-01

    multiway methods, namely PARAFAC, TUCKER3 and N-PLS, to establish simultaneous enzyme activity assays for pectin lyase and pectin methyl esterase. Correlation coefficients Rpred2 for prediction test sets are 0.48, 0.96 and 0.96 for pectin lyase and 0.70, 0.89 and 0.89 for pectin methyl esterase......Enzymes are used in many processes to release fermentable sugars for green production of biofuel, or the refinery of biomass for extraction of functional food ingredients such as pectin or prebiotic oligosaccharides. The complex biomasses may, however, require a multitude of specific enzymes which...... are active on specific substrates generating a multitude of products. In this paper we use the plant polymer, pectin, to present a method to quantify enzyme activity of two pectolytic enzymes by monitoring their superimposed spectral evolutions simultaneously. The data is analyzed by three chemometric...

  16. Determination of the activity signature of key carbohydrate metabolism enzymes in phenolic-rich grapevine tissues

    DEFF Research Database (Denmark)

    Covington, Elizabeth Dunn; Roitsch, Thomas Georg; Dermastia, Marina

    2016-01-01

    Physiological studies in plants often require enzyme extraction from tissues containing high concentrations of phenols and polyphenols. Unless removed or neutralized, such compounds may hinder extraction, inactivate enzymes, and interfere with enzyme detection. The following protocol for activity...... assays for enzymes of primary carbohydrate metabolism, while based on our recently published one for quantitative measurement of activities using coupled spectrophotometric assays in a 96-well format, is tailored to the complexities of phenolic- and anthocyanin-rich extracts from grapevine leaf...

  17. Enzyme activity and kinetics in substrate-amended river sediments

    Energy Technology Data Exchange (ETDEWEB)

    Duddridge, J E; Wainwright, M

    1982-01-01

    In determining the effects of heavy metals in microbial activity and litter degradation in river sediments, one approach is to determine the effects of these pollutants on sediment enzyme activity and synthesis. Methods to assay amylase, cellulase and urease activity in diverse river sediments are reported. Enzyme activity was low in non-amended sediments, but increased markedly when the appropriate substrate was added, paralleling both athropogenic and natural amendment. Linear relationships between enzyme activity, length of incubation, sample size and substrate concentration were established. Sediment enzyme activity generally obeyed Michaelis-Menton kinetics, but of the three enzymes, urease gave least significant correlation coefficients when the data for substrate concentration versus activity was applied to the Eadie-Hofstee transformation of the Michaelis-Menten equation. K/sub m/ and V/sub max/ for amylase, cellulase and urease in sediments are reported. (JMT)

  18. Enzyme Activity Experiments Using a Simple Spectrophotometer

    Science.gov (United States)

    Hurlbut, Jeffrey A.; And Others

    1977-01-01

    Experimental procedures for studying enzyme activity using a Spectronic 20 spectrophotometer are described. The experiments demonstrate the effect of pH, temperature, and inhibitors on enzyme activity and allow the determination of Km, Vmax, and Kcat. These procedures are designed for teaching large lower-level biochemistry classes. (MR)

  19. Activation of interfacial enzymes at membrane surfaces

    DEFF Research Database (Denmark)

    Mouritsen, Ole G.; Andresen, Thomas Lars; Halperin, Avi

    2006-01-01

    A host of water-soluble enzymes are active at membrane surfaces and in association with membranes. Some of these enzymes are involved in signalling and in modification and remodelling of the membranes. A special class of enzymes, the phospholipases, and in particular secretory phospholipase A2 (s...

  20. Activity assessment of microbial fibrinolytic enzymes.

    Science.gov (United States)

    Kotb, Essam

    2013-08-01

    Conversion of fibrinogen to fibrin inside blood vessels results in thrombosis, leading to myocardial infarction and other cardiovascular diseases. In general, there are four therapy options: surgical operation, intake of antiplatelets, anticoagulants, or fibrinolytic enzymes. Microbial fibrinolytic enzymes have attracted much more attention than typical thrombolytic agents because of the expensive prices and the side effects of the latter. The fibrinolytic enzymes were successively discovered from different microorganisms, the most important among which is the genus Bacillus. Microbial fibrinolytic enzymes, especially those from food-grade microorganisms, have the potential to be developed as functional food additives and drugs to prevent or cure thrombosis and other related diseases. There are several assay methods for these enzymes; this may due to the insolubility of substrate, fibrin. Existing assay methods can be divided into three major groups. The first group consists of assay of fibrinolytic activity with natural proteins as substrates, e.g., fibrin plate methods. The second and third groups of assays are suitable for kinetic studies and are based on the determination of hydrolysis of synthetic peptide esters. This review will deal primarily with the microorganisms that have been reported in literature to produce fibrinolytic enzymes and the first review discussing the methods used to assay the fibrinolytic activity.

  1. Changes in activities of tissues enzymes in rats administered Ficus ...

    African Journals Online (AJOL)

    This study evaluates the effects of methanolic extract of Ficus exasperata leaf on the ... measuring the levels of some key enzymes in ... powder using an electrical blender. .... of the cells at these doses. .... activities and acute toxicity of a stem.

  2. Adsorption and enzyme activity of asparaginase at lipid Langmuir and Langmuir-Blodgett films

    International Nuclear Information System (INIS)

    Rocha Junior, Carlos da; Caseli, Luciano

    2017-01-01

    In this present work, the surface activity of the enzyme asparaginase was investigated at the air-water interface, presenting surface activity in high ionic strengths. Asparaginase was incorporated in Langmuir monolayers of the phospholipid dipalmitoylphosphatidylcholine (DPPC), forming a mixed film, which was characterized with surface pressure-area isotherms, surface potential-area isotherms, polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS), and Brewster angle microscopy (BAM). The adsorption of the enzyme at the air-water interface condensed the lipid monolayer and increased the film compressibility at high surface pressures. Amide bands in the PM-IRRAS spectra were identified, with the C−N and C =O dipole moments lying parallel to monolayer plane, revealing the structuring of the enzyme into α-helices and β-sheets. The floating monolayers were transferred to solid supports as Langmuir-Blodgett (LB) films and characterized with fluorescence spectroscopy and atomic force microscopy. Catalytic activities of the films were measured and compared to the homogenous medium. The enzyme accommodated in the LB films preserved more than 78% of the enzyme activity after 30 days, in contrast for the homogeneous medium, which preserved less than 13%. The method presented in this work not only allows for an enhanced catalytic activity, but also can help explain why certain film architectures exhibit better performance. - Highlights: • Biomembranes are mimicked with Langmuir monolayers. • Asparaginase is incorporated into the lipid monolayer. • Enzyme adsorption is confirmed with tensiometry and infrared spectroscopy. • Langmuir-Blodgett films of the enzyme present enzyme activity.

  3. Adsorption and enzyme activity of asparaginase at lipid Langmuir and Langmuir-Blodgett films

    Energy Technology Data Exchange (ETDEWEB)

    Rocha Junior, Carlos da; Caseli, Luciano, E-mail: lcaseli@unifesp.br

    2017-04-01

    In this present work, the surface activity of the enzyme asparaginase was investigated at the air-water interface, presenting surface activity in high ionic strengths. Asparaginase was incorporated in Langmuir monolayers of the phospholipid dipalmitoylphosphatidylcholine (DPPC), forming a mixed film, which was characterized with surface pressure-area isotherms, surface potential-area isotherms, polarization-modulated infrared reflection-absorption spectroscopy (PM-IRRAS), and Brewster angle microscopy (BAM). The adsorption of the enzyme at the air-water interface condensed the lipid monolayer and increased the film compressibility at high surface pressures. Amide bands in the PM-IRRAS spectra were identified, with the C−N and C =O dipole moments lying parallel to monolayer plane, revealing the structuring of the enzyme into α-helices and β-sheets. The floating monolayers were transferred to solid supports as Langmuir-Blodgett (LB) films and characterized with fluorescence spectroscopy and atomic force microscopy. Catalytic activities of the films were measured and compared to the homogenous medium. The enzyme accommodated in the LB films preserved more than 78% of the enzyme activity after 30 days, in contrast for the homogeneous medium, which preserved less than 13%. The method presented in this work not only allows for an enhanced catalytic activity, but also can help explain why certain film architectures exhibit better performance. - Highlights: • Biomembranes are mimicked with Langmuir monolayers. • Asparaginase is incorporated into the lipid monolayer. • Enzyme adsorption is confirmed with tensiometry and infrared spectroscopy. • Langmuir-Blodgett films of the enzyme present enzyme activity.

  4. Detection of enzyme activity in decontaminated spices of industrial use

    International Nuclear Information System (INIS)

    Müller, R.; Theobald, R.

    1995-01-01

    A range of decontaminated spices of industrial use have been examinated for their enzymes (catalase, peroxidase, amylase, lipase activity). The genuine enzymes remain fully active in irradiated spices, whereas the microbial load is clearly reduced. In contrast steam treated spices no longer demonstrate enzyme activities. Steam treatment offers e.g. black pepper without lipase activity, which can no longer cause fat deterioration. Low microbial load in combination with clearly detectable enzyme activity in spices is an indication for irradiation, whereas, reduced microbial contamination combined with enzyme inactivation indicate steam treatment of raw material [de

  5. Evaluation of the organophosphorus hydrolase enzyme activity in creams and investigation of its stability

    Directory of Open Access Journals (Sweden)

    Mariye Rajaie

    2016-06-01

    Full Text Available The main purpose of this project is investigation of the organophosphorus hydrolase (OPH enzyme activity in water in oil (w/o and oil in water (o/w creams and investigation of the OPH enzyme stability in formulated creams. OPH enzyme was extracted and purified from strain flavobacterium. The w/o and o/w creams were prepared using different formulations. In order to achieve an emulsion with maximum stability, appropriate percentage of the cream components was selected by studying different formulations and the physical and chemical stability of the produced cream were considered. 5Uenzyme/90gcream enzyme was used for each formulation. To measure the enzyme activity in creams, extraction method was used and enzyme activity was determined based on parathion hydrolysis. The thermal stability of OPH in both types of w/o and o/w creams was studied at 4 and 30  °C for various time periods. The average enzyme activity was about 0.0065 U/gcream and 0.018 U/gcream for w/o and o/w creams respectivly. According to the results, the relative activity at 4 °C was reduced to 50% after 26 and 45 days in w/o and o/w creams, respectivly. The results showed that the OPH enzyme activity in o/w cream was 2.6 times more than that of w/o cream, because of the higher hydrophobicity of o/w cream compared to w/o. The OPH enzyme stability in o/w cream was greater in comparison to w/o cream. The OPH enzyme was active for nearly 2 months on o/w creams at 4 °C .

  6. Direct measurement of catalase activity in living cells and tissue biopsies

    International Nuclear Information System (INIS)

    Scaglione, Christine N.; Xu, Qijin; Ramanujan, V. Krishnan

    2016-01-01

    Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear

  7. Direct measurement of catalase activity in living cells and tissue biopsies

    Energy Technology Data Exchange (ETDEWEB)

    Scaglione, Christine N.; Xu, Qijin; Ramanujan, V. Krishnan, E-mail: Ramanujanv@csmc.edu

    2016-01-29

    Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies – can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. - Highlights: • A novel, direct measurement of Catalase enzyme activity via, oxygen sensing method. • Steady-stateprofiles of Catalase activity follow the Michaelis-Menten Kinetics. • Catalase-specific activity demonstrated using genetic and pharmacological tools. • Overcomes limitations of spectroscopic methods and indirect calorimetric approaches. • Clear

  8. [Effects of altitudes on soil microbial biomass and enzyme activity in alpine-gorge regions.

    Science.gov (United States)

    Cao, Rui; Wu, Fu Zhong; Yang, Wan Qin; Xu, Zhen Feng; Tani, Bo; Wang, Bin; Li, Jun; Chang, Chen Hui

    2016-04-22

    In order to understand the variations of soil microbial biomass and soil enzyme activities with the change of altitude, a field incubation was conducted in dry valley, ecotone between dry valley and mountain forest, subalpine coniferous forest, alpine forest and alpine meadow from 1563 m to 3994 m of altitude in the alpine-gorge region of western Sichuan. The microbial biomass carbon and nitrogen, and the activities of invertase, urease and acid phosphorus were measured in both soil organic layer and mineral soil layer. Both the soil microbial biomass and soil enzyme activities showed the similar tendency in soil organic layer. They increased from 2158 m to 3028 m, then decreased to the lowest value at 3593 m, and thereafter increased until 3994 m in the alpine-gorge region. In contrast, the soil microbial biomass and soil enzyme activities in mineral soil layer showed the trends as, the subalpine forest at 3028 m > alpine meadow at 3994 m > montane forest ecotone at 2158 m > alpine forest at 3593 m > dry valley at 1563 m. Regardless of altitudes, soil microbial biomass and soil enzyme activities were significantly higher in soil organic layer than in mineral soil layer. The soil microbial biomass was significantly positively correlated with the activities of the measured soil enzymes. Moreover, both the soil microbial biomass and soil enzyme activities were significantly positively correlated with soil water content, organic carbon, and total nitrogen. The activity of soil invertase was significantly positively correlated with soil phosphorus content, and the soil acid phosphatase was so with soil phosphorus content and soil temperature. In brief, changes in vegetation and other environmental factors resulting from altitude change might have strong effects on soil biochemical properties in the alpine-gorge region.

  9. Effect of irradiation on lysosomal enzyme activation in cultured macrophages

    International Nuclear Information System (INIS)

    Clarke, C.; Wills, E.D.

    1980-01-01

    The effect of γrays on lysosomal enzyme activity of normal and immune macrophages of DBA/2 mice cultured in vitro has been studied. A dose of 500 rad did not significantly affect lysosomal enzyme activity 3 hours after irradiation but caused the activity to increase to 1.4 times the control value 22.5 hours after irradiation. 22.5 hours after a dose of 3000 rad the enzyme activity increased to 2.5 times the control. Lysosomal enzyme activity of the macrophages was also markedly increased by immunization of the mice with D lymphoma cells, before culture in vitro, but irradiation of these cells with a dose of 500 rad caused a further increase in lysosomal enzyme activity. The results indicate that immunization and irradiation both cause stimulation of lysosomal enzyme activity in macrophages but that the mechanisms of activation are unlikely to be identical. (author)

  10. Enzyme specific activity in functionalized nanoporous supports

    International Nuclear Information System (INIS)

    Lei Chenghong; Soares, Thereza A; Shin, Yongsoon; Liu Jun; Ackerman, Eric J

    2008-01-01

    Here we reveal that enzyme specific activity can be increased substantially by changing the protein loading density (P LD ) in functionalized nanoporous supports so that the enzyme immobilization efficiency (I e , defined as the ratio of the specific activity of the immobilized enzyme to the specific activity of the free enzyme in solution) can be much higher than 100%. A net negatively charged glucose oxidase (GOX) and a net positively charged organophosphorus hydrolase (OPH) were entrapped spontaneously in NH 2 - and HOOC-functionalized mesoporous silica (300 A, FMS) respectively. The specific activity of GOX entrapped in FMS increased with decreasing P LD . With decreasing P LD , I e of GOX in FMS increased from 150%. Unlike GOX, OPH in HOOC-FMS showed increased specific activity with increasing P LD . With increasing P LD , the corresponding I e of OPH in FMS increased from 100% to>200%. A protein structure-based analysis of the protein surface charges directing the electrostatic interaction-based orientation of the protein molecules in FMS demonstrates that substrate access to GOX molecules in FMS is limited at high P LD , consequently lowering the GOX specific activity. In contrast, substrate access to OPH molecules in FMS remains open at high P LD and may promote a more favorable confinement environment that enhances the OPH activity

  11. Simultaneous measurement of two enzyme activities using infrared spectroscopy: A comparative evaluation of PARAFAC, TUCKER and N-PLS modeling.

    Science.gov (United States)

    Baum, Andreas; Hansen, Per Waaben; Meyer, Anne S; Mikkelsen, Jørn Dalgaard

    2013-08-06

    Enzymes are used in many processes to release fermentable sugars for green production of biofuel, or the refinery of biomass for extraction of functional food ingredients such as pectin or prebiotic oligosaccharides. The complex biomasses may, however, require a multitude of specific enzymes which are active on specific substrates generating a multitude of products. In this paper we use the plant polymer, pectin, to present a method to quantify enzyme activity of two pectolytic enzymes by monitoring their superimposed spectral evolutions simultaneously. The data is analyzed by three chemometric multiway methods, namely PARAFAC, TUCKER3 and N-PLS, to establish simultaneous enzyme activity assays for pectin lyase and pectin methyl esterase. Correlation coefficients Rpred(2) for prediction test sets are 0.48, 0.96 and 0.96 for pectin lyase and 0.70, 0.89 and 0.89 for pectin methyl esterase, respectively. The retrieved models are compared and prediction test sets show that especially TUCKER3 performs well, even in comparison to the supervised regression method N-PLS. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. The effect of hyperthermia and radiation on lysosomal enzyme activity of mouse mammary tumours

    International Nuclear Information System (INIS)

    Barratt, G.M.; Wills, E.D.

    1979-01-01

    The effects of hyperthermia and radiation have been studied on the acid phosphatase and β-glucuronidase activities in lysosomes of C3H mice mammary tumours and of the spleen. Quantitative histochemical methods have been used. Hyperthermic treatment of both spontaneous and transplanted tumours caused an increase in the activity of both acid phosphatase and β-glucuronase when measured immediately after treatment, but the activities returned to normal after 24 hours. In contrast a radiation dose of 3500 rad did not cause an increase in activity of either enzyme immediately, but a large activation was observed after 24 hr. Combination of hyperthermic and radiation treatment caused increases in enzyme activities which were dependent on the time after treatment. Hyperthermic treatment of the lower body of mice bearing tumours also caused activation of lysosomal enzymes in the spleen. This may be hormone mediated. It is considered that the increased lysosomal enzyme activity observed after hyperthermia may be a consequence of increased permeability of the lysosomal membrane caused by hyperthermia. (author)

  13. EVOLUTIONARY TRANSITIONS IN ENZYME ACTIVITY OF ANT FUNGUS GARDENS

    DEFF Research Database (Denmark)

    De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G

    2010-01-01

    an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across...... the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens...... are targeted primarily towards partial degradation of plant cell walls, reflecting a plesiomorphic state of non-domesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major...

  14. Milk enzyme activities and subclinical mastitis among women in Guinea-Bissau

    DEFF Research Database (Denmark)

    Rasmussen, Lill Brith Wium; Hartvig, Ditte Luise; Kæstel, Pernille

    2008-01-01

    research as indicators of SCM, udder health, and milk quality. Study Design: To investigate if milk enzyme activities and the inflammatory interleukin 8 (IL-8) level are increased in women with SCM, we measured sodium, potassium, NAGase, LDH, AcP, AP, and IL-8 in breastmilk samples collected at 2 months......Background: Subclinical mastititis (SCM) is a condition with raised milk concentration of sodium and milk immune factors. The milk enzymes N-acetyl-β-D-glucosaminidase (NAGase), lactate dehydrogenase (LDH), acid phosphatase (AcP), and alkaline phosphatase (AP) have attracted attention in dairy...... in univariate linear regression (p enzymes and IL-8). Conclusions: A positive association between the Na/K ratio and the breastmilk enzymes NAGase, LDH, AcP, and AP was found. Breastmilk enzymes have not previously been investigated in relation to SCM in women, and further...

  15. Detection of Extracellular Enzyme Activities in Ganoderma neo-japonicum

    OpenAIRE

    Jo, Woo-Sik; Park, Ha-Na; Cho, Doo-Hyun; Yoo, Young-Bok; Park, Seung-Chun

    2011-01-01

    The ability of Ganoderma to produce extracellular enzymes, including β-glucosidase, cellulase, avicelase, pectinase, xylanase, protease, amylase, and ligninase was tested in chromogenic media. β-glucosidase showed the highest activity, among the eight tested enzymes. In particular, Ganoderma neo-japonicum showed significantly stronger activity for β-glucosidase than that of the other enzymes. Two Ganoderma lucidum isolates showed moderate activity for avicelase; however, Ganoderma neo-japonic...

  16. An appraisal of the enzyme stability-activity trade-off.

    Science.gov (United States)

    Miller, Scott R

    2017-07-01

    A longstanding idea in evolutionary physiology is that an enzyme cannot jointly optimize performance at both high and low temperatures due to a trade-off between stability and activity. Although a stability-activity trade-off has been observed for well-characterized examples, such a trade-off is not imposed by any physical chemical constraint. To better understand the pervasiveness of this trade-off, I investigated the stability-activity relationship for comparative biochemical studies of purified orthologous enzymes identified by a literature search. The nature of this relationship varied greatly among studies. Notably, studies of enzymes with low mean synonymous nucleotide sequence divergence were less likely to exhibit the predicted negative correlation between stability and activity. Similarly, a survey of directed evolution investigations of the stability-activity relationship indicated that these traits are often uncoupled among nearly identical yet phenotypically divergent enzymes. This suggests that the presumptive trade-off often reported for investigations of enzymes with high mean sequence divergence may in some cases instead be a consequence of the degeneration over time of enzyme function in unselected environments, rather than a direct effect of thermal adaptation. The results caution against the general assertion of a stability-activity trade-off during enzyme adaptation. © 2017 The Author(s). Evolution © 2017 The Society for the Study of Evolution.

  17. IFCC primary reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C. Part 9: reference procedure for the measurement of catalytic concentration of alkaline phosphatase International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Scientific Division, Committee on Reference Systems of Enzymes (C-RSE) (1)).

    Science.gov (United States)

    Schumann, Gerhard; Klauke, Rainer; Canalias, Francesca; Bossert-Reuther, Steffen; Franck, Paul F H; Gella, F-Javier; Jørgensen, Poul J; Kang, Dongchon; Lessinger, Jean-Marc; Panteghini, Mauro; Ceriotti, Ferruccio

    2011-09-01

    Abstract This paper is the ninth in a series dealing with reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C and the certification of reference preparations. Other parts deal with: Part 1. The concept of reference procedures for the measurement of catalytic activity concentrations of enzymes; Part 2. Reference procedure for the measurement of catalytic concentration of creatine kinase; Part 3. Reference procedure for the measurement of catalytic concentration of lactate dehydrogenase; Part 4. Reference procedure for the measurement of catalytic concentration of alanine aminotransferase; Part 5. Reference procedure for the measurement of catalytic concentration of aspartate aminotransferase; Part 6. Reference procedure for the measurement of catalytic concentration of γ-glutamyltransferase; Part 7. Certification of four reference materials for the determination of enzymatic activity of γ-glutamyltransferase, lactate dehydrogenase, alanine aminotransferase and creatine kinase at 37 °C; Part 8. Reference procedure for the measurement of catalytic concentration of α-amylase. The procedure described here is derived from the previously described 30 °C IFCC reference method. Differences are tabulated and commented on in Appendix 1.

  18. A comparison of maximal bioenergetic enzyme activities obtained with commonly used homogenization techniques.

    Science.gov (United States)

    Grace, M; Fletcher, L; Powers, S K; Hughes, M; Coombes, J

    1996-12-01

    Homogenization of tissue for analysis of bioenergetic enzyme activities is a common practice in studies examining metabolic properties of skeletal muscle adaptation to disease, aging, inactivity or exercise. While numerous homogenization techniques are in use today, limited information exists concerning the efficacy of specific homogenization protocols. Therefore, the purpose of this study was to compare the efficacy of four commonly used approaches to homogenizing skeletal muscle for analysis of bioenergetic enzyme activity. The maximal enzyme activity (Vmax) of citrate synthase (CS) and lactate dehydrogenase (LDH) were measured from homogenous muscle samples (N = 48 per homogenization technique) and used as indicators to determine which protocol had the highest efficacy. The homogenization techniques were: (1) glass-on-glass pestle; (2) a combination of a mechanical blender and a teflon pestle (Potter-Elvehjem); (3) a combination of the mechanical blender and a biological detergent; and (4) the combined use of a mechanical blender and a sonicator. The glass-on-glass pestle homogenization protocol produced significantly higher (P pestle homogenization protocol is the technique of choice for studying bioenergetic enzyme activity in skeletal muscle.

  19. High inorganic triphosphatase activities in bacteria and mammalian cells: identification of the enzymes involved.

    Directory of Open Access Journals (Sweden)

    Gregory Kohn

    Full Text Available BACKGROUND: We recently characterized a specific inorganic triphosphatase (PPPase from Nitrosomonas europaea. This enzyme belongs to the CYTH superfamily of proteins. Many bacterial members of this family are annotated as predicted adenylate cyclases, because one of the founding members is CyaB adenylate cyclase from A. hydrophila. The aim of the present study is to determine whether other members of the CYTH protein family also have a PPPase activity, if there are PPPase activities in animal tissues and what enzymes are responsible for these activities. METHODOLOGY/PRINCIPAL FINDINGS: Recombinant enzymes were expressed and purified as GST- or His-tagged fusion proteins and the enzyme activities were determined by measuring the release of inorganic phosphate. We show that the hitherto uncharacterized E. coli CYTH protein ygiF is a specific PPPase, but it contributes only marginally to the total PPPase activity in this organism, where the main enzyme responsible for hydrolysis of inorganic triphosphate (PPP(i is inorganic pyrophosphatase. We further show that CyaB hydrolyzes PPP(i but this activity is low compared to its adenylate cyclase activity. Finally we demonstrate a high PPPase activity in mammalian and quail tissue, particularly in the brain. We show that this activity is mainly due to Prune, an exopolyphosphatase overexpressed in metastatic tumors where it promotes cell motility. CONCLUSIONS AND GENERAL SIGNIFICANCE: We show for the first time that PPPase activities are widespread in bacteria and animals. We identified the enzymes responsible for these activities but we were unable to detect significant amounts of PPP(i in E. coli or brain extracts using ion chromatography and capillary electrophoresis. The role of these enzymes may be to hydrolyze PPP(i, which could be cytotoxic because of its high affinity for Ca(2+, thereby interfering with Ca(2+ signaling.

  20. Direct measurement of catalase activity in living cells and tissue biopsies.

    Science.gov (United States)

    Scaglione, Christine N; Xu, Qijin; Ramanujan, V Krishnan

    2016-01-29

    Spatiotemporal regulation of enzyme-substrate interactions governs the decision-making steps in biological systems. Enzymes, being functional units of every living cell, contribute to the macromolecular stability of cell survival, proliferation and hence are vital windows to unraveling the biological complexity. Experimental measurements capturing this dynamics of enzyme-substrate interactions in real time add value to this understanding. Furthermore these measurements, upon validation in realistic biological specimens such as clinical biopsies - can further improve our capability in disease diagnostics and treatment monitoring. Towards this direction, we describe here a novel, high-sensitive measurement system for measuring diffusion-limited enzyme-substrate kinetics in real time. Using catalase (enzyme) and hydrogen peroxide (substrate) as the example pair, we demonstrate that this system is capable of direct measurement of catalase activity in vitro and the measured kinetics follows the classical Michaelis-Menten reaction kinetics. We further demonstrate the system performance by measuring catalase activity in living cells and in very small amounts of liver biopsies (down to 1 μg total protein). Catalase-specific enzyme activity is demonstrated by genetic and pharmacological tools. Finally we show the clinically-relevant diagnostic capability of our system by comparing the catalase activities in liver biopsies from young and old mouse (liver and serum) samples. We discuss the potential applicability of this system in clinical diagnostics as well as in intraoperative surgical settings. Copyright © 2016 Elsevier Inc. All rights reserved.

  1. General discussion about enzymes activities of radiation injury

    International Nuclear Information System (INIS)

    Vucicevic, M.; Sukalo, I.

    1989-01-01

    Researching reliable and practical indicators of radiation injury, however, is very interesting and considerable department of scientific studies, practical and theoretical. Enzymes activities are among biochemical indicators which are changed after radiation injury. Activity of these specific proteins is important in regulation of every biochemical reaction in existing beings. Biological macromolecules can be damaged by radiation or the cell permeability can be changed. All of these influence directly on enzymes activities. In this paper we present the review of the all important enzymes, indicators of the radiation injury, which variances on reference to normal values are significant of the functional and the structural changes of essential organs (author)

  2. General discussion about enzymes activities of radiation injury

    Energy Technology Data Exchange (ETDEWEB)

    Vucicevic, M; Sukalo, I [Institute of Nuclear Sciences Boris Kidric, Vinca, Beograd (Serbia and Montenegro)

    1989-07-01

    Researching reliable and practical indicators of radiation injury, however, is very interesting and considerable department of scientific studies, practical and theoretical. Enzymes activities are among biochemical indicators which are changed after radiation injury. Activity of these specific proteins is important in regulation of every biochemical reaction in existing beings. Biological macromolecules can be damaged by radiation or the cell permeability can be changed. All of these influence directly on enzymes activities. In this paper we present the review of the all important enzymes, indicators of the radiation injury, which variances on reference to normal values are significant of the functional and the structural changes of essential organs (author)

  3. Actinomycete enzymes and activities involved in straw saccharification

    Energy Technology Data Exchange (ETDEWEB)

    McCarthy, A J; Ball, A S [Liverpool Univ. (UK). Dept. of Genetics and Microbiology

    1990-01-01

    This research programme has been directed towards the analysis of actinomycete enzyme systems involved in the degradation of plant biomass (lignocellulose.) The programme was innovative in that a novel source of enzymes was systematically screened and wheat straw saccharifying activity was the test criterion. Over 200 actinomycete strains representing a broad taxonomic range were screened. A range of specific enzyme activities were involved and included cellulase, xylanase, arabinofuranosidase, acetylesterase, {beta}-xylosidase and {beta}-glucosidase. Since hemicellulose (arabinoxylan) was the primary source of sugar, xylanases were characterized. The xylan-degrading systems of actinomycetes were complex and nonuniform, with up to six separate endoxylanases identified in active strains. Except for microbispora bispora, actinomycetes were found to be a poor source of cellulase activity. Evidence for activity against the lignin fraction of straw was produced for a range of actinomycete strains. While modification reactions were common, cleavage of inter-monomer bonds, and utilization of complex polyphenolic compounds were restricted to two strains: Thermomonospora mesophila and Streptomyces badius. Crude enzyme preparations from actinomycetes can be used to generate sugar, particularly pentoses, directly from cereal straw. The potential for improvements in yield rests with the formulation to cooperative enzyme combinations from different strains. The stability properties of enzymes from thermophilic strains and the general neutral to alkali pH optima offer advantages in certain process situations. Actinomycetes are a particularly rich source of xylanases for commercial application and can rapidly solubilise a lignocarbohydrate fraction of straw which may have both product and pretreatment potential. 31 refs., 4 figs., 5 tabs.

  4. Thermodynamic Activity-Based Progress Curve Analysis in Enzyme Kinetics.

    Science.gov (United States)

    Pleiss, Jürgen

    2018-03-01

    Macrokinetic Michaelis-Menten models based on thermodynamic activity provide insights into enzyme kinetics because they separate substrate-enzyme from substrate-solvent interactions. Kinetic parameters are estimated from experimental progress curves of enzyme-catalyzed reactions. Three pitfalls are discussed: deviations between thermodynamic and concentration-based models, product effects on the substrate activity coefficient, and product inhibition. Copyright © 2017 Elsevier Ltd. All rights reserved.

  5. Immobilized enzyme reactor chromatography: Optimization of protein retention and enzyme activity in monolithic silica stationary phases

    International Nuclear Information System (INIS)

    Besanger, Travis R.; Hodgson, Richard J.; Green, James R.A.; Brennan, John D.

    2006-01-01

    Our group recently reported on the application of protein-doped monolithic silica columns for immobilized enzyme reactor chromatography, which allowed screening of enzyme inhibitors present in mixtures using mass spectrometry for detection. The enzyme was immobilized by entrapment within a bimodal meso/macroporous silica material prepared by a biocompatible sol-gel processing route. While such columns proved to be useful for applications such as screening of protein-ligand interactions, significant amounts of entrapped proteins leached from the columns owing to the high proportion of macropores within the materials. Herein, we describe a detailed study of factors affecting the morphology of protein-doped bioaffinity columns and demonstrate that specific pH values and concentrations of poly(ethylene glycol) can be used to prepare essentially mesoporous columns that retain over 80% of initially loaded enzyme in an active and accessible form and yet still retain sufficient porosity to allow pressure-driven flow in the low μL/min range. Using the enzyme γ-glutamyl transpeptidase (γ-GT), we further evaluated the catalytic constants of the enzyme entrapped in capillary columns with different silica morphologies as a function of flowrate and backpressure using the enzyme reactor assay mode. It was found that the apparent activity of the enzyme was highest in mesoporous columns that retained high levels of enzyme. In such columns, enzyme activity increased by ∼2-fold with increases in both flowrate (from 250 to 1000 nL/min) and backpressure generated (from 500 to 2100 psi) during the chromatographic activity assay owing to increases in k cat and decreases in K M , switching from diffusion controlled to reaction controlled conditions at ca. 2000 psi. These results suggest that columns with minimal macropore volumes (<5%) are advantageous for the entrapment of soluble proteins for bioaffinity and bioreactor chromatography

  6. Inhibition of existing denitrification enzyme activity by chloramphenicol

    Science.gov (United States)

    Brooks, M.H.; Smith, R.L.; Macalady, D.L.

    1992-01-01

    Chloramphenicol completely inhibited the activity of existing denitrification enzymes in acetylene-block incubations with (i) sediments from a nitrate-contaminated aquifer and (ii) a continuous culture of denitrifying groundwater bacteria. Control flasks with no antibiotic produced significant amounts of nitrous oxide in the same time period. Amendment with chloramphenicol after nitrous oxide production had begun resulted in a significant decrease in the rate of nitrous oxide production. Chloramphenicol also decreased (>50%) the activity of existing denitrification enzymes in pure cultures of Pseudomonas denitrificans that were harvested during log- phase growth and maintained for 2 weeks in a starvation medium lacking electron donor. Short-term time courses of nitrate consumption and nitrous oxide production in the presence of acetylene with P. denitrificans undergoing carbon starvation were performed under optimal conditions designed to mimic denitrification enzyme activity assays used with soils. Time courses were linear for both chloramphenicol and control flasks, and rate estimates for the two treatments were significantly different at the 95% confidence level. Complete or partial inhibition of existing enzyme activity is not consistent with the current understanding of the mode of action of chloramphenicol or current practice, in which the compound is frequently employed to inhibit de novo protein synthesis during the course of microbial activity assays. The results of this study demonstrate that chloramphenicol amendment can inhibit the activity of existing denitrification enzymes and suggest that caution is needed in the design and interpretation of denitrification activity assays in which chloramphenicol is used to prevent new protein synthesis.

  7. Modification of polymer surfaces to enhance enzyme activity and stability

    DEFF Research Database (Denmark)

    Hoffmann, Christian

    Enzyme immobilization is an important concept for the development of improved biocatalytic processes, primarily through facilitated separation procedures. However, enzyme immobilization usually comes at a price of reduced biocatalytic activity. For this reason, different immobilization methods have...... already been developed, combining the same goal to improve enzyme activity, stability and selectivity. Polymer materials have shown, due to their easy processibility and versatile properties, high potential as enzyme support. However, in order to achieve improved enzyme performance, the combination...... on their tailored surface modification in order to obtain improved enzyme-support systems. Firstly, an off-stoichiometric thiol-ene (OSTE) thermosetting material was used for the development of a screening platform allowing the investigation of micro-environmental effects and their impact on the activity...

  8. Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan.

    Science.gov (United States)

    Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz Ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

    2015-01-01

    This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract.

  9. Determination of lytic enzyme activities of indigenous Trichoderma isolates from Pakistan

    Science.gov (United States)

    Asad, Saeed Ahmad; Tabassum, Ayesha; Hameed, Abdul; Hassan, Fayyaz ul; Afzal, Aftab; Khan, Sabaz Ali; Ahmed, Rafiq; Shahzad, Muhammad

    2015-01-01

    Abstract This study investigated lytic enzyme activities in three indigenous Trichoderma strains namely, Trichoderma asperellum, Trichoderma harzianum and Trichoderma sp. Native Trichoderma strains and a virulent strain of Rhizoctonia solani isolated from infected bean plants were also included in the study. Enzyme activities were determined by measuring sugar reduction by dinitrosalicylic acid (DNS) method using suitable substrates. The antagonists were cultured in minimal salt medium with the following modifications: medium A (1 g of glucose), medium B (0.5 g of glucose + 0.5 g of deactivated R. solani mycelia), medium C (1.0 g of deactivated respective antagonist mycelium) and medium D (1 g of deactivated R. solani mycelia). T asperellum showed presence of higher amounts of chitinases, β-1, 3-glucanases and xylanases in extracellular protein extracts from medium D as compared to medium A. While, the higher activities of glucosidases and endoglucanses were shown in medium D extracts by T. harzianum. β-glucosidase activities were lower compared with other enzymes; however, activities of the extracts of medium D were significantly different. T. asperellum exhibited maximum inhibition (97.7%). On the other hand, Trichoderma sp. did not show any effect on mycelia growth of R. solani on crude extract. PMID:26691463

  10. [Effects of snow pack on soil nitrogen transformation enzyme activities in a subalpine Abies faxioniana forest of western Sichuan, China].

    Science.gov (United States)

    Xiong, Li; Xu, Zhen-Feng; Wu, Fu-Zhong; Yang, Wan-Qin; Yin, Rui; Li, Zhi-Ping; Gou, Xiao-Lin; Tang, Shi-Shan

    2014-05-01

    This study characterized the dynamics of the activities of urease, nitrate reductase and nitrite reductase in both soil organic layer and mineral soil layer under three depths of snow pack (deep snowpack, moderate snowpack and shallow snowpack) over the three critical periods (snow formed period, snow stable period, and snow melt period) in the subalpine Abies faxoniana forest of western Sichuan in the winter of 2012 and 2013. Throughout the winter, soil temperature under deep snowpack increased by 46.2% and 26.2%, respectively in comparison with moderate snowpack and shallow snowpack. In general, the three nitrogen-related soil enzyme activities under shallow snowpack were 0.8 to 3.9 times of those under deep snowpack during the winter. In the beginning and thawing periods of seasonal snow pack, shallow snowpack significantly increased the activities of urease, nitrate and nitrite reductase enzyme in both soil organic layer and mineral soil layer. Although the activities of the studied enzymes in soil organic layer and mineral soil layer were observed to be higher than those under deep- and moderate snowpacks in deep winter, no significant difference was found under the three snow packs. Meanwhile, the effects of snowpack on the activities of the measured enzymes were related with season, soil layer and enzyme type. Significant variations of the activities of nitrogen-related enzymes were found in three critical periods over the winter, and the three measured soil enzymes were significantly higher in organic layer than in mineral layer. In addition, the activities of the three measured soil enzymes were closely related with temperature and moisture in soils. In conclusion, the decrease of snow pack induced by winter warming might increase the activities of soil enzymes related with nitrogen transformation and further stimulate the process of wintertime nitrogen transformation in soils of the subalpine forest.

  11. Microbial Enzyme Activity and Carbon Cycling in Grassland Soil Fractions

    Science.gov (United States)

    Allison, S. D.; Jastrow, J. D.

    2004-12-01

    Extracellular enzymes are necessary to degrade complex organic compounds present in soils. Using physical fractionation procedures, we tested whether old soil carbon is spatially isolated from degradative enzymes across a prairie restoration chronosequence in Illinois, USA. We found that carbon-degrading enzymes were abundant in all soil fractions, including macroaggregates, microaggregates, and the clay fraction, which contains carbon with a mean residence time of ~200 years. The activities of two cellulose-degrading enzymes and a chitin-degrading enzyme were 2-10 times greater in organic matter fractions than in bulk soil, consistent with the rapid turnover of these fractions. Polyphenol oxidase activity was 3 times greater in the clay fraction than in the bulk soil, despite very slow carbon turnover in this fraction. Changes in enzyme activity across the restoration chronosequence were small once adjusted for increases in soil carbon concentration, although polyphenol oxidase activity per unit carbon declined by 50% in native prairie versus cultivated soil. These results are consistent with a `two-pool' model of enzyme and carbon turnover in grassland soils. In light organic matter fractions, enzyme production and carbon turnover both occur rapidly. However, in mineral-dominated fractions, both enzymes and their carbon substrates are immobilized on mineral surfaces, leading to slow turnover. Soil carbon accumulation in the clay fraction and across the prairie restoration chronosequence probably reflects increasing physical isolation of enzymes and substrates on the molecular scale, rather than the micron to millimeter scale.

  12. [Effects of Different Reclaimed Scenarios on Soil Microbe and Enzyme Activities in Mining Areas].

    Science.gov (United States)

    Li, Jun-jian; Liu, Feng; Zhou, Xiao-mei

    2015-05-01

    Abstract: Ecological degradation in the mining areas is greatly aggravated in recent several decades, and ecological restoration has become the primary measure for the sustainable development. Soil microbe and enzyme activity are sensitive indices to evaluate soil quality. Ecological reconstruction was initiated in Antaibao mining area, and we tested soil physicochemical properties, microbial populations of azotobacteria, nitrifying-bacteria and denitrifying-bacteria, and enzyme activities (including sucrose, polyphenol oxidase, dehydrogenase and urease) under different regeneration scenarios. Regeneration scenarios had significant effects on soil physicochemical properties, microbial population and enzyme activities. Total nitrogen was strongly correlated with azotobacteria and nitrifying-bacteria, however, total nitrogen was not correlated with denitrifying-bacteria. Phenol oxidase activity was negatively correlated with soil organic carbon and total nitrogen, but other enzyme activities were positively correlated with soil organic carbon and total nitrogen. Principal Component Analysis ( PCA) was applied to analyze the integrated fertility index (IFI). The highest and lowest IFIs were in Robinia pseudoacacia-Pinus tabuliformis mixed forests and un-reclaimed area, respectively. R. pseudoacacia-P. tabuliformis mixed forests were feasible for reclaimed mining areas in semi-arid region Northwest Shanxi.

  13. Isolation of a tyrosine-activating enzyme from baker's yeast

    NARCIS (Netherlands)

    Ven, A.M. van de; Koningsberger, V.V.; Overbeek, J.Th.G.

    1958-01-01

    The extracts of ether-CO2-frozen baker's yeast contain enzymes that catalyze the ATP-linked amino acid activation by way of pyrophosphate elimination. From the extract a tyrosine-activating enzyme could be isolated, which, judging from ultracentrifugation and electrophoretic data, was about 70% pure

  14. Evaluation of Macerating Pectinase Enzyme Activity under Various Temperature, pH and Ethanol Regimes

    Directory of Open Access Journals (Sweden)

    Andrew G. Reynolds

    2018-02-01

    Full Text Available The polygalacturonase (PGU, hemicellulase (mannanase and protease enzyme activities in commercial macerating, pectinase-enzyme preparations commonly used by wineries in Ontario (Scottzyme Color X and Color Pro were measured under various simulated process conditions (temperature, pH, and ethanol concentration. Treatments included three temperatures (15, 20 and 30 °C; pH = 3.0, 3.5, 4.0 and 5.0; ethanol = 0%, four pH levels (3.0, 3.5, 4.0 and 5.0; temperature = 15, 20, 30 and 50 °C; ethanol = 0%, and four ethanol concentrations ((2.5, 5, 7.5 and 10%; temperature = 20 °C and pH = 3.5. Polygalacturonase enzyme activity in Color X increased linearly with temperature at all pH levels, and increased with pH at all temperature regimes. Polygalacturonase activity decreased with increasing ethanol. Color X mannanase activity increased with temperatures between 15 and 40 °C, and decreased with increased pH between 3.0 and 5.0. Response of mannanase to ethanol was cubic with a sharp decrease between 8 and 10% ethanol. Protease activity increased linearly with temperatures between 20 and 40 °C. These data suggest that the PGU, mannanase and protease components in these enzyme products provide sufficient activities within the ranges of pH, temperature, and ethanol common during the initial stages of red wine fermentations, although low must temperatures (<20 °C and presence of ethanol would likely lead to sub-optimal enzyme activities.

  15. Chaperone-like activities of α-synuclein: α-Synuclein assists enzyme activities of esterases

    International Nuclear Information System (INIS)

    Ahn, Misun; Kim, SeungBum; Kang, Mira; Ryu, Yeonwoo; Doohun Kim, T.

    2006-01-01

    α-Synuclein, a major constituent of Lewy bodies (LBs), has been implicated to play a critical role in the pathogenesis of Parkinson's disease (PD), although the physiological function of α-synuclein has not yet been known. Here we have shown that α-synuclein, which has no well-defined secondary or tertiary structure, can protect the enzyme activity of microbial esterases against stress conditions such as heat, pH, and organic solvents. In particular, the flexibility of α-synuclein and its C-terminal region seems to be important for complex formation, but the structural integrity of the C-terminal region may not be required for stabilization of enzyme activity. In addition, atomic force microscopy (AFM) and in vivo enzyme assays showed highly specific interactions of esterases with α-synuclein. Our results indicate that α-synuclein not only protects the enzyme activity of microbial esterases in vitro, but also can stabilize the active conformation of microbial esterases in vivo

  16. Rapid shifts in Atta cephalotes fungus-garden enzyme activity after a change in fungal substrate (Attini, Formicidae)

    DEFF Research Database (Denmark)

    Kooij, P W; Schiøtt, M; Boomsma, J J

    2011-01-01

    Fungus gardens of the basidiomycete Leucocoprinus gongylophorus sustain large colonies of leaf-cutting ants by degrading the plant material collected by the ants. Recent studies have shown that enzyme activity in these gardens is primarily targeted toward starch, proteins and the pectin matrix......, we measured the changes in enzyme activity after a controlled shift in fungal substrate offered to six laboratory colonies of Atta cephalotes. An ant diet consisting exclusively of grains of parboiled rice rapidly increased the activity of endo-proteinases and some of the pectinases attacking...... from the rice diet, relative to the leaf diet controls. Enzyme activity in the older, bottom sections of fungus gardens decreased, indicating a faster processing of the rice substrate compared to the leaf diet. These results suggest that leaf-cutting ant fungus gardens can rapidly adjust enzyme...

  17. Influence of 2. 45 GHz microwave radiation on enzyme activity

    Energy Technology Data Exchange (ETDEWEB)

    Galvin, M J; Parks, D L; McRee, D I

    1981-05-01

    The in vitro activity of acetylcholinesterase and creatine phosphokinase was determined during in vitro exposure to 2.45 GHz microwave radiation. The enzyme activities were examined during exposure to microwave radiation at specific absorption rates (SAR) of 1, 10, 50, and 100 mW/g. These specific absorption rates had no effect on the activity of either enzyme when the temperature of the control and exposed samples were similar. These data demonstrate that the activity of these two enzymes is not affected by microwave radiation at the SARs and frequency employed in this study.

  18. HPRT Enzyme Activity of Blood Cells From Patients With Downs Syndrome

    International Nuclear Information System (INIS)

    Sbubber, E.K.; Abdul-Rahman, M.H.; Sultan, A.F.; Hamamy, H.A.

    1998-01-01

    Hypoxanthine phosphoribosyl transferase (HPRT) enzyme activity was determined in erythrocytes from 16 children (aged below one year to 11 year) with down s syndrome using 8-C 14 Hypoxanthine and radioeleelrophorsis techniques. Significant (P<0.01) reduction in HPRT enzyme activity was seen in D S children compared to that of 18 (age and sex matched) healthy children. Pure 21 - trisomic erythrocytes expressed lower enzyme activity than mosaic cell. Mothers of D S children showed significantly (P<0.01) lower enzyme activity than mothers of normal children . Reduced activity of HPRT enzyme was also observed in PHA-stimulated lymphocytes of DS children and their mothers. These results indicated that deficiency of HPRT in D S patients may contribute to the abnormal purine metabolism associated with the symptomatology of this syndrome

  19. Correlation of secretory phospholipase-A2 activity and fatty acids in cerebrospinal fluid with liver enzymes tests

    Directory of Open Access Journals (Sweden)

    Sepideh Ghodoosifar

    2016-02-01

    Full Text Available Introduction: The aim was to determine whether secretory phospholipase-A2 (sPLA2 activity and fatty acids in cerebrospinal fluid (CSF are correlated with liver enzymes tests. Methods: CSF and serum samples were collected from 49 patients (age 18-65 as part of routine diagnostic testing. Along with serum liver enzymes aspartate aminotransferase (AST, alanine aminotransferase (ALT and alkaline phosphatase (ALP, the fatty acid composition of CSF was measured by gas liquid chromatography. CSF enzyme activities of sPLA2 were measured using the standard assay with diheptanoyl thio-phosphatidylcholin as substrate. Results: The saturated fatty acids (SFAs including palmitic acid and stearic acid were positively, and the unsaturated fatty acids including oleic acid and linoleic acid were negatively correlated with liver enzymes tests. In regression analysis with adjustment for body mass index (BMI, the elevated liver enzymes tests were positively associated with activity of sPLA2 (β > 0.31, P 0.38, P < 0.010 and negatively with total monounsaturated fatty acids (MUFAs (β < -0.40, P < 0.001 contents of CSF. Conclusion: CSF activity of sPLA2 and fatty acids may be linked to peripheral markers of liver function, suggesting an indirect impact of central fatty acids on hepatocytes function and metabolism.

  20. Enzyme-immobilized SiO2-Si electrode: Fast interfacial electron transfer with preserved enzymatic activity

    Science.gov (United States)

    Wang, Gang; Yau, Siu-Tung

    2005-12-01

    The enzyme, glucose oxidase (GOx), is immobilized using electrostatic interaction on the native oxide of heavily doped n-type silicon. Voltammetric measurement shows that the immobilized GOx gives rise to a very fast enzyme-silicon interfacial electron transfer rate constant of 7.9s-1. The measurement also suggests that the enzyme retains its native conformation when immobilized on the silicon surface. The preserved native conformation of GOx is further confirmed by testing the enzymatic activity of the immobilized GOx using glucose. The GOx-immobilized silicon is shown to behave as a glucose sensor that detects glucose with concentrations as low as 50μM.

  1. Concentration profiles near an activated enzyme.

    Science.gov (United States)

    Park, Soohyung; Agmon, Noam

    2008-09-25

    When a resting enzyme is activated, substrate concentration profile evolves in its vicinity, ultimately tending to steady state. We use modern theories for many-body effects on diffusion-influenced reactions to derive approximate analytical expressions for the steady-state profile and the Laplace transform of the transient concentration profiles. These show excellent agreement with accurate many-particle Brownian-dynamics simulations for the Michaelis-Menten kinetics. The steady-state profile has a hyperbolic dependence on the distance of the substrate from the enzyme, albeit with a prefactor containing the complexity of the many-body effects. These are most conspicuous for the substrate concentration at the surface of the enzyme. It shows an interesting transition as a function of the enzyme turnover rate. When it is high, the contact concentration decays monotonically to steady state. However, for slow turnover it is nonmonotonic, showing a minimum due to reversible substrate binding, then a maximum due to diffusion of new substrate toward the enzyme, and finally decay to steady state. Under certain conditions one can obtain a good estimate for the critical value of the turnover rate constant at the transition.

  2. Extraction of Active Enzymes from "Hard-to-Break-Cells"

    DEFF Research Database (Denmark)

    Ottaviani, Alessio; Tesauro, Cinzia; Fjelstrup, S

    We present the utilization of a rolling circle amplification (RCA) based assay to investigate the extraction efficiency of active enzymes from a class of “hard-to-break” cells, yeast Saccaramyces cerevisiae. Current analyses of microorganisms, such as pathogenic bacteria, parasites or particular...... life stages of microorganisms (e.g. spores from bacteria or fungi) is hampered by the lack of efficient lysis protocols that preserve the activity and integrity of the cellular content. Presented herein is a flexible scheme to screen lysis protocols for active enzyme extraction. We also report a gentle...... yet effective approach for extraction of active enzymes by entrapping cells in microdroplets. Combined effort of optimized extraction protocols and effective analytical approaches is expected to generate impact in future disease diagnosis and environmental safety....

  3. Regulation of antioxidant enzyme activities in male and female rat macrophages by sex steroids

    Directory of Open Access Journals (Sweden)

    Azevedo R.B.

    2001-01-01

    Full Text Available Human and animal immune functions present sex dimorphism that seems to be mainly regulated by sex hormones. In the present study, the activities of the antioxidant enzymes total superoxide dismutase (SOD, catalase (CAT, and glutathione peroxidase (GSH-Px were measured in intraperitoneal resident macrophages from adult male and female rats. In addition to comparing males and females, we also examined the regulation of these enzyme activities in macrophages by sex steroids. GSH-Px activity did not differ between male and female macrophages. However, both total SOD and CAT activities were markedly higher in females than in males (83 and 180%. Removal of the gonads in both males and females (comparison between castrated groups increased the difference in SOD activity from 83 to 138% and reduced the difference in CAT activity from 180 to 86%. Castration and testosterone administration did not significantly modify the activities of the antioxidant enzymes in male macrophages. Ovariectomy did not affect SOD or GSH-Px activity but markedly reduced (48% CAT activity. This latter change was fully reversed by estrogen administration, whereas progesterone had a smaller effect. These results led us to conclude that differences in the SOD and CAT activities may partially explain some of the differences in immune function reported for males and females. Also, estrogen is a potent regulator of CAT in macrophages and therefore this enzyme activity in macrophages may vary considerably during the menstrual cycle.

  4. PENGARUH DEGRADASI ENZIM PROTEOLITIK TERHADAP AKTIVITAS ANGIOTENSIN CONVERTING ENZYME INHIBITOR BEKASAM DENGAN Lactobacillus plantarum B1765 (The Effect of Degradation of Proteolitic Enzyme on Angiotensin Converting Enzyme Inhibitor Activity of Bekasam with Lactobacillus plantarum B1765

    Directory of Open Access Journals (Sweden)

    Prima Retno Wikandari

    2016-10-01

    Full Text Available This research studied the effect of digestive enzyme degradation on the Angiotensin Converting Enzyme Inhibitor (ACEI activity and the stability of bekasam peptide and ACEI activity. Water extract of bekasam was subjected to pepsin and trypsin. The stability of peptide was measured from the changes of peptide concentration before and after treatment by those enzymes. The stability of ACEI activity was measured by hypuric acid liberated from Hip-His-Leu as ACE substrate and determined by spectrophotometer. The results showed that proteolytic enzyme degradation did not affect the concentration of peptide (p>0,05 and the mean concentration 36.72. It was closely related with the ACEI activity that did not change significantly before and after digestion by pepsin and trypsin (p>0,05 and the mean ACEI activity was 70.73. It showed that ACEI activity of bekasam did not change by the degradation of digestive enzyme. Keywords: bekasam, fermented fish, peptides, ACEI activity ABSTRAK Penelitian ini bertujuan untuk mengkaji pengaruh degradasi enzim pencernaan proteolitik terhadap stabilitas peptida dan aktivitas Angiotensin Converting Enzyme Inhibitor (ACEI bekasam yang difermentasi dengan kultur starter Lactobacillus plantarum B1765. Terhadap ekstrak bekasam diberi perlakuan enzim proteolitik pepsin dan tripsin. Pengujian stabilitas peptida diukur dengan ada tidaknya perubahan jumlah peptida setelah perlakuan enzim menggunakan metode formol, sedangkan aktivitas ACEI dilakukan dengan mengetahui jumlah asam hipurat dari substrat Hip-His-Leu yang dibebaskan oleh ACE diukur dengan spektrofotometer. Hasil pengujian menunjukkan perlakuan enzim proteolitik tidak berpengaruh pada konsentrasi peptida dengan p>0,05 dengan nilai rata-rata konsentrasi peptida sebesar 36,72. Hal ini berkorelasi dengan aktivitas ACEI yang juga menunjukkan tidak ada pengaruh antara perlakuan sebelum dan setelah degradasi enzim (p>0,05 dengan rata-rata aktivitas ACEI sebesar 70,73. Hasil

  5. [Study on soil enzyme activities and microbial biomass carbon in greenland irrigated with reclaimed water].

    Science.gov (United States)

    Pan, Neng; Hou, Zhen-An; Chen, Wei-Ping; Jiao, Wen-Tao; Peng, Chi; Liu, Wen

    2012-12-01

    The physicochemical properties of soils might be changed under the long-term reclaimed water irrigation. Its effects on soil biological activities have received great attentions. We collected surface soil samples from urban green spaces and suburban farmlands of Beijing. Soil microbial biomass carbon (SMBC), five types of soil enzyme activities (urease, alkaline phosphatase, invertase, dehydrogenase and catalase) and physicochemical indicators in soils were measured subsequently. SMBC and enzyme activities from green land soils irrigated with reclaimed water were higher than that of control treatments using drinking water, but the difference is not significant in farmland. The SMBC increased by 60.1% and 14.2% than those control treatments in 0-20 cm soil layer of green land and farmland, respectively. Compared with their respective controls, the activities of enzymes in 0-20 cm soil layer of green land and farmland were enhanced by an average of 36.7% and 7.4%, respectively. Investigation of SMBC and enzyme activities decreased with increasing of soil depth. Significantly difference was found between 0-10 cm and 10-20 cm soil layer in green land. Soil biological activities were improved with long-term reclaimed water irrigation in Beijing.

  6. Alpha-Glucosidase Enzyme Biosensor for the Electrochemical Measurement of Antidiabetic Potential of Medicinal Plants.

    Science.gov (United States)

    Mohiuddin, M; Arbain, D; Islam, A K M Shafiqul; Ahmad, M S; Ahmad, M N

    2016-12-01

    A biosensor for measuring the antidiabetic potential of medicinal plants was developed by covalent immobilization of α-glucosidase (AG) enzyme onto amine-functionalized multi-walled carbon nanotubes (MWCNTs-NH2). The immobilized enzyme was entrapped in freeze-thawed polyvinyl alcohol (PVA) together with p-nitrophenyl-α-D-glucopyranoside (PNPG) on the screen-printed carbon electrode at low pH to prevent the premature reaction between PNPG and AG enzyme. The enzymatic reaction within the biosensor is inhibited by bioactive compounds in the medicinal plant extracts. The capability of medicinal plants to inhibit the AG enzyme on the electrode correlates to the potential of the medicinal plants to inhibit the production of glucose from the carbohydrate in the human body. Thus, the inhibition indicates the antidiabetic potential of the medicinal plants. The performance of the biosensor was evaluated to measure the antidiabetic potential of three medicinal plants such as Tebengau (Ehretis laevis), Cemumar (Micromelum pubescens), and Kedondong (Spondias dulcis) and acarbose (commercial antidiabetic drug) via cyclic voltammetry, amperometry, and spectrophotometry. The cyclic voltammetry (CV) response for the inhibition of the AG enzyme activity by Tebengau plant extracts showed a linear relation in the range from 0.423-8.29 μA, and the inhibition detection limit was 0.253 μA. The biosensor exhibited good sensitivity (0.422 μA/mg Tebengau plant extracts) and rapid response (22 s). The biosensor retains approximately 82.16 % of its initial activity even after 30 days of storage at 4 °C.

  7. Evolutionary transitions in enzyme activity of ant fungus gardens.

    Science.gov (United States)

    De Fine Licht, Henrik H; Schiøtt, Morten; Mueller, Ulrich G; Boomsma, Jacobus J

    2010-07-01

    Fungus-growing (attine) ants and their fungal symbionts passed through several evolutionary transitions during their 50 million year old evolutionary history. The basal attine lineages often shifted between two main cultivar clades, whereas the derived higher-attine lineages maintained an association with a monophyletic clade of specialized symbionts. In conjunction with the transition to specialized symbionts, the ants advanced in colony size and social complexity. Here we provide a comparative study of the functional specialization in extracellular enzyme activities in fungus gardens across the attine phylogeny. We show that, relative to sister clades, gardens of higher-attine ants have enhanced activity of protein-digesting enzymes, whereas gardens of leaf-cutting ants also have increased activity of starch-digesting enzymes. However, the enzyme activities of lower-attine fungus gardens are targeted primarily toward partial degradation of plant cell walls, reflecting a plesiomorphic state of nondomesticated fungi. The enzyme profiles of the higher-attine and leaf-cutting gardens appear particularly suited to digest fresh plant materials and to access nutrients from live cells without major breakdown of cell walls. The adaptive significance of the lower-attine symbiont shifts remains unclear. One of these shifts was obligate, but digestive advantages remained ambiguous, whereas the other remained facultative despite providing greater digestive efficiency.

  8. Genetic ontogeny of pancreatic enzymes in Labrus bergylta larvae and the effect of feed type on enzyme activities and gene expression.

    Science.gov (United States)

    Hansen, Truls Wergeland; Folkvord, Arild; Grøtan, Espen; Sæle, Øystein

    2013-03-01

    A newly cultivated wrasse species, Labrus bergylta, have shown great potential for use in Atlantic salmon (Salmo salar) farms in the battle against sea lice (Lepeoptheirus salmonis) infections. Hatchery reared L. bergylta were studied from 2 to 55 DPH to examine the molecular basis of digestive ontogeny related to the pancreas. An isolated feeding trial was performed on 27-34 DPH larvae to compare the effect of diet on enzyme activity and the possible exogenous contribution by live feed. The following genes coding for key pancreatic enzymes were analyzed by qPCR: trypsin, Cyp7 A1, BAL, sPLA(2) 1B, amylase and pancreatic chitinase. Enzyme activity was measured on trypsin, neutral lipase, sPLA(2), amylase and chitinase in fed and unfed larvae. We did not observe any effects of the formulated diet v.s. rotifers on enzyme activities of neutral lipase, chitinase and sPLA(2). However, a probable feed-dependency was observed at a transcriptional level, where rotifers seem to stimulate upregulation. The regulation of BAL was the only exception, where an upregulation was observed after weaning both in the ontogeny series and the experimental part. Our data on pancreatic chitinase and amylase mRNA levels suggest the importance of carbohydrates in the diet of early larval and juvenile L. bergylta. Copyright © 2012 Elsevier Inc. All rights reserved.

  9. Proteomic and activity profiles of ascorbate-glutathione cycle enzymes in germinating barley embryo

    DEFF Research Database (Denmark)

    Bønsager, Birgit Christine; Shahpiri, Azar; Finnie, Christine

    2010-01-01

    Enzymes involved in redox control are important during seed germination and seedling growth. Ascorbate-glutathione cycle enzymes in barley embryo extracts were monitored both by 2D-gel electrophoresis and activity measurements from 4 to 144 h post imbibition (PI). Strikingly different activity...... profiles were observed. No ascorbate peroxidase (APX) activity was present in mature seeds but activity was detected after 24 h PI and increased 14-fold up to 144 h PI. In contrast, dehydroascorbate reductase (DHAR) activity was present at 4 h PI and first decreased by 9-fold until 72 h PI followed by a 5......-fold increase at 144 h PI. Glutathione reductase and monodehydroascorbate reductase activities were also detected at 4 h PI, and showed modest increases of 1.8- and 2.7-fold, respectively, by 144 h PI. The combination of functional analysis with the proteomics approach enabled correlation...

  10. Light-regulation of enzyme activity in anacystis nidulans (Richt.).

    Science.gov (United States)

    Duggan, J X; Anderson, L E

    1975-01-01

    The effect of light on the levels of activity of six enzymes which are light-modulated in higher plants was examined in the photosynthetic procaryot Anacystis nidulans. Ribulose-5-phosphate kinase (EC 2.7.1.19) was found to be light-activated in vivo and dithiothreitol-activated in vitro while glucose-6-phosphate dehydrogenase (EC 1.1.1.49) was light-inactivated and dithiothreitol-inactivated. The enzymes fructose-1,6-diphosphate phosphatase (EC 3.1.3.11), sedoheptulose-1,7-diphosphate phosphatase, NAD- and NADP-linked glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12; EC 1.2.1.13) were not affected by light treatment of the intact algae, but sedoheptulose-diphosphate phosphatase and the glyceraldehyde-3-phosphate dehydrogenases were dithiothreitol-activated in crude extracts. Light apparently controls the activity of the reductive and oxidative pentose phosphate pathway in this photosynthetic procaryot as in higher plants, through a process which probably involves reductive modulation of enzyme activity.

  11. Activity enhancement of ligninolytic enzymes of Trametes versicolor ...

    African Journals Online (AJOL)

    Suspended cultures of white-rot fungus, Trametes versicolor, supplemented with bagasse powder showed a concentration dependent enhancement in the ligninolytic enzymes activity in liquid shake cultures. 2% (w/v) bagasse powder improved greater stability to the enzymes. The optimum pH is 3.5 and the optimum ...

  12. Study of cellulase enzymes self-assembly in aqueous-acetonitrile solvent: Viscosity measurements

    Energy Technology Data Exchange (ETDEWEB)

    Ghaouar, N., E-mail: naoufel-ghaouar@lycos.co [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia); Institut National des Sciences Appliquees et de Technologie, INSAT, Centre Urbain Nord, BP. 676, Tunis (Tunisia); Aschi, A. [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia); Belbahri, L. [School of Engineering of Lullier, University of Applied Sciences of Western Switzerland, 150, Route de Presinge, 1254 Jussy (Switzerland); Trabelsi, S.; Gharbi, A. [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia)

    2009-11-15

    The present study extends the viscosity measurements performed by Ghaouar et al. [Physica B, submitted for publication.] to study the conformational change of the cellulase enzymes in aqueous-acetonitrile mixture. We aim to investigate: (i) the denaturation process by measuring the specific viscosity for temperatures varying between 25 and 65 deg. C and acetonitrile concentrations between 0% and 50%, (ii) the enzyme-enzyme interaction by calculating the Huggins coefficient and (iii) the enzyme sizes by following the hydrodynamic radius for various temperatures. The precipitation of cellulases versus acetonitrile concentration is also considered. We show from all physical quantities measured in this work that the precipitation and the denaturation processes of cellulase enzymes exist together.

  13. Study of cellulase enzymes self-assembly in aqueous-acetonitrile solvent: Viscosity measurements

    International Nuclear Information System (INIS)

    Ghaouar, N.; Aschi, A.; Belbahri, L.; Trabelsi, S.; Gharbi, A.

    2009-01-01

    The present study extends the viscosity measurements performed by Ghaouar et al. [Physica B, submitted for publication.] to study the conformational change of the cellulase enzymes in aqueous-acetonitrile mixture. We aim to investigate: (i) the denaturation process by measuring the specific viscosity for temperatures varying between 25 and 65 deg. C and acetonitrile concentrations between 0% and 50%, (ii) the enzyme-enzyme interaction by calculating the Huggins coefficient and (iii) the enzyme sizes by following the hydrodynamic radius for various temperatures. The precipitation of cellulases versus acetonitrile concentration is also considered. We show from all physical quantities measured in this work that the precipitation and the denaturation processes of cellulase enzymes exist together.

  14. A Review on the Effects of Supercritical Carbon Dioxide on Enzyme Activity

    Directory of Open Access Journals (Sweden)

    Marie Zarevúcka

    2010-01-01

    Full Text Available Different types of enzymes such as lipases, several phosphatases, dehydrogenases, oxidases, amylases and others are well suited for the reactions in SC-CO2. The stability and the activity of enzymes exposed to carbon dioxide under high pressure depend on enzyme species, water content in the solution and on the pressure and temperature of the reaction system. The three-dimensional structure of enzymes may be significantly altered under extreme conditions, causing their denaturation and consequent loss of activity. If the conditions are less adverse, the protein structure may be largely retained. Minor structural changes may induce an alternative active protein state with altered enzyme activity, specificity and stability.

  15. Markers of oxidative stress and erythrocyte antioxidant enzyme activity in older men and women with differing physical activity.

    Science.gov (United States)

    Rowiński, Rafał; Kozakiewicz, Mariusz; Kędziora-Kornatowska, Kornelia; Hübner-Woźniak, Elżbieta; Kędziora, Józef

    2013-11-01

    The aim of the present study was to examine the relationship between markers of oxidative stress and erythrocyte antioxidant enzyme activity and physical activity in older men and women. The present study included 481 participants (233 men and 248 women) in the age group 65-69 years (127 men and 125 women) and in the age group 90 years and over (106 men and 123 women). The classification of respondents by physical activity was based on answers to the question if, in the past 12 months, they engaged in any pastimes which require physical activity. The systemic oxidative stress status was assessed by measuring plasma iso-PGF2α and protein carbonyl concentration as well as erythrocyte antioxidant enzymes activity, i.e., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione reductase (GR). The concentration of plasma iso-PGF2α and protein carbonyls (CP) was lower in groups of younger men and women compared to the respective older groups. In all examined groups, physical activity resulted in decrease of these oxidative stress markers and simultaneously caused adaptive increase in the erythrocyte SOD activity. Additionally, in active younger men CAT, GPx, and GR activities were higher than in sedentary ones. In conclusion, oxidative stress increase is age-related, but physical activity can reduce oxidative stress markers and induce adaptive increase in the erythrocyte antioxidant enzyme activity, especially SOD, even in old and very old men and women. © 2013.

  16. Superoxide Dismutase (SOD Enzyme Activity Assay in Fasciola spp. Para-sites and Liver Tissue Extract

    Directory of Open Access Journals (Sweden)

    M Assady

    2011-09-01

    Full Text Available Background: The purpose of this comparative study was to detect superoxide dismutase (SOD activities in Fasciola hepatica, F. gigantica parasites, infected and healthy liver tissues in order to determine of species effects and liver infection on SODs activity level.Methods: Fasciola spp. parasites and sheep liver tissues (healthy and infected liver tissues, 10 samples for each, were collected, homogenized and investigated for protein measurement, protein detection and SOD enzyme activity assay. Protein concentration was measured by Bradford method and SODs band protein was detected on SDS-PAGE. SODs activity was determined by iodonitrotetrazolium chloride, INT, and xanthine substrates. Independent samples t-test was conducted for analysis of SODs activities difference.Results: Protein concentration means were detected for F. hepatica 1.3 mg/ ml, F. gigantica 2.9 mg/ml, healthy liver tissue 5.5 mg/ml and infected liver tissue 1.6 mg/ml (with similar weight sample mass. Specific enzyme activities in the samples were obtained 0.58, 0.57, 0.51, 1.43 U/mg for F. hepatica, F. gigantica, healthy liver and infected liver respectively. Gel electrophoresis of Fasciola spp. and sheep liver tissue extracts revealed a band protein with MW of 60 kDa. The statistical analysis revealed significant difference between SOD activities of Fasciola species and also between SOD activity of liver tissues (P<.05.Conclusion: Fasciola species and liver infection are effective causes on SOD enzyme activity level.

  17. Enzyme Stability and Activity in Non-Aqueous Reaction Systems: A Mini Review

    Directory of Open Access Journals (Sweden)

    Shihui Wang

    2016-02-01

    Full Text Available Enormous interest in biocatalysis in non-aqueous phase has recently been triggered due to the merits of good enantioselectivity, reverse thermodynamic equilibrium, and no water-dependent side reactions. It has been demonstrated that enzyme has high activity and stability in non-aqueous media, and the variation of enzyme activity is attributed to its conformational modifications. This review comprehensively addresses the stability and activity of the intact enzymes in various non-aqueous systems, such as organic solvents, ionic liquids, sub-/super-critical fluids and their combined mixtures. It has been revealed that critical factors such as Log P, functional groups and the molecular structures of the solvents define the microenvironment surrounding the enzyme molecule and affect enzyme tertiary and secondary structure, influencing enzyme catalytic properties. Therefore, it is of high importance for biocatalysis in non-aqueous media to elucidate the links between the microenvironment surrounding enzyme surface and its stability and activity. In fact, a better understanding of the correlation between different non-aqueous environments and enzyme structure, stability and activity can contribute to identifying the most suitable reaction medium for a given biotransformation.

  18. Visualization of enzyme activities inside earthworm biopores by in situ soil zymography

    Science.gov (United States)

    Thu Duyen Hoang, Thi; Razavi, Bahar. S.; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    Earthworms can strongly activate microorganisms, increase microbial and enzyme activities and consequently the turnover of native soil organic matter. In extremely dynamic microhabitats and hotspots as biopores made by earthworms, the in situ enzyme activities are a footprint of complex biotic interactions. The effect of earthworms on the alteration of enzyme activities inside biopores and the difference between bio-pores and earthworm-free soil was visualized by in situ soil zymography (Spohn and Kuzyakov, 2014). For the first time, we prepared quantitative imaging of enzyme activities in biopores. Furthermore, we developed the zymography technique by direct application of a substrate saturated membrane to the soil to obtain better spatial resolution. Lumbricus terrestris L. was placed into transparent box (15×20×15cm). Simultaneously, maize seed was sown in the soil. Control soil box with maize and without earthworm was prepared in the same way. After two weeks when bio-pore systems were formed by earthworm, we visualized in situ enzyme activities of five hydrolytic enzymes (β-glucosidase, cellobiohydrolase, chitinase, xylanase, leucine aminopeptidase) and phosphatase. Followed by non-destructive zymography, biopore samples and control soil were destructively collected to assay enzyme kinetics by fluorogenically labeled substrates method. Zymography showed higher activity of β-glucosidase, chitinase, xylanase and phosphatase in biopores comparing to bulk soil. These differences were further confirmed by fluorimetric microplate enzyme assay detected significant difference of Vmax in four above mentioned enzymes. Vmax of β-glucosidase, chitinase, xylanase and phosphatase in biopores is 68%, 108%, 50% and 49% higher than that of control soil. However, no difference in cellobiohydrolase and leucine aminopeptidase kinetics between biopores and control soil were detected. This indicated little effect of earthworms on protein and cellulose transformation in soil

  19. Inhibitory activities of Moringa oleifera leaf extract against α-glucosidase enzyme in vitro

    Science.gov (United States)

    Natsir, H.; Wahab, A. W.; Laga, A.; Arif, A. R.

    2018-03-01

    Alpha-glucosidase is a key enzyme in the final process of breaking carbohydrates into glucose. Inhibition of α-glucosidase affected more absorption of glucose, so it can reduce hyperglycemia condition. The aims of this study is to determine the effectiveness of inhibition wet and dried Moringa oleifera leaf extract through α-glucosidase activity in vitro. The effectiveness study of inhibition on the activity of α-glucosidase enzyme obtained from white glutinous rice (Oryza sativa glutinosa) was carried out using wet and dried kelor leaf extract of 13% (w/v) with 10 mM α-D-glucopyranoside (PNPG) substrate. A positive control used 1% acarbose and substrate without addition of extract was a negative control. Inhibitory activity was measured using spectrophotometers at a wavelength of 400 nm. The result showed that the inhibition activity against α-glucosidase enzyme of dried leaf extract, wet leaf extract and acarbose was 81,39%, 83,94%, and 95,4%, respectively on pH 7,0. The effectiveness inhibition of the wet Moringa leaf extract was greater than the dried leaf extract. The findings suggest that M. oleifera leaf has the potential to be developed as an alternative food therapy for diabetics.

  20. Detoxification enzymes activities in deltamethrin and bendiocarb ...

    African Journals Online (AJOL)

    Detoxification enzymes activities in deltamethrin and bendiocarb resistant and susceptible malarial vectors ( Anopheles gambiae ) breeding in Bichi agricultural and residential sites, Kano state, Nigeria.

  1. Visualization of Enzyme Activities in Earthworm Biopores by In Situ Soil Zymography.

    Science.gov (United States)

    Razavi, Bahar S; Hoang, Duyen; Kuzyakov, Yakov

    2017-01-01

    Earthworms produce biopores with strongly increased microbial and enzyme activities and consequently they form microbial hotspots in soil. In extremely dynamic microhabitats and hotspots such as earthworm biopores, the in situ enzyme activities are a footprint of process rates and complex biotic interactions. The effect of earthworms on enzyme activities inside biopores, relative to earthworm-free soil, can be visualized by in situ soil zymography. Here, we describe the details of the approach and discuss its advantages and limitations. Direct zymography provides high spatial resolution for quantitative images of enzyme activities in biopores.

  2. Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

    Directory of Open Access Journals (Sweden)

    Lin Yuan

    Full Text Available Three hundred one-day-old male broiler chickens (Ross-308 were fed corn-soybean basal diets containing non-starch polysaccharide (NSP enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI and average daily gain (ADG were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05. Feed-to-gain ratio (FGR was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05. Apparent digestibility of crude protein (ADCP was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05. Cholecystokinin (CCK level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05, but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05, respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05. However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05. The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05. Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

  3. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    Science.gov (United States)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-01-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology. PMID:26861509

  4. Nanocaged enzymes with enhanced catalytic activity and increased stability against protease digestion

    Science.gov (United States)

    Zhao, Zhao; Fu, Jinglin; Dhakal, Soma; Johnson-Buck, Alexander; Liu, Minghui; Zhang, Ting; Woodbury, Neal W.; Liu, Yan; Walter, Nils G.; Yan, Hao

    2016-02-01

    Cells routinely compartmentalize enzymes for enhanced efficiency of their metabolic pathways. Here we report a general approach to construct DNA nanocaged enzymes for enhancing catalytic activity and stability. Nanocaged enzymes are realized by self-assembly into DNA nanocages with well-controlled stoichiometry and architecture that enabled a systematic study of the impact of both encapsulation and proximal polyanionic surfaces on a set of common metabolic enzymes. Activity assays at both bulk and single-molecule levels demonstrate increased substrate turnover numbers for DNA nanocage-encapsulated enzymes. Unexpectedly, we observe a significant inverse correlation between the size of a protein and its activity enhancement. This effect is consistent with a model wherein distal polyanionic surfaces of the nanocage enhance the stability of active enzyme conformations through the action of a strongly bound hydration layer. We further show that DNA nanocages protect encapsulated enzymes against proteases, demonstrating their practical utility in functional biomaterials and biotechnology.

  5. Activity of certain enzymes in cadmium-poisoned chicks

    Energy Technology Data Exchange (ETDEWEB)

    Kench, J E; Gubb, P J.D.

    1970-01-01

    Activities of a number of enzymes in the liver and other tissues of newly hatched cadmium poisoned chicks have been compared with those of normal controls before and after incubation with Cd/sup +2/ at a concentration similar to that present in vivo. Concentrations of Cd/sup +2/ in the various cellular fractions were determined, after wet oxidation, by atomic absorption spectrophotometry. Interaction of Cd/sup +2/ with enzymes may provide information on the localization of enzymes within mitochondria and other cellular structures. 7 references.

  6. Vitality Improvement of the Mediterranean Fruit Fly, Ceratitis capitata Wied 2- Measured by using ME and At Pase Enzyme Activities and Total Protein Content

    International Nuclear Information System (INIS)

    Salama, M.S.; Shoman, A.A.; Elbermawy, S.M.; Abul Yazid, I.

    2000-01-01

    The present investigation aims at producing sterile adult Mediterranean fruit fly, Ceratitis capitata Wied. Having the best possible vitality through the use of irradiation and /or a mutagenic substances to be used in a sterile insect technique program. Several types of mutagenic that were thought to cause mutations were used as IGR's, temperature, formaldehyde, colchicine, alcohols, serve ral types of larval rearing media and gamma-rays. In a common pathway, malic enzyme (ME) activity, adenosine triphosphatase (ATPase) enzyme activity and the total protein contents are studied as direct parameters for measuring vitality of the insect. It was found that there is an increment at levels of these parameters due to the treatment of egg stage by the previously mentioned treatments specially the usage of the rice hulls as a bulking component in the larval rearing media alone or followed by irradiation of the pupal stage with 90 Gy

  7. Pathogenicity and cell wall-degrading enzyme activities of some ...

    African Journals Online (AJOL)

    Dr. J. T. Ekanem

    2005-12-17

    Dec 17, 2005 ... be attributed to the activities of these cell wall degrading enzymes. Keywords: Cowpea ... bacteria have long been known to produce enzymes capable of ... Inoculated seeds were sown in small plastic pots filled with steam- ...

  8. Plant carbohydrate binding module enhances activity of hybrid microbial cellulase enzyme

    Directory of Open Access Journals (Sweden)

    Caitlin Siobhan Byrt

    2012-11-01

    Full Text Available A synthetic, highly active cellulase enzyme suitable for in planta production may be a valuable tool for biotechnological approaches to develop transgenic biofuel crops with improved digestibility. Here, we demonstrate that the addition of a plant derived carbohydrate binding module (CBM to a synthetic glycosyl hydrolase (GH improved the activity of the hydrolase in releasing sugar from plant biomass. A CEL-HYB1-CBM enzyme was generated by fusing a hybrid microbial cellulase, CEL-HYB1, with the carbohydrate-binding module (CBM of the tomato (Solanum lycopersicum SlCel9C1 cellulase. CEL-HYB1 and CEL-HYB1-CBM enzymes were produced in vitro using Pichia pastoris and the activity of these enzymes was tested using CMC, MUC and native crystalline cellulose assays. The presence of the CBM substantially improved the endo-glucanase activity of CEL-HYB1, especially against the native crystalline cellulose encountered in Sorghum plant cell walls. These results indicate that addition of an endogenous plant derived CBM to cellulase enzymes may enhance hydrolytic activity.

  9. Activities of cholinesterase enzyme among diazinon and sevin insecticides sprayers in the western part of Iran

    Directory of Open Access Journals (Sweden)

    Ali Jalilian

    2016-10-01

    Full Text Available Objective: To measure the activities of cholinesterase enzyme among farmers who used the selected insecticides for the purpose of preventing the growth of agricultural pests on their farms. Methods: A total of 21 people used diazinon to spray their agricultural lands and 13 people also used sevin to spray theirs in western part of Iran. Lovi Bond method was used for the measurement of cholinesterase activity. Results: Results revealed that the enzyme level before spraying with diazinon was 100.0% among 3 workers and 87.5% in 18 of them. This level decreased to 75.0% among 13 workers and 67.5% in 5 workers. The number of workers that had headache, pale, dizziness with headache, nausea, diarrhea with cramps and stomachache were 5, 9, 5, 3, 4 and 7 respectively. These symptoms decreased after 72 h. Out of 13 workers who sprayed with sevin, the enzyme level before spraying was normal (100.0% among 5 workers and 87.5% in 8 workers. After spraying, the enzyme level was 87.5% in 5 workers, 75.0% in 5 workers and 67.5% in 3 workers. Conclusions: These workers were in danger of chemical poisoning. Measurement of precholinesterase and post-cholinesterase exposures is recommended in order to compare the values after pesticide application.

  10. Reveal the response of enzyme activities to heavy metals through in situ zymography.

    Science.gov (United States)

    Duan, Chengjiao; Fang, Linchuan; Yang, Congli; Chen, Weibin; Cui, Yongxing; Li, Shiqing

    2018-07-30

    Enzymes in the soil are vital for assessing heavy metal soil pollution. Although the presence of heavy metals is thought to change the soil enzyme system, the distribution of enzyme activities in heavy metal polluted-soil is still unknown. For the first time, using soil zymography, we analyzed the distribution of enzyme activities of alfalfa rhizosphere and soil surface in the metal-contaminated soil. The results showed that the growth of alfalfa was significantly inhibited, and an impact that was most pronounced in seedling biomass and chlorophyll content. Catalase activity (CAT) in alfalfa decreased with increasing heavy metal concentrations, while malondialdehyde (MDA) content continually increased. The distribution of enzyme activities showed that both phosphatase and β-glucosidase activities were associated with the roots and were rarely distributed throughout the soil. In addition, the total hotspot areas of enzyme activities were the highest in extremely heavy pollution soil. The hotspot areas of phosphatase were 3.4%, 1.5% and 7.1% under none, moderate and extremely heavy pollution treatment, respectively, but increased from 0.1% to 0.9% for β-glucosidase with the increasing pollution levels. Compared with the traditional method of enzyme activities, zymography can directly and accurately reflect the distribution and extent of enzyme activity in heavy metals polluted soil. The results provide an efficient research method for exploring the interaction between enzyme activities and plant rhizosphere. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. Digestive enzyme activities and gastrointestinal fermentation in wood-eating catfishes.

    Science.gov (United States)

    German, Donovan P; Bittong, Rosalie A

    2009-11-01

    To determine what capabilities wood-eating and detritivorous catfishes have for the digestion of refractory polysaccharides with the aid of an endosymbiotic microbial community, the pH, redox potentials, concentrations of short-chain fatty acids (SCFAs), and the activity levels of 14 digestive enzymes were measured along the gastrointestinal (GI) tracts of three wood-eating taxa (Panaque cf. nigrolineatus "Marañon", Panaque nocturnus, and Hypostomus pyrineusi) and one detritivorous species (Pterygoplichthys disjunctivus) from the family Loricariidae. Negative redox potentials (-600 mV) were observed in the intestinal fluids of the fish, suggesting that fermentative digestion was possible. However, SCFA concentrations were low (<3 mM in any intestinal region), indicating that little GI fermentation occurs in the fishes' GI tracts. Cellulase and xylanase activities were low (<0.03 U g(-1)), and generally decreased distally in the intestine, whereas amylolytic and laminarinase activities were five and two orders of magnitude greater, respectively, than cellulase and xylanase activities, suggesting that the fish more readily digest soluble polysaccharides. Furthermore, the Michaelis-Menten constants (K(m)) of the fishes' beta-glucosidase and N-acetyl-beta-D-glucosaminidase enzymes were significantly lower than the K(m) values of microbial enzymes ingested with their food, further suggesting that the fish efficiently digest soluble components of their detrital diet rather than refractory polysaccharides. Coupled with rapid gut transit and poor cellulose digestibility, the wood-eating catfishes appear to be detritivores reliant on endogenous digestive mechanisms, as are other loricariid catfishes. This stands in contrast to truly "xylivorous" taxa (e.g., beavers, termites), which are reliant on an endosymbiotic community of microorganisms to digest refractory polysaccharides.

  12. DNA-directed control of enzyme-inhibitor complex formation: a modular approach to reversibly switch enzyme activity

    NARCIS (Netherlands)

    Janssen, B.M.G.; Engelen, W.; Merkx, M.

    2015-01-01

    DNA-templated reversible assembly of an enzyme–inhibitor complex is presented as a new and highly modular approach to control enzyme activity. TEM1-ß-lactamase and its inhibitor protein BLIP were conjugated to different oligonucleotides, resulting in enzyme inhibition in the presence of template

  13. Effect of benzo[a]pyrene on detoxification and the activity of antioxidant enzymes of marine microalgae

    Science.gov (United States)

    Shen, Chen; Miao, Jingjing; Li, Yun; Pan, Luqing

    2016-04-01

    The objective of this study was to examine the effect of benzo[a]pyrene (BaP) on the detoxification and antioxidant systems of two microalgae, Isochrysis zhanjiangensis and Platymonas subcordiformis. In our study, these two algae were exposed to BaP for 4 days at three different concentrations including 0.5 μg L-1 (low), 3 μg L-1 (mid) and 18 μg L-1 (high). The activity of detoxification enzymes, ethoxyresorufin O-deethylase (EROD) and glutathione S-transferase (GST) increased in P. subcordiformis in all BaP-treated groups. In I. zhanjiangensis, the activity of these two enzymes increased at the beginning of exposure, and then decreased in the groups treated with mid- and high BaP. The activity of antioxidant enzyme superoxide dismutase (SOD) increased in I. zhanjiangensis in all BaP-treated groups, and then decreased in high BaP-treated group, while no significant change was observed in P. subcordiformis. The activity of antioxidant enzyme catalase (CAT) increased in I. zhanjiangensis and P. subcordiformis in all BaPtreated groups. The content of malondialdehyde (MDA) in Isochrysis zhanjiangensis increased first, and then decreased in high BaP-treated group, while no change occurred in P. subcordiformis. These results demonstrated that BaP significantly influenced the activity of detoxifying and antioxidant enzymes in microalgae. The metabolic related enzymes (EROD, GST and CAT) may serve as sensitive biomarkers of measuring the contamination level of BaP in marine water.

  14. Enzyme Activities in Oleaginous Yeasts Accumulating and Utilizing Exogenous or Endogenous Lipids

    NARCIS (Netherlands)

    Holdsworth, Jane E.; Veenhuis, Marten; Ratledge, Colin

    1988-01-01

    The activities of ATP:citrate lyase (ACL; EC 4.1.3.8), carnitine acetyltransferase (CAT; EC 2.3.1.7), NADP+-dependent isocitrate dehydrogenase (ICDH; EC 1.1.1.42), isocitrate lyase (ICL; EC 4.1.3.1) and malic enzyme (malate dehydrogenase; EC 1.1.1.40) were measured in four oleaginous yeasts, Candida

  15. Micropollutant degradation via extracted native enzymes from activated sludge.

    Science.gov (United States)

    Krah, Daniel; Ghattas, Ann-Kathrin; Wick, Arne; Bröder, Kathrin; Ternes, Thomas A

    2016-05-15

    A procedure was developed to assess the biodegradation of micropollutants in cell-free lysates produced from activated sludge of a municipal wastewater treatment plant (WWTP). This proof-of-principle provides the basis for further investigations of micropollutant biodegradation via native enzymes in a solution of reduced complexity, facilitating downstream protein analysis. Differently produced lysates, containing a variety of native enzymes, showed significant enzymatic activities of acid phosphatase, β-galactosidase and β-glucuronidase in conventional colorimetric enzyme assays, whereas heat-deactivated controls did not. To determine the enzymatic activity towards micropollutants, 20 compounds were spiked to the cell-free lysates under aerobic conditions and were monitored via LC-ESI-MS/MS. The micropollutants were selected to span a wide range of different biodegradabilities in conventional activated sludge treatment via distinct primary degradation reactions. Of the 20 spiked micropollutants, 18 could be degraded by intact sludge under assay conditions, while six showed reproducible degradation in the lysates compared to the heat-deactivated negative controls: acetaminophen, N-acetyl-sulfamethoxazole (acetyl-SMX), atenolol, bezafibrate, erythromycin and 10,11-dihydro-10-hydroxycarbamazepine (10-OH-CBZ). The primary biotransformation of the first four compounds can be attributed to amide hydrolysis. However, the observed biotransformations in the lysates were differently influenced by experimental parameters such as sludge pre-treatment and the addition of ammonium sulfate or peptidase inhibitors, suggesting that different hydrolase enzymes were involved in the primary degradation, among them possibly peptidases. Furthermore, the transformation of 10-OH-CBZ to 9-CA-ADIN was caused by a biologically-mediated oxidation, which indicates that in addition to hydrolases further enzyme classes (probably oxidoreductases) are present in the native lysates. Although the

  16. Measurement and characterization of cellulase activity in sclerophyllous forest litter.

    Science.gov (United States)

    Criquet, Stéven

    2002-07-01

    Cellulases are enzymatic proteins which hydrolyze cellulose polymers to smaller oligosaccharides, cellobiose and glucose. They consist in three major types of enzymes: endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91) and beta-glucosidases (EC 3.2.1.21) which play an essential role in carbon turnover of forest ecosystem. The aim of this study was firstly to determine the parameters (i.e. buffer type, pH, temperature, quantity of litter, incubation time and reagent type) which affect the measurement of cellulase activity in a sclerophyllous forest litter, and secondly to compare two methods for measuring cellulase activity: a direct method and an extraction method. In the direct method, the litter was directly incubated with a buffered solution containing the enzyme substrate, whereas in the extraction method, the cellulases were firstly extracted before measuring their activity. The results were compared with other studies about soil cellulase activity, and it appeared that several parameters (buffer type, pH, temperature and sample quantity) which influence the measurement of cellulase activity differ according to whether a soil or a litter is considered. Concerning the procedure used for the measurement of cellulase activity, results showed that the activity values were higher when using an extraction procedure than when using a direct procedure. The extraction procedure, combined with a concentration stage of the extract, also allowed electrophoretic analysis (PAGE) of the cellulases extracted from the litter. The electrophoretic pattern revealed two cellulase isoenzymes which may be related to the occurrence of two pH-activity peaks of these enzymes when citrate buffer was used for the measurement of cellulase activity in the litter.

  17. A method to measure hydrolytic activity of adenosinetriphosphatases (ATPases.

    Directory of Open Access Journals (Sweden)

    Gianluca Bartolommei

    Full Text Available The detection of small amounts (nanomoles of inorganic phosphate has a great interest in biochemistry. In particular, phosphate detection is useful to evaluate the rate of hydrolysis of phosphatases, that are enzymes able to remove phosphate from their substrate by hydrolytic cleavage. The hydrolysis rate is correlated to enzyme activity, an extremely important functional parameter. Among phosphatases there are the cation transporting adenosinetriphosphatases (ATPases, that produce inorganic phosphate by cleavage of the γ-phosphate of ATP. These membrane transporters have many fundamental physiological roles and are emerging as potential drug targets. ATPase hydrolytic activity is measured to test enzyme functionality, but it also provides useful information on possible inhibitory effects of molecules that interfere with the hydrolytic process. We have optimized a molybdenum-based protocol that makes use of potassium antimony (III oxide tartrate (originally employed for phosphate detection in environmental analysis to allow its use with phosphatase enzymes. In particular, the method was successfully applied to native and recombinant ATPases to demonstrate its reliability, validity, sensitivity and versatility. Our method introduces significant improvements to well-established experimental assays, which are currently employed for ATPase activity measurements. Therefore, it may be valuable in biochemical and biomedical investigations of ATPase enzymes, in combination with more specific tests, as well as in high throughput drug screening.

  18. NRSA enzyme decomposition model data

    Data.gov (United States)

    U.S. Environmental Protection Agency — Microbial enzyme activities measured at more than 2000 US streams and rivers. These enzyme data were then used to predict organic matter decomposition and microbial...

  19. Hfq stimulates the activity of the CCA-adding enzyme

    Directory of Open Access Journals (Sweden)

    Betat Heike

    2007-10-01

    Full Text Available Abstract Background The bacterial Sm-like protein Hfq is known as an important regulator involved in many reactions of RNA metabolism. A prominent function of Hfq is the stimulation of RNA polyadenylation catalyzed by E. coli poly(A polymerase I (PAP. As a member of the nucleotidyltransferase superfamily, this enzyme shares a high sequence similarity with an other representative of this family, the tRNA nucleotidyltransferase that synthesizes the 3'-terminal sequence C-C-A to all tRNAs (CCA-adding enzyme. Therefore, it was assumed that Hfq might not only influence the poly(A polymerase in its specific activity, but also other, similar enzymes like the CCA-adding enzyme. Results Based on the close evolutionary relation of these two nucleotidyltransferases, it was tested whether Hfq is a specific modulator acting exclusively on PAP or whether it also influences the activity of the CCA-adding enzyme. The obtained data indicate that the reaction catalyzed by this enzyme is substantially accelerated in the presence of Hfq. Furthermore, Hfq binds specifically to tRNA transcripts, which seems to be the prerequisite for the observed effect on CCA-addition. Conclusion The increase of the CCA-addition in the presence of Hfq suggests that this protein acts as a stimulating factor not only for PAP, but also for the CCA-adding enzyme. In both cases, Hfq interacts with RNA substrates, while a direct binding to the corresponding enzymes was not demonstrated up to now (although experimental data indicate a possible interaction of PAP and Hfq. So far, the basic principle of these stimulatory effects is not clear yet. In case of the CCA-adding enzyme, however, the presented data indicate that the complex between Hfq and tRNA substrate might enhance the product release from the enzyme.

  20. Carotenoid-cleavage activities of crude enzymes from Pandanous amryllifolius.

    Science.gov (United States)

    Ningrum, Andriati; Schreiner, Matthias

    2014-11-01

    Carotenoid degradation products, known as norisoprenoids, are aroma-impact compounds in several plants. Pandan wangi is a common name of the shrub Pandanus amaryllifolius. The genus name 'Pandanus' is derived from the Indonesian name of the tree, pandan. In Indonesia, the leaves from the plant are used for several purposes, e.g., as natural colorants and flavor, and as traditional treatments. The aim of this study was to determine the cleavage of β-carotene and β-apo-8'-carotenal by carotenoid-cleavage enzymes isolated from pandan leaves, to investigate dependencies of the enzymatic activities on temperature and pH, to determine the enzymatic reaction products by using Headspace Solid Phase Microextraction Gas Chromatography/Mass Spectrophotometry (HS-SPME GC/MS), and to investigate the influence of heat treatment and addition of crude enzyme on formation of norisoprenoids. Crude enzymes from pandan leaves showed higher activity against β-carotene than β-apo-8'-carotenal. The optimum temperature of crude enzymes was 70°, while the optimum pH value was 6. We identified β-ionone as the major volatile reaction product from the incubations of two different carotenoid substrates, β-carotene and β-apo-8'-carotenal. Several treatments, e.g., heat treatment and addition of crude enzymes in pandan leaves contributed to the norisoprenoid content. Our findings revealed that the crude enzymes from pandan leaves with carotenoid-cleavage activity might provide a potential application, especially for biocatalysis, in natural-flavor industry. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

  1. Enzyme activities by indicator of quality in organic soil

    Science.gov (United States)

    Raigon Jiménez, Mo; Fita, Ana Delores; Rodriguez Burruezo, Adrián

    2016-04-01

    The analytical determination of biochemical parameters, as soil enzyme activities and those related to the microbial biomass is growing importance by biological indicator in soil science studies. The metabolic activity in soil is responsible of important processes such as mineralization and humification of organic matter. These biological reactions will affect other key processes involved with elements like carbon, nitrogen and phosphorus , and all transformations related in soil microbial biomass. The determination of biochemical parameters is useful in studies carried out on organic soil where microbial processes that are key to their conservation can be analyzed through parameters of the metabolic activity of these soils. The main objective of this work is to apply analytical methodologies of enzyme activities in soil collections of different physicochemical characteristics. There have been selective sampling of natural soils, organic farming soils, conventional farming soils and urban soils. The soils have been properly identified conserved at 4 ° C until analysis. The enzyme activities determinations have been: catalase, urease, cellulase, dehydrogenase and alkaline phosphatase, which bring together a representative group of biological transformations that occur in the soil environment. The results indicate that for natural and agronomic soil collections, the values of the enzymatic activities are within the ranges established for forestry and agricultural soils. Organic soils are generally higher level of enzymatic, regardless activity of the enzyme involved. Soil near an urban area, levels of activities have been significantly reduced. The vegetation cover applied to organic soils, results in greater enzymatic activity. So the quality of these soils, defined as the ability to maintain their biological productivity is increased with the use of cover crops, whether or spontaneous species. The practice of cover based on legumes could be used as an ideal choice

  2. Are spontaneous conformational interconversions a molecular basis for long-period oscillations in enzyme activity?

    Science.gov (United States)

    Queiroz-Claret, C; Valon, C; Queiroz, O

    1988-01-01

    An unconventional hypothesis to the molecular basis of enzyme rhythms is that the intrinsic physical instability of the protein molecules which, in an aqueous medium, tend to move continuously from one conformational state to another could lead, in the population of enzyme molecules, to sizeable long-period oscillations in affinity for substrate and sensitivity to ligands and regulatory effects. To investigate this hypothesis, malate dehydrogenase was extracted and purified from leaves of the plant Kalanchoe blossfeldiana. The enzyme solutions were maintained under constant conditions and sampled at regular intervals for up to 40 or 70 h for measurements of activity as a function of substrate concentration, Km for oxaloacetic acid and sensitivity to the action of 2,3-butanedione, a modifier of active site arginyl residues. The results show that continuous slow oscillations in the catalytic capacity of the enzyme occur in all the extracts checked, together with fluctuations in Km. Apparent circadian periodicities were observed in accordance with previous data established during long run (100 h) experiments. The saturation curves for substrate showed multiple kinetic functions, with various pronounced intermediary plateaus and "bumps" depending on the time of sampling. Variation in the response to the effect of butanedione indicated fluctuation in the accessibility to the active site. Taken together, the results suggest that, under constant conditions, the enzyme in solution shifts continuously and reversibly between different configurations. This was confirmed by parallel studies on the proton-NMR spectrum of water aggregates in the enzyme solution and proton exchange rates.(ABSTRACT TRUNCATED AT 250 WORDS)

  3. Effect of Barley and Enzyme on Performance, Carcass, Enzyme Activity and Digestion Parameters of Broilers

    Directory of Open Access Journals (Sweden)

    majid kalantar

    2016-04-01

    Ross-308 broiler chickens were allocated randomly to 3 treatments with 5 replicates using a CRD statistical design. Treatments were included control, barley and barley+ enzyme. The experimental diets were formulated to have similar contents of crude protein, metabolizable energy, total non-starch polysaccharides (NSP. Results and Discussion According to the results, effect of barley with or without enzyme on growth performance at starter, grower and the entire period and also on carcass characteristics, pancreas enzyme activity and measures of ileal acidity and viscosity at the age of 42 were significant (P

  4. Effect of bleaching on mercury release from amalgam fillings and antioxidant enzyme activities: a pilot study.

    Science.gov (United States)

    Cakir, Filiz Yalcin; Ergin, Esra; Gurgan, Sevil; Sabuncuoglu, Suna; Arpa, Cigdem Sahin; Tokgoz, İlknur; Ozgunes, Hilal; Kiremitci, Arlin

    2015-01-01

    The aim of this pilot clinical study was to determine the mercury release from amalgam fillings and antioxidant enzyme activities (Superoxide Dismutase [SOD] and Catalase[CAT] ) in body fluids after exposure to two different vital tooth bleaching systems. Twenty eight subjects with an average age of 25.6 years (18-41) having at least two but not more than four Class II amalgam fillings on each quadrant arch in the mouth participated in the study. Baseline concentrations of mercury levels in whole blood, urine, and saliva were measured by a Vapor Generation Accessory connected to an Atomic Absorption Spectrometer. Erythrocyte enzymes, SOD, and CAT activities in blood were determined kinetically. Subjects were randomly assigned to two groups of 14 volunteers. Group 1 was treated with an at-home bleaching system (Opalescence PF 35% Carbamide Peroxide, Ultradent), and Group 2 was treated with a chemically activated office bleaching system (Opalescence Xtra Boost 38% Hydrogen Peroxide, Ultradent) according to the manufacturer's recommendations. Twenty-four hours after bleaching treatments, concentrations of mercury and enzymes were remeasured. There were no significant differences on mercury levels in blood, urine, and saliva before and after bleaching treatments (p > 0.05). No differences were also found in the level of antioxidant enzyme activities (SOD and CAT) before and after treatments (p > 0.05). Mercury release did not affect the enzyme activities (p > 0.05). Bleaching treatments either office or home did not affect the amount of mercury released from amalgam fillings in blood, urine, and saliva and the antioxidant-enzyme activities in blood. Bleaching treatments with the systems tested in this pilot study have no deleterious effect on the mercury release from amalgam fillings and antioxidant enzymes in body fluids. © 2014 Wiley Periodicals, Inc.

  5. Effect of diffusion on enzyme activity in a microreactor

    NARCIS (Netherlands)

    Swarts, J.W.; Kolfschoten, R.C.; Jansen, M.C.A.A.; Janssen, A.E.M.; Boom, R.M.

    2010-01-01

    To establish general rules for setting up an enzyme microreactor system, we studied the effect of diffusion on enzyme activity in a microreactor. As a model system we used the hydrolysis of ortho-nitrophenyl-ß-d-galactopyranoside by ß-galactosidase from Kluyveromyces lactis. We found that the

  6. Differentiation between activity of digestive enzymes of Brachionus calyciflorus and extracellular enzymes of its epizooic bacteria

    Directory of Open Access Journals (Sweden)

    Wilko H. AHLRICHS

    2009-08-01

    Full Text Available The rotifer Brachionus calyciflorus was examined by scanning electron microscopy (SEM for surface-attached, i.e. epizootic, bacteria to ascertain their specific localization and thus find out if we could discern between rotifer and bacterial enzyme activity. The lorica of B. calyciflorus was colonized by one distinct type of bacteria, which originated from the algal culture used for rotifer feeding. The corona, posterior epidermis and foot of all inspected individuals were always without attached bacteria. The density of the attached bacteria was higher with the increasing age of B. calyciflorus: while young individuals were colonized by ~ tens of bacterial cells, older ones had on average hundreds to thousands of attached bacteria. We hypothesize that epizooic bacteria may produce the ectoenzymes phosphatases and β-N-acetylhexosaminidases on the lorica, but not on the corona of B. calyciflorus. Since enzyme activities of epizooic bacteria may influence the values and interpretation of bulk rotifer enzyme activities, we should take the bacterial contribution into account.

  7. Enzyme activities in reclaimed coal mine spoils and soils

    Energy Technology Data Exchange (ETDEWEB)

    Fresquez, P R; Aldon, E F; Lindemann, W C

    1987-11-01

    The segregation and stockpiling of topsoil material may reduce enzymatic activities that may hinder normal nutrient cycling processes in reclaimed minelands. The effects of topsoiling and reclamation age on dehydrogenase, nitrogenase, phosphatase, arylsulphatase, amylase, cellulase, invertase and urease activities were evaluated on three reclaimed non-top-soiled and five reclaimed topsoiled areas and compared with an indisturbed reference soil. Three months after topsoiling and revegetation, activities of the enzymes in the reclaimed areas, with the exception of dehydrogenase, were statistically equal to activities of the undisturbed soil. Most enzymes, including dehydrogenase, peaked in the next 1 or 2 years after reclamation with topsoiling and declined thereafter. A 4-year-old topsoiled site (revegetated in 1978) was statistically similar to the undisturbed soil. Amylase activity, however, was significantly lower after the fourth year compared to the undisturbed soil. The non-topsoiled areas, even after 6, 7 and 8 years, appeared to have lower enzyme activities than the younger topsoiled areas or the undisturbed soil. This trend was supported by the finding that the 4-year-old topsoiled site was more enzymatically similar to the undisturbed soil than was the 8-year-old non-topsoiled site (revegetated in 1974). The low enzyme acitivities found in the non-topsoiled areas may be a result of their adverse chemical and physical properties, as well as the low diversity of microorganisms. These studies demonstrate the value of topsoil use for early establishment of soil processes in reclaimed areas. 3 figs., 19 refs., 8 tabs.

  8. Proteolytic Enzymes in Detergents: Evidence of Their Presence through Activity Measurements Based on Electrophoresis

    Science.gov (United States)

    Saperas, Nuria; Fonfria-Subiros, Elsa

    2011-01-01

    This laboratory exercise uses a problem-based approach to expose students to some basic concepts relating to proteins and enzymes. One of the main applications of enzymes at the industrial level is their use in the detergent market. The students examine a detergent sample to ascertain whether proteolytic enzymes are a component and, if so, which…

  9. [The restoration of the enzyme activity of chernozem soil after gamma-irradiation].

    Science.gov (United States)

    Denisova, T V; Kazeev, K Sh

    2006-01-01

    The Influence of gamma-radiation by dozes 1, 5, 10 and 20 kGy on enzyme activity of ordinary chemozem were studied. Dynamics of the restoration of the enzyme activity after the influence of gamma-radiation in model experiments in 3, 30, 90 and 180 days was investigated. The doze 1 kGy did no statistically significant influence on the investigated enzymes. Dehydrogenase is more radiosensitive enzyme than catalase. Values of the saccharase activity differed a significant variation so in most cases it has not been registered statistically significant changes. In 90-180 days of the incubation enzymes activity was restored up to control values. Dehydrogenase activity in 180 days in variants with dozes 10 and 20 kGy was restored up to a level of the control, over variants with dozes 1 and 5 kGy--is higher than the control over 78% and 23% accordingly. Saccharase activity in 180 days after the influence of gamma-radiation with a doze 20 kGy was on 61% lower than the control.

  10. Remote enzyme activation using gold coated magnetite as antennae for radio frequency fields

    Science.gov (United States)

    Collins, Christian B.; Ackerson, Christopher J.

    2018-02-01

    The emerging field of remote enzyme activation, or the ability to remotely turn thermophilic increase enzyme activity, could be a valuable tool for understanding cellular processes. Through exploitation of the temperature dependence of enzymatic processes and high thermal stability of thermophilic enzymes these experiments utilize nanoparticles as `antennae' that convert radiofrequency (RF) radiation into local heat, increasing activity of the enzymes without increasing the temperature of the surrounding bulk solution. To investigate this possible tool, thermolysin, a metalloprotease was covalently conjugated to 4nm gold coated magnetite particles via peptide bond formation with the protecting ligand shell. RF stimulated protease activity at 17.76 MHz in a solenoid shaped antenna, utilizing both electric and magnetic field interactions was investigated. On average 40 percent higher protease activity was observed in the radio frequency fields then when bulk heating the sample to the same temperature. This is attributed to electrophoretic motion of the nanoparticle enzyme conjugates and local regions of heat generated by the relaxation of the magnetite cores with the oscillating field. Radio frequency local heating of nanoparticles conjugated to enzymes as demonstrated could be useful in the activation of specific enzymes in complex cellular environments.

  11. Ligninolytic enzyme activities in mycelium of some wild and ...

    African Journals Online (AJOL)

    Lignin is probably one of the most recalcitrant compounds synthesized by plants. This compound is degraded by few microorganisms. White-rot fungi have been extensively studied due to its powerful ligninolytic enzymes. In this study, ligninolytic enzyme activities of different fungal species (six commercial and 13 wild) were ...

  12. 3D Restoration Microscopy Improves Quantification of Enzyme-Labeled Fluorescence-Based Single-Cell Phosphatase Activity in Plankton

    OpenAIRE

    Diaz-de-Quijano, Daniel; Palacios, Pilar; Hornák, Karel; Felip, Marisol

    2014-01-01

    The ELF or fluorescence-labeled enzyme activity (FLEA) technique is a culture-independent single-cell tool for assessing plankton enzyme activity in close-to-in situ conditions. We demonstrate that single-cell FLEA quantifications based on two-dimensional (2D) image analysis were biased by up to one order of magnitude relative to deconvolved 3D. This was basically attributed to out-of-focus light, and partially to object size. Nevertheless, if sufficient cells were measured (25-40 cells), bia...

  13. Cellulase activities in biomass conversion: measurement methods and comparison.

    Science.gov (United States)

    Dashtban, Mehdi; Maki, Miranda; Leung, Kam Tin; Mao, Canquan; Qin, Wensheng

    2010-12-01

    Cellulose, the major constituent of all plant materials and the most abundant organic molecule on the Earth, is a linear biopolymer of glucose molecules, connected by β-1,4-glycosidic bonds. Enzymatic hydrolysis of cellulose requires mixtures of hydrolytic enzymes including endoglucanases, exoglucanases (cellobiohydrolases), and β-glucosidases acting in a synergistic manner. In biopolymer hydrolysis studies, enzyme assay is an indispensable part. The most commonly used assays for the individual enzymes as well as total cellulase activity measurements, including their advantages and limitations, are summarized in this review article. In addition, some novel approaches recently used for enzyme assays are summarized.

  14. Activity of selected hydrolytic enzymes in Allium sativum L. anthers.

    Science.gov (United States)

    Winiarczyk, Krystyna; Gębura, Joanna

    2016-05-01

    The aim of the study was to determine enzymatic activity in sterile Allium sativum anthers in the final stages of male gametophyte development (the stages of tetrads and free microspores). The analysed enzymes were shown to occur in the form of numerous isoforms. In the tetrad stage, esterase activity was predominant, which was manifested by the greater number of isoforms of the enzyme. In turn, in the microspore stage, higher numbers of isoforms of acid phosphatases and proteases were detected. The development of sterile pollen grains in garlic is associated with a high level of protease and acid phosphatase activity and lower level of esterase activities in the anther locule. Probably this is the first description of the enzymes activity (ACPH, EST, PRO) in the consecutives stages of cell wall formation which is considered to be one of the causes of male sterility in flowering plant. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  15. Optimization of Enzyme Co-Immobilization with Sodium Alginate and Glutaraldehyde-Activated Chitosan Beads.

    Science.gov (United States)

    Gür, Sinem Diken; İdil, Neslihan; Aksöz, Nilüfer

    2018-02-01

    In this study, two different materials-alginate and glutaraldehyde-activated chitosan beads-were used for the co-immobilization of α-amylase, protease, and pectinase. Firstly, optimization of multienzyme immobilization with Na alginate beads was carried out. Optimum Na alginate and CaCl 2 concentration were found to be 2.5% and 0.1 M, respectively, and optimal enzyme loading ratio was determined as 2:1:0.02 for pectinase, protease, and α-amylase, respectively. Next, the immobilization of multiple enzymes on glutaraldehyde-activated chitosan beads was optimized (3% chitosan concentration, 0.25% glutaraldehyde with 3 h of activation and 3 h of coupling time). While co-immobilization was successfully performed with both materials, the specific activities of enzymes were found to be higher for the enzymes co-immobilized with glutaraldehyde-activated chitosan beads. In this process, glutaraldehyde was acting as a spacer arm. SEM and FTIR were used for the characterization of activated chitosan beads. Moreover, pectinase and α-amylase enzymes immobilized with chitosan beads were also found to have higher activity than their free forms. Three different enzymes were co-immobilized with these two materials for the first time in this study.

  16. Revealing hidden effect of earthworm on C distribution and enzyme activity

    Science.gov (United States)

    Razavi, Bahar S.; Hoang, Duyen; Kuzyakov, Yakov

    2017-04-01

    Despite its importance for terrestrial nutrient and carbon cycling, the spatial organization and localization of microbial activity in soil and in biopores is poorly understood. We hypothesized that biopores created by earthworm play a critical role in reducing the gap of SOM input and microbial activities between topsoil and subsoil. Accordingly, Carbon (C) allocation by earthworms was related to enzyme distribution along soil profile. For the first time we visualized spatial distribution of enzyme activities (β-glucosidase, chitinase and acid phosphatase) and C allocation (by 14C imaging) in earthworm biopores in topsoil and subsoil. Soil zymography (an in situ method for the analysis of the two-dimensional distribution of enzyme activity in soil) was accompanied with 14C imaging (a method that enables to trace distribution of litter and C in soil profile) to visualize change of enzyme activities along with SOM incorporation by earthworms from topsoil to subsoil. Experiment was set up acquiring rhizoboxes (9×1×50 cm) filled up with fresh soil and 3 earthworms (L. terrestris), which were then layered with 14C-labeled plant-litter of 0.3 MBq on the soil surface. 14C imaging and zymography have been carried out after one month. Activities of all enzymes regardless of their nutrient involvement (C, N, P) were higher in the biopores than in bulk soil, but the differences were larger in topsoil compared to subsoil. Among three enzymes, Phosphatase activity was 4-times higher in the biopore than in the bulk soil. Phosphatase activity was closely associated with edge of burrows and correlate positively with 14C activity. These results emphasized especial contribution of hotspheres such as biopores to C allocation in subsoil - which is limited in C input and nutrients - and in stimulation of microbial and enzymatic activity by input of organic residues, e.g. by earthworms. In conclusion, biopore increased enzymatic mobilization of nutrients (e.g. P) inducing allocation

  17. Molecular dynamics explorations of active site structure in designed and evolved enzymes.

    Science.gov (United States)

    Osuna, Sílvia; Jiménez-Osés, Gonzalo; Noey, Elizabeth L; Houk, K N

    2015-04-21

    This Account describes the use of molecular dynamics (MD) simulations to reveal how mutations alter the structure and organization of enzyme active sites. As proposed by Pauling about 70 years ago and elaborated by many others since then, biocatalysis is efficient when functional groups in the active site of an enzyme are in optimal positions for transition state stabilization. Changes in mechanism and covalent interactions are often critical parts of enzyme catalysis. We describe our explorations of the dynamical preorganization of active sites using MD, studying the fluctuations between active and inactive conformations normally concealed to static crystallography. MD shows how the various arrangements of active site residues influence the free energy of the transition state and relates the populations of the catalytic conformational ensemble to the enzyme activity. This Account is organized around three case studies from our laboratory. We first describe the importance of dynamics in evaluating a series of computationally designed and experimentally evolved enzymes for the Kemp elimination, a popular subject in the enzyme design field. We find that the dynamics of the active site is influenced not only by the original sequence design and subsequent mutations but also by the nature of the ligand present in the active site. In the second example, we show how microsecond MD has been used to uncover the role of remote mutations in the active site dynamics and catalysis of a transesterase, LovD. This enzyme was evolved by Tang at UCLA and Codexis, Inc., and is a useful commercial catalyst for the production of the drug simvastatin. X-ray analysis of inactive and active mutants did not reveal differences in the active sites, but relatively long time scale MD in solution showed that the active site of the wild-type enzyme preorganizes only upon binding of the acyl carrier protein (ACP) that delivers the natural acyl group to the active site. In the absence of bound ACP

  18. Spatial distribution of enzyme activities along the root and in the rhizosphere of different plants

    Science.gov (United States)

    Razavi, Bahar S.; Zarebanadkouki, Mohsen; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2015-04-01

    Extracellular enzymes are important for decomposition of many biological macromolecules abundant in soil such as cellulose, hemicelluloses and proteins. Activities of enzymes produced by both plant roots and microbes are the primary biological drivers of organic matter decomposition and nutrient cycling. So far acquisition of in situ data about local activity of different enzymes in soil has been challenged. That is why there is an urgent need in spatially explicit methods such as 2-D zymography to determine the variation of enzymes along the roots in different plants. Here, we developed further the zymography technique in order to quantitatively visualize the enzyme activities (Spohn and Kuzyakov, 2013), with a better spatial resolution We grew Maize (Zea mays L.) and Lentil (Lens culinaris) in rhizoboxes under optimum conditions for 21 days to study spatial distribution of enzyme activity in soil and along roots. We visualized the 2D distribution of the activity of three enzymes:β-glucosidase, leucine amino peptidase and phosphatase, using fluorogenically labelled substrates. Spatial resolution of fluorescent images was improved by direct application of a substrate saturated membrane to the soil-root system. The newly-developed direct zymography shows different pattern of spatial distribution of enzyme activity along roots and soil of different plants. We observed a uniform distribution of enzyme activities along the root system of Lentil. However, root system of Maize demonstrated inhomogeneity of enzyme activities. The apical part of an individual root (root tip) in maize showed the highest activity. The activity of all enzymes was the highest at vicinity of the roots and it decreased towards the bulk soil. Spatial patterns of enzyme activities as a function of distance from the root surface were enzyme specific, with highest extension for phosphatase. We conclude that improved zymography is promising in situ technique to analyze, visualize and quantify

  19. Interaction of on-site and near real time measured turbidity and enzyme activity in stream water.

    Science.gov (United States)

    Stadler, Philipp; Farnleitner, Andreas H.; Zessner, Matthias

    2013-04-01

    influence of turbidity on rapid GLUC measurements of stream water. During event run off conditions with high sediment load, accuracy of the GLUC determination was assayed. Various on-site set ups were tested to ascertain the use of sample prefiltration. We would like acknowledge financial support from the Austrian Science Funds (FWF) as part of the Vienna Doctoral Programme on Water Resource Systems (DK-plus W1219-N22). References: Cabral, J. P. S. 2010. "Water Microbiology. Bacterial Pathogens and Water." International Journal of Environmental Research and Public Health 7 (10): 3657-3703. Biswal , N. , S. Gupta, N. Ghosh, and A. Pradhan 2003. "Recovery of turbidity free fluorescence from measured fluorescence: An experimental approach. Optics Express 11, (24): 3320. Molina-Munoz, M., J. M. Poyatos, R. Vilchez, E. Hontoria, B. Rodelas, and J. Gonzalez-Lopez. 2007. "Effect of the Concentration of Suspended Solids on the Enzymatic Activities and Biodiversity of a Submerged Membrane Bioreactor for Aerobic Treatment of Domestic Wastewater." Applied Microbiology and Biotechnology 73 (6): 1441-1451. Tryland, I., and L. Fiksdal. 1998. "Enzyme Characteristics of β-d-Galactosidase-and β-d-Glucuronidase-Positive Bacteria and Their Interference in Rapid Methods for Detection of Waterborne Coliforms andEscherichia Coli." Applied and Environmental Microbiology 64 (3): 1018-1023.

  20. Enzymes extracted from apple peels have activity in reducing higher alcohols in Chinese liquors.

    Science.gov (United States)

    Han, Qi'an; Shi, Junling; Zhu, Jing; Lv, Hongliang; Du, Shuangkui

    2014-10-01

    As the unavoidable byproducts of alcoholic fermentation, higher alcohols are unhealthy compounds widespread in alcoholic drinks. To investigate the activity of apple crude enzymes toward higher alcohols in liquors, five kinds of apple peels, namely, Fuji, Gala, Golden Delicious, Red Star, and Jonagold, were chosen to prepare enzymes, and three kinds of Chinese liquors, namely, Xifeng (containing 45% ethanol), Taibai (containing 50% ethanol), and Erguotou (containing 56% ethanol), were tested. Enzymes were prepared in the forms of liquid solution, powder, and immobilized enzymes using sodium alginate (SA) and chitosan. The treatment was carried out at 37 °C for 1 h. The relative amounts of different alcohols (including ethanol, 1-propanol, isobutanol, 1-butanol, isoamylol, and 1-hexanol) were measured using gas chromatography (GC). Conditions for preparing SA-immobilized Fuji enzymes (SA-IEP) were optimized, and the obtained SA-IEP (containing 0.3 g of enzyme) was continuously used to treat Xifeng liquor eight times, 20 mL per time. Significant degradation rates (DRs) of higher alcohols were observed at different degrees, and it also showed enzyme specificity according to the apple varieties and enzyme preparations. After five repeated treatments, the DRs of the optimized Fuji SA-IEP remained 70% for 1-hexanol and >15% for other higher alcohols.

  1. Triclabendazole Effect on Protease Enzyme Activity in the Excretory- Secretory Products of Fasciola hepatica in Vitro.

    Directory of Open Access Journals (Sweden)

    Yosef Shrifi

    2014-03-01

    Full Text Available Fasciola hepatica is one of the most important helminthes parasites and triclabendazole (TCBZ is routinely used for treatment of infected people and animals. Secreted protease enzymes by the F. hepatica plays a critical role in the invasion, migration, nutrition and the survival of parasite and are key targets for novel drugs and vaccines. The aim of study was to determine the protease activity of excretory- secretory products (ESP of F. hepatica in the presence of TCBZ anthelmintic.F. hepatica helminthes were collected and cultured within RPMI 1640 [TCBZ treated (test and untreated (control] for 6 h at 37 °C. ESP of treated and control were collected, centrifuged and supernatants were stored at -20°C. Protein concentrations were measured according to Bradford method. Protease enzymes activities of ESP samples were estimated by using sigma's non-specific protease activity assay. ESP protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE.Mean protein concentrations in control and treated of ESP samples were determined 196.1 ±14.52 and 376.4 ±28.20 μg/ml, respectively. Mean protease enzymes activities in control and treated were 0.37 ±0.1 and 0.089 ±0.03 U/ml, respectively. Significant difference between proteins concentrations and protease enzymes activities of two groups was observed (P<0.05. SDS-PAGE showed different patterns of protein bands between treated and control samples.The TCBZ reduced secreted protease enzymes activities and possibly effects on invasion, migration, nutrition and particularly survival of the parasite in the host tissues.

  2. A virus-based single-enzyme nanoreactor

    NARCIS (Netherlands)

    Comellas Aragones, M.; Engelkamp, H.; Claessen, V.I.; Sommerdijk, N.A.J.M.; Rowan, A.E.; Christianen, P.C.M.; Maan, J.C.; Verduin, B.J.M.; Cornelissen, J.J.L.M.; Nolte, R.J.M.

    2007-01-01

    Most enzyme studies are carried out in bulk aqueous solution, at the so-called ensemble level, but more recently studies have appeared in which enzyme activity is measured at the level of a single molecule, revealing previously unseen properties. To this end, enzymes have been chemically or

  3. Influence of long-term fertilization on soil enzyme activities

    Directory of Open Access Journals (Sweden)

    Alina Dora SAMUEL

    2009-05-01

    Full Text Available Soil enzyme activities (actual and potential dehydrogenase, catalase, acid and alkaline phosphatase were determined in the 0–10, 10–20, and 20–30 cm layers of a brown luvic soil submitted to a complex fertilization experiment with different types of green manure. It was found that each activity decreased with increasing sampling depth. It should be emphasized that greenmanuring of maize led to a significant increase in each of the five enzymatic activities determined. The enzymatic indicators of soil quality calculated from the values of enzymatic activities showed the order: lupinus + rape + oat > lupinus > vetch + oat + ryegrass > lupinus + oat + vetch > unfertilized plot. This order means that by determination of enzymatic activities valuable information can be obtained regarding fertility status of soils. There were significant correlations of soil enzyme activities with chemical properties.

  4. Functionalization of PDMS modified and plasma activated two-component polyurethane coatings by surface attachment of enzymes

    International Nuclear Information System (INIS)

    Kreider, Alexej; Richter, Katharina; Sell, Stephan; Fenske, Mandus; Tornow, Christian; Stenzel, Volkmar; Grunwald, Ingo

    2013-01-01

    This article describes a new strategy for coupling the enzyme horseradish peroxidase to a two-component polyurethane (2C-PUR) coating. A stable polymer conjugate was achieved by combining the enzyme and the 2C-PUR coating which was modified with poly(dimethylsiloxane) (PDMS), located at the surface. An atmospheric pressure plasma jet system was used to convert alkyl groups from the PDMS into polar silanol functionalities. This conversion was proven by X-ray photoelectron spectroscopy and dynamic contact angle measurements. In addition, the stability of the activated 2C-PUR surface containing silanol groups was determined by measuring the contact angle as a function of time. Compared to the non-modified 2C-PUR systems the one with PDMS displayed a higher stability over a time period over 28 h. In a silanization process the coating was treated with (3-aminopropyl) trimethoxysilane and the enzyme was subsequently immobilized to the coating via the cross linker glutaraldehyde to receive new biomimetic catalytic/enzymatic functions. The chemical immobilization (chemisorption) of the enzyme to the surface showed statistically significant higher biological activity as compared to references samples without using a cross linker (physisorption). The presented technique offers the opportunity to design new and smart multifunctional surface coatings which employ biomimetic capabilities.

  5. Functionalization of PDMS modified and plasma activated two-component polyurethane coatings by surface attachment of enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Kreider, Alexej; Richter, Katharina; Sell, Stephan; Fenske, Mandus; Tornow, Christian; Stenzel, Volkmar [Fraunhofer Institute for Manufacturing Technology and Advanced Materials - IFAM, Wiener Strasse 12, 28359 Bremen (Germany); Grunwald, Ingo, E-mail: ingo.grunwald@ifam.fraunhofer.de [Fraunhofer Institute for Manufacturing Technology and Advanced Materials - IFAM, Wiener Strasse 12, 28359 Bremen (Germany)

    2013-05-15

    This article describes a new strategy for coupling the enzyme horseradish peroxidase to a two-component polyurethane (2C-PUR) coating. A stable polymer conjugate was achieved by combining the enzyme and the 2C-PUR coating which was modified with poly(dimethylsiloxane) (PDMS), located at the surface. An atmospheric pressure plasma jet system was used to convert alkyl groups from the PDMS into polar silanol functionalities. This conversion was proven by X-ray photoelectron spectroscopy and dynamic contact angle measurements. In addition, the stability of the activated 2C-PUR surface containing silanol groups was determined by measuring the contact angle as a function of time. Compared to the non-modified 2C-PUR systems the one with PDMS displayed a higher stability over a time period over 28 h. In a silanization process the coating was treated with (3-aminopropyl) trimethoxysilane and the enzyme was subsequently immobilized to the coating via the cross linker glutaraldehyde to receive new biomimetic catalytic/enzymatic functions. The chemical immobilization (chemisorption) of the enzyme to the surface showed statistically significant higher biological activity as compared to references samples without using a cross linker (physisorption). The presented technique offers the opportunity to design new and smart multifunctional surface coatings which employ biomimetic capabilities.

  6. Influence of probiotics on the growth and digestive enzyme activity of white Pacific shrimp ( Litopenaeus vannamei)

    Science.gov (United States)

    Gómez, R. Geovanny D.; Shen, M. A.

    2008-05-01

    The influence of Bacillus probiotics on the digestive enzyme activity and the growth of Litopenaeus vannamei were determined in this study. The shrimp was treated with five percentages (1.5, 3.0, 4.5, 6.0 and 7.5) of probiotics ( Bacillus spp.) supplemented to the feed and cultured for 45d. The growth measured as the weight gain at the end of culturing was significantly ( Pshrimps than that of the control (without receiving probiotics). Activities of protease and amylase, two digestive enzymes of the midgut gland and the intestine were significantly ( Pshrimp than in the control.

  7. A metal-based inhibitor of NEDD8-activating enzyme.

    Directory of Open Access Journals (Sweden)

    Hai-Jing Zhong

    Full Text Available A cyclometallated rhodium(III complex [Rh(ppy(2(dppz](+ (1 (where ppy=2-phenylpyridine and dppz=dipyrido[3,2-a:2',3'-c]phenazine dipyridophenazine has been prepared and identified as an inhibitor of NEDD8-activating enzyme (NAE. The complex inhibited NAE activity in cell-free and cell-based assays, and suppressed the CRL-regulated substrate degradation and NF-κB activation in human cancer cells with potency comparable to known NAE inhibitor MLN4924. Molecular modeling analysis suggested that the overall binding mode of 1 within the binding pocket of the APPBP1/UBA3 heterodimer resembled that for MLN4924. Complex 1 is the first metal complex reported to suppress the NEDDylation pathway via inhibition of the NEDD8-activating enzyme.

  8. Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.

    Science.gov (United States)

    van Munster, Jolanda M; Thomas, Baptiste; Riese, Michel; Davis, Adrienne L; Gray, Christopher J; Archer, David B; Flitsch, Sabine L

    2017-02-21

    Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.

  9. Understanding drivers of peatland extracellular enzyme activity in the PEATcosm experiment: mixed evidence for enzymic latch hypothesis

    Science.gov (United States)

    Karl J. Romanowicz; Evan S. Kane; Lynette R. Potvin; Aleta L. Daniels; Randy Kolka; Erik A. Lilleskov

    2015-01-01

    Aims. Our objective was to assess the impacts of water table position and plant functional groups on peatland extracellular enzyme activity (EEA) framed within the context of the enzymic latch hypothesis. Methods. We utilized a full factorial experiment with 2 water table (WT) treatments (high and low) and 3 plant functional...

  10. Root carbon inputs to the rhizosphere stimulate extracellular enzyme activity and increase nitrogen availability in temperate forest soils

    Science.gov (United States)

    Brzostek, E. R.; Phillips, R.; Dragoni, D.; Drake, J. E.; Finzi, A. C.

    2011-12-01

    The mobilization of nitrogen (N) from soil organic matter in temperate forest soils is controlled by the microbial production and activity of extracellular enzymes. The exudation of carbon (C) by tree roots into the rhizosphere may subsidize the microbial production of extracellular enzymes in the rhizosphere and increase the access of roots to N. The objective of this research was to investigate whether rates of root exudation and the resulting stimulation of extracellular enzyme activity in the rhizosphere (i.e., rhizosphere effect) differs between tree species that form associations with ectomycorrhizal (ECM) or arbuscular mycorrhizal (AM) fungi. This research was conducted at two temperate forest sites, the Harvard Forest (HF) in Central MA and the Morgan Monroe State Forest (MMSF) in Southern IN. At the HF, we measured rates of root exudation and the rhizosphere effects on enzyme activity, N cycling, and C mineralization in AM and ECM soils. At the MMSF, we recently girdled AM and ECM dominated plots to examine the impact of severing belowground C allocation on rhizosphere processes. At both sites, the rhizosphere effect on proteolytic, chitinolytic and ligninolytic enzyme activities was greater in ECM soils than in AM soils. In particular, higher rates of proteolytic enzyme activity increased the availability of amino acid-N in ECM rhizospheres relative to the bulk soils. Further, this stimulation of enzyme activity was directly correlated with higher rates of C mineralization in the rhizosphere than in the bulk soil. Although not significantly different between species, root exudation of C comprised 3-10% of annual gross primary production at the HF. At the MMSF, experimental girdling led to a larger decline in soil respiration and enzyme activity in ECM plots than in AM plots. In both ECM and AM soils, however, girdling resulted in equivalent rates of enzyme activity in rhizosphere and corresponding bulk soils. The results of this study contribute to the

  11. Subcellular distribution of histone-degrading enzyme activities from rat liver

    International Nuclear Information System (INIS)

    Heinrich, P.C.; Raydt, G.; Puschendorf, B.; Jusic, M.

    1976-01-01

    Chromatin prepared from liver tissue contains a histone-degrading enzyme activity with a pH optimum of 7.5-8.0, whereas chromatin isolated from purified nuclei is devoid of it. The histone-degrading enzyme activity was assayed with radioactively labelled total histones from Ehrlich ascites tumor cells. Among the different subcellular fractions assayed, only lysosomes and mitochondria exhibited histone-degrading enzymes. A pH optimum around 4.0-5.0 was found for the lysosomal fraction, whereas 7.5-8.0 has been found for mitochondria. Binding studies of frozen and thawed lysosomes or mitochondria to proteinase-free chromatin demonstrate that the proteinase associated with chromatin isolated from frozen tissue originates from damaged mitochondria. The protein degradation patterns obtained after acrylamide gel electrophoresis are similar for the chromatin-associated and the mitochondrial proteinase and different from that obtained after incubation with lysosomes. The chromatin-associated proteinase as well as the mitochondrial proteinase are strongly inhibited by 1.0 mM phenylmethanesulfonyl fluoride. Weak inhibition is found for lysosomal proteinases at pH 5. Kallikrein-trypsin inhibitor, however, inhibits lysosomal proteinase activity and has no effect on either chromatin-associated or mitochondrial proteinases. The higher template activity of chromatin isolated from a total homogenate compared to chromatin prepared from nuclei may be due to the presence of this histone-degrading enzyme activity. (orig.) [de

  12. Gaseous environment of plants and activity of enzymes of carbohydrate catabolism

    International Nuclear Information System (INIS)

    Ivanov, B.F.; Zemlyanukhin, A.A.; Igamberdiev, A.U.; Salam, A.M.M.

    1989-01-01

    The authors investigated the action of hypoxia and high CO 2 concentration in the atmosphere on activity of phosphofructokinase, aldolase, glucose phosphate isomerase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase, alcohol dehydrogenase, and isocitrate lyase in pea seedlings (Pisum sativum L.), corn scutella (Zea mays L.), and hemp cotyledons (Cannabis sativa L.). The first 4-12h of hypoxia witnessed suppression of enzymes of the initial stages of glycolysis (glucose-6-phosphate isomerase, phosphofructokinase)and activation of enzymes of its final stages (alcohol dehydrogenase and lactate dehydrogenase) and enzymes linking glycolysis and the pentose phosphate pathway (aldolase and glucose-6-phosphate dehydrogenase). An excess of CO 2 in the environment accelerated and amplified this effect. At the end of a 24-h period of anaerobic incubation, deviations of enzyme activity from the control were leveled in both gaseous environments. An exception was observed in the case of phosphofructokinase, whose activity increased markedly at this time in plants exposed to CO 2 . Changes in activity of the enzymes were coupled with changes in their kinetic parameters (apparent K m and V max values). The activity of isocitrate lyase was suppressed in both variants of hypoxic gaseous environments, a finding that does not agree with the hypothesis as to participation of the glyoxylate cycle in the metabolic response of plants to oxygen stress. Thus, temporary inhibition of the system of glycolysis and activation of the pentose phosphate pathway constituted the initial response of the plants to O 2 stress, and CO 2 intensified this metabolic response

  13. Protective Antioxidant Enzyme Activities are Affected by Drought in Quinoa (Chenopodium Quinoa Willd)

    DEFF Research Database (Denmark)

    Fghire, Rachid; Ali, Oudou Issa; Anaya, Fatima

    2013-01-01

    Changes in water availability are responsible for a variety of biochemical stress responses in plant organisms. Stress induced by this factor may be associated with enhanced reactive oxygen species (ROS) generations, which cause oxidative damage. In the present study we investigated the activities...... increased in all treatments. These results suggest that antioxidant enzymes play important roles in reducing oxidative stress in quinoa plant exposed to drought stress....... of antioxidant enzymes superoxide dismutase (SOD), polyphenoloxydase (PPO), peroxidase (POD) and catalase (CAT), measured at flowering in quinoa, subjected to varying levels of drought stress. Drought levels were 100, 50 and 33% of evapotranspiration (ETc), and rainfed. Compared to full water supply (100%ETc...

  14. Ultrasound assisted intensification of enzyme activity and its properties: a mini-review.

    Science.gov (United States)

    Nadar, Shamraja S; Rathod, Virendra K

    2017-08-22

    Over the last decade, ultrasound technique has emerged as the potential technology which shows large applications in food and biotechnology processes. Earlier, ultrasound has been employed as a method of enzyme inactivation but recently, it has been found that ultrasound does not inactivate all enzymes, particularly, under mild conditions. It has been shown that the use of ultrasonic treatment at appropriate frequencies and intensity levels can lead to enhanced enzyme activity due to favourable conformational changes in protein molecules without altering its structural integrity. The present review article gives an overview of influence of ultrasound irradiation parameters (intensity, duty cycle and frequency) and enzyme related factors (enzyme concentration, temperature and pH) on the catalytic activity of enzyme during ultrasound treatment. Also, it includes the effect of ultrasound on thermal kinetic parameters and Michaelis-Menten kinetic parameters (k m and V max ) of enzymes. Further, in this review, the physical and chemical effects of ultrasound on enzyme have been correlated with thermodynamic parameters (enthalpy and entropy). Various techniques used for investigating the conformation changes in enzyme after sonication have been highlighted. At the end, different techniques of immobilization for ultrasound treated enzyme have been summarized.

  15. Interrogating the activities of conformational deformed enzyme by single-molecule fluorescence-magnetic tweezers microscopy

    Science.gov (United States)

    Guo, Qing; He, Yufan; Lu, H. Peter

    2015-01-01

    Characterizing the impact of fluctuating enzyme conformation on enzymatic activity is critical in understanding the structure–function relationship and enzymatic reaction dynamics. Different from studying enzyme conformations under a denaturing condition, it is highly informative to manipulate the conformation of an enzyme under an enzymatic reaction condition while monitoring the real-time enzymatic activity changes simultaneously. By perturbing conformation of horseradish peroxidase (HRP) molecules using our home-developed single-molecule total internal reflection magnetic tweezers, we successfully manipulated the enzymatic conformation and probed the enzymatic activity changes of HRP in a catalyzed H2O2–amplex red reaction. We also observed a significant tolerance of the enzyme activity to the enzyme conformational perturbation. Our results provide a further understanding of the relation between enzyme behavior and enzymatic conformational fluctuation, enzyme–substrate interactions, enzyme–substrate active complex formation, and protein folding–binding interactions. PMID:26512103

  16. Heterogeneity of hydrolytic enzyme activities under drought: imaging and quantitative analysis

    Science.gov (United States)

    Sanaullah, Muhammad; Razavi, Bahar S.; Kuzyakov, Yakov

    2015-04-01

    The zymography-based "snap-shoot" of enzyme activities in the rhizosphere is challenging to detect the in situ microbial response to global climate change. We developed in situ soil zymography and used it for identification and localization of hotspots of β-glucosidase activity in the rhizosphere of maize under drought stress (30% of field capacity). The zymographic signals were especially high at root tips and were much stronger for activity of β-glucosidase under drought as compared with optimal moisture (70% of field capacity). This distribution of enzyme activity was confirmed by fluorogenically labelled substrates applied directly to the root exudates. The activity of β-glucosidase in root exudates (produced by root and microorganism associated on the root surface), sampled within 1 hour after zymography was significantly higher by drought stressed plants as compared with optimal moisture. In contrast, the β-glucosidase activity in destructively sampled rhizosphere soil was lower under drought stress compared with optimal moisture. Furthermore, drought stress did not affected β-glucosidase activity in bulk soil, away from rhizosphere. Consequently, we conclude that higher release of mucilage by roots und drought stimulated β-glucosidase activity in the rhizosphere. Thus, the zymography revealed plant-mediated mechanisms accelerating β-glucosidase activity under drought at the root-soil interface. So, coupling of zymography and enzyme assays in the rhizosphere and non-rhizosphere soil enables precise mapping the changes in two-dimensional distribution of enzyme activities due to climate change within dynamic soil interfaces.

  17. Measurement of the enzymes lactate dehydrogenase and creatine kinase using reflectance spectroscopy and reagent strips.

    OpenAIRE

    Stevens, J F; Tsang, W; Newall, R G

    1983-01-01

    Two new methods for the assay of total activities of lactate dehydrogenase and creatine kinase are described, in which the enzyme activities are measured from a solid-state reagent strip during a kinetic reaction, the reaction being monitored in the ultra-violet region of the spectrum by reflectance spectroscopy. The performances of these methods are evaluated, and compared to conventional "wet" chemistry methods. The solid-phase reagent methods demonstrated precision and accuracy acceptable ...

  18. Experimental Strategy to Discover Microbes with Gluten-degrading Enzyme Activities.

    Science.gov (United States)

    Helmerhorst, Eva J; Wei, Guoxian

    2014-05-05

    Gluten proteins contained in the cereals barley, rye and wheat cause an inflammatory disorder called celiac disease in genetically predisposed individuals. Certain immunogenic gluten domains are resistant to degradation by mammalian digestive enzymes. Enzymes with the ability to target such domains are potentially of clinical use. Of particular interest are gluten-degrading enzymes that would be naturally present in the human body, e.g. associated with resident microbial species. This manuscript describes a selective gluten agar approach and four enzyme activity assays, including a gliadin zymogram assay, designed for the selection and discovery of novel gluten-degrading microorganisms from human biological samples. Resident and harmless bacteria and/or their derived enzymes could potentially find novel applications in the treatment of celiac disease, in the form of a probiotic agent or as a dietary enzyme supplement.

  19. Hydrolytic enzyme activity enhanced by Barium supplementation

    Directory of Open Access Journals (Sweden)

    Camilo Muñoz

    2016-10-01

    Full Text Available Hydrolysis of polymers is a first and often limiting step during the degradation of plant residues. Plant biomass is generally a major component of waste residues and a major renewable resource to obtain a variety of secondary products including biofuels. Improving the performance of enzymatic hydrolysis of plant material with minimum costs and limiting the use of additional microbial biomass or hydrolytic enzymes directly influences competitiveness of these green biotechnological processes. In this study, we cloned and expressed a cellulase and two esterases recovered from environmental thermophilic soil bacterial communities and characterize their optimum activity conditions including the effect of several metal ions. Results showed that supplementing these hydrolytic reactions with Barium increases the activity of these extracellular hydrolytic enzymes. This observation represents a simple but major improvement to enhance the efficiency and competitiveness of this process within an increasingly important biotechnological sector.

  20. Keratocyte apoptosis and corneal antioxidant enzyme activities after refractive corneal surgery.

    Science.gov (United States)

    Bilgihan, K; Bilgihan, A; Adiguzel, U; Sezer, C; Yis, O; Akyol, G; Hasanreisoglu, B

    2002-01-01

    Refractive corneal surgery induces keratocyte apoptosis and generates reactive oxygen radicals (ROS) in the cornea. The purpose of the present study is to evaluate the correlation between keratocyte apoptosis and corneal antioxidant enzyme activities after different refractive surgical procedures in rabbits. Rabbits were divided into six groups. All groups were compared with the control group (Group 1), after epithelial scraping (Group 2), epithelial scrape and photorefractive keratectomy (PRK) (traditional PRK: Group 3), transepithelial PRK (Group 4), creation of a corneal flap with microkeratome (Group 5) and laser-assisted in situ keratomileusis (LASIK, Group 6). Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay (to detect DNA fragmentation in situ) and light microscopy were used to detect apoptosis in rabbit eyes. Glutathione peroxidase (Gpx) and superoxide dismutase (SOD) activities of the corneal tissues were measured with spectrophotometric methods. Corneal Gpx and SOD activities decreased significantly in all groups when compared with the control group (P<0.05) and groups 2, 3 and 6 showed a significantly higher amount of keratocyte apoptosis (P<0.05). Not only a negative correlation was observed between corneal SOD activity and keratocyte apoptosis (cc: -0.3648) but Gpx activity also showed negative correlation with keratocyte apoptosis (cc: -0.3587). The present study illustrates the negative correlation between keratocyte apoptosis and corneal antioxidant enzyme activities. This finding suggests that ROS may be partly responsible for keratocyte apoptosis after refractive surgery.

  1. Temperature sensitivity differences with depth and season between carbon, nitrogen, and phosphorus cycling enzyme activities in an ombrotrophic peatland system

    Science.gov (United States)

    Steinweg, J. M.; Kostka, J. E.; Hanson, P. J.; Schadt, C. W.

    2017-12-01

    Northern peatlands have large amounts of soil organic matter due to reduced decomposition. Breakdown of organic matter is initially mediated by extracellular enzymes, the activity of which may be controlled by temperature, moisture, and substrate availability, all of which vary seasonally throughout the year and with depth. In typical soils the majority of the microbial biomass and decomposition occurs within the top 30cm due to reduced organic matter inputs in the subsurface however peatlands by their very nature contain large amounts of organic matter throughout their depth profile. We hypothesized that potential enzyme activity would be greatest at the surface of the peat due to a larger microbial biomass compared to 40cm and 175cm below the surface and that temperature sensitivity would be greatest at the surface during winter but lowest during the summer due to high temperatures and enzyme efficiency. Peat samples were collected in February, July, and August 2012 from the DOE Spruce and Peatland Responses Under Climatic and Environmental Change project at Marcell Experimental Forest S1 bog. We measured potential activity of hydrolytic enzymes involved in three different nutrient cycles: beta-glucosidase (carbon), leucine amino peptidase (nitrogen), and phosphatase (phosphorus) at 15 temperature points ranging from 3°C to 65°C. Enzyme activity decreased with depth as expected but there was no concurrent change in activation energy (Ea). The reduction in enzyme activity with depth indicates a smaller pool which coincided with a decreased microbial biomass. Differences in enzyme activity with depth also mirrored the changes in peat composition from the acrotelm to the catotelm. Season did play a role in temperature sensitivity with Ea of β-glucosidase and phosphatase being the lowest in August as expected but leucine amino peptidase (a nitrogen acquiring enzyme) Ea was not influenced by season. As temperatures rise, especially in winter months, enzymatic

  2. Mineralogical impact on long-term patterns of soil nitrogen and phosphorus enzyme activities

    Science.gov (United States)

    Mikutta, Robert; Turner, Stephanie; Meyer-Stüve, Sandra; Guggenberger, Georg; Dohrmann, Reiner; Schippers, Axel

    2014-05-01

    Soil chronosequences provide a unique opportunity to study microbial activity over time in mineralogical diverse soils of different ages. The main objective of this study was to test the effect of mineralogical properties, nutrient and organic matter availability over whole soil pro-files on the abundance and activity of the microbial communities. We focused on microbio-logical processes involved in nitrogen and phosphorus cycling at the 120,000-year Franz Josef soil chronosequence. Microbial abundances (microbial biomass and total cell counts) and enzyme activities (protease, urease, aminopeptidase, and phosphatase) were determined and related to nutrient contents and mineralogical soil properties. Both, microbial abundances and enzyme activities decreased with soil depth at all sites. In the organic layers, microbial biomass and the activities of N-hydrolyzing enzymes showed their maximum at the intermediate-aged sites, corresponding to a high aboveground biomass. In contrast, the phosphatase activity increased with site age. The activities of N-hydrolyzing enzymes were positively correlated with total carbon and nitrogen contents, whereas the phosphatase activity was negatively correlated with the phosphorus content. In the mineral soil, the enzyme activities were generally low, thus reflecting the presence of strongly sorbing minerals. Sub-strate-normalized enzyme activities correlated negatively to clay content as well as poorly crystalline Al and Fe oxyhydroxides, supporting the view that the evolution of reactive sec-ondary mineral phases alters the activity of the microbial communities by constraining sub-strate availability. Our data suggest a strong mineralogical influence on nutrient cycling par-ticularly in subsoil environments.

  3. Seasonal variation in the temperature sensitivity of proteolytic enzyme activity in temperate forest soils

    Science.gov (United States)

    Brzostek, Edward R.; Finzi, Adrien C.

    2012-03-01

    Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.

  4. Muscle enzyme activities in a deep-sea squaloid shark, Centroscyllium fabricii, compared with its shallow-living relative, Squalus acanthias.

    Science.gov (United States)

    Treberg, Jason R; Martin, R Aidan; Driedzic, William R

    2003-12-01

    The activities of several enzymes of energy metabolism were measured in the heart, red muscle, and white muscle of a deep and a shallow living squaloid shark, Centroscyllium fabricii and Squalus acanthias, respectively. The phylogenetic closeness of these species, combined with their active predatory nature, similar body form, and size makes them well matched for comparison. This is the first time such a comparison has been made involving a deep-sea elasmobranch. Enzyme activities were similar in the heart, but generally lower in the red muscle of C. fabricii. Paralleling the trend seen in deep-sea teleosts, the white muscle of C. fabricii had substantially lower activities of key glycolytic enzymes, pyruvate kinase and lactate dehydrogenase, relative to S. acanthias or other shallow living elasmobranchs. Unexpectedly, between the squaloid sharks examined, creatine phosphokinase activity was higher in all tissues of the deep living C. fabricii. Low white muscle glycolytic enzyme activities in the deep-sea species coupled with high creatine phosphokinase activity suggests that the capacity for short burst swimming is likely limited once creatine phosphate supplies have been exhausted. Copyright 2003 Wiley-Liss, Inc.

  5. Arabinogalactan proteins: focus on carbohydrate active enzymes

    Directory of Open Access Journals (Sweden)

    Eva eKnoch

    2014-06-01

    Full Text Available Arabinogalactan proteins (AGPs are a highly diverse class of cell surface proteoglycans that are commonly found in most plant species. AGPs play important roles in many cellular processes during plant development, such as reproduction, cell proliferation, pattern formation and growth, and in plant-microbe interaction. However, little is known about the molecular mechanisms of their function. Numerous studies using monoclonal antibodies that recognize different AGP glycan epitopes have shown the appearance of a slightly altered AGP glycan in a specific stage of development in plant cells. Therefore, it is anticipated that the biosynthesis and degradation of AGP glycan is tightly regulated during development. Until recently, however, little was known about the enzymes involved in the metabolism of AGP glycans. In this review, we summarize recent discoveries of carbohydrate active enzymes (CAZy; http://www.cazy.org/ involved in the biosynthesis and degradation of AGP glycans, and we discuss the biological role of these enzymes in plant development.

  6. Applicability of rapid and on-site measured enzyme activity for surface water quality monitoring in an agricultural catchment

    Science.gov (United States)

    Stadler, Philipp; Farnleitner, Andreas H.; Sommer, Regina; Kumpan, Monika; Zessner, Matthias

    2014-05-01

    For the near real time and on-site detection of microbiological fecal pollution of water, the measurement of beta-D- Glucuronidase (GLUC) enzymatic activity has been suggested as a surrogate parameter and has been already successfully operated for water quality monitoring of ground water resources (Ryzinska-Paier et al. 2014). Due to possible short measure intervals of three hours, this method has high potential as a water quality monitoring tool. While cultivation based standard determination takes more than one working day (Cabral 2010) the potential advantage of detecting the GLUC activity is the high temporal measuring resolution. Yet, there is still a big gap of knowledge on the fecal indication capacity of GLUC (specificity, sensitivity, persistence, etc.) in relation to potential pollution sources and catchment conditions (Cabral 2010, Ryzinska-Paier et al. 2014). Furthermore surface waters are a big challenge for automated detection devices in a technical point of view due to the high sediment load during event conditions. This presentation shows results gained form two years of monitoring in an experimental catchment (HOAL) dominated by agricultural land use. Two enzymatic measurement devices are operated parallel at the catchment outlet to test the reproducibility and precision of the method. Data from continuous GLUC monitoring under both base flow and event conditions is compared with reference samples analyzed by standardized laboratory methods for fecal pollution detection (e.g. ISO 16649-1, Colilert18). It is shown that rapid enzymatic on-site GLUC determination can successfully be operated from a technical point of view for surface water quality monitoring under the observed catchment conditions. The comparison of enzyme activity with microbiological standard analytics reveals distinct differences in the dynamic of the signals during event conditions. Cabral J. P. S. (2010) "Water Microbiology. Bacterial Pathogens and Water" International Journal of

  7. Purification and characterization of extracellular amylolytic enzyme ...

    African Journals Online (AJOL)

    In the present study, the amylase enzyme producing potential of four different Aspergillus species was analyzed. The extracted amylase enzyme was purified by diethyl amino ethyl (DEAE) cellulose and Sephadex G-50 column chromatography and the enzyme activity was measured by using synthetic substrate starch.

  8. Enzyme activity of a Phanerochaete chrysosporium cellobiohydrolase

    African Journals Online (AJOL)

    The aim of this study was to produce a secreted, heterologously expressed Phanerochaete chrysosporium cellobiohydrolase (CBHI.1) protein that required no in vitro chemical refolding and to investigate the cellulolytic activity of the clone expressing the glutathione S-transferase (GST) fused CBHI.1 protein. Plate enzyme ...

  9. Enzyme activity in bioregulator-treated tomato (Solanum ...

    African Journals Online (AJOL)

    USER

    2010-05-31

    May 31, 2010 ... African Journal of Biotechnology Vol. 9(22), pp. 3264-3271, 31 ... In this work, spectrophotometric analysis ... most stable enzymes in vegetables and its thermal destruc-tion ... proteins, carbohydrates, lipids and allelochemicals (Hedin et al., 1995) ..... activities isolated from corn root plasma membrane. Plant.

  10. The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme.

    Science.gov (United States)

    Tsume, Yasuhiro; Amidon, Gordon L

    2012-03-26

    The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5'-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0-105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5'-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5'-O-L-phenylalanyl-L-tyrosylfloxuridine and 5'-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enzymes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.

  11. Effect of Robola and Cabernet Sauvignon extracts on platelet activating factor enzymes activity on U937 cells.

    Science.gov (United States)

    Xanthopoulou, M N; Asimakopoulos, D; Antonopoulou, S; Demopoulos, C A; Fragopoulou, E

    2014-12-15

    A number of studies support the anti-atherogenic effect of wine compounds. The scope of this study was to examine the effect of a red (Cabernet Sauvignon-CS) and a white (Robola-R) wine, as well as resveratrol and quercetin, on the platelet activating factor (PAF) biosynthetic enzymes, acetyl-CoA:lyso-PAF acetyltransferase (lyso-PAF-AT) and DTT-insensitive CDP-choline 1-alkyl-2-acetyl-sn-glycerol cholinephosphotransferase (PAF-CPT), and its main catabolic enzyme (PAF acetylhydrolase; PAF-AH), on U937 cells, in cell free and in intact cell experiments. In cell free experiments, phenolic compounds and wine extracts inhibited PAF biosynthetic enzymes, however in higher concentrations than intact cell experiments. In the latter cases, polar lipids of both wines inhibited in the same order of magnitude the action of lyso-PAF-AT and of PAF-CPT. The water fractions possessed a dual action, in lower concentrations they activated both enzymes, while in higher concentrations only inhibited PAF-CPT. All fractions either did not affect or slightly activated PAF-AH activity. In conclusion, wine compounds may exert their anti-inflammatory activity by reducing PAF levels through modulation of the PAF metabolic enzymes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. Enzyme activity and allosteric characteristics of gamma-irradiated solid aspartate transcarbamylase

    International Nuclear Information System (INIS)

    Bigler, W.N.; Tolbert, B.M.

    1977-01-01

    Aspartate transcarbamylase purified from E. coli was lyophilized, irradiated in vacuo with γ radiation from a cesium-137 source, redissolved in buffer under a nitrogen atmosphere, and assayed for enzyme activity. Lyophilized and redissolved enzyme had normal catalytic and allosteric kinetic characteristics. The average D 37 observed with saturating substrate, 25 mM aspartate, was 4.1 Mrad. With less than saturating substrate, 5 mM aspartate, the activity increases from zero to 1.6 Mrad and then decreases with a D 37 of 7.2 Mrad. Inclusion of 1 mM CTP, an allosteric inhibitor, in the 5 mM aspartate assays results in a more pronounced maximum in the activity curve occurring at slightly higher dose, 2.2 Mrad. Inhibitability by CTP has a D 37 of 2.3 Mrad with doses below the activity maximum. Enzyme lyophilized in the presence of 1 mM CTP has a D 37 of 2.9 Mrad. ATCase activity changes caused by irradiation of lyophylized bacteria were qualitatively like the changes observed in the detailed studies with the purified enzyme. Apparent radiation sensitivities of ATCase in lyophilized bacteria were observed to vary with the technique used to disrupt the resuspended bacteria

  13. Disturbances in lysosomal enzymes activity in rats, following experimental postradiation disease

    International Nuclear Information System (INIS)

    Drozdz, M.; Piwowarczyk, B.; Olczyk, K.; Pikula-Zachara, M.

    1981-01-01

    The studies were aimed at detecting the biological effects of radiation on rat's organism, through studying the activity of lysosomal enzymes in blood plasma and some organs. The contemporary studies suggest that lysosomes play an important role in the occurrence and course of postradiation disease. The obtained results suggest the multidirectional gamma-rays effects on lysosomal enzymes response in serum, leucocytes, liver lysosomes and in liver, kidneys, lungs, heart. Increased activity of acid phosphatase, beta-glucoronidase and beta-acetyl-glucosaminase in the tissues of irradiated animals indicates that gamma rays labilizate the lysosomal membrane. The range of changes indicates a selective nature of this phenomenon. Kidneys, lungs and liver appeared the most ray-sensitive organs. The activity of acid phosphatase was found to be most increased in blood serum and leucocytes. The activity of all examined enzymes in liver lysosomes was decreased. Acid phosphatase exhibited the greatest activity increases. Lysosomal responses are indicative of the degree of destructive or regenerative changes in the organism. (author)

  14. Characterization of inhibitor(s) of β-glucuronidase enzyme activity in GUS-transgenic wheat

    KAUST Repository

    Ramadan, Ahmed M Ali

    2011-06-26

    The uidA gene, encoding for β-glucuronidase (GUS), is the most frequently used reporter gene in plants. As a reporter enzyme, GUS can be assayed both qualitatively and quantitatively. In wheat, there are numerous reports of failure in detecting GUS enzyme activity in tissues of transgenic plants, while other reports have suggested presence of β-glucuronidase inhibitor(s) in wheat tissues. In the present study, we show that the β-glucuronidase enzyme activity is not only tissue-specific but also genotype-dependent. Our data demonstrate that the glucuronic acid could be the candidate inhibitor for β-glucuronidase enzyme activity in wheat leaves and roots. It should be noted that the assays to detect β-glucuronidase enzyme activity in wheat should be interpreted carefully. Based on the data of our present study, we recommend studying the chemical pathways, the unintended effects and the possible loss-of-function of any candidate transgene prior to transformation experiments. © 2011 Springer Science+Business Media B.V.

  15. Characterization of inhibitor(s) of β-glucuronidase enzyme activity in GUS-transgenic wheat

    KAUST Repository

    Ramadan, Ahmed M Ali; Eissa, Hala F.; El-Domyati, Fotouh M.; Saleh, Osama Mesilhy; Ibrahim, Nasser E.; Salama, M. I.; Mahfouz, Magdy M.; Bahieldin, Ahmed M.

    2011-01-01

    The uidA gene, encoding for β-glucuronidase (GUS), is the most frequently used reporter gene in plants. As a reporter enzyme, GUS can be assayed both qualitatively and quantitatively. In wheat, there are numerous reports of failure in detecting GUS enzyme activity in tissues of transgenic plants, while other reports have suggested presence of β-glucuronidase inhibitor(s) in wheat tissues. In the present study, we show that the β-glucuronidase enzyme activity is not only tissue-specific but also genotype-dependent. Our data demonstrate that the glucuronic acid could be the candidate inhibitor for β-glucuronidase enzyme activity in wheat leaves and roots. It should be noted that the assays to detect β-glucuronidase enzyme activity in wheat should be interpreted carefully. Based on the data of our present study, we recommend studying the chemical pathways, the unintended effects and the possible loss-of-function of any candidate transgene prior to transformation experiments. © 2011 Springer Science+Business Media B.V.

  16. Antibacterial and glucosyltransferase enzyme inhibitory activity of helmyntostachyszelanica

    Science.gov (United States)

    Kuspradini, H.; Putri, AS; Mitsunaga, T.

    2018-04-01

    Helminthostachyszeylanica is a terrestrial, herbaceous, fern-like plant of southeastern Asia and Australia, commonly known as tunjuk-langit. This kind of plant have a medicinal properties such as treatment of malaria, dysentery and can be eaten with betel in the treatment of whooping cough. To evaluate the scientific basis for the use of the plant, the antimicrobial activities of extracts of the stem and leaves were evaluated. The bacteria used in this study is Streptococcus sobrinus, a species of gram-positive, that may be associated with human dental caries. The dried powdered plant parts were extracted using methanol and 50% aqueous extract and screened for their antibacterial effects of Streptococcus sobrinus using the 96 well-plate microdilution broth method. The inhibitory activities of its related enzyme were also determined. The plant extracts showed variable antibacterial and Glucosyltransferase enzyme inhibitory activity while some extracts could not cause any inhibition. It was shown that 50% ethanolics of Helminthostachyzeylanica stem have a potency as anti dental caries agents.

  17. Optimization of ultrasound-assisted extraction of pectinase enzyme from guava (Psidium guajava) peel: Enzyme recovery, specific activity, temperature, and storage stability.

    Science.gov (United States)

    Amid, Mehrnoush; Murshid, Fara Syazana; Manap, Mohd Yazid; Islam Sarker, Zaidul

    2016-01-01

    This study aimed to investigate the effects of the ultrasound-assisted extraction conditions on the yield, specific activity, temperature, and storage stability of the pectinase enzyme from guava peel. The ultrasound variables studied were sonication time (10-30 min), ultrasound temperature (30-50 °C), pH (2.0-8.0), and solvent-to-sample ratio (2:1 mL/g to 6:1 mL/g). The main goal was to optimize the ultrasound-assisted extraction conditions to maximize the recovery of pectinase from guava peel with the most desirable enzyme-specific activity and stability. Under the optimum conditions, a high yield (96.2%), good specific activity (18.2 U/mg), temperature stability (88.3%), and storage stability (90.3%) of the extracted enzyme were achieved. The optimal conditions were 20 min sonication time, 40 °C temperature, at pH 5.0, using a 4:1 mL/g solvent-to-sample ratio. The study demonstrated that optimization of ultrasound-assisted process conditions for the enzyme extraction could improve the enzymatic characteristics and yield of the enzyme.

  18. An easy and efficient permeabilization protocol for in vivo enzyme activity assays in cyanobacteria

    DEFF Research Database (Denmark)

    Rasmussen, Randi Engelberth; Erstad, Simon Matthé; Ramos Martinez, Erick Miguel

    2016-01-01

    microbial cell factories. Better understanding of the activities of enzymes involved in the central carbon metabolism would lead to increasing product yields. Currently cell-free lysates are the most widely used method for determination of intracellular enzyme activities. However, due to thick cell walls...... used directly in the assays, the permeabilized cells exhibited the enzyme activities that are comparable or even higher than those detected for cell-free lysates. Moreover, the permeabilized cells could be stored at -20 °C without losing the enzyme activities. The permeabilization process...... for permeabilization of the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803, and determination of two intracellular enzymes, ribulose-1,5-bisphosphate carboxylase/decarboxylase (Rubisco) and glucose-6-phosphate dehydrogenase (G6PDH), that play pivotal roles in the central carbon metabolism...

  19. Enzymatic activity measured by microcalorimetry in soil amended with organic residues

    Directory of Open Access Journals (Sweden)

    Karina Cenciani

    2011-08-01

    Full Text Available Enzymatic activity is an important property for soil quality evaluation. Two sequences of experiments were carried out in order to evaluate the enzymatic activity in a soil (Rhodic Eutrudox amended with cattle manure, earthworm casts, or sewage sludges from the municipalities of Barueri and Franca. The activity of commercial enzymes was measured by microcalorimetry in the same soil samples after sterilization. In the first experiment, the enzyme activities of cellulase, protease, and urease were determined in the soil samples during a three month period. In the second sequence of experiments, the thermal effect of the commercial enzymes cellulase, protease, and urease on sterilized soil samples under the same tretaments was monitored for a period of 46 days. The experimental design was randomized and arranged as factorial scheme in five treatments x seven samplings with five replications. The treatment effects were statistically evaluated by one-way analysis of variance. Tukey´s test was used to compare means at p < 0.05. The presence of different sources of organic residues increased the enzymatic activity in the sampling period. Cattle manure induced the highest enzymatic activity, followed by municipal sewage sludge, whereas earthworm casts induced the lowest activity, but differed from control treatment. The thermal effect on the enzyme activity of commercial cellulase, protease, and urease showed a variety of time peaks. These values probably oscillated due to soil physical-chemical factors affecting the enzyme activity on the residues.

  20. Seasonality of fibrolytic enzyme activity in herbivore microbial ...

    African Journals Online (AJOL)

    2012-08-21

    Aug 21, 2012 ... liberating end-products such as volatile fatty acids. Cellulase enzyme ... All the other common chemicals such as glacial acetic acid, sodium azide .... specific activity was observed among animal species and between seasons ...

  1. Changes in growth, survival and digestive enzyme activities of Asian ...

    African Journals Online (AJOL)

    A study was conducted to determine the effects of different dietary treatments on the growth, survival and digestive enzyme activities of Mystus nemurus larvae. Newly hatched larvae were reared for 14 days in twelve 15 L glass aquaria (for growth and survival) and eight 300 L fiberglass tanks (for enzyme samples) at a ...

  2. Early bichemical markers of effects: Enzyme induction, oncogene activation and markers of oxidative damage

    DEFF Research Database (Denmark)

    Poulsen, Henrik E.; Loft, Steffen

    1995-01-01

    Early bichemical marker, enzyme induction, oncogene activation, oxidative damage, low-density lipoprotein......Early bichemical marker, enzyme induction, oncogene activation, oxidative damage, low-density lipoprotein...

  3. Improving enzymatic activities and thermostability of a tri-functional enzyme with SOD, catalase and cell-permeable activities.

    Science.gov (United States)

    Luangwattananun, Piriya; Eiamphungporn, Warawan; Songtawee, Napat; Bülow, Leif; Isarankura Na Ayudhya, Chartchalerm; Prachayasittikul, Virapong; Yainoy, Sakda

    2017-04-10

    Synergistic action of major antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) is known to be more effective than the action of any single enzyme. Recently, we have engineered a tri-functional enzyme, 6His-MnSOD-TAT/CAT-MnSOD (M-TAT/CM), with SOD, CAT and cell-permeable activities. The protein actively internalized into the cells and showed superior protection against oxidative stress-induced cell death over native enzymes fused with TAT. To improve its molecular size, enzymatic activity and stability, in this study, MnSOD portions of the engineered protein were replaced by CuZnSOD, which is the smallest and the most heat resistant SOD isoform. The newly engineered protein, CAT-CuZnSOD/6His-CuZnSOD-TAT (CS/S-TAT), had a 42% reduction in molecular size and an increase in SOD and CAT activities by 22% and 99%, respectively. After incubation at 70°C for 10min, the CS/S-TAT retained residual SOD activity up to 54% while SOD activity of the M-TAT/CM was completely abolished. Moreover, the protein exhibited a 5-fold improvement in half-life at 70°C. Thus, this work provides insights into the design and synthesis of a smaller but much more stable multifunctional antioxidant enzyme with ability to enter mammalian cells for further application as protective/therapeutic agent against oxidative stress-related conditions. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Extracellular enzyme activity in a willow sewage treatment system.

    Science.gov (United States)

    Brzezinska, Maria Swiontek; Lalke-Porczyk, Elżbieta; Kalwasińska, Agnieszka

    2012-12-01

    This paper presents the results of studies on the activity of extra-cellular enzymes in soil-willow vegetation filter soil which is used in the post-treatment of household sewage in an onsite wastewater treatment system located in central Poland. Wastewater is discharged from the detached house by gravity into the onsite wastewater treatment system. It flows through a connecting pipe into a single-chamber septic tank and is directed by the connecting pipe to a control well to be further channelled in the soil-willow filter by means of a subsurface leaching system. Soil samples for the studies were collected from two depths of 5 cm and 1 m from three plots: close to the wastewater inflow, at mid-length of the plot and close to its terminal part. Soil samples were collected from May to October 2009. The activity of the extra-cellular enzymes was assayed by the fluorometric method using 4-methylumbelliferyl and 7-amido-4-methylcoumarin substrate. The ranking of potential activity of the assayed enzymes was the same at 5 cm and 1 m soil depths, i.e. esterase > phosphmomoesterase > leucine-aminopeptidase > β-glucosidase > α-glucosidase. The highest values of enzymatic activity were recorded in the surface layer of the soil at the wastewater inflow and decreased with increasing distance from that point.

  5. Enzyme Enzyme activities in relation to sugar accumulation in tomato

    International Nuclear Information System (INIS)

    Alam, M.J.; Rahman, M.H.; Mamun, M.A.; Islam, K.

    2006-01-01

    Enzyme activities in tomato juice of five different varieties viz. Ratan, Marglove, BARI-1, BARI-5 and BARI-6, in relation to sugar accumulation were investigated at different maturity stages. The highest amount of invertase and beta-galactosidase was found in Marglove and the lowest in BARI- 6 at all maturity stages. Total soluble sugar and sucrose contents were highest in BARI-1 and lowest in BARI-6. The activity of amylase was maximum in Ratan and minimum in Marglove. Protease activity was highest in Ratan and lowest in BARI-6. BARI-1 contained the highest cellulase activity and the lowest in BARI-5. The amount of total soluble sugar and sucrose increased moderately from premature to ripe stage. The activities of amylase and cellulase increased up to the mature stage and then decreased drastically in the ripe stage. The activities of invertase and protease increased sharply from the premature to the ripe stage while the beta-galactosidase activity decreased remarkably. No detectable amount of reducing sugar was present in the premature stage in all cultivars of tomato but increased thereafter upto the ripe stage. The highest reducing sugar was present in BARI-5 in all of the maturity stages. (author)

  6. Enzymic oxidation of carbon monoxide. II

    Energy Technology Data Exchange (ETDEWEB)

    Yagi, T

    1959-01-01

    An enzyme which catalyzes the oxidation of carbon monoxide into carbon dioxide was obtained in a cell free state from Desulfovibrio desulfuricans. The enzyme activity was assayed manometrically by measuring the rate of gas uptake under the atmosphere of carbon monoxide in the presence of benzyl-viologen as an oxidant. The optimum pH range was 7 to 8. The activity was slightly suppressed by illumination. The enzyme was more stable than hydrogenase or formate dehydrogenase against the heat treatment, suggesting that it is a different entity from these enzymes. In the absence of an added oxidant, the enzyme preparation produced hydrogen gas under the atmosphere of carbon monoxide. The phenomenon can be explained assuming the reductive decomposition of water. 17 references, 4 figures, 2 tables.

  7. Rapid intranasal delivery of chloramphenicol acetyltransferase in the active form to different brain regions as a model for enzyme therapy in the CNS.

    Science.gov (United States)

    Appu, Abhilash P; Arun, Peethambaran; Krishnan, Jishnu K S; Moffett, John R; Namboodiri, Aryan M A

    2016-02-01

    The blood brain barrier (BBB) is critical for maintaining central nervous system (CNS) homeostasis by restricting entry of potentially toxic substances. However, the BBB is a major obstacle in the treatment of neurotoxicity and neurological disorders due to the restrictive nature of the barrier to many medications. Intranasal delivery of active enzymes to the brain has therapeutic potential for the treatment of numerous CNS enzyme deficiency disorders and CNS toxicity caused by chemical threat agents. The aim of this work is to provide a sensitive model system for analyzing the rapid delivery of active enzymes into various regions of the brain with therapeutic bioavailability. We tested intranasal delivery of chloramphenicol acetyltransferase (CAT), a relatively large (75kD) enzyme, in its active form into different regions of the brain. CAT was delivered intranasally to anaesthetized rats and enzyme activity was measured in different regions using a highly specific High Performance Thin Layer Chromatography (HP-TLC)-radiometry coupled assay. Active enzyme reached all examined areas of the brain within 15min (the earliest time point tested). In addition, the yield of enzyme activity in the brain was almost doubled in the brains of rats pre-treated with matrix metalloproteinase-9 (MMP-9). Intranasal administration of active enzymes in conjunction with MMP-9 to the CNS is both rapid and effective. The present results suggest that intranasal enzyme therapy is a promising method for counteracting CNS chemical threat poisoning, as well as for treating CNS enzyme deficiency disorders. Published by Elsevier B.V.

  8. In vitro enzyme-mimic activity and in vivo therapeutic potential of HSJ-0017, a novel Mn porphyrin-based antioxidant enzyme mimic.

    Science.gov (United States)

    Li, Bao-qiu; Dong, Xin; Li, Na; Gao, Ji-you; Yuan, Qiang; Fang, Shi-hong; Gong, Xian-chang; Wang, Shu-juan; Wang, Feng-shan

    2014-10-01

    Manganese (III) 5, 10, 15, 20-tetrakis [3-(2-(2-methoxy)-ethoxy) ethoxy] phenyl porphyrin chloride, designated HSJ-0017, is a novel antioxidant enzyme mimic. The aim of the present study was to investigate the enzyme-mimic activity and the therapeutic potential of HSJ-0017 in free radical-related diseases. Superoxide dismutase (SOD) mimic activity was measured by the nitroblue tetrazolium chloride monohydrate reduction assay. Catalase (CAT) mimic activity was measured based on the decomposition of hydrogen peroxide. The antitumor, radioprotective and chemoprotective effects of HSJ-0017 were evaluated in H22 or S180 tumor-bearing Kunming mice. The anti-inflammatory and hepatoprotective effects were, respectively, evaluated in histamine-induced edema model and CCl4-induced hepatic damage model in Wistar rats. HSJ-0017 over a concentration range of 0.001-10 µmol/L significantly inhibited the generation of superoxide anion. Significant hydrogen peroxide scavenging activity was observed when the concentration of HSJ-0017 was higher than 0.01 µmol/L. HSJ-0017 at a dose of 3.0 mg/kg exhibited significant antitumor effect on S180 tumor xenografts, whereas no significant antitumor effect was observed in H22 tumor xenografts. HSJ-0017 at a dose of 3.0 mg/kg enhanced the antitumor effects of radiotherapy and chemotherapy, and reduced their toxicity. However, HSJ-0017 counteracted the antitumor effects of radiotherapy when administered simultaneously with radiotherapy. HSJ-0017 showed significant anti-inflammatory and hepatoprotective effects. Our results demonstrate that HSJ-0017 exhibits antioxidant, antitumor, anti-inflammatory, radioprotective, chemoprotective, and hepatoprotective effects. It is a potent dual SOD/CAT mimic. © 2014 by the Society for Experimental Biology and Medicine.

  9. Substrate-Wrapped, Single-Walled Carbon Nanotube Probes for Hydrolytic Enzyme Characterization.

    Science.gov (United States)

    Kallmyer, Nathaniel E; Musielewicz, Joseph; Sutter, Joel; Reuel, Nigel F

    2018-04-17

    Hydrolytic enzymes are a topic of continual study and improvement due to their industrial impact and biological implications; however, the ability to measure the activity of these enzymes, especially in high-throughput assays, is limited to an established, few enzymes and often involves the measurement of secondary byproducts or the design of a complex degradation probe. Herein, a versatile single-walled carbon nanotube (SWNT)-based biosensor that is straightforward to produce and measure is described. The hydrolytic enzyme substrate is rendered as an amphiphilic polymer, which is then used to solubilize the hydrophobic nanotubes. When the target enzyme degrades the wrapping, the SWNT fluorescent signal is quenched due to increased solvent accessibility and aggregation, allowing quantitative measurement of hydrolytic enzyme activity. Using (6,5) chiral SWNT suspended with polypeptides and polysaccharides, turnover frequencies are estimated for cellulase, pectinase, and bacterial protease. Responses are recorded for concentrations as low as 5 fM using a well-characterized protease, Proteinase K. An established trypsin-based plate reader assay is used to compare this nanotube probe assay with standard techniques. Furthermore, the effect of freeze-thaw cycles and elevated temperature on enzyme activity is measured, suggesting freezing to have minimal impact even after 10 cycles and heating to be detrimental above 60 °C. Finally, rapid optimization of enzyme operating conditions is demonstrated by generating a response surface of cellulase activity with respect to temperature and pH to determine optimal conditions within 2 h of serial scans.

  10. Measurement of the enzymes lactate dehydrogenase and creatine kinase using reflectance spectroscopy and reagent strips.

    Science.gov (United States)

    Stevens, J F; Tsang, W; Newall, R G

    1983-01-01

    Two new methods for the assay of total activities of lactate dehydrogenase and creatine kinase are described, in which the enzyme activities are measured from a solid-state reagent strip during a kinetic reaction, the reaction being monitored in the ultra-violet region of the spectrum by reflectance spectroscopy. The performances of these methods are evaluated, and compared to conventional "wet" chemistry methods. The solid-phase reagent methods demonstrated precision and accuracy acceptable for diagnostic purposes, and were easy to use by trained operators. PMID:6655069

  11. Activity of angiotensin-converting enzyme and risk of severe hypoglycaemia in type 1 diabetes mellitus

    DEFF Research Database (Denmark)

    Pedersen-Bjergaard, U; Agerholm-Larsen, Birgit; Pramming, S

    2001-01-01

    BACKGROUND: The insertion (I) allele of the angiotensin-converting-enzyme (ACE) gene occurs at increased frequency in endurance athletes. This association suggests that low ACE activity is favourable for performance in conditions with limited substrate availability. Such conditions occur in endur......BACKGROUND: The insertion (I) allele of the angiotensin-converting-enzyme (ACE) gene occurs at increased frequency in endurance athletes. This association suggests that low ACE activity is favourable for performance in conditions with limited substrate availability. Such conditions occur...... by diabetes history, degree of hypoglycaemia awareness, measurement of C-peptide, haemoglobin A(1c), and serum ACE concentrations, and determination of ACE genotype. FINDINGS: Patients with the DD genotype had a relative risk of severe hypoglycaemia in the preceding 2 years of 3.2 (95% CI 1.4-7.4) compared...

  12. High activity and low temperature optima of extracellular enzymes in Arctic sediments: implications for carbon cycling by heterotrophic microbial communities

    DEFF Research Database (Denmark)

    Arnosti, C.; Jørgensen, BB

    2003-01-01

    (chondroitin sulfate, fucoidan, xylan and pullulan) to determine the temperature-activity responses of hydrolysis of a related class of compounds. All 4 enzyme activities showed similarly low temperature optima in the range of 15 to 18degreesC. These temperature optima are considerably lower than most previous......The rate of the initial step in microbial remineralization of organic carbon, extracellular enzymatic hydrolysis, was investigated as a function of temperature in permanently cold sediments from 2 fjords on the west coast of Svalbard (Arctic Ocean). We used 4 structurally distinct polysaccharides...... reports of temperature optima for enzyme activities in marine sediments. At 0degreesC, close to the in situ temperature, these enzyme activities achieved 13 to 38% of their rates at optimum temperatures. In one experiment, sulfate reduction rates were measured in parallel with extracellular enzymatic...

  13. The Feasibility of Enzyme Targeted Activation for Amino Acid/Dipeptide Monoester Prodrugs of Floxuridine; Cathepsin D as a Potential Targeted Enzyme

    Directory of Open Access Journals (Sweden)

    Gordon L. Amidon

    2012-03-01

    Full Text Available The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5¢-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0–105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5¢-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5¢-O-L-phenylalanyl-L-tyrosylfloxuridine and 5¢-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enyzmes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5¢-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.

  14. Monoamine oxidase A gene polymorphisms and enzyme activity associated with risk of gout in Taiwan aborigines.

    Science.gov (United States)

    Tu, Hung-Pin; Ko, Albert Min-Shan; Wang, Shu-Jung; Lee, Chien-Hung; Lea, Rod A; Chiang, Shang-Lun; Chiang, Hung-Che; Wang, Tsu-Nai; Huang, Meng-Chuan; Ou, Tsan-Teng; Lin, Gau-Tyan; Ko, Ying-Chin

    2010-02-01

    Taiwanese aborigines have a high prevalence of hyperuricemia and gout. Uric acid levels and urate excretion have correlated with dopamine-induced glomerular filtration response. MAOs represent one of the major renal dopamine metabolic pathways. We aimed to identify the monoamine oxidase A (MAOA, Xp11.3) gene variants and MAO-A enzyme activity associated with gout risk. This study was to investigate the association between gout and the MAOA single-nucleotide polymorphisms (SNPs) rs5953210, rs2283725, and rs1137070 as well as between gout and the COMT SNPs rs4680 Val158Met for 374 gout cases and 604 controls. MAO-A activity was also measured. All three MAOA SNPs were significantly associated with gout. A synonymous MAOA SNP, rs1137070 Asp470Asp, located in exon 14, was associated with the risk of having gout (P = 4.0 x 10(-5), adjusted odds ratio 1.46, 95% confidence intervals [CI]: 1.11-1.91). We also showed that, when compared to individuals with the MAOA GAT haplotype, carriers of the AGC haplotype had a 1.67-fold (95% CI: 1.28-2.17) higher risk of gout. Moreover, we found that MAOA enzyme activity correlated positively with hyperuricemia and gout (P for trend = 2.00 x 10(-3) vs. normal control). We also found that MAOA enzyme activity by rs1137070 allele was associated with hyperuricemia and gout (P for trend = 1.53 x 10(-6) vs. wild-type allele). Thus, our results show that some MAOA alleles, which have a higher enzyme activity, predispose to the development of gout.

  15. Characterization of Carbohydrate Active Enzymes Involved in Arabinogalactan Protein Metabolism

    DEFF Research Database (Denmark)

    Knoch, Eva

    and tissues, their functions and synthesis are still poorly understood. The aim of the research presented in the thesis was to characterize carbohydrate active enzymes involved in AGP biosynthesis and modification to gain insights into the biosynthesis of the glycoproteins in plants. Candidate...... glycosyltransferases and glycoside hydrolases were selected based on co-expression profiles from a transcriptomics analysis. Reverse genetics approach on a novel glucuronosyltransferase involved in AGP biosynthesis has revealed that the enzyme activity is required for normal cell elongation in etiolated seedlings....... The enzymatic activity of a hydrolase from GH family 17 was investigated, without successful determination of the activity. Members of hydrolase family 43 appeared to be localized in the Golgi-apparatus, which is also the compartment for glycan biosynthesis. The localization of these glycoside hydrolases...

  16. Elevated serum angiotensin-converting enzyme (SACE) activity in acute pulmonary histoplasmosis.

    Science.gov (United States)

    Davies, S F; Rohrbach, M S; Thelen, V; Kuritsky, J; Gruninger, R; Simpson, M L; DeRemee, R A

    1984-03-01

    Serum angiotensin-converting enzyme (SACE) levels were measured in 44 subjects six weeks after acute pulmonary histoplasmosis. All patients were infected in a common-source outbreak of histoplasmosis which occurred on one day. All patients had both strictly defined clinical and serologic evidence of infection. The SACE activity was elevated at six weeks compared to normal controls, and seven of the 44 had levels more than 2 SD above the normal mean. SACE levels were also measured at three and 24 weeks after acute infection in a smaller number of the same subjects. Serial observations demonstrated that all subjects (including those with normal and elevated SACE at six weeks) had a rise and fall in SACE activity following symptomatic acute pulmonary histoplasmosis. Our findings suggest that elevated SACE does not reliably separate sarcoidosis from histoplasmosis, although elevations in histoplasmosis are much less common and may occur only briefly following acute pulmonary histoplasmosis. More important, it seems that SACE activity rises acutely in all patients with symptomatic acute histoplasmosis and then falls gradually toward baseline over several months, coinciding temporally with the granulomatous response.

  17. Deletion of creB in Aspergillus oryzae increases secreted hydrolytic enzyme activity.

    Science.gov (United States)

    Hunter, A J; Morris, T A; Jin, B; Saint, C P; Kelly, J M

    2013-09-01

    Aspergillus oryzae has been used in the food and beverage industry for centuries, and industrial strains have been produced by multiple rounds of selection. Targeted gene deletion technology is particularly useful for strain improvement in such strains, particularly when they do not have a well-characterized meiotic cycle. Phenotypes of an Aspergillus nidulans strain null for the CreB deubiquitinating enzyme include effects on growth and repression, including increased activity levels of various enzymes. We show that Aspergillus oryzae contains a functional homologue of the CreB deubiquitinating enzyme and that a null strain shows increased activity levels of industrially important secreted enzymes, including cellulases, xylanases, amylases, and proteases, as well as alleviated inhibition of spore germination on glucose medium. Reverse transcription-quantitative PCR (RT-qPCR) analysis showed that the increased levels of enzyme activity in both Aspergillus nidulans and Aspergillus oryzae are mirrored at the transcript level, indicating transcriptional regulation. We report that Aspergillus oryzae DAR3699, originally isolated from soy fermentation, has a similar phenotype to that of a creB deletion mutant of the RIB40 strain, and it contains a mutation in the creB gene. Collectively, the results for Aspergillus oryzae, Aspergillus nidulans, Trichoderma reesei, and Penicillium decumbens show that deletion of creB may be broadly useful in diverse fungi for increasing production of a variety of enzymes.

  18. Effect of different nutrient supply and other growth factors on the activity of the oxidizing enzymes in plants

    Energy Technology Data Exchange (ETDEWEB)

    Amberger, A

    1960-01-01

    Among the plants studied were french beans and peas; the oxidizing enzymes examined were ascorbic acid oxidase, cytochrome oxidase, phenol oxidase, peroxidase and catalase. Increasing the K dosage reduced enzyme activity and raised dry matter contents until at a very high dosage this action was reversed. Both N and P increased enzyme activity and yields. With B high enzyme activity and low dry matter content were both associated with deficiency and toxicity levels. Increasing the Fe dosage led to a rise in both dry matter content and enzyme activity, whereas F depressed yields and raised enzyme activity. Lack of water increased respiration. Light inhibited all enzyme activity.

  19. Cadmium Phytoavailability and Enzyme Activity under Humic Acid Treatment in Fluvo-aquic Soil

    Science.gov (United States)

    Liu, Borui; Huang, Qing; Su, Yuefeng

    2018-01-01

    A pot experiment was conducted to investigate the cadmium (Cd) availability to pakchois (Brassica chinensis L.) as well as the enzyme activities in fluvo-aquic soil under humic acid treatment. The results showed that the phytoavailability of Cd in soil decreased gradually as humic acid concentration rose (0 to 12 g·kg-1), while the activities of urease (UE), alkaline phosphatase (ALP) and catalase (CAT) kept increasing (P enzymes due to the Cd pollution. In conclusion, humic acid is effective for the reduction of both Cd phytoavailability and the damage to enzyme activities due to Cd pollution in fluvo-aquic soil

  20. Is there any role of prolidase enzyme activity in the etiology of preeclampsia?

    Science.gov (United States)

    Pehlivan, Mustafa; Ozün Ozbay, Pelin; Temur, Muzaffer; Yılmaz, Ozgur; Verit, Fatma Ferda; Aksoy, Nurten; Korkmazer, Engin; Üstünyurt, Emin

    2017-05-01

    To evaluate a relationship between preeclampsia and prolidase enzyme activity. A prospective cohort study of 41 pregnant women diagnosed with preeclampsia and 31 healthy pregnant women as control group was selected at Harran University Hospital Department of Obstetrics and Gynecology. The prolidase enzyme activity was analyzed in maternal and umbilical cord plasma, amniotic fluid and placental and umbilical cord tissues by Chinard method in addition to maternal serum levels of lactate dehydrogenase (LDH), serum glutamate pyruvate transaminase (SGPT) and serum glutamate oxaloacetate transaminase (SGOT). A significant relationship was found between plasma prolidase activity (635 ± 83 U/L) (p  = 0.007), umbilical cord plasma prolidase activity (610 ± 90 U/L) (p = 0.013), amniotic fluid prolidase activity (558 ± 100 U/L) (p  = 0.001), umbilical cord tissue prolidase activity (4248 ± 1675 U/gr protein) (p  = 0.013) and placental tissue prolidase activity (2116 ± 601 U/gr protein) (p  = 0.001) in preeclamptic group when compared to healthy pregnant women. There is a strong correlation between prolidase enzyme activity and preeclampsia. Prolidase enzyme activity may play a role in preeclampsia.

  1. Protein Hydrolysis from Catfish Prepared by Papain Enzyme and Antioxidant Activity of Hydrolyzate

    Directory of Open Access Journals (Sweden)

    Ace Baehaki

    2015-12-01

    Full Text Available The objective of this research was to make a protein hydrolysates from catfish (Pangasius pangasius enzymatically using papain enzyme and analyzed the antioxidant activity of protein hydrolysates produced. The research used the method completely randomized design with two replications the treatment were the difference concentration of the papain enzyme (0%, 1%, 2%, 3%, 4%, 5%, and 6%. The parameters of research were antioxidative activity using DPPH (2,2-difenil-1–pikrilhidrazil, protein content, and molecular weight using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis. The results showed that catfish protein hydrolysates prepared by papain enzyme has antioxidative activity. The highest degree of hydrolysis was 71.98% at enzyme concentration of 6%. Based on the DPPH scavenging method catfish protein hydrolysates has the antioxidative activity with the value 37.85-67.62%. The protein content of catfish protein hydrolysates were 20.86-54.47 mg/ml. The molecular weight of catfish protein hydrolyzates were 11.90-65.20 kDa.

  2. Effect of citric acid and microbial phytase on serum enzyme activities ...

    African Journals Online (AJOL)

    Effect of citric acid and microbial phytase on serum enzyme activities and plasma minerals retention in broiler chicks. ... African Journal of Biotechnology ... An experiment was conducted to study the effect of microbial phytase supplementation and citric acid in broiler chicks fed corn-soybean meal base diets on enzyme ...

  3. Ratio Imaging of Enzyme Activity Using Dual Wavelength Optical Reporters

    Directory of Open Access Journals (Sweden)

    Moritz F. Kircher

    2002-04-01

    Full Text Available The design of near-infrared fluorescent (NIRF probes that are activated by specific proteases has, for the first time, allowed enzyme activity to be imaged in vivo. In the current study, we report on a method of imaging enzyme activity using two fluorescent probes that, together, provide improved quantitation of enzymatic activity. The method employs two chemically similar probes that differ in their degradability by cathepsin B. One probe consists of the NIRF dye Cy5.5 attached to a particulate carrier, a crosslinked iron oxide nanoparticle (CLIO, through cathepsin B cleavable l-arginyl peptides. A second probe consists of Cy3.5 attached to a CLIO through proteolytically resistant d-arginyl peptides. Using mixtures of the two probes, we have shown that the ratio of Cy5.5 to Cy3.5 fluorescence can be used to determine levels of cathepsin B in the environment of nanoparticles with macrophages in suspension. After intravenous injection, tissue fluorescence from the nondegradable Cy3.5–d-arginyl probe reflected nanoparticle accumulation, while fluorescence of the Cy5.5–l-arginyl probe was dependent on both accumulation and activation by cathepsin B. Dual wavelength ratio imaging can be used for the quantitative imaging of a variety of enzymes in clinically important settings, while the magnetic properties of the probes allow their detection by MR imaging.

  4. Enzyme activity deviates due to spatial and temporal temperature profiles in commercial microtiter plate readers.

    Science.gov (United States)

    Grosch, Jan-Hendrik; Sieben, Michaela; Lattermann, Clemens; Kauffmann, Kira; Büchs, Jochen; Spieß, Antje C

    2016-03-01

    Microtiter plates (MTP) and automatized techniques are increasingly applied in the field of biotechnology. However, the susceptibility of MTPs to edge effects such as thermal gradients can lead to high variation of measured enzyme activities. In an effort to enhance experimental reliability, to quantify, and to minimize instrument-caused deviations in enzyme kinetics between two MTP-readers, we comprehensively quantified temperature distribution in 96-well MTPs. We demonstrated the robust application of the absorbance dye cresol red as easily applicable temperature indicator in cuvettes and MTPs and determined its accuracy to ±0.16°C. We then quantified temperature distributions in 96-well MTPs revealing temperature deviations over single MTP of up to 2.2°C and different patterns in two commercial devices (BioTek Synergy 4 and Synergy Mx). The obtained liquid temperature was shown to be substantially controlled by evaporation. The temperature-induced enzyme activity variation within MTPs amounted to about 20 %. Activity deviations between MTPs and to those in cuvettes were determined to 40 % due to deviations from the set temperature in MTPs. In conclusion, we propose a better control of experimental conditions in MTPs or alternative experimental systems for reliable determination of kinetic parameters for bioprocess development. Copyright © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Metabolic organization and effects of feeding on enzyme activities of the dogfish shark (Squalus acanthias) rectal gland.

    Science.gov (United States)

    Walsh, Patrick J; Kajimura, Makiko; Mommsen, Thomas P; Wood, Chris M

    2006-08-01

    In order to investigate the metabolic poise of the elasmobranch rectal gland, we conducted two lines of experimentation. First, we examined the effects of feeding on plasma metabolites and enzyme activities from several metabolic pathways in several tissues of the dogfish shark, Squalus acanthias, after starvation and at 6, 20, 30 and 48 h post-feeding. We found a rapid and sustained ten-fold decrease in plasma beta-hydroxybutyrate at 6 h and beyond compared with starved dogfish, suggesting an upregulation in the use of this substrate, a decrease in production, or both. Plasma acetoacetate levels remain unchanged, whereas there was a slight and transient decrease in plasma glucose levels at 6 h. Several enzymes showed a large increase in activity post-feeding, including beta-hydroxybutyrate dehydrogenase in rectal gland and liver, and in rectal gland, isocitrate dehydrogenase, citrate synthase, lactate dehydrogenase, aspartate amino transferase, alanine amino transferase, glutamine synthetase and Na(+)/K(+) ATPase. Also notable in these enzyme measurements was the overall high level of activity in the rectal gland in general. For example, activity of the Krebs' TCA cycle enzyme citrate synthase (over 30 U g(-1)) was similar to activities in muscle from other species of highly active fish. Surprisingly, lactate dehydrogenase activity in the gland was also high (over 150 U g(-1)), suggesting either an ability to produce lactate anaerobically or use lactate as an aerobic fuel. Given these interesting observations, in the second aspect of the study we examined the ability of several metabolic substrates (alone and in combination) to support chloride secretion by the rectal gland. Among the substrates tested at physiological concentrations (glucose, beta-hydroxybutyrate, lactate, alanine, acetoacetate, and glutamate), only glucose could consistently maintain a viable preparation. Whereas beta-hydroxybutyrate could enhance gland activity when presented in combination

  6. Tissue and plasma enzyme activities in juvenile green iguanas.

    Science.gov (United States)

    Wagner, R A; Wetzel, R

    1999-02-01

    To determine activities of intracellular enzymes in 8 major organs in juvenile green iguanas and to compare tissue and plasma activities. 6 green iguanas iguanas, but high values may not always indicate overt muscle disease. The AMS activity may be specific for the pancreas, but the wide range of plasma activity would likely limit its diagnostic usefulness. Activities of AST and LDH may reflect tissue damage or inflammation, but probably do not reflect damage to specific tissues or organs.

  7. Enzyme activity assays within microstructured optical fibers enabled by automated alignment.

    Science.gov (United States)

    Warren-Smith, Stephen C; Nie, Guiying; Schartner, Erik P; Salamonsen, Lois A; Monro, Tanya M

    2012-12-01

    A fluorescence-based enzyme activity assay has been demonstrated within a small-core microstructured optical fiber (MOF) for the first time. To achieve this, a reflection-based automated alignment system has been developed, which uses feedback and piezoelectric actuators to maintain optical alignment. The auto-alignment system provides optical stability for the time required to perform an activity assay. The chosen assay is based on the enzyme proprotein convertase 5/6 (PC6) and has important applications in women's health.

  8. [Estimation of adaptive capacities in Magnitogorsk children from the activity of some detoxification enzymes].

    Science.gov (United States)

    koganova, Z I; Ingel', F I; Antipanova, N A; Legostoeva, T B; Poliakova, O V

    2010-01-01

    The paper provides the first fragment of a multiparameter study analyzing the influence of environmental pollution, the social and psychological features of a family, and some endogenous factors on genome stability and sensitivity in a developed ferrous metallurgy town. It also gives data on the urine and serum activity of the lysosomal enzyme N-acetyl-b-D-glucosaminidase (NAG) and the serum activity of catalase in an organized contingent of apparently healthy children (n = 178; 6 kindergartens) aged 5-7 years, who live permanently in Magnitogorsk at different distances from the metallurgical works. More than 70% of children selected for examination were found to have average normal levels of activity of the enzymes studied. According to the average levels of enzyme activity, there were only 2 kindergartens (both from the left-bank region). In the children from the left-bank area, enzyme activities varied more greatly, which suggests the higher prevalence of tense adaptation. Correlation analysis revealed association between the children's serum activity of enzymes and some components of snow pollution. It is anticipated that the found changes in serum activities of N-acetyl-beta-D-glucosaminidase and catalase may be determined by individual differences in a child's response to ambient air pollutants.

  9. A fluorescence-based hydrolytic enzyme activity assay for quantifying toxic effects of Roundup® to Daphnia magna

    DEFF Research Database (Denmark)

    Ørsted, Michael; Roslev, Peter

    2015-01-01

    Daphnia magna is a widely used model organism for aquatic toxicity testing. In the present study, we investigated the hydrolytic enzyme activity of D. magna after exposure to toxicant stress. In vivo enzyme activity was quantified using 15 fluorogenic enzyme probes based on 4-methylumbelliferyl...... or 7-amino-4-methylcoumarin. Probing D. magna enzyme activity was evaluated using short-term exposure (24-48 h) to the reference chemical K2Cr2O7, or the herbicide formulation Roundup®. Toxicant induced changes in hydrolytic enzyme activity were compared to changes in mobility (ISO 6341). The results...... showed that hydrolytic enzyme activity was quantifiable as a combination of whole body fluorescence of D. magna, and fluorescence of the surrounding water. Exposure of D. magna to lethal and sublethal concentrations of Roundup® resulted in loss of whole body enzyme activity, and release of cell...

  10. Spatial characterization of proteolytic enzyme activity in the foregut region of the adult necrophagous fly, Protophormia terraenovae.

    Science.gov (United States)

    Rivers, David B; Acca, Gillian; Fink, Marc; Brogan, Rebecca; Schoeffield, Andrew

    2014-08-01

    The spatial distribution of proteolytic enzymes in the adult foregut of Protophormia terraenovae was studied in the context of protein digestion and regurgitation. Based on substrate specificity, pH optima, and use of specific protease inhibitors, all adults tested displayed enzyme activity in the foregut consistent with pepsin, trypsin and chymotrypsin. Chymotrypsin-like and trypsin-like enzyme activity were detected in all gut fluids and tissues tested, with chymotrypsin displaying the highest activity in saliva and salivary gland tissue, whereas maximal trypsin activity was evident in the crop. Pepsin-like activity was only evident in crop fluids and tissues. The activity of all three enzymes was low or undetectable (pepsin) in the fluids and tissue homogenates derived from the esophagus and cardia of any of the adults assayed. Fed adult females displayed higher enzyme activities than fed males, and the activity of all three enzymes were much more prevalent in fed adults than starved. The pH optimum of the trypsin-like enzyme was between pH 7.0 and 8.0; chymotrypsin was near pH 8.0; and maximal pepsin-like activity occurred between pH 1.0 and 2.0. Regurgitate from fed adult females displayed enzyme activity consistent with the proteolytic enzymes detected in crop gut fluids. Enzymes in regurgitate were not derived from food sources based on assays of bovine liver samples. These latter observations suggest that adult flies release fluids from foregut when encountering dry foods, potentially as a means to initiate extra-oral digestion. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. Lead action on activity of some enzymes of plants

    International Nuclear Information System (INIS)

    Korolyov, A.N.; Koshkaryova, A.I.

    2008-01-01

    Lead action on activity of some enzymes of young plants of barley double-row (Hordeum distichon L.) families of cereals (Grominea). It is established that activity urease, catalase, ascorbatoxidase is in dependence as from a lead dose in a nutritious solution, and term ontogenesis. At later stages ontogenesis the increase in concentration of lead in an inhabitancy leads to sharp decrease in activity ascorbatoxidase. In the same conditions activity urease and catalase raises.

  12. Activity of an enzyme immobilized on superparamagnetic particles in a rotational magnetic field

    Energy Technology Data Exchange (ETDEWEB)

    Mizuki, Toru; Watanabe, Noriyuki; Nagaoka, Yutaka [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan); Fukushima, Tadamasa [Shimadzu GLC Ltd., Phenomenex Support Centre, Tokyo 110-0016 (Japan); Morimoto, Hisao; Usami, Ron [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan); Maekawa, Toru, E-mail: maekawa@toyonet.toyo.ac.jp [Bio-Nano Electronics Research Centre, Toyo University, Saitama 350-8585 (Japan)

    2010-03-19

    We immobilize {alpha}-amylase extracted from Bacillus Iicheniformis on the surfaces of superparamagnetic particles and investigate the effect of a rotational magnetic field on the enzyme's activity. We find that the activity of the enzyme molecules immobilized on superparamagnetic particles increases in the rotational magnetic field and reaches maximum at a certain frequency. We clarify the effect of the cluster structures formed by the superparamagnetic particles on the activity. Enzyme reactions are enhanced even in a tiny volume of solution using the present method, which is very important for the development of efficient micro reactors and micro total analysis systems ({mu}-TAS).

  13. In vivo enzyme activity in inborn errors of metabolism

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, G.N.; Walter, J.H.; Leonard, J.V.; Halliday, D. (Clinical Research Centre, Harrow (England))

    1990-08-01

    Low-dose continuous infusions of (2H5)phenylalanine, (1-13C)propionate, and (1-13C)leucine were used to quantitate phenylalanine hydroxylation in phenylketonuria (PKU, four subjects), propionate oxidation in methylmalonic acidaemia (MMA, four subjects), and propionic acidaemia (PA, four subjects) and leucine oxidation in maple syrup urine disease (MSUD, four subjects). In vivo enzyme activity in PKU, MMA, and PA subjects was similar to or in excess of that in adult controls (range of phenylalanine hydroxylation in PKU, 3.7 to 6.5 mumol/kg/h, control 3.2 to 7.9, n = 7; propionate oxidation in MMA, 15.2 to 64.8 mumol/kg/h, and in PA, 11.1 to 36.0, control 5.1 to 19.0, n = 5). By contrast, in vivo leucine oxidation was undetectable in three of the four MSUD subjects (less than 0.5 mumol/kg/h) and negligible in the remaining subject (2 mumol/kg/h, control 10.4 to 15.7, n = 6). These results suggest that significant substrate removal can be achieved in some inborn metabolic errors either through stimulation of residual enzyme activity in defective enzyme systems or by activation of alternate metabolic pathways. Both possibilities almost certainly depend on gross elevation of substrate concentrations. By contrast, only minimal in vivo oxidation of leucine appears possible in MSUD.

  14. In vivo enzyme activity in inborn errors of metabolism

    International Nuclear Information System (INIS)

    Thompson, G.N.; Walter, J.H.; Leonard, J.V.; Halliday, D.

    1990-01-01

    Low-dose continuous infusions of [2H5]phenylalanine, [1-13C]propionate, and [1-13C]leucine were used to quantitate phenylalanine hydroxylation in phenylketonuria (PKU, four subjects), propionate oxidation in methylmalonic acidaemia (MMA, four subjects), and propionic acidaemia (PA, four subjects) and leucine oxidation in maple syrup urine disease (MSUD, four subjects). In vivo enzyme activity in PKU, MMA, and PA subjects was similar to or in excess of that in adult controls (range of phenylalanine hydroxylation in PKU, 3.7 to 6.5 mumol/kg/h, control 3.2 to 7.9, n = 7; propionate oxidation in MMA, 15.2 to 64.8 mumol/kg/h, and in PA, 11.1 to 36.0, control 5.1 to 19.0, n = 5). By contrast, in vivo leucine oxidation was undetectable in three of the four MSUD subjects (less than 0.5 mumol/kg/h) and negligible in the remaining subject (2 mumol/kg/h, control 10.4 to 15.7, n = 6). These results suggest that significant substrate removal can be achieved in some inborn metabolic errors either through stimulation of residual enzyme activity in defective enzyme systems or by activation of alternate metabolic pathways. Both possibilities almost certainly depend on gross elevation of substrate concentrations. By contrast, only minimal in vivo oxidation of leucine appears possible in MSUD

  15. Phosphorus fractions, microbial biomass and enzyme activities in ...

    African Journals Online (AJOL)

    Potohar, northern Punjab, Pakistan in September, 2008 and analysed for P fractions and microbial parameters including microbial biomass C, microbial biomass N, microbial biomass P, and activities of dehydrogenase and alkaline phosphatase enzymes. The average size of different P fractions (% of total P) in the soils ...

  16. First glycoside hydrolase family 2 enzymes from Thermus antranikianii and Thermus brockianus with β-glucosidase activity

    Directory of Open Access Journals (Sweden)

    Carola eSchröder

    2015-06-01

    Full Text Available Two genes tagh2 and tbgh2 coding for enzymes with hydrolytic activity towards esculin were identified from the extreme thermophilic, aerobic bacteria Thermus antranikianii (Ta and T. brockianus (Tb. Shortened conserved domains predicted a membership of the enzymes of glycoside hydrolase (GH family 2. At present, β-galactosidase activity is found frequently in GH family 2 but β-glucosidase activity has not been reported in this family before. The enzymes TaGH2 and TbGH2 preferred hydrolysis of nitrophenol-linked β-D-glucopyranosides with specific activities of 3,966 U/mg and 660 U/mg, respectively. Residual activities of 40 % (TaGH2 and 51 % (TbGH2 towards 4-NP-β-D-galactopyranoside were observed. Furthermore, TaGH2 hydrolyzed cellobiose. TbGH2, however, showed no activity on cellobiose or lactose. The enzymes exhibited highest activity at 95 °C (TaGH2 and 90 °C (TbGH2 at pH 6.5. Both enzymes were extremely thermostable and thermal activation up to 250 % was observed at temperatures between 50 and 60 °C. Accordingly, the first thermoactive glycoside hydrolase family 2 enzymes with β glucosidase activity have been identified and characterized. The hydrolysis of cellobiose is a unique property of TaGH2 when compared to the enzymes of GH family 2.

  17. activity of enzyme trypsin immobilized onto macroporous poly(epoxy

    African Journals Online (AJOL)

    dell

    consequential effects of covalent immobilization. EXPERIMENTAL. Materials .... immersed into water bath. ... storage stability of the enzyme was studied ... pore size range of about 10 to 150 µm. ... figures, the differences in activities (slopes.

  18. Changes in photosynthesis and activities of enzymes involved in ...

    African Journals Online (AJOL)

    tolerance, respectively were used to investigate the oxygen consumption rate of photosystem I, the oxygen evolution rate of photosystem II, cab transcript levels, and activities of enzymes involved in photosynthetic carbon reduction cycle.

  19. Enzyme-activity mutations detected in mice after paternal fractionated irradiation

    International Nuclear Information System (INIS)

    Charles, D.J.; Pretsch, W.

    1986-01-01

    (101/E1 X C3H/E1)F 1 -hybrid male mice were exposed in a 24-h fractionation interval to either 3.0 + 3.0-Gy or 5.1 + 5.1-Gy X-irradiation, and mated to untreated Test-stock females. The offspring were examined for mutations at 7 recessive specific loci and for activity alterations of erythrocyte enzymes controlled presumably by 12 loci. No enzyme-activity mutant was found in 3610 F 1 -offspring of the control group. In the experimental groups, no mutant was detected in 533 (3.0 + 3.0 Gy) and 173 (5.1 + 5.1 Gy) offspring from postspermatogonial germ cells treated. After treatment of spermatogonia, 1 mutant in 3388 F 1 -offspring of the 3.0 + 3.0-Gy group, and 5 mutants in 3187 F 1 offspring of the 5.1 + 5.1-Gy group were found. The mutants were all genetically confirmed. The frequency (expressed as mutants/locus/gamete) of enzyme-activity mutations is 2 (5.1 + 5.1-Gy group) to 10 (3.0 + 3.0-Gy group) times lower than the frequency of recessive specific-locus mutations. (Auth.)

  20. Effect of irradiation on immobilized enzymes compared with that on enzymes in solution

    International Nuclear Information System (INIS)

    Schachinger, L.; Schippel, C.; Altmann, E.; Diepold, B.; Yang, C.; Jaenike, M.; Hochhaeuser, E.

    1985-01-01

    Glucose oxidase and catalase were immobilized by attaching them to nylon fibers that had been treated with triethyloxonium-tetrafluoroborate, diaminohexane and glutaraldialdehyde according to Morris, Campell and Hornby (1975). This method assures that the enzymes are bound to a side chain of the polyamide structure. Enzyme activity (as measured by the O 2 -uptake and by microcalorimetry) was found to be unchanged after 2 years. The apparent Ksub(m)-constants of the immobilized enzymes with glucose were the same as those for enzymes in solution. GOD and catalase immobilized in poly(acrylamide) gel had the same Ksub(m)-value. Despite the high stability during storage, the radiation induced inactivation of enzymes immobilized on gel or chromosorb, an inorganic carrier, was of the same order of magnitude as that of the dissolved enzymes. The enzymes bound to nylon fibers showed a higher radiation sensitivity. This might have been caused by an additional attack on the binding site of the carrier. (orig.)

  1. Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens

    International Nuclear Information System (INIS)

    Perrone, Carmen E.; Duan, Jian Dong; Jeffrey, Alan M.; Williams, Gary M.; Ahr, Hans-Juergen; Schmidt, Ulrich; Enzmann, Harald H.

    2004-01-01

    Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovoturkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using 32 P-postlabeling for DNA adducts. In ovoexposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4'-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had 32 P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity. (orig.)

  2. Embryonic turkey liver: activities of biotransformation enzymes and activation of DNA-reactive carcinogens

    Energy Technology Data Exchange (ETDEWEB)

    Perrone, Carmen E.; Duan, Jian Dong; Jeffrey, Alan M.; Williams, Gary M. [New York Medical College, Department of Pathology, Valhalla (United States); Ahr, Hans-Juergen; Schmidt, Ulrich [Bayer AG, Institute of Toxicology, Wuppertal (Germany); Enzmann, Harald H. [Federal Institute for Drugs and Medical Devices, Bonn (Germany)

    2004-10-01

    Avian embryos are a potential alternative model for chemical toxicity and carcinogenicity research. Because the toxic and carcinogenic effects of some chemicals depend on bioactivation, activities of biotransformation enzymes and formation of DNA adducts in embryonic turkey liver were examined. Biochemical analyses of 22-day in ovoturkey liver post-mitochondrial fractions revealed activities of the biotransformation enzymes 7-ethoxycoumarin de-ethylase (ECOD), 7-ethoxyresorufin de-ethylase (EROD), aldrin epoxidase (ALD), epoxide hydrolase (EH), glutathione S-transferase (GST), and UDP-glucuronyltransferase (GLUT). Following the administration of phenobarbital (24 mg/egg) on day 21, enzyme activities of ECOD, EROD, ALD, EH and GLUT, but not of GST, were increased by two-fold or higher levels by day 22. In contrast, acute administration of 3-methylcholanthrene (5 mg/egg) induced only ECOD and EROD activities. Bioactivation of structurally diverse pro-carcinogens was also examined using {sup 32}P-postlabeling for DNA adducts. In ovoexposure of turkey embryos on day 20 of gestation to 2-acetylaminofluorene (AAF), 4,4'-methylenebis(2-chloroaniline) (MOCA), benzo[a]pyrene (BaP), and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) resulted in the formation of DNA adducts in livers collected by day 21. Some of the DNA adducts had {sup 32}P-postlabeling chromatographic migration patterns similar to DNA adducts found in livers from Fischer F344 rats exposed to the same pro-carcinogens. We conclude that 21-day embryonic turkey liver is capable of chemical biotransformation and activation of genotoxic carcinogens to form DNA adducts. Thus, turkey embryos could be utilized to investigate potential chemical toxicity and carcinogenicity. (orig.)

  3. Probiotic activity of lignocellulosic enzyme as bioactivator for rice husk degradation

    Science.gov (United States)

    Lamid, Mirni; Al-Arif, Anam; Warsito, Sunaryo Hadi

    2017-02-01

    The utilization of lignocellulosic enzyme will increase nutritional value of rice husk. Cellulase consists of C1 (β-1, 4-glucan cellobiohydrolase or exo-β-1,4glucanase), Cc (endo-β-1,4-glucanase) and component and cellobiose (β-glucocidase). Hemicellulase enzyme consists of endo-β-1,4-xilanase, β-xilosidase, α-L arabinofuranosidase, α-D-glukuronidaseand asetil xilan esterase. This research aimed to study the activity of lignocellulosic enzyme, produced by cows in their rumen, which can be used as a bioactivator in rice husk degradation. This research resulted G6 and G7 bacteria, producing xylanase and cellulase with the activity of 0.004 U mL-1 and 0.021 U mL-1; 0.003 ( U mL-1) and 0.026 (U mL-1) respectively.

  4. Microbial dynamics and enzyme activities in tropical Andosols depending on land use and nutrient inputs

    Science.gov (United States)

    Mganga, Kevin; Razavi, Bahar; Kuzyakov, Yakov

    2015-04-01

    Microbial decomposition of soil organic matter is mediated by enzymes and is a key source of terrestrial CO2 emissions. Microbial and enzyme activities are necessary to understand soil biochemical functioning and identify changes in soil quality. However, little is known about land use and nutrients availability effects on enzyme activities and microbial processes, especially in tropical soils of Africa. This study was conducted to examine how microbial and enzyme activities differ between different land uses and nutrient availability. As Andosols of Mt. Kilimanjaro are limited by nutrient concentrations, we hypothesize that N and P additions will stimulate enzyme activity. N and P were added to soil samples (0-20 cm) representing common land use types in East Africa: (1) savannah, (2) maize fields, (3) lower montane forest, (4) coffee plantation, (5) grasslands and (6) traditional Chagga homegardens. Total CO2 efflux from soil, microbial biomass and activities of β-glucosidase, cellobiohydrolase, chitinase and phosphatase involved in C, N and P cycling, respectively was monitored for 60 days. Total CO2 production, microbial biomass and enzyme activities varied in the order forest soils > grassland soils > arable soils. Increased β-glucosidase and cellobiohydrolase activities after N addition of grassland soils suggest that microorganisms increased N uptake and utilization to produce C-acquiring enzymes. Low N concentration in all soils inhibited chitinase activity. Depending on land use, N and P addition had an inhibitory or neutral effect on phosphatase activity. We attribute this to the high P retention of Andosols and low impact of N and P on the labile P fractions. Enhanced CO2 production after P addition suggests that increased P availability could stimulate soil organic matter biodegradation in Andosols. In conclusion, land use and nutrients influenced soil enzyme activities and microbial dynamics and demonstrated the decline in soil quality after landuse

  5. An amperometric enzyme biosensor for real-time measurements of cellobiohydrolase activity on insoluble cellulose

    DEFF Research Database (Denmark)

    Cruys-Bagger, Nicolaj; Guilin, Ren; Tatsumi, Hirosuke

    2012-01-01

    An amperometric enzyme biosensor for continuous detection of cellobiose has been implemented as an enzyme assay for cellulases. We show that the initial kinetics for cellobiohydrolase I, Cel7A from Trichoderma reesei, acting on different types of cellulose substrates, semi-crystalline and amorphous......, can be monitored directly and in real-time by an enzyme-modified electrode based on cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium (Pc). PcCDH was cross-linked and immobilized on the surface of a carbon paste electrode which contained a mediator, benzoquinone. An oxidation current...... of the reduced mediator, hydroquinone, produced by the CDH-catalyzed reaction with cellobiose, was recorded under constant-potential amperometry at +0.5 V (vs. Ag/AgCl). The CDH-biosensors showed high sensitivity (87.7 µA mM−1 cm−2), low detection limit (25 nM), and fast response time (t95% ∼ 3 s...

  6. Co-ordinate activation of lipogenic enzymes in hepatocellular carcinoma.

    Science.gov (United States)

    Yahagi, Naoya; Shimano, Hitoshi; Hasegawa, Kiyoshi; Ohashi, Kenichi; Matsuzaka, Takashi; Najima, Yuho; Sekiya, Motohiro; Tomita, Sachiko; Okazaki, Hiroaki; Tamura, Yoshiaki; Iizuka, Yoko; Ohashi, Ken; Nagai, Ryozo; Ishibashi, Shun; Kadowaki, Takashi; Makuuchi, Masatoshi; Ohnishi, Shin; Osuga, Jun-ichi; Yamada, Nobuhiro

    2005-06-01

    Hepatocellular carcinoma is a very common neoplastic disease in countries where hepatitis viruses B and/or C are prevalent. Small hepatocellular carcinoma lesions detected by ultrasonography at an early stage are often hyperechoic because they are composed of well-differentiated cancer cells that are rich in triglyceride droplets. The triglyceride content of hepatocytes depends in part on the rate of lipogenesis. Key lipogenic enzymes, such as fatty acid synthase, are co-ordinately regulated at the transcriptional level. We therefore examined the mRNA expression of lipogenic enzymes in human hepatocellular carcinoma samples from 10 patients who had undergone surgical resection. All of the samples exhibited marked elevation of expression of mRNA for lipogenic enzymes, such as fatty acid synthase, acetyl-CoA carboxylase and ATP citrate lyase, compared with surrounding non-cancerous liver tissue. In contrast, the changes in mRNA expression of SREBP-1, a transcription factor that regulates a battery of lipogenic enzymes, did not show a consistent trend. In some cases where SREBP-1 was elevated, the main contributing isoform was SREBP-1c rather than SREBP-1a. Thus, lipogenic enzymes are markedly induced in hepatocellular carcinomas, and in some cases SREBP-1c is involved in this activation.

  7. Effects of electrokinetic treatment of a heavy metal contaminated soil on soil enzyme activities

    International Nuclear Information System (INIS)

    Cang Long; Zhou Dongmei; Wang Quanying; Wu Danya

    2009-01-01

    There is a growing concern on the potential application of a direct current (DC) electric field to soil for removing contaminants, but little is known about its impact on soil enzyme activities. This study investigated the change of enzyme activities of a heavy metal contaminated soil before and after electrokinetic (EK) treatments at lab-scale and the mechanisms of EK treatment to affect soil enzyme activities were explored. After treatments with 1-3 V cm -1 of voltage gradient for 420 h, soil pH, electrical conductivity (EC), soil organic carbon, dissolved organic carbon (DOC), soil heavy metal concentration and enzyme activities were analyzed. The results showed that the average removal efficiencies of soil copper were about 65% and 83% without and with pH control of catholyte, respectively, and all the removal efficiencies of cadmium were above 90%. The soil invertase and catalase activities increased and the highest invertase activity was as 170 times as the initial one. The activities of soil urease and acidic phosphatase were lower than the initial ones. Bivariate correlation analyses indicated that the soil invertase and acidic phosphatase activities were significantly correlated with soil pH, EC, and DOC at P < 0.05, but the soil urease activities had no correlation with the soil properties. On the other hand, the effects of DC electric current on solution invertase and catalase enzyme protein activities indicated that it had negative effect on solution catalase activity and little effect on solution invertase activity. From the change of invertase and catalase activities in soil and solution, the conclusion can be drawn that the dominant effect mechanism is the change of soil properties by EK treatments.

  8. Effects of electrokinetic treatment of a heavy metal contaminated soil on soil enzyme activities

    Energy Technology Data Exchange (ETDEWEB)

    Cang Long [State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China); Graduate School of the Chinese Academy of Sciences, Beijing 100049 (China); Zhou Dongmei, E-mail: dmzhou@issas.ac.cn [State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China); Wang Quanying; Wu Danya [State Key Laboratory of Soil and Sustainable Agriculture, Institute of Soil Science, Chinese Academy of Sciences, Nanjing 210008 (China); Graduate School of the Chinese Academy of Sciences, Beijing 100049 (China)

    2009-12-30

    There is a growing concern on the potential application of a direct current (DC) electric field to soil for removing contaminants, but little is known about its impact on soil enzyme activities. This study investigated the change of enzyme activities of a heavy metal contaminated soil before and after electrokinetic (EK) treatments at lab-scale and the mechanisms of EK treatment to affect soil enzyme activities were explored. After treatments with 1-3 V cm{sup -1} of voltage gradient for 420 h, soil pH, electrical conductivity (EC), soil organic carbon, dissolved organic carbon (DOC), soil heavy metal concentration and enzyme activities were analyzed. The results showed that the average removal efficiencies of soil copper were about 65% and 83% without and with pH control of catholyte, respectively, and all the removal efficiencies of cadmium were above 90%. The soil invertase and catalase activities increased and the highest invertase activity was as 170 times as the initial one. The activities of soil urease and acidic phosphatase were lower than the initial ones. Bivariate correlation analyses indicated that the soil invertase and acidic phosphatase activities were significantly correlated with soil pH, EC, and DOC at P < 0.05, but the soil urease activities had no correlation with the soil properties. On the other hand, the effects of DC electric current on solution invertase and catalase enzyme protein activities indicated that it had negative effect on solution catalase activity and little effect on solution invertase activity. From the change of invertase and catalase activities in soil and solution, the conclusion can be drawn that the dominant effect mechanism is the change of soil properties by EK treatments.

  9. Intrinsic Xenobiotic Metabolizing Enzyme Activities in Early Life Stages of Zebrafish (Danio rerio).

    Science.gov (United States)

    Otte, Jens C; Schultz, Bernadette; Fruth, Daniela; Fabian, Eric; van Ravenzwaay, Bennard; Hidding, Björn; Salinas, Edward R

    2017-09-01

    Early life stages of zebrafish (Danio rerio, zf) are gaining attention as an alternative invivo test system for drug discovery, early developmental toxicity screenings and chemical testing in ecotoxicological and toxicological testing strategies. Previous studies have demonstrated transcriptional evidence for xenobiotic metabolizing enzymes (XME) during early zf development. However, elaborate experiments on XME activities during development are incomplete. In this work, the intrinsic activities of representative phase I and II XME were monitored by transformation of putative zf model substrates analyzed using photometry and high pressure liquid chromatography techniques. Six different defined stages of zf development (between 2.5 h postfertilization (hpf) to 120 hpf) were investigated by preparing a subcellular fraction from whole organism homogenates. We demonstrated that zf embryos as early as 2.5 hpf possess intrinsic metabolic activities for esterase, Aldh, Gst, and Cyp1a above the methodological detection limit. The activities of the enzymes Cyp3a and Nat were measurable during later stages in development. Activities represent dynamic patterns during development. The role of XME activities revealed in this work is relevant for the assessing toxicity in this test system and therefore contributes to a valuable characterization of zf embryos as an alternative testing organism in toxicology. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  10. Ambient Ionization Mass Spectrometry Measurement of Aminotransferase Activity

    Science.gov (United States)

    Yan, Xin; Li, Xin; Zhang, Chengsen; Xu, Yang; Cooks, R. Graham

    2017-06-01

    A change in enzyme activity has been used as a clinical biomarker for diagnosis and is useful in evaluating patient prognosis. Current laboratory measurements of enzyme activity involve multi-step derivatization of the reaction products followed by quantitative analysis of these derivatives. This study simplified the reaction systems by using only the target enzymatic reaction and directly detecting its product. A protocol using paper spray mass spectrometry for identifying and quantifying the reaction product has been developed. Evaluation of the activity of aspartate aminotransferase (AST) was chosen as a proof-of-principle. The volume of sample needed is greatly reduced compared with the traditional method. Paper spray has a desalting effect that avoids sprayer clogging problems seen when examining serum samples by nanoESI. This very simple method does not require sample pretreatment and additional derivatization reactions, yet it gives high quality kinetic data, excellent limits of detection (60 ppb from serum), and coefficients of variation <10% in quantitation. [Figure not available: see fulltext.

  11. Digestive enzymes in Rhinolophus euryale (Rhinolophidae, Chiroptera are active also during hibernation

    Directory of Open Access Journals (Sweden)

    Maxinová Edita

    2017-11-01

    Full Text Available During the winter, bats use hibernation as a means of surviving the period of low prey offer. However, the Mediterranean horseshoe bat (Rhinolophus euryale arouses from torpor quite frequently. Based on the actual climatic conditions, it can profit from occasional foraging oportunities, when they occur. We analysed faeces collected on four nights during the period from November 2012 to February 2013 from the Domica-Baradla cave system (Slovakia and Hungary. In mid-November, the largest proportion of faecal contents were from Lepidoptera. Later on, the proportion of non-consumptive mass in the faeces increased and prey remnants disappeared. We analysed the activity of digestive enzymes (amylase, chitobiase, endochitinase and glukosaminidase in faeces. The activity of these enzymes was detected in fresh faeces throughout the whole winter. The faecal activity of the chitinases was relatively stable during the monitored period, whilst the activity of amylase was highest during late November and December. Some level of active digestive enzymes during the winter could be an adaptation to occasional winter foraging.

  12. Mesoporous silica-encapsulated gold nanoparticles as artificial enzymes for self-activated cascade catalysis.

    Science.gov (United States)

    Lin, Youhui; Li, Zhenhua; Chen, Zhaowei; Ren, Jinsong; Qu, Xiaogang

    2013-04-01

    A significant challenge in chemistry is to create synthetic structures that mimic the complexity and function of natural systems. Here, a self-activated, enzyme-mimetic catalytic cascade has been realized by utilizing expanded mesoporous silica-encapsulated gold nanoparticles (EMSN-AuNPs) as both glucose oxidase- and peroxidase-like artificial enzymes. Specifically, EMSN helps the formation of a high degree of very small and well-dispersed AuNPs, which exhibit an extraordinarily stability and dual enzyme-like activities. Inspired by these unique and attractive properties, we further piece them together into a self-organized artificial cascade reaction, which is usually completed by the oxidase-peroxidase coupled enzyme system. Our finding may pave the way to use matrix as the structural component for the design and development of biomimetic catalysts and to apply enzyme mimics for realizing higher functions. Copyright © 2013 Elsevier Ltd. All rights reserved.

  13. Responses of absolute and specific enzyme activity to consecutive application of composted sewage sludge in a Fluventic Ustochrept.

    Directory of Open Access Journals (Sweden)

    Xiao Liu

    Full Text Available Composted sewage sludge (CS is considered a rich source of soil nutrients and significantly affects the physical, chemical, and biological characteristics of soil, but its effect on specific enzyme activity in soil is disregarded. The present experiment examined the absolute and specific enzyme activity of the enzymes involved in carbon, nitrogen, and phosphorus cycles, the diversity of soil microbial functions, and soil community composition in a Fluventic Ustochrept under a maize-wheat rotation system in North China during 2012-2015. Application of CS led to increase in MBC and in its ratio to both total organic carbon (TOC and microbial biomass nitrogen (MBN. Absolute enzyme activity, except that of phosphatase, increased in CS-treated soils, whereas specific activity of all the enzymes declined, especially at the highest dose of CS (45 t ha-1. The diversity of soil microbial community also increased in CS-treated soils, whereas its functional diversity declined at higher doses of CS owing to the lowered specific enzyme activity. These changes indicate that CS application induced the domination of microorganisms that are not metabolically active and those that use resources more efficiently, namely fungi. Redundancy analysis showed that fundamental alterations in soil enzyme activity depend on soil pH. Soil specific enzyme activity is affected more than absolute enzyme activity by changes in soil properties, especially soil microbial activity and composition of soil microflora (as judged by the following ratios: MBC/TOC, MBC/MBN, and TOC/LOC, that is labile organic carbon through the Pearson Correlation Coefficient. Specific enzyme activity is thus a more accurate parameter than absolute enzyme activity for monitoring the effect of adding CS on the activities and structure of soil microbial community.

  14. Impact of chlortetracycline and sulfapyridine antibiotics on soil enzyme activities

    Science.gov (United States)

    Molaei, Ali; Lakzian, Amir; Datta, Rahul; Haghnia, Gholamhosain; Astaraei, Alireza; Rasouli-Sadaghiani, MirHassan; Ceccherini, Maria T.

    2017-10-01

    Pharmaceutical antibiotics are frequently used in the livestock and poultry industries to control infectious diseases. Due to the lack of proper guidance for use, the majority of administrated antibiotics and their metabolites are excreted to the soil environment through urine and feces. In the present study, we used chlortetracycline and sulfapyridine antibiotics to screen out their effects on dehydrogenase, alkaline phosphatase and urease activity. Factorial experiments were conducted with different concentrations of antibiotic (0, 10, 25 and 100 mg kg-1 of soil) mixed with soil samples, and the enzyme activity was measured at intervals of 1, 4 and 21 days. The results show that the chlortetracycline and sulfapyridine antibiotics negatively affect the dehydrogenase activity, but the effect of sulfapyridine decreases with time of incubation. Indeed, sulfapyridine antibiotic significantly affect the alkaline phosphatase activity for the entire three-time interval, while chlortetracycline seems to inhibit its activity within 1 and 4 days of incubation. The effects of chlortetracycline and sulfapyridine antibiotics on urease activity appear similar, as they both significantly affect the urease activity on day 1 of incubation. The present study concludes that chlortetracycline and sulfapyridine antibiotics have harmful effects on soil microbes, with the extent of effects varying with the duration of incubation and the type of antibiotics used.

  15. Regulatory proteins (inhibitors or activators) affect estimates of Msub(r) of enzymes and receptors by radiation inactivation

    International Nuclear Information System (INIS)

    Potier, M.; Giroux, S.

    1985-01-01

    The radiation-inactivation method allows the determination of the Msub(r) of enzymes and receptors by monitoring the decay of biological activity as a function of absorbed dose. The presence of regulatory or effector proteins (inhibitors or activators) associated with an enzyme or receptor, or released in the preparation after tissue homogenization, may affect the decay of biological activity. How the activity is affected, however, will depend on the type of inhibition (competitive or non-competitive), the inhibitor or activator concentration, the dissociation constant of the enzyme-effector system, and the effector Msub(r) relative to that of the enzyme. Since little is known on how effector proteins influence radiation inactivation of enzymes and receptors, we have considered a theoretical model in an effort to provide a framework for the interpretation of experimentally obtained data. Our model predicts that competitive and non-competitive inhibitors of enzymes could be distinguished by analysing irradiated samples with various substrate concentrations. Inhibitors will decrease whereas activators will increase the apparent target size of enzymes or receptors. (author)

  16. Differences in forage-acquisition and fungal enzyme activity contribute to niche segregation in Panamanian leaf-cutting ants

    DEFF Research Database (Denmark)

    Kooij, Pepijn Wilhelmus; Liberti, Joanito; Giampoudakis, Konstantinos

    2014-01-01

    activities of twelve fungus garden decomposition enzymes, belonging to the amylases, cellulases, hemicellulases, pectinases and proteinases, and show that average enzyme activity per unit of fungal mass in Atta gardens is lower than in Acromyrmex gardens. Expression profiles of fungal enzymes in Atta also...... for decomposition enzymes....

  17. A function-based screen for seeking RubisCO active clones from metagenomes: novel enzymes influencing RubisCO activity.

    Science.gov (United States)

    Böhnke, Stefanie; Perner, Mirjam

    2015-03-01

    Ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) is a key enzyme of the Calvin cycle, which is responsible for most of Earth's primary production. Although research on RubisCO genes and enzymes in plants, cyanobacteria and bacteria has been ongoing for years, still little is understood about its regulation and activation in bacteria. Even more so, hardly any information exists about the function of metagenomic RubisCOs and the role of the enzymes encoded on the flanking DNA owing to the lack of available function-based screens for seeking active RubisCOs from the environment. Here we present the first solely activity-based approach for identifying RubisCO active fosmid clones from a metagenomic library. We constructed a metagenomic library from hydrothermal vent fluids and screened 1056 fosmid clones. Twelve clones exhibited RubisCO activity and the metagenomic fragments resembled genes from Thiomicrospira crunogena. One of these clones was further analyzed. It contained a 35.2 kb metagenomic insert carrying the RubisCO gene cluster and flanking DNA regions. Knockouts of twelve genes and two intergenic regions on this metagenomic fragment demonstrated that the RubisCO activity was significantly impaired and was attributed to deletions in genes encoding putative transcriptional regulators and those believed to be vital for RubisCO activation. Our new technique revealed a novel link between a poorly characterized gene and RubisCO activity. This screen opens the door to directly investigating RubisCO genes and respective enzymes from environmental samples.

  18. Assessment of digestive enzymes activity during the fry development of the endangered Caspian brown trout Salmo caspius.

    Science.gov (United States)

    Zamani, A; Hajimoradloo, A; Madani, R; Farhangi, M

    2009-09-01

    The study of digestive enzymes activity at Salmo caspius fry showed that enzymes were available at the moment of mouth opening on the first day post hatching (dph) and the activity of enzymes showed no significant difference from the hatching day 28 dph. An increased activity was seen between 32 and 43 dph and this activity was significantly higher than the activity during the first 28 days. In the primary stages after yolk sac resorption (43-58 dph), enzymes activity showed an increased profile, however none of them showed a significant difference between 43 and 58 dph.

  19. Alternate-Day High-Fat Diet Induces an Increase in Mitochondrial Enzyme Activities and Protein Content in Rat Skeletal Muscle.

    Science.gov (United States)

    Li, Xi; Higashida, Kazuhiko; Kawamura, Takuji; Higuchi, Mitsuru

    2016-04-06

    Long-term high-fat diet increases muscle mitochondrial enzyme activity and endurance performance. However, excessive calorie intake causes intra-abdominal fat accumulation and metabolic syndrome. The purpose of this study was to investigate the effect of an alternating day high-fat diet on muscle mitochondrial enzyme activities, protein content, and intra-abdominal fat mass in rats. Male Wistar rats were given a standard chow diet (CON), high-fat diet (HFD), or alternate-day high-fat diet (ALT) for 4 weeks. Rats in the ALT group were fed a high-fat diet and standard chow every other day for 4 weeks. After the dietary intervention, mitochondrial enzyme activities and protein content in skeletal muscle were measured. Although body weight did not differ among groups, the epididymal fat mass in the HFD group was higher than those of the CON and ALT groups. Citrate synthase and beta-hydroxyacyl CoA dehydrogenase activities in the plantaris muscle of rats in HFD and ALT were significantly higher than that in CON rats, whereas there was no difference between HFD and ALT groups. No significant difference was observed in muscle glycogen concentration or glucose transporter-4 protein content among the three groups. These results suggest that an alternate-day high-fat diet induces increases in mitochondrial enzyme activities and protein content in rat skeletal muscle without intra-abdominal fat accumulation.

  20. Life-history evolution and the microevolution of intermediary metabolism: activities of lipid-metabolizing enzymes in life-history morphs of a wing-dimorphic cricket.

    Science.gov (United States)

    Zera, Anthony J; Zhao, Zhangwu

    2003-03-01

    Although a considerable amount of information is available on the ecology, genetics, and physiology of life-history traits, much more limited data are available on the biochemical and genetic correlates of life-history variation within species. Specific activities of five enzymes of lipid biosynthesis and two enzymes of amino acid catabolism were compared among lines selected for flight-capable (LW[f]) versus flightless (SW) morphs of the cricket Gryllus firmus. These morphs, which exist in natural populations, differ genetically in ovarian growth (100-400% higher in SW) and aspects of flight capability including the size of wings and flight muscles, and the concentration of triglyceride flight fuel (40% greater in LW[f]). Consistently higher activity of each enzyme in LW(f) versus SW-selected lines, and strong co-segregation between morph and enzyme activity, demonstrated genetically based co-variance between wing morph and enzyme activity. Developmental profiles of enzyme activities strongly paralleled profiles of triglyceride accumulation during adulthood and previous measures of in vivo lipid biosynthesis. These data strongly imply that genetically based elevation in activities of lipogenic enzymes, and enzymes controlling the conversion of amino acids into lipids, is an important cause underlying the elevated accumulation of triglyceride in the LW(f) morph, a key biochemical component of the trade-off between elevated early fecundity and flight capability. Global changes in lipid and amino-acid metabolism appear to have resulted from microevolutionary alteration of regulators of metabolism. Finally, strong genotype x environment (diet) interactions were observed for most enzyme activities. Future progress in understanding the functional causes of life-history evolution requires a more detailed synthesis of the fields of life-history evolution and metabolic biochemistry. Wing polymorphism is a powerful experimental model in such integrative studies.

  1. Fungal enzymes in the attine ant symbiosis

    DEFF Research Database (Denmark)

    de Fine Licht, Henrik Hjarvard; Schiøtt, Morten; Boomsma, Jacobus Jan

    the more basal attine genera use substrates such as flowers, plant debris, small twigs, insect feces and insect carcasses. This diverse array of fungal substrates across the attine lineage implies that the symbiotic fungus needs different enzymes to break down the plant material that the ants provide...... or different efficiencies of enzyme function. Fungal enzymes that degrade plant cell walls may have functionally co-evolved with the ants in this scenario. We explore this hypothesis with direct measurements of enzyme activity in fungus gardens in 12 species across 8 genera spanning the entire phylogeny...... and diversity of life-styles within the attine clade. We find significant differences in enzyme activity between different genera and life-styles of the ants. How these findings relate to attine ant coevolution and crop optimization are discussed....

  2. Real-time monitoring of enzyme activity in a mesoporous silicon double layer

    Science.gov (United States)

    Orosco, Manuel M.; Pacholski, Claudia; Sailor, Michael J.

    2009-04-01

    The activity of certain proteolytic enzymes is often an indicator of disease states such as cancer, stroke and neurodegeneracy, so there is a need for rapid assays that can characterize the kinetics and substrate specificity of enzymatic reactions. Nanostructured membranes can efficiently separate biomolecules, but coupling a sensitive detection method to such a membrane remains difficult. Here, we demonstrate a single mesoporous nanoreactor that can isolate and quantify in real time the reaction products of proteases. The reactor consists of two layers of porous films electrochemically prepared from crystalline silicon. The upper layer, with large pore sizes (~100 nm in diameter), traps the protease and acts as the reactor. The lower layer, with smaller pore sizes (~6 nm), excludes the proteases and other large proteins and captures the reaction products. Infiltration of the digested fragments into the lower layer produces a measurable change in optical reflectivity, and this allows label-free quantification of enzyme kinetics in real time within a volume of ~5 nl.

  3. Carbohydrate-active enzymes in Trichoderma harzianum: a bioinformatic analysis bioprospecting for key enzymes for the biofuels industry.

    Science.gov (United States)

    Ferreira Filho, Jaire Alves; Horta, Maria Augusta Crivelente; Beloti, Lilian Luzia; Dos Santos, Clelton Aparecido; de Souza, Anete Pereira

    2017-10-12

    Trichoderma harzianum is used in biotechnology applications due to its ability to produce powerful enzymes for the conversion of lignocellulosic substrates into soluble sugars. Active enzymes involved in carbohydrate metabolism are defined as carbohydrate-active enzymes (CAZymes), and the most abundant family in the CAZy database is the glycoside hydrolases. The enzymes of this family play a fundamental role in the decomposition of plant biomass. In this study, the CAZymes of T. harzianum were identified and classified using bioinformatic approaches after which the expression profiles of all annotated CAZymes were assessed via RNA-Seq, and a phylogenetic analysis was performed. A total of 430 CAZymes (3.7% of the total proteins for this organism) were annotated in T. harzianum, including 259 glycoside hydrolases (GHs), 101 glycosyl transferases (GTs), 6 polysaccharide lyases (PLs), 22 carbohydrate esterases (CEs), 42 auxiliary activities (AAs) and 46 carbohydrate-binding modules (CBMs). Among the identified T. harzianum CAZymes, 47% were predicted to harbor a signal peptide sequence and were therefore classified as secreted proteins. The GH families were the CAZyme class with the greatest number of expressed genes, including GH18 (23 genes), GH3 (17 genes), GH16 (16 genes), GH2 (13 genes) and GH5 (12 genes). A phylogenetic analysis of the proteins in the AA9/GH61, CE5 and GH55 families showed high functional variation among the proteins. Identifying the main proteins used by T. harzianum for biomass degradation can ensure new advances in the biofuel production field. Herein, we annotated and characterized the expression levels of all of the CAZymes from T. harzianum, which may contribute to future studies focusing on the functional and structural characterization of the identified proteins.

  4. Viral Pseudo Enzymes Activate RIG-I via Deamidation to Evade Cytokine Production

    Science.gov (United States)

    He, Shanping; Zhao, Jun; Song, Shanshan; He, Xiaojing; Minassian, Arlet; Zhou, Yu; Zhang, Junjie; Brulois, Kevin; Wang, Yuqi; Cabo, Jackson; Zandi, Ebrahim; Liang, Chengyu; Jung, Jae U; Zhang, Xuewu; Feng, Pinghui

    2015-01-01

    SUMMARY RIG-I is a pattern recognition receptor that senses viral RNA and is crucial for host innate immune defense. Here we describe a mechanism of RIG-I activation through amidotransferase-mediated deamidation. We show that viral homologues of phosphoribosylformyglycinamide synthase (PFAS), although lacking intrinsic enzyme activity, recruit cellular PFAS to deamidate and activate RIG-I. Accordingly, depletion and biochemical inhibition of PFAS impair RIG-I deamidation and concomitant activation. Purified PFAS and viral homologue thereof deamidate RIG-I in vitro. Ultimately, herpesvirus hijacks activated RIG-I to avoid antiviral cytokine production; loss of RIG-I or inhibition of RIG-I deamidation results in elevated cytokine production. Together, these findings demonstrate a surprising mechanism of RIG-I activation that is mediated by an enzyme. PMID:25752576

  5. Induction of antioxidant enzyme activity and lipid peroxidation level in ion-beam-bombarded rice seeds

    Science.gov (United States)

    Semsang, Nuananong; Yu, LiangDeng

    2013-07-01

    Low-energy ion beam bombardment has been used to mutate a wide variety of plant species. To explore the indirect effects of low-energy ion beam on biological damage due to the free radical production in plant cells, the increase in antioxidant enzyme activities and lipid peroxidation level was investigated in ion-bombarded rice seeds. Local rice seeds were bombarded with nitrogen or argon ion beams at energies of 29-60 keV and ion fluences of 1 × 1016 ions cm-2. The activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST) and lipid peroxidation level were assayed in the germinated rice seeds after ion bombardment. The results showed most of the enzyme activities and lipid peroxidation levels in both the argon and nitrogen bombarded samples were higher than those in the natural control. N-ion bombardment could induce higher levels of antioxidant enzyme activities in the rice samples than the Ar-ion bombardment. Additional effects due to the vacuum condition were found to affect activities of some antioxidant enzymes and lipid peroxidation level. This study demonstrates that ion beam bombardment and vacuum condition could induce the antioxidant enzyme activity and lipid peroxidation level which might be due to free radical production in the bombarded rice seeds.

  6. Hydrolytic and ligninolytic enzyme activities in the Pb contaminated soil inoculated with litter-decomposing fungi.

    Science.gov (United States)

    Kähkönen, Mika A; Lankinen, Pauliina; Hatakka, Annele

    2008-06-01

    The impact of Pb contamination was tested to five hydrolytic (beta-glucosidase, beta-xylosidase, beta-cellobiosidase, alpha-glucosidase and sulphatase) and two ligninolytic (manganese peroxidase, MnP and laccase) enzyme activities in the humus layer in the forest soil. The ability of eight selected litter-degrading fungi to grow and produce extracellular enzymes in the heavily Pb (40 g Pb of kg ww soil(-1)) contaminated and non-contaminated soil in the non-sterile conditions was also studied. The Pb content in the test soil was close to that of the shooting range at Hälvälä (37 g Pb of kg ww soil(-1)) in Southern Finland. The fungi were Agaricus bisporus, Agrocybe praecox, Gymnopus peronatus, Gymnopilus sapineus, Mycena galericulata, Gymnopilus luteofolius, Stropharia aeruginosa and Stropharia rugosoannulata. The Pb contamination (40 g Pb of kg ww soil(-1)) was deleterious to all five studied hydrolytic enzyme activities after five weeks of incubation. All five hydrolytic enzyme activities were significantly higher in the soil than in the extract of the soil indicating that a considerable part of enzymes were particle bound in the soils. Hydrolytic enzyme activities were higher in the non-contaminated soil than in the Pb contaminated soil. Fungal inocula increased the hydrolytic enzyme activities beta-cellobiosidase and beta-glucosidase in non-contaminated soils. All five hydrolytic enzyme activities were similar with fungi and without fungi in the Pb contaminated soil. This was in line that Pb contamination (40 g Pb of kg ww soil(-1)) depressed the growth of all fungi compared to those grown without Pb in the soil. Laccase and MnP activities were low in both Pb contaminated and non-contaminated soil cultures. MnP activities were higher in soil cultures containing Pb than without Pb. Our results showed that Pb in the shooting ranges decreased fungal growth and microbial functioning in the soil.

  7. Quantitative enzyme activity determination with zeptomole sensitivity by microfluidic gradient-gel zymography.

    Science.gov (United States)

    Hughes, Alex J; Herr, Amy E

    2010-05-01

    We describe a sensitive zymography technique that utilizes an automated microfluidic platform to report enzyme molecular weight, amount, and activity (including k(cat) and K(m)) from dilute protein mixtures. Calf intestinal alkaline phosphatase (CIP) is examined in detail as a model enzyme system, and the method is also demonstrated for horseradish peroxidase (HRP). The 40 min assay has a detection limit of 5 zmol ( approximately 3 000 molecules) of CIP. Two-step pore-limit electrophoresis with enzyme assay (PLENZ) is conducted in a single, straight microchannel housing a polyacrylamide (PA) pore-size gradient gel. In the first step, pore limit electrophoresis (PLE) sizes and pseudoimmobilizes resolved proteins. In the second step, electrophoresis transports both charged and neutral substrates into the PLE channel to the entrapped proteins. Arrival of substrate at the resolved enzyme band generates fluorescent product that reveals enzyme molecular weight against a fluorescent protein ladder. Additionally, the PLENZ zymography assay reports the kinetic properties of CIP in a fully quantitative manner. In contrast to covalent enzyme immobilization, physical pseudoimmobilization of CIP in the PA gel does not significantly reduce its maximum substrate turnover rate. However, an 11-fold increase in the Michaelis constant (over the free solution value) is observed, consistent with diffusional limitations on substrate access to the enzyme active site. PLENZ offers a robust platform for rapid and multiplexed functional analysis of heterogeneous protein samples in drug discovery, clinical diagnostics, and biocatalyst engineering.

  8. High-Throughput Analysis of Enzyme Activities

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Guoxin [Iowa State Univ., Ames, IA (United States)

    2007-01-01

    High-throughput screening (HTS) techniques have been applied to many research fields nowadays. Robot microarray printing technique and automation microtiter handling technique allows HTS performing in both heterogeneous and homogeneous formats, with minimal sample required for each assay element. In this dissertation, new HTS techniques for enzyme activity analysis were developed. First, patterns of immobilized enzyme on nylon screen were detected by multiplexed capillary system. The imaging resolution is limited by the outer diameter of the capillaries. In order to get finer images, capillaries with smaller outer diameters can be used to form the imaging probe. Application of capillary electrophoresis allows separation of the product from the substrate in the reaction mixture, so that the product doesn't have to have different optical properties with the substrate. UV absorption detection allows almost universal detection for organic molecules. Thus, no modifications of either the substrate or the product molecules are necessary. This technique has the potential to be used in screening of local distribution variations of specific bio-molecules in a tissue or in screening of multiple immobilized catalysts. Another high-throughput screening technique is developed by directly monitoring the light intensity of the immobilized-catalyst surface using a scientific charge-coupled device (CCD). Briefly, the surface of enzyme microarray is focused onto a scientific CCD using an objective lens. By carefully choosing the detection wavelength, generation of product on an enzyme spot can be seen by the CCD. Analyzing the light intensity change over time on an enzyme spot can give information of reaction rate. The same microarray can be used for many times. Thus, high-throughput kinetic studies of hundreds of catalytic reactions are made possible. At last, we studied the fluorescence emission spectra of ADP and obtained the detection limits for ADP under three different

  9. Effect of Cereal Type and Enzyme Addition on Performance, Pancreatic Enzyme Activity, Intestinal Microflora and Gut Morphology of Broilers

    Directory of Open Access Journals (Sweden)

    Kalantar M

    2016-06-01

    Full Text Available The effects of grain and carbohydrase enzyme supplementation were investigated on digestive physiology of chickens. A total of 625 one-day-old chicks (Ross 308 were randomly assigned to five treatments in a completely randomized design. Treatments included two different types of grains (wheat, and barley with or without a multi-carbohydrase supplement. A corn-based diet was also considered to serve as a control. Feeding barley-based diet with multi-carbohydrase led to higher feed intake (P < 0.01 than those fed corn- and wheat-based diets. Birds fed on barley and wheat diets had lower weight gain despite a higher feed conversion ratio (P < 0.01. Total count and number of different type of bacteria including Gram-negative, E. coli, and Clostridia increased after feeding wheat and barley but the number of Lactobacilli and Bifidobacteria decreased (P < 0.01. Feeding barley and wheat diets reduced villus height in different parts of the small intestine when compared to those fed on a corn diet. However, enzyme supplementation of barley and wheat diets improved weight gain and feed conversion ratio and resulted in reduced number of E. coli and Clostridia and increased number of Lactobacilli and Bifidobacteria, and also restored the negative effects on intestinal villi height (P < 0.01. The activities of pancreatic α-amylase and lipase were (P < 0.01 increased in chickens fed wheat and barley diets when compared to the control fed on a corn diet. Enzyme supplementation reduced the activities of pancreatic α-amylase and lipase (P < 0.01. In conclusion, various dietary non-starch polysaccharides without enzyme supplementation have an adverse effect on digesta viscosity, ileal microflora, villi morphology, and pancreatic enzyme activity.

  10. Increased activities of mitochondrial enzymes in white adipose tissue in trained rats

    DEFF Research Database (Denmark)

    Stallknecht, B; Vinten, J; Ploug, T

    1991-01-01

    of 8-12 rats were swim trained for 10 wk or served as either sedentary, sham swim-trained, or cold-stressed controls. White adipose tissue was removed, and the activities of the respiratory chain enzyme cytochrome-c oxidase (CCO) and of the enzyme malate dehydrogenase (MDH), which participates...... 0.05). In female rats the CCO activity expressed per milligram protein was increased 4.5-fold in the trained compared with the sedentary control rats (P less than 0.01). Neither cold stress nor sham swim training increased CCO or MDH activities in white adipose tissue (P greater than 0...

  11. Properties of latent and thiol-activated rat hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase and regulation of enzyme activity.

    Science.gov (United States)

    Dotan, I; Shechter, I

    1983-10-15

    The effect of the thiols glutathione (GSH), dithiothreitol (DTT), and dithioerythritol (DTE) on the conversion of an inactive, latent form (El) of rat liver 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, EC 1.1.1.34) to a catalyticaly active form (Ea) is examined. Latent hepatic microsomal HMG-CoA reductase is activated to a similar degree of activation by DTT and DTE and to a lower extent by GSH. All three thiols affect both Km and Vmax values of the enzyme toward HMG-CoA and NADPH. Studies of the effect of DTT on the affinity binding of HMG-CoA reductase to agarose-hexane-HMG-CoA (AG-HMG-CoA) resin shows that thiols are necessary for the binding of the enzyme to the resin. Removal of DTT from AG-HMG-CoA-bound soluble Ea (active enzyme) does not cause dissociation of the enzyme from the resin at low salt concentrations. Substitution of DTT by NADPH does not promote binding of soluble El (latent enzyme) to AG-HMG-CoA. The enzymatic activity of Ea in the presence of DTT and GSH indicates that these thiols compete for the same binding site on the enzyme. Diethylene glycol disulfide (ESSE) and glutathione disulfide (GSSG) inhibit the activity of Ea. ESSE is more effective for the inhibition of Ea than GSSG, causing a higher degree of maximal inhibition and affecting the enzymatic activity at lower concentrations. A method is described for the rapid conversion of soluble purified Ea to El using gel-filtration chromatography on Bio-Gel P-4 columns. These combined results point to the importance of the thiol/disulfide ratio for the modulation of hepatic HMG-CoA reductase activity.

  12. Virtual Biochemistry – pH effect on enzyme activity

    Directory of Open Access Journals (Sweden)

    D.N. Heidrich

    2011-04-01

    Full Text Available Protocols of laboratory experiments, followed by teacher's explanation, not always clearly translate to the student the dynamics to beadopted for the implementation of the proposed practice. One of these cases is related to the study of the effect of pH on enzyme activity. For better help the understanding of the technical procedure, a hypermedia was built based on a protocol adopted at the Department of Biochemistry, UFSC. The hypermedia shows how theeffect of variations in pH can be observed  in vitro. Taking as example salivary amylase and the consumption of starch (substrate by means of iodine staining, a set of pH buffers was tested to identify the best pH for this enzyme  activity. This hypermedia as introductory tool for such practice was tested on aNutrition course classroom. Students agree that the hypermedia provided a better understanding of the proposed activities. Teachers also notice a smallerreagents consumption and reduction of the time spent by the students in the achievement of the experiment.

  13. Protein immobilization on epoxy-activated thin polymer films: effect of surface wettability and enzyme loading.

    Science.gov (United States)

    Chen, Bo; Pernodet, Nadine; Rafailovich, Miriam H; Bakhtina, Asya; Gross, Richard A

    2008-12-02

    A series of epoxy-activated polymer films composed of poly(glycidyl methacrylate/butyl methacrylate/hydroxyethyl methacrylate) were prepared. Variation in comonomer composition allowed exploration of relationships between surface wettability and Candida antartica lipase B (CALB) binding to surfaces. By changing solvents and polymer concentrations, suitable conditions were developed for preparation by spin-coating of uniform thin films. Film roughness determined by AFM after incubation in PBS buffer for 2 days was less than 1 nm. The occurrence of single CALB molecules and CALB aggregates at surfaces was determined by AFM imaging and measurements of volume. Absolute numbers of protein monomers and multimers at surfaces were used to determine values of CALB specific activity. Increased film wettability, as the water contact angle of films increased from 420 to 550, resulted in a decreased total number of immobilized CALB molecules. With further increases in the water contact angle of films from 55 degrees to 63 degrees, there was an increased tendency of CALB molecules to form aggregates on surfaces. On all flat surfaces, two height populations, differing by more than 30%, were observed from height distribution curves. They are attributed to changes in protein conformation and/or orientation caused by protein-surface and protein-protein interactions. The fraction of molecules in these populations changed as a function of film water contact angle. The enzyme activity of immobilized films was determined by measuring CALB-catalyzed hydrolysis of p-nitrophenyl butyrate. Total enzyme specific activity decreased by decreasing film hydrophobicity.

  14. Predicting novel substrates for enzymes with minimal experimental effort with active learning.

    Science.gov (United States)

    Pertusi, Dante A; Moura, Matthew E; Jeffryes, James G; Prabhu, Siddhant; Walters Biggs, Bradley; Tyo, Keith E J

    2017-11-01

    Enzymatic substrate promiscuity is more ubiquitous than previously thought, with significant consequences for understanding metabolism and its application to biocatalysis. This realization has given rise to the need for efficient characterization of enzyme promiscuity. Enzyme promiscuity is currently characterized with a limited number of human-selected compounds that may not be representative of the enzyme's versatility. While testing large numbers of compounds may be impractical, computational approaches can exploit existing data to determine the most informative substrates to test next, thereby more thoroughly exploring an enzyme's versatility. To demonstrate this, we used existing studies and tested compounds for four different enzymes, developed support vector machine (SVM) models using these datasets, and selected additional compounds for experiments using an active learning approach. SVMs trained on a chemically diverse set of compounds were discovered to achieve maximum accuracies of ~80% using ~33% fewer compounds than datasets based on all compounds tested in existing studies. Active learning-selected compounds for testing resolved apparent conflicts in the existing training data, while adding diversity to the dataset. The application of these algorithms to wide arrays of metabolic enzymes would result in a library of SVMs that can predict high-probability promiscuous enzymatic reactions and could prove a valuable resource for the design of novel metabolic pathways. Copyright © 2017 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

  15. Predicting novel substrates for enzymes with minimal experimental effort with active learning

    Energy Technology Data Exchange (ETDEWEB)

    Pertusi, Dante A.; Moura, Matthew E.; Jeffryes, James G.; Prabhu, Siddhant; Walters Biggs, Bradley; Tyo, Keith E. J.

    2017-11-01

    Enzymatic substrate promiscuity is more ubiquitous than previously thought, with significant consequences for understanding metabolism and its application to biocatalysis. This realization has given rise to the need for efficient characterization of enzyme promiscuity. Enzyme promiscuity is currently characterized with a limited number of human-selected compounds that may not be representative of the enzyme's versatility. While testing large numbers of compounds may be impractical, computational approaches can exploit existing data to determine the most informative substrates to test next, thereby more thoroughly exploring an enzyme's versatility. To demonstrate this, we used existing studies and tested compounds for four different enzymes, developed support vector machine (SVM) models using these datasets, and selected additional compounds for experiments using an active learning approach. SVMs trained on a chemically diverse set of compounds were discovered to achieve maximum accuracies of similar to 80% using similar to 33% fewer compounds than datasets based on all compounds tested in existing studies. Active learning-selected compounds for testing resolved apparent conflicts in the existing training data, while adding diversity to the dataset. The application of these algorithms to wide arrays of metabolic enzymes would result in a library of SVMs that can predict high-probability promiscuous enzymatic reactions and could prove a valuable resource for the design of novel metabolic pathways.

  16. Induction of antioxidant enzyme activity and lipid peroxidation level in ion-beam-bombarded rice seeds

    Energy Technology Data Exchange (ETDEWEB)

    Semsang, Nuananong, E-mail: nsemsang@gmail.com [Molecular Biology Laboratory, Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Yu, LiangDeng [Plasma and Beam Physics Research Facility, Department of Physics and Materials Science, Faculty of Science, Chiang Mai University, Chiang Mai 50200 (Thailand); Thailand Center of Excellence in Physics, Commission on Higher Education, 328 Si Ayutthaya Road, Bangkok 10400 (Thailand)

    2013-07-15

    Highlights: ► Ion beam bombarded rice seeds in vacuum. ► Studied seed survival from the ion bombardment. ► Determined various antioxidant enzyme activities and lipid peroxidation level. ► Discussed vacuum, ion species and ion energy effects. ► Attributed the changes to free radical formation due to ion bombardment. -- Abstract: Low-energy ion beam bombardment has been used to mutate a wide variety of plant species. To explore the indirect effects of low-energy ion beam on biological damage due to the free radical production in plant cells, the increase in antioxidant enzyme activities and lipid peroxidation level was investigated in ion-bombarded rice seeds. Local rice seeds were bombarded with nitrogen or argon ion beams at energies of 29–60 keV and ion fluences of 1 × 10{sup 16} ions cm{sup −2}. The activities of the antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR), glutathione S-transferase (GST) and lipid peroxidation level were assayed in the germinated rice seeds after ion bombardment. The results showed most of the enzyme activities and lipid peroxidation levels in both the argon and nitrogen bombarded samples were higher than those in the natural control. N-ion bombardment could induce higher levels of antioxidant enzyme activities in the rice samples than the Ar-ion bombardment. Additional effects due to the vacuum condition were found to affect activities of some antioxidant enzymes and lipid peroxidation level. This study demonstrates that ion beam bombardment and vacuum condition could induce the antioxidant enzyme activity and lipid peroxidation level which might be due to free radical production in the bombarded rice seeds.

  17. Metrological aspects of enzyme production

    International Nuclear Information System (INIS)

    Kerber, T M; Pereira-Meirelles, F V; Dellamora-Ortiz, G M

    2010-01-01

    Enzymes are frequently used in biotechnology to carry out specific biological reactions, either in industrial processes or for the production of bioproducts and drugs. Microbial lipases are an important group of biotechnologically valuable enzymes that present widely diversified applications. Lipase production by microorganisms is described in several published papers; however, none of them refer to metrological evaluation and the estimation of the uncertainty in measurement. Moreover, few of them refer to process optimization through experimental design. The objectives of this work were to enhance lipase production in shaken-flasks with Yarrowia lipolytica cells employing experimental design and to evaluate the uncertainty in measurement of lipase activity. The highest lipolytic activity obtained was about three- and fivefold higher than the reported activities of CRMs BCR-693 and BCR-694, respectively. Lipase production by Y. lipolytica cells aiming the classification as certified reference material is recommended after further purification and stability studies

  18. Diverse effects of arsenic on selected enzyme activities in soil-plant-microbe interactions.

    Science.gov (United States)

    Lyubun, Yelena V; Pleshakova, Ekaterina V; Mkandawire, Martin; Turkovskaya, Olga V

    2013-11-15

    Under the influence of pollutants, enzyme activities in plant-microbe-soil systems undergo changes of great importance in predicting soil-plant-microbe interactions, regulation of metal and nutrient uptake, and, ultimately, improvement of soil health and fertility. We evaluated the influence of As on soil enzyme activities and the effectiveness of five field crops for As phytoextraction. The initial As concentration in soil was 50mg As kg(-1) soil; planted clean soil, unplanted polluted soil, and unplanted clean soil served as controls. After 10 weeks, the growth of the plants elevated soil dehydrogenase activity relative to polluted but unplanted control soils by 2.4- and 2.5-fold for sorghum and sunflower (respectively), by 3-fold for ryegrass and sudangrass, and by 5.2-fold for spring rape. Soil peroxidase activity increased by 33% with ryegrass and rape, while soil phosphatase activity was directly correlated with residual As (correlation coefficient R(2)=0.7045). We conclude that soil enzyme activities should be taken into account when selecting plants for phytoremediation. Copyright © 2013 Elsevier B.V. All rights reserved.

  19. Cyanide does more to inhibit heme enzymes, than merely serving as an active-site ligand

    Energy Technology Data Exchange (ETDEWEB)

    Parashar, Abhinav [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Venkatachalam, Avanthika [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India); Gideon, Daniel Andrew [Center for Biomedical Research, VIT University, Vellore, Tamil Nadu, 632014 India (India); Manoj, Kelath Murali, E-mail: satyamjayatu@yahoo.com [REDOx Lab, PSG Institute of Advanced Studies, Avinashi Road, Peelamedu, Coimbatore, Tamil Nadu, 641004 (India)

    2014-12-12

    Highlights: • Cyanide (CN) is a well-studied toxic principle, known to inhibit heme-enzymes. • Inhibition is supposed to result from CN binding at the active site as a ligand. • Diverse heme enzymes’ CN inhibition profiles challenge prevailing mechanism. • Poor binding efficiency of CN at low enzyme concentrations and ligand pressures. • CN-based diffusible radicals cause ‘non-productive electron transfers’ (inhibition). - Abstract: The toxicity of cyanide is hitherto attributed to its ability to bind to heme proteins’ active site and thereby inhibit their activity. It is shown herein that the long-held interpretation is inadequate to explain several observations in heme-enzyme reaction systems. Generation of cyanide-based diffusible radicals in heme-enzyme reaction milieu could shunt electron transfers (by non-active site processes), and thus be detrimental to the efficiency of oxidative outcomes.

  20. Impact on enzyme activity as a new quality index of wastewater.

    Science.gov (United States)

    Balestri, Francesco; Moschini, Roberta; Cappiello, Mario; Del-Corso, Antonella; Mura, Umberto

    2013-03-15

    The aim of this study was to define a new indicator for the quality of wastewaters that are released into the environment. A quality index is proposed for wastewater samples in terms of the inertness of wastewater samples toward enzyme activity. This involves taking advantage of the sensitivity of enzymes to pollutants that may be present in the waste samples. The effect of wastewater samples on the rate of a number of different enzyme-catalyzed reactions was measured, and the results for all the selected enzymes were analyzed in an integrated fashion (multi-enzymatic sensor). This approach enabled us to define an overall quality index, the "Impact on Enzyme Function" (IEF-index), which is composed of three indicators: i) the Synoptic parameter, related to the average effect of the waste sample on each component of the enzymatic sensor; ii) the Peak parameter, related to the maximum effect observed among all the effects exerted by the sample on the sensor components; and, iii) the Interference parameter, related to the number of sensor components that are affected less than a fixed threshold value. A number of water based samples including public potable tap water, fluids from urban sewage systems, wastewater disposal from leather, paper and dye industries were analyzed and the IEF-index was then determined. Although the IEF-index cannot discriminate between different types of wastewater samples, it could be a useful parameter in monitoring the improvement of the quality of a specific sample. However, by analyzing an adequate number of waste samples of the same type, even from different local contexts, the profile of the impact of each component of the multi-enzymatic sensor could be typical for specific types of waste. The IEF-index is proposed as a supplementary qualification score for wastewaters, in addition to the certification of the waste's conformity to legal requirements. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Human beta-glucuronidase. Measurement of its activity in gallbladder bile devoid of intrinsic interference.

    Science.gov (United States)

    Ho, Y C; Ho, K J

    1988-04-01

    Our purpose is to develop a standard method for preparing the bile for beta-glucuronidase determination by removal of bile acids and conjugated bilirubin which interfere with its activity. The bile acids and conjugated bilirubin in their purified solutions and in the diluted gallbladder biles could be extracted completely with cholestyramine in powder form or tetrahexylammonium chloride (THAC) in chloroform or ethyl acetate. The enzyme was, however, partially precipitated with cholestyramine and denatured by chloroform but not by ethyl acetate. A standard procedure, therefore, includes extraction of the diluted gallbladder bile with THAC in ethyl acetate, followed by determination of the maximal velocity (Vmax) of the enzyme by a kinetic method employing phenolphthalein glucuronide as the substrate. The average Vmax of beta-glucuronidase in the 20 normal gallbladder biles was 165 +/- 86 nmol/min/ml (mean +/- SD), a 23.5-fold increase over the activity before extraction. The measured activity represented the true activity of the enzyme in the bile for recovery of activity of the enzyme added to the bile was practically complete.

  2. Studies about the enzyme activity changes of the skin caused by X-rays. Pt. 2

    International Nuclear Information System (INIS)

    Bathori, E.; Soltesz, L.

    1975-01-01

    After application of soft X-rays with 100, 500 and 1,000 R, the activity change of the acid and alcaline phosphatase in the skin of mice has been studied. The studies happened immediatly after the irradiation and at the 1st, 2nd, 4th, 8th and 16th day. The measurement of the enzyme activity washable out of the skin and remaining in the skin after the washing and the total activity of the skin have been taken up. With the acid phosphatase it would be determined that the quantity washable out of the skin is unusually low; the activity values of the wash solution increase at the 2nd day after the irradiation in dependence on the applicated dose; at the moment 0 after an irradiation with 1,000 R, a significative activity increase in the wash solution appears; the homogenate activity at the 2nd day only gets a significative activity increase after the irradiation with 500 R. The alcaline phosphatase was measurable in the wash solution at no moment of the measurement. However, the alcaline phosphatase has been decreased in the homogenate with few exceptions. (orig.) [de

  3. A Sensitive and Robust Enzyme Kinetic Experiment Using Microplates and Fluorogenic Ester Substrates

    Science.gov (United States)

    Johnson, R. Jeremy; Hoops, Geoffrey C.; Savas, Christopher J.; Kartje, Zachary; Lavis, Luke D.

    2015-01-01

    Enzyme kinetics measurements are a standard component of undergraduate biochemistry laboratories. The combination of serine hydrolases and fluorogenic enzyme substrates provides a rapid, sensitive, and general method for measuring enzyme kinetics in an undergraduate biochemistry laboratory. In this method, the kinetic activity of multiple protein…

  4. DNA-Based Sensor for Real-Time Measurement of the Enzymatic Activity of Human Topoisomerase I

    DEFF Research Database (Denmark)

    Marcussen, Lærke Bay; Jepsen, Morten Leth; Kristoffersen, Emil Laust

    2013-01-01

    Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I....... The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor....... The cytotoxic effect of camptothecins correlates directly with the intracellular topoisomerase I activity. We therefore envision that the presented sensor may find use for the prediction of cellular drug response. Moreover, inhibition of topoisomerase I by camptothecin is readily detectable using the presented...

  5. Angiotensin-I Converting Enzyme (ACE Inhibitory and Anti-Oxidant Activities of Sea Cucumber (Actinopyga lecanora Hydrolysates

    Directory of Open Access Journals (Sweden)

    Raheleh Ghanbari

    2015-12-01

    Full Text Available In recent years, food protein-derived hydrolysates have received considerable attention because of their numerous health benefits. Amongst the hydrolysates, those with anti-hypertensive and anti-oxidative activities are receiving special attention as both activities can play significant roles in preventing cardiovascular diseases. The present study investigated the angiotensin-I converting enzyme (ACE inhibitory and anti-oxidative activities of Actinopyga lecanora (A. lecanora hydrolysates, which had been prepared by alcalase, papain, bromelain, flavourzyme, pepsin, and trypsin under their optimum conditions. The alcalase hydrolysate showed the highest ACE inhibitory activity (69.8% after 8 h of hydrolysis while the highest anti-oxidative activities measured by 2,2-diphenyl 1-1-picrylhydrazyl radical scavenging (DPPH (56.00% and ferrous ion-chelating (FIC (59.00% methods were exhibited after 24 h and 8 h of hydrolysis, respectively. The ACE-inhibitory and anti-oxidative activities displayed dose-dependent trends, and increased with increasing protein hydrolysate concentrations. Moreover, strong positive correlations between angiotensin-I converting enzyme (ACE inhibitory and anti-oxidative activities were also observed. This study indicates that A. lecanora hydrolysate can be exploited as a source of functional food owing to its anti-oxidant as well as anti-hypertension functions.

  6. Radiation effects on the parotid gland of mammals. Pt. 3. Behaviour of enzyme activity after irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Tomassi, I; Balzi, M; Cremonini, D; Becciolini, A; Giannardi, G [Florence Univ. (Italy). Istituto di Radiologia; Pelu, G [I.N.R.C.A., Florence (Italy). Inst. of Radiology

    1979-08-01

    Modifications of some enzyme activities in parotid tissue homogenates have been studied in animals which were also examined for morphological changes and for plasma and parotid amylase activity. Results from irradiated animals show a certain increase in maltase activity. Alkaline phosphatase and LAP show no significant variations; a similar behaviour is shown by lysosomal enzymes and protein content. A different pattern was seen by comparing the curves of these enzymes with those of the same activity in the small intestine. This result appears to be due to the different radiosensitivity of these tissues.

  7. Prostaglandin levels and lysosomal enzyme activities in irradiated rats

    International Nuclear Information System (INIS)

    Trocha, P.J.; Catravas, G.N.

    1980-01-01

    Whole-body irradiation of rats results in the release of hydrolases from lysosomes, an increase in lysosomal enzyme activities, and changes in the prostaglandin levels in spleen and liver tissues. A transient increase in the concentration of prostaglandins E and F and leakage of lysosomal hydrolases occurred in both spleen and liver tissues 3-6 hours after the animals were irradiated. Maximal values for hydrolase activities, prostaglandin E and F content, and release of lysosomal enzymes were found 4 days postirradiation in rat spleens whereas in the liver only slight increases were observed at this time period for prostaglandin F levels. On day 7 there was a final rise in the spleen's prostaglandin E and F concentrations and leakage of hydrolases from the lysosomes before returning to near normal values on day 11. The prostaglandin F concentration in liver was also slightly elevated on the 7th day after irradiation and then decreased to control levels. (author)

  8. Contrasting effects of ammonium and nitrate additions on the biomass of soil microbial communities and enzyme activities in subtropical China

    Directory of Open Access Journals (Sweden)

    C. Zhang

    2017-10-01

    Full Text Available The nitrate to ammonium ratios in nitrogen (N compounds in wet atmospheric deposits have increased over the recent past, which is a cause for some concern as the individual effects of nitrate and ammonium deposition on the biomass of different soil microbial communities and enzyme activities are still poorly defined. We established a field experiment and applied ammonium (NH4Cl and nitrate (NaNO3 at monthly intervals over a period of 4 years. We collected soil samples from the ammonium and nitrate treatments and control plots in three different seasons, namely spring, summer, and fall, to evaluate the how the biomass of different soil microbial communities and enzyme activities responded to the ammonium (NH4Cl and nitrate (NaNO3 applications. Our results showed that the total contents of phospholipid fatty acids (PLFAs decreased by 24 and 11 % in the ammonium and nitrate treatments, respectively. The inhibitory effects of ammonium on Gram-positive bacteria (G+ and bacteria, fungi, actinomycetes, and arbuscular mycorrhizal fungi (AMF PLFA contents ranged from 14 to 40 % across the three seasons. We also observed that the absolute activities of C, N, and P hydrolyses and oxidases were inhibited by ammonium and nitrate, but that nitrate had stronger inhibitory effects on the activities of acid phosphatase (AP than ammonium. The activities of N-acquisition specific enzymes (enzyme activities normalized by total PLFA contents were about 21 and 43 % lower in the ammonium and nitrate treatments than in the control, respectively. However, the activities of P-acquisition specific enzymes were about 19 % higher in the ammonium treatment than in the control. Using redundancy analysis (RDA, we found that the measured C, N, and P hydrolysis and polyphenol oxidase (PPO activities were positively correlated with the soil pH and ammonium contents, but were negatively correlated with the nitrate contents. The PLFA biomarker contents were positively

  9. Contrasting effects of ammonium and nitrate additions on the biomass of soil microbial communities and enzyme activities in subtropical China

    Science.gov (United States)

    Zhang, Chuang; Zhang, Xin-Yu; Zou, Hong-Tao; Kou, Liang; Yang, Yang; Wen, Xue-Fa; Li, Sheng-Gong; Wang, Hui-Min; Sun, Xiao-Min

    2017-10-01

    The nitrate to ammonium ratios in nitrogen (N) compounds in wet atmospheric deposits have increased over the recent past, which is a cause for some concern as the individual effects of nitrate and ammonium deposition on the biomass of different soil microbial communities and enzyme activities are still poorly defined. We established a field experiment and applied ammonium (NH4Cl) and nitrate (NaNO3) at monthly intervals over a period of 4 years. We collected soil samples from the ammonium and nitrate treatments and control plots in three different seasons, namely spring, summer, and fall, to evaluate the how the biomass of different soil microbial communities and enzyme activities responded to the ammonium (NH4Cl) and nitrate (NaNO3) applications. Our results showed that the total contents of phospholipid fatty acids (PLFAs) decreased by 24 and 11 % in the ammonium and nitrate treatments, respectively. The inhibitory effects of ammonium on Gram-positive bacteria (G+) and bacteria, fungi, actinomycetes, and arbuscular mycorrhizal fungi (AMF) PLFA contents ranged from 14 to 40 % across the three seasons. We also observed that the absolute activities of C, N, and P hydrolyses and oxidases were inhibited by ammonium and nitrate, but that nitrate had stronger inhibitory effects on the activities of acid phosphatase (AP) than ammonium. The activities of N-acquisition specific enzymes (enzyme activities normalized by total PLFA contents) were about 21 and 43 % lower in the ammonium and nitrate treatments than in the control, respectively. However, the activities of P-acquisition specific enzymes were about 19 % higher in the ammonium treatment than in the control. Using redundancy analysis (RDA), we found that the measured C, N, and P hydrolysis and polyphenol oxidase (PPO) activities were positively correlated with the soil pH and ammonium contents, but were negatively correlated with the nitrate contents. The PLFA biomarker contents were positively correlated with soil

  10. A new methodology for the determination of enzyme activity based on carbon nanotubes and glucose oxidase.

    Science.gov (United States)

    Yeşiller, Gülden; Sezgintürk, Mustafa Kemal

    2015-11-10

    In this research, a novel enzyme activity analysis methodology is introduced as a new perspective for this area. The activity of elastase enzyme, which is a digestive enzyme mostly of found in the digestive system of vertebrates, was determined by an electrochemical device composed of carbon nanotubes and a second enzyme, glucose oxidase, which was used as a signal generator enzyme. In this novel methodology, a complex bioactive layer was constructed by using carbon nanotubes, glucose oxidase and a supporting protein, gelatin on a solid, conductive substrate. The activity of elastase was determined by monitoring the hydrolysis rate of elastase enzyme in the bioactive layer. As a result of this hydrolysis of elastase, glucose oxidase was dissociated from the bioactive layer, and following this the electrochemical signal due to glucose oxidase was decreased. The progressive elastase-catalyzed digestion of the bioactive layer containing glucose oxidase decreased the layer's enzymatic efficiency, resulting in a decrease of the glucose oxidation current as a function of the enzyme activity. The ratio of the decrease was correlated to elastase activity level. In this study, optimization experiments of bioactive components and characterization of the resulting new electrochemical device were carried out. A linear calibration range from 0.0303U/mL to 0.0729U/mL of elastase was reported. Real sample analyses were also carried out by the new electrochemical device. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Association between Antioxidant Enzyme Activities and Enterovirus-Infected Type 1 Diabetic Children.

    Science.gov (United States)

    Abdel-Moneim, Adel; El-Senousy, Waled M; Abdel-Latif, Mahmoud; Khalil, Rehab G

    2018-01-01

    To examine the effect of infection with Enterovirus (EV) in children with type 1 diabetes (T1D) on the activities of serum antioxidant enzymes in diabetic and nondiabetic controls. Three hundred and eighty-two diabetic and 100 nondiabetic children were tested for EV RNA using reverse transcriptase (RT)-PCR. The activities of serum superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase (CAT) were also estimated in diabetic patients infected with EV (T1D-EV+), those not infected with EV (T1D-EV-), and in nondiabetic controls. The frequency of EV was higher in diabetic children (100/382; 26.2%) than in healthy controls (0/100). Levels of fasting blood glucose (FBG), glycosylated hemoglobin (HbA1c) and C-reactive protein (CRP) were significantly higher but C-peptide was significantly lower in diabetic children than in controls. CRP levels were higher in the T1D-EV+ group than in the T1D-EV- group, and higher in all diabetic children than in nondiabetic controls. The activities of the antioxidant enzymes GPx, SOD, and CAT decreased significantly in diabetic children compared to in controls. Moreover, the activities of the enzymes tested were significantly reduced in the T1D-EV+ group compared to in the T1D-EV- group. Our data indicate that EV infection correlated with a decrease in the activity of antioxidant enzymes in the T1D-EV+ group compared to in the T1D-EV- group; this may contribute to β cell damage and increased inflammation. © 2018 The Author(s) Published by S. Karger AG, Basel.

  12. The influence of carbon nanotubes on enzyme activity and structure: investigation of different immobilization procedures through enzyme kinetics and circular dichroism studies

    International Nuclear Information System (INIS)

    Cang-Rong, Jason Teng; Pastorin, Giorgia

    2009-01-01

    In the last decade, many environmental organizations have devoted their efforts to identifying renewable biosystems, which could provide sustainable fuels and thus enhance energy security. Amidst the myriad of possibilities, some biofuels make use of different types of waste biomasses, and enzymes are often employed to hydrolyze these biomasses and produce sugars that will be subsequently converted into ethanol. In this project, we aimed to bridge nanotechnology and biofuel production: here we report on the activity and structure of the enzyme amyloglucosidase (AMG), physically adsorbed or covalently immobilized onto single-walled carbon nanotubes (SWNTs) and multi-walled carbon nanotubes (MWNTs). In fact, carbon nanotubes (CNTs) present several properties that render them ideal support systems, without the diffusion limitations displayed by porous material and with the advantage of being further functionalizable at their surface. Chemical ligation was achieved both on oxidized nanotubes (via carbodiimide chemistry), as well as on amino-functionalized nanotubes (via periodate-oxidized AMG). Results showed that AMG retained a certain percentage of its specific activity for all enzyme-carbon nanotubes complexes prepared, with the physically adsorbed samples displaying better catalytic efficiency than the covalently immobilized samples. Analysis of the enzyme's structure through circular dichroism (CD) spectroscopy revealed significant structural changes in all samples, the degree of change being consistent with the activity profiles. This study proves that AMG interacts differently with carbon nanotubes depending on the method employed. Due to the higher activity reported by the enzyme physically adsorbed onto CNTs, these samples demonstrated a vast potential for further development. At the same time, the possibility of inducing magnetic properties into CNTs offers the opportunity to easily separate them from the original solution. Hence, substances to which they

  13. Superoxide dismutase and catalase conjugated to polyethylene glycol increases endothelial enzyme activity and oxidant resistance

    International Nuclear Information System (INIS)

    Beckman, J.S.; Minor, R.L. Jr.; White, C.W.; Repine, J.E.; Rosen, G.M.; Freeman, B.A.

    1988-01-01

    Covalent conjugation of superoxide dismutase and catalase with polyethylene glycol (PEG) increases the circulatory half-lives of these enzymes from 125 I-PEG-catalase or 125 I-PEG-superoxide dismutase produced a linear, concentration-dependent increase in cellular enzyme activity and radioactivity. Fluorescently labeled PEG-superoxide dismutase incubated with endothelial cells showed a vesicular localization. Mechanical injury to cell monolayers, which is known to stimulate endocytosis, further increased the uptake of fluorescent PEG-superoxide dismutase. Addition of PEG and PEG-conjugated enzymes perturbed the spin-label binding environment, indicative of producing an increase in plasma membrane fluidity. Thus, PEG conjugation to superoxide dismutase and catalase enhances cell association of these enzymes in a manner which increases cellular enzyme activities and provides prolonged protection from partially reduced oxygen species

  14. Quantitative Characterization of Major Hepatic UDP-Glucuronosyltransferase Enzymes in Human Liver Microsomes: Comparison of Two Proteomic Methods and Correlation with Catalytic Activity.

    Science.gov (United States)

    Achour, Brahim; Dantonio, Alyssa; Niosi, Mark; Novak, Jonathan J; Fallon, John K; Barber, Jill; Smith, Philip C; Rostami-Hodjegan, Amin; Goosen, Theunis C

    2017-10-01

    Quantitative characterization of UDP-glucuronosyltransferase (UGT) enzymes is valuable in glucuronidation reaction phenotyping, predicting metabolic clearance and drug-drug interactions using extrapolation exercises based on pharmacokinetic modeling. Different quantitative proteomic workflows have been employed to quantify UGT enzymes in various systems, with reports indicating large variability in expression, which cannot be explained by interindividual variability alone. To evaluate the effect of methodological differences on end-point UGT abundance quantification, eight UGT enzymes were quantified in 24 matched liver microsomal samples by two laboratories using stable isotope-labeled (SIL) peptides or quantitative concatemer (QconCAT) standard, and measurements were assessed against catalytic activity in seven enzymes ( n = 59). There was little agreement between individual abundance levels reported by the two methods; only UGT1A1 showed strong correlation [Spearman rank order correlation (Rs) = 0.73, P quantitative proteomic data should be validated against catalytic activity whenever possible. In addition, metabolic reaction phenotyping exercises should consider spurious abundance-activity correlations to avoid misleading conclusions. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  15. Cell age dependent variations in oxidative protective enzymes

    International Nuclear Information System (INIS)

    Blakely, E.A.; Chang, P.Y.; Lommel, L.; Tobias, C.A.

    1986-01-01

    Activity levels of antioxidant enzymes were correlated before and after heavy-ion exposures with cellular radiosensitivity. In preliminary feasibility experiments with human T-1 cells relatively high antioxidant enzyme levels were shown in the unirradiated G 1 phase prior to the normal DNA synthetic phase. Endogenous cellular levels of three antioxidant enzymes were measured at various times in the unirradiated human T-1 cell division cycle. The enzymes measured were: catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSHPX). Unlike the case in Chinese hamster V79 cells the early data with the synchronized human cell show that in very early G 1 phase (e.g., approximately 1.5 hours after mitotic selection) there are significant peaks in the levels (U/mg cell protein) of both CAT and SOD. Both enzymes show increases as the unirradiated cells progressed from mitosis into G 1 phase while the levels of GSHPX measured in duplicate samples were somewhat more variable than was the case for the other two enzymes. Studies were made in collaboration with the Armed Forces Radiobiology Research Institute

  16. Crystallization of Hevamine, an Enzyme with Lysozyme/Chitinase Activity from Hevea brasiliensis Latex

    NARCIS (Netherlands)

    ROZEBOOM, HJ; BUDIANI, A; BEINTEMA, JJ

    1990-01-01

    Hevamine, an enzyme with both lysozyme and chitinase activity, was isolated and purified from Hevea brasiliensis (rubber tree) latex. The enzyme (molecular weight 29,000) is homologous to certain “pathogenesis-related” proteins from plants, but not to hen egg-white or phage T4 lysozyme. To

  17. Enzyme activity and seedling growth of soybean seeds under accelerated aging

    Directory of Open Access Journals (Sweden)

    Yadollhhi Nooshabadi S.J.

    2013-11-01

    Full Text Available Seed aging is the main problem of seed storage. Changes of bio-chemical and reduction of seedling growth are consequence of seed deterioration. An experiment was conducted to evaluate the effects of accelerated aging on soybean seed germination indexes and enzyme activity. Seeds were incubated in closed plastic boxes for the accelerated aging treatments. Three accelerate aging regimes were performed by placing seeds at 41°C and relative humidity (RH of 90-100 % for 0, 2, 4, 6 and 8 days periods. Our results showed that increasing aging duration resulted higher reduction in germination characteristics, catalase and ascorbate peroxidase. Germination percentage, means time to germination, germination index, normal seedling percentage and enzyme activity decrease significantly.

  18. Ecotoxicological effects of copper and selenium combined pollution on soil enzyme activities in planted and unplanted soils.

    Science.gov (United States)

    Hu, Bin; Liang, Dongli; Liu, Juanjuan; Xie, Junyu

    2013-04-01

    The present study explored the joint effects of Cu and Se pollution mechanisms on soil enzymes to provide references for the phytoremediation of contaminated areas and agricultural environmental protection. Pot experiments and laboratory analyses were carried out to study the individual and combined influences of Cu and Se on soil enzyme activities. The activities of four soil enzymes (urease, catalase, alkaline phosphatase, and nitrate reductase) were chosen. All soil enzyme activities tested were inhibited by Cu and Se pollution, either individually or combined, in varying degrees, following the order nitrate reductase>urease>catalase>alkaline phosphatase. Growing plants stimulated soil enzyme activity in a similar trend compared with treatments without plants. The joint effects of Cu and Se on catalase activity showed synergism at low concentrations and antagonism at high concentrations, whereas the opposite was observed for urease activity. However, nitrate reductase activity showed synergism both with and without plant treatments. The half maximal effective concentration (EC50) of exchangeable fractions had a similar trend with the EC50 of total content and was lower than that of total content. The EC50 values of nitrate reductase and urease activities were significantly lower for both Se and Cu (p<0.05), which indicated that they were more sensitive than the other two enzymes. Copyright © 2013 SETAC.

  19. Artificial Enzymes, "Chemzymes"

    DEFF Research Database (Denmark)

    Bjerre, Jeannette; Rousseau, Cyril Andre Raphaël; Pedersen, Lavinia Georgeta M

    2008-01-01

    Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models that successf......Enzymes have fascinated scientists since their discovery and, over some decades, one aim in organic chemistry has been the creation of molecules that mimic the active sites of enzymes and promote catalysis. Nevertheless, even today, there are relatively few examples of enzyme models...... that successfully perform Michaelis-Menten catalysis under enzymatic conditions (i.e., aqueous medium, neutral pH, ambient temperature) and for those that do, very high rate accelerations are seldomly seen. This review will provide a brief summary of the recent developments in artificial enzymes, so called...... "Chemzymes", based on cyclodextrins and other molecules. Only the chemzymes that have shown enzyme-like activity that has been quantified by different methods will be mentioned. This review will summarize the work done in the field of artificial glycosidases, oxidases, epoxidases, and esterases, as well...

  20. Puromycin-sensitive aminopeptidase: an antiviral prodrug activating enzyme.

    Science.gov (United States)

    Tehler, Ulrika; Nelson, Cara H; Peterson, Larryn W; Provoda, Chester J; Hilfinger, John M; Lee, Kyung-Dall; McKenna, Charles E; Amidon, Gordon L

    2010-03-01

    Cidofovir (HPMPC) is a broad-spectrum antiviral agent, currently used to treat AIDS-related human cytomegalovirus retinitis. Cidofovir has recognized therapeutic potential for orthopox virus infections, although its use is hampered by its inherent low oral bioavailability. Val-Ser-cyclic HPMPC (Val-Ser-cHPMPC) is a promising peptide prodrug which has previously been shown by us to improve the permeability and bioavailability of the parent compound in rodent models (Eriksson et al., 2008. Molecular Pharmaceutics 5, 598-609). Puromycin-sensitive aminopeptidase was partially purified from Caco-2 cell homogenates and identified as a prodrug activating enzyme for Val-Ser-cHPMPC. The prodrug activation process initially involves an enzymatic step where the l-Valine residue is removed by puromycin-sensitive aminopeptidase, a step that is bestatin-sensitive. Subsequent chemical hydrolysis results in the generation of cHPMPC. A recombinant puromycin-sensitive aminopeptidase was generated and its substrate specificity investigated. The k(cat) for Val-pNA was significantly lower than that for Ala-pNA, suggesting that some amino acids are preferred over others. Furthermore, the three-fold higher k(cat) for Val-Ser-cHPMPC as compared to Val-pNA suggests that the leaving group may play an important role in determining hydrolytic activity. In addition to its ability to hydrolyze a variety of substrates, these observations strongly suggest that puromycin-sensitive aminopeptidase is an important enzyme for activating Val-Ser-cHPMPC in vivo. Taken together, our data suggest that puromycin-sensitive aminopeptidase makes an attractive target for future prodrug design.

  1. A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity.

    Science.gov (United States)

    Lam, Sonia Y; Yeung, Rachel C Y; Yu, Tsz-Ha; Sze, Kong-Hung; Wong, Kam-Bo

    2011-03-01

    Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity. Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy. Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures.

  2. A rigidifying salt-bridge favors the activity of thermophilic enzyme at high temperatures at the expense of low-temperature activity.

    Directory of Open Access Journals (Sweden)

    Sonia Y Lam

    2011-03-01

    Full Text Available Thermophilic enzymes are often less active than their mesophilic homologues at low temperatures. One hypothesis to explain this observation is that the extra stabilizing interactions increase the rigidity of thermophilic enzymes and hence reduce their activity. Here we employed a thermophilic acylphosphatase from Pyrococcus horikoshii and its homologous mesophilic acylphosphatase from human as a model to study how local rigidity of an active-site residue affects the enzymatic activity.Acylphosphatases have a unique structural feature that its conserved active-site arginine residue forms a salt-bridge with the C-terminal carboxyl group only in thermophilic acylphosphatases, but not in mesophilic acylphosphatases. We perturbed the local rigidity of this active-site residue by removing the salt-bridge in the thermophilic acylphosphatase and by introducing the salt-bridge in the mesophilic homologue. The mutagenesis design was confirmed by x-ray crystallography. Removing the salt-bridge in the thermophilic enzyme lowered the activation energy that decreased the activation enthalpy and entropy. Conversely, the introduction of the salt-bridge to the mesophilic homologue increased the activation energy and resulted in increases in both activation enthalpy and entropy. Revealed by molecular dynamics simulations, the unrestrained arginine residue can populate more rotamer conformations, and the loss of this conformational freedom upon the formation of transition state justified the observed reduction in activation entropy.Our results support the conclusion that restricting the active-site flexibility entropically favors the enzymatic activity at high temperatures. However, the accompanying enthalpy-entropy compensation leads to a stronger temperature-dependency of the enzymatic activity, which explains the less active nature of the thermophilic enzymes at low temperatures.

  3. Spatial localization of the first and last enzymes effectively connects active metabolic pathways in bacteria.

    Science.gov (United States)

    Meyer, Pablo; Cecchi, Guillermo; Stolovitzky, Gustavo

    2014-12-14

    Although much is understood about the enzymatic cascades that underlie cellular biosynthesis, comparatively little is known about the rules that determine their cellular organization. We performed a detailed analysis of the localization of E.coli GFP-tagged enzymes for cells growing exponentially. We found that out of 857 globular enzymes, at least 219 have a discrete punctuate localization in the cytoplasm and catalyze the first or the last reaction in 60% of biosynthetic pathways. A graph-theoretic analysis of E.coli's metabolic network shows that localized enzymes, in contrast to non-localized ones, form a tree-like hierarchical structure, have a higher within-group connectivity, and are traversed by a higher number of feed-forward and feedback loops than their non-localized counterparts. A Gene Ontology analysis of these enzymes reveals an enrichment of terms related to essential metabolic functions in growing cells. Given that these findings suggest a distinct metabolic role for localization, we studied the dynamics of cellular localization of the cell wall synthesizing enzymes in B. subtilis and found that enzymes localize during exponential growth but not during stationary growth. We conclude that active biochemical pathways inside the cytoplasm are organized spatially following a rule where their first or their last enzymes localize to effectively connect the different active pathways and thus could reflect the activity state of the cell's metabolic network.

  4. Activity of metabolic enzymes and muscle-specific gene expression in parr and smolts Atlantic salmon Salmo salar L. of different age groups.

    Science.gov (United States)

    Churova, Maria V; Meshcheryakova, Olga V; Veselov, Aleksey E; Efremov, Denis A; Nemova, Nina N

    2017-08-01

    This study was conducted to characterize the energy metabolism level and the features of muscle growth regulation during the development of Atlantic salmon (Salmo salar) inhabiting the Indera River (Kola Peninsula, Russia). The activities of aerobic and anaerobic enzymes (cytochrome c oxidase and lactate dehydrogenase) and carbohydrate metabolism enzymes (glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, and aldolase) were measured in muscle and liver tissue. Gene expression levels of myosin heavy chain (MyHC), myostatin (MSTN-1a), and myogenic regulatory factors (MRFs-MyoD1a, MyoD1b, MyoD1c, Myf5, myogenin) were measured in the white muscles of salmon parr of ages 0+, 1+, 2+, and 3+ and smolts of ages 2+ and 3+. Multidirectional changes in the activity of enzymes involved in aerobic and anaerobic energy metabolism with age were shown in the white muscles of the parr. The cytochrome c oxidase activity was higher in muscles of underyearlings (0+) and yearlings (1+) and decreased in 2+ and 3+ age groups. The activity of lactate dehydrogenase, in contrast, increased with age. The patterns of changes in expression levels of MyoD1a, MyoD1b, myogenin, MyHC, and MSTN-1a at different ages of the parr were similar. Particularly, the expression of these genes peaked in the yearling parr (1+) and then decreased in elder groups. The differences were revealed in parameters studied between the parr and smolts. The level of aerobic and anaerobic metabolism enzyme activities was higher in the white muscles of smolts than in parr. The activity of carbohydrate metabolism enzymes was decreased in the smolts' livers. The expression levels of MyHC, MyoD1a, MyoD1b, and myogenin were lower in smolts at age 2+ compared to parr. These findings expand our knowledge of age-related and stage-related features of energy metabolism and muscle development regulation in young Atlantic salmon in their natural habitat. The results might be used for monitoring of the salmon

  5. The Effects of Fenarimol and Methyl Parathion on Glucose 6-Phosphate Dehydrogenase Enzyme Activity in Rats

    Directory of Open Access Journals (Sweden)

    Ferda ARI

    2017-10-01

    Full Text Available Fenarimol and methyl parathion are pesticides that have been used in agriculture for several years. These pesticides have significant effects on environmental and human health. Therefore, we investigated the effects of methyl parathion and fenarimol on glucose 6-phosphate dehydrogenase (EC 1.1.1.49 enzyme activity in rats. The glucose 6- phosphate dehydrogenase is the first enzyme of the pentose phosphate pathway and it is important in detoxifying reactions by NADPH generated. In this study, wistar albino rats administrated with methyl parathion (7 mg kg–1 and fenarimol (200 mg kg−1 by intraperitoneally for different periods (2, 4, 8, 16, 32, 64, and 72 h. The glucose 6-phosphate dehydrogenase enzyme activity was assayed in liver, kidney, brain, and small intestine in male and female rats. The exposure of fenarimol and methyl parathion caused increase of glucose 6-phosphate dehydrogenase enzyme activity in rat tissues, especially at last periods. We suggest that this increment of enzyme activity may be the reason of toxic effects of fenarimol and methyl parathion.

  6. Controlled immobilisation of active enzymes on the cowpea mosaic virus capsid

    Science.gov (United States)

    Aljabali, Alaa A. A.; Barclay, J. Elaine; Steinmetz, Nicole F.; Lomonossoff, George P.; Evans, David J.

    2012-08-01

    Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors.Immobilisation of horseradish peroxidase (HRP) and glucose oxidase (GOX) via covalent attachment of modified enzyme carbohydrate to the exterior of the cowpea mosaic virus (CPMV) capsid gave high retention of enzymatic activity. The number of enzymes bound per virus was determined to be about eleven for HRP and 2-3 for GOX. This illustrates that relatively large biomacromolecules can be readily coupled to the virus surface using simple conjugation strategies. Virus-biomacromolecule hybrids have great potential for uses in catalysis, diagnostic assays or biosensors. Electronic supplementary information (ESI) available: Alternative conjugation strategies, agarose gel electrophoresis of CPMV and CPMV-HRP conjugates, UV-vis spectrum of HRP-ADHCPMV, agarose gel electrophoresis of GOX-ADHCPMV particles and corresponding TEM image, calibration curves for HRP-ADHCPMV and GOX-ADHCPMV, DLS data for GOX-ADHCPMV are made available. See DOI: 10.1039/c2nr31485a

  7. Angiotensin-converting enzyme activity in Cavalier King Charles Spaniels with an ACE gene polymorphism and myxomatous mitral valve disease

    DEFF Research Database (Denmark)

    Meurs, Kathryn M.; Olsen, Lisbeth H.; Reimann, Maria J.

    2018-01-01

    a canine ACE gene polymorphism associated with a decrease in angiotensin-converting enzyme (ACE) activity. The aim of this study was to evaluate for the prevalence of the ACE polymorphism in CKCS with mitral valve disease and to determine whether the presence of the polymorphism is associated......Objectives Myxomatous mitral valve disease (MMVD) is the most common heart disease in the dog. It is particularly common in the Cavalier King Charles Spaniel (CKCS) breed and affected dogs are frequently managed with angiotensin-converting enzyme inhibitors (ACE-I). We have previously identified...... with alterations in ACE activity at different stages of cardiac disease. Methods Seventy-three dogs with a diagnosis of mitral valve disease were evaluated and a blood sample was drawn for ACE polymorphism genotyping and ACE activity measurement. Results Forty-three dogs were homozygous for the ACE polymorphism...

  8. Survival of Bemisia tabaci and activity of plant defense-related enzymes in genotypes of Capsicum annuum L.

    Directory of Open Access Journals (Sweden)

    Luis Latournerie-Moreno

    2015-03-01

    Full Text Available The whitefly Bemisia tabaci (Gennadius, 1889 is a major plant pest of horticultural crops from the families Solanaceae, Fabaceae and Cucurbitaceae in Neotropical areas. The exploration of host plant resistance and their biochemical mechanisms offers an excellent alternative to better understand factors affecting the interaction between phytophagous insect and host plant. We evaluated the survival of B. tabaci in landrace genotypes of Capsicum annuum L., and the activity of plant defense-related enzymes (chitinase, polyphenoloxidase, and peroxidase. The landrace genotypes Amaxito, Tabaquero, and Simojovel showed resistance to B. tabaci, as we observed more than 50% nymphal mortality, while in the commercial susceptible genotype Jalapeño mortality of B. tabaci nymphs was not higher than 20%. The activities of plant defense-related enzymes were significantly different among pepper genotypes (P < 0.05. Basal activities of chitinase, polyphenoloxidase and peroxidase were significantly lower or equal in landrace genotypes than that of the commercial genotype Jalapeño. The activity of plant enzymes was differential among pepper genotypes (P < 0.05. For example, the activity of chitinase enzyme generally was higher in non-infested plants with B. tabaci than those infested. Instead polyphenoloxidase ('Amaxito' and 'Simojovel' and peroxidase enzymes activities ('Tabaquero' increased in infested plants (P < 0.05. We conclude that basal activities of plant defense-related enzymes could be act through other mechanism plant induction, since plant defense-related enzymes showed a different induction response to B. tabaci. We underlined the role of polyphenoloxidase as plant defense in the pepper genotype Simojovel related to B. tabaci.

  9. Impaired redox state and respiratory chain enzyme activities in the cerebellum of vitamin A-treated rats

    International Nuclear Information System (INIS)

    Oliveira, Marcos Roberto de; Fonseca Moreira, Jose Claudio

    2008-01-01

    Vitamin A is a micronutrient that participates in the maintenance of the mammalian cells homeostasis. However, excess of vitamin A, which may be achieved through increased intake of the vitamin either therapeutically or inadvertently, induces several deleterious effects in a wide range of mammalian cells, including neuronal cells. Vitamin A is a redox-active molecule, and it was previously demonstrated that it induces oxidative stress in several cell types. Therefore, in the present work, we investigated the effects of vitamin A supplementation at clinical doses (1000-9000 IU/(kg day)) on redox environment and respiratory chain activity in the adult rat cerebellum. Glutathione-S-transferase (GST) enzyme activity was also measured here. The animals were treated for 3, 7, or 28 days with vitamin A as retinol palmitate. We found increased levels of molecular markers of oxidative damage in the rat cerebellum in any period analyzed. Additionally, vitamin A supplementation impaired cerebellar mitochondrial electron transfer chain (METC) activity. GST enzyme activity was increased in the cerebellum of rats chronically treated with vitamin A. Based on our results and data previously published, we recommend more caution in prescribing vitamin A at high doses even clinically, since there is a growing concern regarding toxic effects associated to vitamin A intake

  10. Mycobacterium tuberculosis lipolytic enzymes as potential biomarkers for the diagnosis of active tuberculosis.

    Directory of Open Access Journals (Sweden)

    Belinda Brust

    Full Text Available BACKGROUND: New diagnosis tests are urgently needed to address the global tuberculosis (TB burden and to improve control programs especially in resource-limited settings. An effective in vitro diagnostic of TB based on serological methods would be regarded as an attractive progress because immunoassays are simple, rapid, inexpensive, and may offer the possibility to detect cases missed by standard sputum smear microscopy. However, currently available serology tests for TB are highly variable in sensitivity and specificity. Lipolytic enzymes have recently emerged as key factors in lipid metabolization during dormancy and/or exit of the non-replicating growth phase, a prerequisite step of TB reactivation. The focus of this study was to analyze and compare the potential of four Mycobacterium tuberculosis lipolytic enzymes (LipY, Rv0183, Rv1984c and Rv3452 as new markers in the serodiagnosis of active TB. METHODS: Recombinant proteins were produced and used in optimized ELISA aimed to detect IgG and IgM serum antibodies against the four lipolytic enzymes. The capacity of the assays to identify infection was evaluated in patients with either active TB or latent TB and compared with two distinct control groups consisting of BCG-vaccinated blood donors and hospitalized non-TB individuals. RESULTS: A robust humoral response was detected in patients with active TB whereas antibodies against lipolytic enzymes were infrequently detected in either uninfected groups or in subjects with latent infection. High specifity levels, ranging from 93.9% to 97.5%, were obtained for all four antigens with sensitivity values ranging from 73.4% to 90.5%, with Rv3452 displaying the highest performances. Patients with active TB usually exhibited strong IgG responses but poor IgM responses. CONCLUSION: These results clearly indicate that the lipolytic enzymes tested are strongly immunogenic allowing to distinguish active from latent TB infections. They appear as potent

  11. Relief of autoinhibition by conformational switch explains enzyme activation by a catalytically dead paralog

    Energy Technology Data Exchange (ETDEWEB)

    Volkov, Oleg A.; Kinch, Lisa; Ariagno, Carson; Deng, Xiaoyi; Zhong, Shihua; Grishin, Nick; Tomchick, Diana R.; Chen, Zhe; Phillips, Margaret A.

    2016-12-15

    Catalytically inactive enzyme paralogs occur in many genomes. Some regulate their active counterparts but the structural principles of this regulation remain largely unknown. We report X-ray structures ofTrypanosoma brucei S-adenosylmethionine decarboxylase alone and in functional complex with its catalytically dead paralogous partner, prozyme. We show monomericTbAdoMetDC is inactive because of autoinhibition by its N-terminal sequence. Heterodimerization with prozyme displaces this sequence from the active site through a complex mechanism involving acis-to-transproline isomerization, reorganization of a β-sheet, and insertion of the N-terminal α-helix into the heterodimer interface, leading to enzyme activation. We propose that the evolution of this intricate regulatory mechanism was facilitated by the acquisition of the dimerization domain, a single step that can in principle account for the divergence of regulatory schemes in the AdoMetDC enzyme family. These studies elucidate an allosteric mechanism in an enzyme and a plausible scheme by which such complex cooperativity evolved.

  12. Defoliation effects on enzyme activities of the ectomycorrhizal fungus Suillus granulatus in a Pinus contorta (lodgepole pine) stand in Yellowstone National Park.

    Science.gov (United States)

    Cullings, Ken; Ishkhanova, Galina; Henson, Joan

    2008-11-01

    Ectomycorrhizal (EM) basidiomycete fungi are obligate mutualists of pines and hardwoods that receive fixed C from the host tree. Though they often share most recent common ancestors with wood-rotting fungi, it is unclear to what extent EM fungi retain the ability to express enzymes that break down woody substrates. In this study, we tested the hypothesis that the dominant EM fungus in a pure pine system retains the ability to produce enzymes that break down woody substrates in a natural setting, and that this ability is inducible by reduction of host photosynthetic potential via partial defoliation. To achieve this, pines in replicate blocks were defoliated 50% by needle removal, and enzyme activities were measured in individual EM root tips that had been treated with antibiotics to prevent possible bacterial activity. Results indicate that the dominant EM fungal species (Suillus granulatus) expressed all enzymes tested (endocellulase D: -glucosidase, laccase, manganese peroxidase, lignin peroxidase, phosphatase and protease), and that activities of these enzymes increased significantly (P pine) has the potential to play a significant role in C, N and P cycling in this forested ecosystem. Therefore, many above-ground factors that reduce photosynthetic potential or divert fixed C from roots may have wide-reaching ecosystem effects.

  13. Enzyme activity and reserve mobilization during Macaw palm ( Acrocomia aculeata seed germination

    Directory of Open Access Journals (Sweden)

    Elisa Monteze Bicalho

    2016-01-01

    Full Text Available ABSTRACT Reserve mobilization in seeds occurs after visible germination, which is marked by the protrusion of the radicle or cotyledonary petiole, as in species of Arecaceae. Acrocomia aculeata (macaw palm, usually produces hard seeds whose endosperm has mannan-rich cell walls. We investigated the composition of storage compounds in macaw palm seed and the roles of two enzymes (endo-β-mannanase, α-galactosidase during and after germination. The seeds were firstly submitted to pre-established protocol to overcome dormancy and promote germination. Enzyme activity in both embryo and endosperm were assayed from the initiation of germinative activities until leaf sheath appearance, and the status of seed structures and reserve compounds were evaluated. Protein content of the embryo decreased with the initiation of imbibition while the lipid content began decreasing six days after removal of the operculum. Increases in enzyme activity and starch content were both observed after visible germination. We suggest that endo-β-mannanase and α-galactosidase become active immediately at germination, facilitating haustorium expansion and providing carbohydrates for initial seedling development. Protein is the first storage compound mobilized during early imbibition, and the observed increase in the starch content of the haustorium was related to lipid degradation in that organ and mannan degradation in the adjacent endosperm.

  14. Soil Enzyme Activities in Pinus tabuliformis (Carriére Plantations in Northern China

    Directory of Open Access Journals (Sweden)

    Weiwei Wang

    2016-05-01

    Full Text Available Changes in forest stand structure may alter the activity of invertase, urease, catalase and phenol oxidase after thinning Pinus tabuliformis (Carriére plantations in Yanqing County of Beijing, China. We examined changes in these soil enzymes as influenced by time since thinning (24, 32, and 40 years since thinning for 3 seasons (spring, summer and autumn following harvesting at two depths in the mineral soil (0–10 cm and 10–20 cm. Invertase and urease increased significantly with time since thinning. Catalase activity was highest in the 24-year-old stand and there were no statistically significant differences between the 32- and 40-year-old stands. In addition, maximum invertase, urease, catalase, and phenol oxidase activities occurred during the summer; minimum activities occurred in autumn. Invertase and urease were positively correlated with each other, as were catalase and phenol oxidase. Most soil enzyme activity was higher in the 0–10 cm layer than at the 10–20 cm depth. As time from thinning increased, differences among soil depth became less significant. These results suggest that seasonal changes of these enzymes have different roles, as the time since thinning and thinning treatments may have both short- and long-term impacts on soil microbial activity.

  15. Metagenomics as a Tool for Enzyme Discovery: Hydrolytic Enzymes from Marine-Related Metagenomes.

    Science.gov (United States)

    Popovic, Ana; Tchigvintsev, Anatoly; Tran, Hai; Chernikova, Tatyana N; Golyshina, Olga V; Yakimov, Michail M; Golyshin, Peter N; Yakunin, Alexander F

    2015-01-01

    This chapter discusses metagenomics and its application for enzyme discovery, with a focus on hydrolytic enzymes from marine metagenomic libraries. With less than one percent of culturable microorganisms in the environment, metagenomics, or the collective study of community genetics, has opened up a rich pool of uncharacterized metabolic pathways, enzymes, and adaptations. This great untapped pool of genes provides the particularly exciting potential to mine for new biochemical activities or novel enzymes with activities tailored to peculiar sets of environmental conditions. Metagenomes also represent a huge reservoir of novel enzymes for applications in biocatalysis, biofuels, and bioremediation. Here we present the results of enzyme discovery for four enzyme activities, of particular industrial or environmental interest, including esterase/lipase, glycosyl hydrolase, protease and dehalogenase.

  16. Direct comparison of enzyme histochemical and immunohistochemical methods to localize an enzyme

    NARCIS (Netherlands)

    van Noorden, Cornelis J. F.

    2002-01-01

    Immunohistochemical localization of enzymes is compared directly with localization of enzyme activity with (catalytic) enzyme histochemical methods. The two approaches demonstrate principally different aspects of an enzyme. The immunohistochemical method localizes the enzyme protein whether it is

  17. Activity of trypsin-like enzymes and gelatinases in rats with doxorubicin cardiomyopathy

    Directory of Open Access Journals (Sweden)

    Iu. А. Gordiienko

    2014-12-01

    Full Text Available Activity of trypsin-like enzymes (ATLE and gelatinases A and B were studied in the blood plasma and extracts from cardiac muscle, cerebral cortex and cerebellum of rats with cardiomyopathy caused by anthracycline antibiotic doxorubicin against the background of preventive application of corvitin and α-ketoglutarate. ATLE significantly increased in blood plasma and extracts from cerebral cortex but decreased in extracts from cardiac muscle and cerebellum in doxorubicin cardiomyopathy (DCMP. In addition, a significant increase of activity of both gelatinases in plasma and tissue extracts was observed. Preventive administration of corvitin and α-ketoglutarate resulted in differently directed changes of activity of the above mentioned enzymes in heart and brain tissues. Obtained data confirm the hypothesis about activation of proteolysis under the influence of anthracycline antibiotics and testify to selective effect of corvitin and α-ketoglutarate on ATLE and gelatinases.

  18. Activity of trypsin-like enzymes and gelatinases in rats with doxorubicin cardiomyopathy.

    Science.gov (United States)

    Gordiienko, Iu A; Babets, Ya V; Kulinich, A O; Shevtsova, A I; Ushakova, G O

    2014-01-01

    Activity of trypsin-like enzymes (ATLE) and gelatinases A and B were studied in the blood plasma and extracts from cardiac muscle, cerebral cortex and cerebellum of rats with cardiomyopathy caused by anthracycline antibiotic doxorubicin against the background of preventive application of corvitin and α-ketoglutarate. ATLE significantly increased in blood plasma and extracts from cerebral cortex but decreased in extracts from cardiac muscle and cerebellum in doxorubicin cardiomyopathy (DCMP). In addition, a significant increase of activity of both gelatinases in plasma and tissue extracts was observed. Preventive administration of corvitin and α-ketoglutarate resulted in differently directed changes of activity of the above mentioned enzymes in heart and brain tissues. Obtained data confirm the hypothesis about activation of proteolysis under the influence of anthracycline antibiotics and testify to selective effect of corvitin and α-ketoglutarate on ATLE and gelatinases.

  19. DETERMINATION of OPTIMUM CONDITION of PAPAIN ENZYME FROM PAPAYA VAR JAVA (Carica papaya

    Directory of Open Access Journals (Sweden)

    Aline Puspita Kusumadjaja

    2010-06-01

    Full Text Available A study to investigate the optimum condition of papain enzyme has been carried out. The condition that are investigated are pH and temperature, based on measurement of enzyme activity which is defined as mmole tyrosin that are released in reaction between papain enzyme and casein as substrat per minute. In this research, the papain enzyme was isolated from pepaya burung varietas Java. The enzyme was partially purified by precipitation method using 30% - 50% saturated acetone. The result showed that the optimum conditions of papain enzyme are in pH 6 with activity 2,606 U/mL, and temperature at 50 oC with activity 2,469 U/mL. Keywords : Papaya var Java, papain, optimum condition, enzymatic activity

  20. Effects of long-term elevated CO2 on N2-fixing, denitrifying and nitrifying enzyme activities in forest soils under Pinus sylvestriformis in Changbai Mountain

    Institute of Scientific and Technical Information of China (English)

    ZHENG Jun-Qiang; HAN Shi-Jie; REN Fei-Rong; ZHOU Yu-Mei; ZHANG Yan

    2008-01-01

    A study was conducted to determine the effects of elevated CO2 on soil N process at Changbai Mountain in Jilin Province,northeastern China (42o24'N,128o06'E,and 738 m elevation).A randomized complete block design of ambient and elevated CO2 was established in an open-top chamber facility in the spring of 1999.Changpai Scotch pine (Pinus sylvestris var.sylvestriformis seeds were sowed in May,1999 and CO2 fumigation treatments began after seeds germination.In each year,the exposure started at the end of April and stopped at the end of October.Soil samples were collected in June and August 2006 and in June 2007,and soil nitrifying,denitrifying and N2-fixing enzyme activities were measured.Results show that soil nitrifying enzyme activities (NEA) in the 5-10 cm soil layer were significantly increased at elevated CO2 by 30.3% in June 2006,by 30.9% in August 2006 and by 11.3% in June 2007.Soil denitrifying enzyme activities (DEA) were significantly decreased by elevated CO2 treatment in June 2006 (P < 0.012) and August 2006 (P < 0.005) samplings in our study; no significant difference was detected in June 2007,and no significant changes in N2-fixing enzyme activity were found.This study suggests that elevated CO2 can alter soil nitrifying enzyme and denitrifying enzyme activities.

  1. Effects of culture conditions on monosaccharide composition of Ganoderma lucidum exopolysaccharide and on activities of related enzymes.

    Science.gov (United States)

    Peng, Lin; Qiao, Shuangkui; Xu, Zhenghong; Guan, Feng; Ding, Zhongyang; Gu, Zhenghua; Zhang, Liang; Shi, Guiyang

    2015-11-20

    We investigated the relationship between monosaccharide composition of Ganoderma lucidum exopolysaccharide (EPS) and activities of EPS synthesis enzymes under various culture temperatures and initial pH values. The mole percentages of three major EPS monosaccharides, glucose, galactose and mannose, varied depending on culture conditions and the resulting EPS displayed differing anti-tumor activities. In nine tested enzymes, higher enzyme activities were correlated with higher temperature and lower initial pH. Altered mole percentages of galactose and mannose under various culture conditions were associated with activities of α-phosphoglucomutase (PGM) and phosphoglucose isomerase (PGI), respectively, and that of mannose was also associated with phosphomannose isomerase (PMI) activity only under various pH. Our findings suggest that mole percentages of G. lucidum EPS monosaccharides can be manipulated by changes of culture conditions that affect enzyme activities, and that novel fermentation strategies based on this approach may enhance production and biological activity of EPS. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Post-cold-storage conditioning time affects soil denitrifying enzyme activity

    DEFF Research Database (Denmark)

    Chirinda, Ngonidzashe; Olesen, Jørgen Eivind; Porter, John Roy

    2011-01-01

    Soil denitrifying enzyme activity (DEA) is often assessed after cold storage. Previous studies using the short-term acetylene inhibition method have not considered conditioning time (post-cold-storage warm-up time prior to soil analysis) as a factor influencing results. We observed fluctuations...

  3. Dietary effects of marine food intake on intestinal and hepatic enzyme activities in rats.

    Science.gov (United States)

    González, M; Caride, B; Lamas, A; Taboada, C

    2001-03-01

    Dietary effects of two diets high in protein from two marine species (Haliotis tuberculata and Anemonia viridis) as compared to a high-quality patron protein such as casein (or casein supplemented with olive oil) on intestinal and hepatic enzymes were studied. After 23 days, the two marine species as diet compared to casein increased the disaccharidase and alkaline phosphatase activities. Feeding Haliotis tuberculata meal produced a decrease on intestinal leucine aminopeptidase activity. The hepatic gamma-glutamyltranspeptidase activity decreased slightly in animals fed Haliotis tuberculata meal. Supplementation of casein with olive oil tended to decrease the intestinal and hepatic enzyme activity.

  4. Real-time investigation of human topoisomerase I reaction kinetics using an optical sensor: a fast method for drug screening and determination of active enzyme concentrations

    Science.gov (United States)

    Kristoffersen, Emil L.; Jørgensen, Line A.; Franch, Oskar; Etzerodt, Michael; Frøhlich, Rikke; Bjergbæk, Lotte; Stougaard, Magnus; Ho, Yi-Ping; Knudsen, Birgitta R.

    2015-05-01

    Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment.Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using

  5. Does estradiol have an impact on the dipeptidyl peptidase IV enzyme activity of the Prevotella intermedia group bacteria?

    Science.gov (United States)

    Fteita, Dareen; Könönen, Eija; Gürsoy, Mervi; Söderling, Eva; Gürsoy, Ulvi Kahraman

    2015-12-01

    Initiation and development of pregnancy-associated gingivitis is seemingly related to the microbial shift towards specific gram-negative anaerobes in subgingival biofilms. It is known that Prevotella intermedia sensu lato is able to use estradiol as an alternative source of growth instead of vitamin K. The aim of the present study was to investigate the impact of estradiol on the bacterial dipeptidyl peptidase IV (DPPIV) enzyme activity in vitro as a virulent factor of the Prevotella intermedia group bacteria, namely P. intermedia, Prevotella nigrescens, Prevotella pallens, and Prevotella aurantiaca. In all experiments, 2 strains of each Prevotella species were used. Bacteria were incubated with the concentrations of 0, 30, 90, and 120 nmol/L of estradiol and were allowed to build biofilms at an air-solid interface. DPPIV activities of biofilms were measured kinetically during 20 min using a fluorometric assay. The enzyme activity was later related to the amount of protein produced by the same biofilm, reflecting the biofilm mass. Estradiol significantly increased DPPIV activities of the 8 Prevotella strains in a strain- and dose-dependent manner. In conclusion, our in vitro experiments indicate that estradiol regulates the DPPIV enzyme activity of P. intermedia, P. nigrescens, P. pallens, and P. aurantiaca strains differently. Our results may, at least partly, explain the role of estradiol to elicit a virulent state which contributes to the pathogenesis of pregnancy-related gingivitis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  6. Selected soil enzymes: Examples of their potential roles in the ...

    African Journals Online (AJOL)

    SERVER

    2008-02-05

    Feb 5, 2008 ... Soil enzymes regulate ecosystem functioning and in particular play a key role in nutrient cycling. In ... A better understanding of the role of these soil enzyme- es activity ..... measure of any disruption caused by pesticides, trace.

  7. A small-molecule inhibitor of the ubiquitin activating enzyme for cancer treatment.

    Science.gov (United States)

    Hyer, Marc L; Milhollen, Michael A; Ciavarri, Jeff; Fleming, Paul; Traore, Tary; Sappal, Darshan; Huck, Jessica; Shi, Judy; Gavin, James; Brownell, Jim; Yang, Yu; Stringer, Bradley; Griffin, Robert; Bruzzese, Frank; Soucy, Teresa; Duffy, Jennifer; Rabino, Claudia; Riceberg, Jessica; Hoar, Kara; Lublinsky, Anya; Menon, Saurabh; Sintchak, Michael; Bump, Nancy; Pulukuri, Sai M; Langston, Steve; Tirrell, Stephen; Kuranda, Mike; Veiby, Petter; Newcomb, John; Li, Ping; Wu, Jing Tao; Powe, Josh; Dick, Lawrence R; Greenspan, Paul; Galvin, Katherine; Manfredi, Mark; Claiborne, Chris; Amidon, Benjamin S; Bence, Neil F

    2018-02-01

    The ubiquitin-proteasome system (UPS) comprises a network of enzymes that is responsible for maintaining cellular protein homeostasis. The therapeutic potential of this pathway has been validated by the clinical successes of a number of UPS modulators, including proteasome inhibitors and immunomodulatory imide drugs (IMiDs). Here we identified TAK-243 (formerly known as MLN7243) as a potent, mechanism-based small-molecule inhibitor of the ubiquitin activating enzyme (UAE), the primary mammalian E1 enzyme that regulates the ubiquitin conjugation cascade. TAK-243 treatment caused depletion of cellular ubiquitin conjugates, resulting in disruption of signaling events, induction of proteotoxic stress, and impairment of cell cycle progression and DNA damage repair pathways. TAK-243 treatment caused death of cancer cells and, in primary human xenograft studies, demonstrated antitumor activity at tolerated doses. Due to its specificity and potency, TAK-243 allows for interrogation of ubiquitin biology and for assessment of UAE inhibition as a new approach for cancer treatment.

  8. Profiling the orphan enzymes

    Science.gov (United States)

    2014-01-01

    The emergence of Next Generation Sequencing generates an incredible amount of sequence and great potential for new enzyme discovery. Despite this huge amount of data and the profusion of bioinformatic methods for function prediction, a large part of known enzyme activities is still lacking an associated protein sequence. These particular activities are called “orphan enzymes”. The present review proposes an update of previous surveys on orphan enzymes by mining the current content of public databases. While the percentage of orphan enzyme activities has decreased from 38% to 22% in ten years, there are still more than 1,000 orphans among the 5,000 entries of the Enzyme Commission (EC) classification. Taking into account all the reactions present in metabolic databases, this proportion dramatically increases to reach nearly 50% of orphans and many of them are not associated to a known pathway. We extended our survey to “local orphan enzymes” that are activities which have no representative sequence in a given clade, but have at least one in organisms belonging to other clades. We observe an important bias in Archaea and find that in general more than 30% of the EC activities have incomplete sequence information in at least one superkingdom. To estimate if candidate proteins for local orphans could be retrieved by homology search, we applied a simple strategy based on the PRIAM software and noticed that candidates may be proposed for an important fraction of local orphan enzymes. Finally, by studying relation between protein domains and catalyzed activities, it appears that newly discovered enzymes are mostly associated with already known enzyme domains. Thus, the exploration of the promiscuity and the multifunctional aspect of known enzyme families may solve part of the orphan enzyme issue. We conclude this review with a presentation of recent initiatives in finding proteins for orphan enzymes and in extending the enzyme world by the discovery of new

  9. Immobilized enzymes: understanding enzyme - surface interactions at the molecular level.

    Science.gov (United States)

    Hoarau, Marie; Badieyan, Somayesadat; Marsh, E Neil G

    2017-11-22

    Enzymes immobilized on solid supports have important and industrial and medical applications. However, their uses are limited by the significant reductions in activity and stability that often accompany the immobilization process. Here we review recent advances in our understanding of the molecular level interactions between proteins and supporting surfaces that contribute to changes in stability and activity. This understanding has been facilitated by the application of various surface-sensitive spectroscopic techniques that allow the structure and orientation of enzymes at the solid/liquid interface to be probed, often with monolayer sensitivity. An appreciation of the molecular interactions between enzyme and surface support has allowed the surface chemistry and method of enzyme attachement to be fine-tuned such that activity and stability can be greatly enhanced. These advances suggest that a much wider variety of enzymes may eventually be amenable to immobilization as green catalysts.

  10. [Effects of bio-crust on soil microbial biomass and enzyme activities in copper mine tailings].

    Science.gov (United States)

    Chen, Zheng; Yang, Gui-de; Sun, Qing-ye

    2009-09-01

    Bio-crust is the initial stage of natural primary succession in copper mine tailings. With the Yangshanchong and Tongguanshan copper mine tailings in Tongling City of Anhui Province as test objects, this paper studied the soil microbial biomass C and N and the activities of dehydrogenase, catalase, alkaline phosphatase, and urease under different types of bio-crust. The bio-crusts improved the soil microbial biomass and enzyme activities in the upper layer of the tailings markedly. Algal crust had the best effect in improving soil microbial biomass C and N, followed by moss-algal crust, and moss crust. Soil microflora also varied with the type of bio-crust. No'significant difference was observed in the soil enzyme activities under the three types of bio-crust. Soil alkaline phosphatase activity was significantly positively correlated with soil microbial biomass and dehydrogenase and urease activities, but negatively correlated with soil pH. In addition, moss rhizoid could markedly enhance the soil microbial biomass and enzyme activities in moss crust rhizoid.

  11. Microbial respiration and kinetics of extracellular enzymes activities through rhizosphere and detritusphere at agricultural site

    Science.gov (United States)

    Löppmann, Sebastian; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2014-05-01

    detritivore communities in the soil. The kinetics (Km and Vmax) of four extracellular hydrolytic enzymes responsible for C- and phosphorous-cycle (β-glucosidase, β-xylosidase, β-cellobiohydrolase and acid phosphatase), microbial biomass, basal respiration (BR) and substrate-induced respiration (SIR) were measured in rhizosphere, detritusphere and control from 0 - 10 and 10 - 20 cm. The metabolic quotient (qCO2) was calculated as specific indicator for efficiency of microbial substrate utilization. We observed clear differences in enzymes activities at low and high concentrations of substrate. At substrate saturation enzyme activity rates of were significantly higher in rooted plots compared to litter amended plots, whereas at lower concentration no treatment effect could be found. The BR, SIR and qCO2 values were significantly higher at 0 - 10 cm of the planted treatment compared to litter and control plots, revealing a significantly higher respiration at lower efficiency of microbial substrate utilization in the rhizosphere. The Michaelis-Menten constant (Km) decreased with depth, especially for β-glucosidase, acid phosphatase and β-xylosidase, indicating higher substrate affinity of microorganisms in deeper soil and therefore different enzyme systems functioning. The substrate affinity factor (Vmax/Km) increased 2-fold with depth for various enzymes, reflecting a switch of predominantly occurring microbial strategies. Vmax/Km ratio indicated relative domination of zymogenous microbial communities (r-strategists) in 0 - 10 cm depth as compared with 10 - 20 cm depth where the K-strategists dominated.

  12. Functional diversity of carbohydrate-active enzymes enabling a bacterium to ferment plant biomass.

    Science.gov (United States)

    Boutard, Magali; Cerisy, Tristan; Nogue, Pierre-Yves; Alberti, Adriana; Weissenbach, Jean; Salanoubat, Marcel; Tolonen, Andrew C

    2014-11-01

    Microbial metabolism of plant polysaccharides is an important part of environmental carbon cycling, human nutrition, and industrial processes based on cellulosic bioconversion. Here we demonstrate a broadly applicable method to analyze how microbes catabolize plant polysaccharides that integrates carbohydrate-active enzyme (CAZyme) assays, RNA sequencing (RNA-seq), and anaerobic growth screening. We apply this method to study how the bacterium Clostridium phytofermentans ferments plant biomass components including glucans, mannans, xylans, galactans, pectins, and arabinans. These polysaccharides are fermented with variable efficiencies, and diauxies prioritize metabolism of preferred substrates. Strand-specific RNA-seq reveals how this bacterium responds to polysaccharides by up-regulating specific groups of CAZymes, transporters, and enzymes to metabolize the constituent sugars. Fifty-six up-regulated CAZymes were purified, and their activities show most polysaccharides are degraded by multiple enzymes, often from the same family, but with divergent rates, specificities, and cellular localizations. CAZymes were then tested in combination to identify synergies between enzymes acting on the same substrate with different catalytic mechanisms. We discuss how these results advance our understanding of how microbes degrade and metabolize plant biomass.

  13. Enzyme inhibitory and antioxidant activities of traditional medicinal plants: Potential application in the management of hyperglycemia

    Directory of Open Access Journals (Sweden)

    Gulati Vandana

    2012-06-01

    Full Text Available Abstract Background Traditional Indian and Australian medicinal plant extracts were investigated to determine their therapeutic potential to inhibit key enzymes in carbohydrate metabolism, which has relevance to the management of hyperglycemia and type 2 diabetes. The antioxidant activities were also assessed. Methods The evaluation of enzyme inhibitory activity of seven Australian aboriginal medicinal plants and five Indian Ayurvedic plants was carried out against α-amylase and α-glucosidase. Antioxidant activity was determined by measuring (i the scavenging effect of plant extracts against 2, 2-diphenyl-1-picryl hydrazyl (DPPH and 2, 2′-azinobis-3-ethylbenzothiazoline-6-sulfonate (ABTS and (ii ferric reducing power. Total phenolic and total flavonoid contents were also determined. Results Of the twelve plant extracts evaluated, the highest inhibitory activity against both α-amylase and α-glucosidase enzymes was exerted by Santalum spicatum and Pterocarpus marsupium with IC50 values of 5.43 μg/ml and 0.9 μg/ml, respectively, and 5.16 μg/ml and 1.06 μg/ml, respectively. However, the extracts of Acacia ligulata (IC50 = 1.01 μg/ml, Beyeria leshnaultii (0.39 μg/ml, Mucuna pruriens (0.8 μg/ml and Boerhaavia diffusa (1.72 μg/ml exhibited considerable activity against α-glucosidase enzyme only. The free radical scavenging activity was found to be prominent in extracts of Acacia kempeana, Acacia ligulata followed by Euphorbia drummondii against both DPPH and ABTS. The reducing power was more pronounced in Euphorbia drummondii and Pterocarpus marsupium extracts. The phenolic and flavonoid contents ranged from 0.42 to 30.27 μg/mg equivalent of gallic acid and 0.51 to 32.94 μg/mg equivalent of quercetin, respectively, in all plant extracts. Pearson’s correlation coefficient between total flavonoids and total phenolics was 0.796. Conclusion The results obtained in this study showed that most of the plant extracts

  14. Changes In Certain Enzymes Activities In Tribolium CONFUSUM As Affected By Vanillin Or GAMMA Irradiation

    International Nuclear Information System (INIS)

    MOHAMED, S.A.; SHOMAN, A.A.; AHMED, Z.A.

    2009-01-01

    The effect of 1 or 4 g vanillin/100 g whole wheat flour on the alkaline phosphatase of one day old larvae revealed that the mean enzyme activity was highly significantly increased in male and non-significant in female Triboluim confusum. As pupae were irradiated, the mean enzyme activity was significantly decreased in males and females (except at dose 300 Gy). Alanine transaminase (ALT or GPT) activity was decreased in males due to the effect of 4% vanillin and increased by irradiation while in female, the activity of ALT was increased when the larvae were reared on flour containing 1% or 4% vanillin and increased when pupae were irradiated at all doses used. There was a positive relationship between all treatments and the activity of aspartate transaminase (AST or GOT) in both sexes. The activity of AST was increased when the male or female larvae were reared on wheat flour containing 1 or 4 % vanillin and when pupae of males or females were irradiated. The choline esterase enzyme in T. confusum adults of both sexes was inhibited according to the effect of treatments with vanillin or gamma irradiation. Treated larvae with 1 or 4 % vanillin or irradiated as pupae at 300, 600 and 800 Gy led to decrease in the activity of choline esterase enzyme with the same pattern in both sexes.

  15. A modern mode of activation for nucleic acid enzymes.

    Directory of Open Access Journals (Sweden)

    Dominique Lévesque

    2007-07-01

    Full Text Available Through evolution, enzymes have developed subtle modes of activation in order to ensure the sufficiently high substrate specificity required by modern cellular metabolism. One of these modes is the use of a target-dependent module (i.e. a docking domain such as those found in signalling kinases. Upon the binding of the target to a docking domain, the substrate is positioned within the catalytic site. The prodomain acts as a target-dependent module switching the kinase from an off state to an on state. As compared to the allosteric mode of activation, there is no need for the presence of a third partner. None of the ribozymes discovered to date have such a mode of activation, nor does any other known RNA. Starting from a specific on/off adaptor for the hepatitis delta virus ribozyme, that differs but has a mechanism reminiscent of this signalling kinase, we have adapted this mode of activation, using the techniques of molecular engineering, to both catalytic RNAs and DNAs exhibiting various activities. Specifically, we adapted three cleaving ribozymes (hepatitis delta virus, hammerhead and hairpin ribozymes, a cleaving 10-23 deoxyribozyme, a ligating hairpin ribozyme and an artificially selected capping ribozyme. In each case, there was a significant gain in terms of substrate specificity. Even if this mode of control is unreported for natural catalytic nucleic acids, its use needs not be limited to proteinous enzymes. We suggest that the complexity of the modern cellular metabolism might have been an important selective pressure in this evolutionary process.

  16. Study of thermal and chemical effects on cellulase enzymes: Viscosity measurements

    International Nuclear Information System (INIS)

    Ghaouar, N.; Aschi, A.; Belbahri, L.; Trabelsi, S.; Gharbi, A.

    2009-01-01

    The behaviour of cellulase enzymes in phosphate saline buffer has been studied over a wide range of temperatures and enzyme concentrations by using viscosity measurements. To characterize the conformation change of cellulase versus temperature and chemical denaturants, such as guanidinium chloride (GdmCl) and urea, the information about the intrinsic viscosity and the hydrodynamic radius are necessary. The dependence of the intrinsic viscosity and the hydrodynamic radius in its random coil conformation on temperature and denaturant concentration were studied. Our results and discussions are limited to the dilute regime of concentration because of abnormalities in conformation observed in the very dilute regime due to the presence of capillary absorption effects.

  17. Study of thermal and chemical effects on cellulase enzymes: Viscosity measurements

    Energy Technology Data Exchange (ETDEWEB)

    Ghaouar, N., E-mail: naoufel-ghaouar@lycos.co [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia); Institut National des Sciences Appliquees et de Technologie, INSAT, Centre Urbain Nord, BP. 676, Tunis (Tunisia); Aschi, A. [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia); Belbahri, L. [Agronomy Department, School of Engineering of Lullier, University of Applied Sciences of Western Switzerland, 150, Route de Presinge, 1254 Jussy (Switzerland); Trabelsi, S.; Gharbi, A. [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 2092 (Tunisia)

    2009-11-15

    The behaviour of cellulase enzymes in phosphate saline buffer has been studied over a wide range of temperatures and enzyme concentrations by using viscosity measurements. To characterize the conformation change of cellulase versus temperature and chemical denaturants, such as guanidinium chloride (GdmCl) and urea, the information about the intrinsic viscosity and the hydrodynamic radius are necessary. The dependence of the intrinsic viscosity and the hydrodynamic radius in its random coil conformation on temperature and denaturant concentration were studied. Our results and discussions are limited to the dilute regime of concentration because of abnormalities in conformation observed in the very dilute regime due to the presence of capillary absorption effects.

  18. Nuclear Localization of Mitochondrial TCA Cycle Enzymes as a Critical Step in Mammalian Zygotic Genome Activation.

    Science.gov (United States)

    Nagaraj, Raghavendra; Sharpley, Mark S; Chi, Fangtao; Braas, Daniel; Zhou, Yonggang; Kim, Rachel; Clark, Amander T; Banerjee, Utpal

    2017-01-12

    Transcriptional control requires epigenetic changes directed by mitochondrial tricarboxylic acid (TCA) cycle metabolites. In the mouse embryo, global epigenetic changes occur during zygotic genome activation (ZGA) at the 2-cell stage. Pyruvate is essential for development beyond this stage, which is at odds with the low activity of mitochondria in this period. We now show that a number of enzymatically active mitochondrial enzymes associated with the TCA cycle are essential for epigenetic remodeling and are transiently and partially localized to the nucleus. Pyruvate is essential for this nuclear localization, and a failure of TCA cycle enzymes to enter the nucleus correlates with loss of specific histone modifications and a block in ZGA. At later stages, however, these enzymes are exclusively mitochondrial. In humans, the enzyme pyruvate dehydrogenase is transiently nuclear at the 4/8-cell stage coincident with timing of human embryonic genome activation, suggesting a conserved metabolic control mechanism underlying early pre-implantation development. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. Activation and thermostabilization effects of cyclic 2, 3-diphosphoglycerate on enzymes from the hyperthermophilic Methanopyrus kandleri.

    Science.gov (United States)

    Shima, S; Hérault, D A; Berkessel, A; Thauer, R K

    1998-11-01

    Enzymes involved in methane formation from carbon dioxide and dihydrogen in Methanopyrus kandleri require high concentrations (> 1 M) of lyotropic salts such as K2HPO4/KH2PO4 or (NH4)2SO4 for activity and for thermostability. The requirement correlates with high intracellular concentrations of cyclic 2,3-diphosphoglycerate (cDPG; approximately 1 M) in this hyperthermophilic organism. We report here on the effects of potassium cDPG on the activity and thermostability of the two methanogenic enzymes cyclohydrolase and formyltransferase and show that at cDPG concentrations prevailing in the cells the investigated enzymes are highly active and completely thermostable. At molar concentrations also the potassium salts of phosphate and of 2,3-bisphosphoglycerate, the biosynthetic precursor of cDPG, were found to confer activity and thermostability to the enzymes. Thermodynamic arguments are discussed as to why cDPG, rather than these salts, is present in high concentrations in the cells of Mp. kandleri.

  20. Nanomaterials with enzyme-like characteristics (nanozymes): next-generation artificial enzymes.

    Science.gov (United States)

    Wei, Hui; Wang, Erkang

    2013-07-21

    Over the past few decades, researchers have established artificial enzymes as highly stable and low-cost alternatives to natural enzymes in a wide range of applications. A variety of materials including cyclodextrins, metal complexes, porphyrins, polymers, dendrimers and biomolecules have been extensively explored to mimic the structures and functions of naturally occurring enzymes. Recently, some nanomaterials have been found to exhibit unexpected enzyme-like activities, and great advances have been made in this area due to the tremendous progress in nano-research and the unique characteristics of nanomaterials. To highlight the progress in the field of nanomaterial-based artificial enzymes (nanozymes), this review discusses various nanomaterials that have been explored to mimic different kinds of enzymes. We cover their kinetics, mechanisms and applications in numerous fields, from biosensing and immunoassays, to stem cell growth and pollutant removal. We also summarize several approaches to tune the activities of nanozymes. Finally, we make comparisons between nanozymes and other catalytic materials (other artificial enzymes, natural enzymes, organic catalysts and nanomaterial-based catalysts) and address the current challenges and future directions (302 references).

  1. Effect of whole body neutron irradiation on certain enzyme activities in different brain areas in mice

    International Nuclear Information System (INIS)

    Kotb, M.A.; Ashour, A.M.; El-Bassiouni, E.A.

    1994-01-01

    Male swiss albino mice were exposed to whole-body irradiation by fast neutrons of 14 MeV average energy. Two single doses of 0.08 sievert and 0.16 sievert were used, corresponding to fluences of 1.27 X 10 8 and 2.54 X 10 8 n/cm 2 respectively. Two enzymes were assessed in different layers of the cerebrum and cerebellum of mouse brain. Changes in the activities of acid phosphatase (ACP) and succinic dehydrogenase (SDH) were taken to measure alterations in lysosomal and mitochondrial functions respectively. The degrees of lysosomal affection in different layers of the cerebrum were not uniform, while changes in A activity were very prominent in certain layers (e.g. external pyramidal layer, polymorphous cells layer and white matter), they were practically absent in others (e.g. internal pyramidal layer). Stronger effect was noted in the tissue layers of the cerebellum. The activity of SDH decreased as result of fast neutron irradiation. The response was more apparent for this enzyme than for ACP. This indicates more liability for a decrease in energy metabolism with consequent effect on behavioural and physiological functions under central nervous system control. 4 figs., 4 tabs

  2. Changes of Enzymes Activity and Production of Secondary Metabolites of Artemisia aucheri in Different Altitudes and Its Relation to Adaptation

    Directory of Open Access Journals (Sweden)

    Hassan Zare-maivan

    2014-08-01

    Full Text Available Artemisia plants are the most abundant plants species in Iran which contain strong antioxidant properties and as such, have medicinal and economic value. Despite wide distribution of Artemisisa species, ecophysiology of its adaptation to changes in altitude and soil property had not been investigated. In this study, the relationships between ecophysiological and adaptation capabilities of A. aucheri to altitude changes through measuring changes in the activity of its antioxidant enzymes and secondary metabolites in situ was investigated based on a completely randomized experiment. The enzyme activities of superoxide dismutase, catalase, peroxidase, and the amount of total phenolics, flavonoids, anthocyanins, malondialdehyde and chlorophylls A and B were measured in A. aucheri plants growing in three different altitudes at and above the 36° latitude on the southern slopes of Eastern Alborz Mountain ranges in triplicate 10*10 m quadrates. Statistical analysis of data showed that soil type was loamy significantly becoming more sandy- loam with lowering in altitude and the soil contained greater amounts of oxides of silicone, aluminum, magnesium, sodium, potassium and phosphorus in upper altitude except calcium which was present in greater quantity in lower altitude. With increasing altitude, activity of superoxide dismutase and quantities of chlorophylls and total phenols in leaves increased. Some biochemical factors in A. aucheri showed significant positive correlation(P ≤ 0.05 between them. Adaptation of A. aucheri to changes in altitude occurred through changing its antioxidant enzymes activity and production of secondary metabolites in response to factors related to the altitude including soil type and texture, moisture level, temperature and most importantly radiation

  3. Modeling the effects of tree species and incubation temperature on soil's extracellular enzyme activity in 78-year-old tree plantations

    Science.gov (United States)

    Zhou, Xiaoqi; Wang, Shen S. J.; Chen, Chengrong

    2017-12-01

    Forest plantations have been widely used as an effective measure for increasing soil carbon (C), and nitrogen (N) stocks and soil enzyme activities play a key role in soil C and N losses during decomposition of soil organic matter. However, few studies have been carried out to elucidate the mechanisms behind the differences in soil C and N cycling by different tree species in response to climate warming. Here, we measured the responses of soil's extracellular enzyme activity (EEA) to a gradient of temperatures using incubation methods in 78-year-old forest plantations with different tree species. Based on a soil enzyme kinetics model, we established a new statistical model to investigate the effects of temperature and tree species on soil EEA. In addition, we established a tree species-enzyme-C/N model to investigate how temperature and tree species influence soil C/N contents over time without considering plant C inputs. These extracellular enzymes included C acquisition enzymes (β-glucosidase, BG), N acquisition enzymes (N-acetylglucosaminidase, NAG; leucine aminopeptidase, LAP) and phosphorus acquisition enzymes (acid phosphatases). The results showed that incubation temperature and tree species significantly influenced all soil EEA and Eucalyptus had 1.01-2.86 times higher soil EEA than coniferous tree species. Modeling showed that Eucalyptus had larger soil C losses but had 0.99-2.38 times longer soil C residence time than the coniferous tree species over time. The differences in the residual soil C and N contents between Eucalyptus and coniferous tree species, as well as between slash pine (Pinus elliottii Engelm. var. elliottii) and hoop pine (Araucaria cunninghamii Ait.), increase with time. On the other hand, the modeling results help explain why exotic slash pine can grow faster, as it has 1.22-1.38 times longer residual soil N residence time for LAP, which mediate soil N cycling in the long term, than native coniferous tree species like hoop pine and

  4. In vitro inhibitory activities of selected Australian medicinal plant extracts against protein glycation, angiotensin converting enzyme (ACE) and digestive enzymes linked to type II diabetes.

    Science.gov (United States)

    Deo, Permal; Hewawasam, Erandi; Karakoulakis, Aris; Claudie, David J; Nelson, Robert; Simpson, Bradley S; Smith, Nicholas M; Semple, Susan J

    2016-11-04

    There is a need to develop potential new therapies for the management of diabetes and hypertension. Australian medicinal plants collected from the Kuuku I'yu (Northern Kaanju) homelands, Cape York Peninsula, Queensland, Australia were investigated to determine their therapeutic potential. Extracts were tested for inhibition of protein glycation and key enzymes relevant to the management of hyperglycaemia and hypertension. The inhibitory activities were further correlated with the antioxidant activities. Extracts of five selected plant species were investigated: Petalostigma pubescens, Petalostigma banksii, Memecylon pauciflorum, Millettia pinnata and Grewia mesomischa. Enzyme inhibitory activity of the plant extracts was assessed against α-amylase, α-glucosidase and angiotensin converting enzyme (ACE). Antiglycation activity was determined using glucose-induced protein glycation models and formation of protein-bound fluorescent advanced glycation endproducts (AGEs). Antioxidant activity was determined by measuring the scavenging effect of plant extracts against 1, 1-diphenyl-2-picryl hydrazyl (DPPH) and using the ferric reducing anti-oxidant potential assay (FRAP). Total phenolic and flavonoid contents were also determined. Extracts of the leaves of Petalostigma banksii and P. pubescens showed the strongest inhibition of α-amylase with IC 50 values of 166.50 ± 5.50 μg/mL and 160.20 ± 27.92 μg/mL, respectively. The P. pubescens leaf extract was also the strongest inhibitor of α-glucosidase with an IC 50 of 167.83 ± 23.82 μg/mL. Testing for the antiglycation potential of the extracts, measured as inhibition of formation of protein-bound fluorescent AGEs, showed that P. banksii root and fruit extracts had IC 50 values of 34.49 ± 4.31 μg/mL and 47.72 ± 1.65 μg/mL, respectively, which were significantly lower (p < 0.05) than other extracts. The inhibitory effect on α-amylase, α-glucosidase and the antiglycation potential of

  5. EFFECTS OF CIGARETTE SMOKING ON ERYTHROCYTE ANTIOXIDATIVE ENZYME ACTIVITIES AND PLASMA CONCENTRATIONS OF THEIR COFACTORS

    Directory of Open Access Journals (Sweden)

    M. Zahraie

    2005-07-01

    Full Text Available Tobacco smoke contains numerous compounds, many ‎of which are oxidants and capable of producing free radical and enhancing ‎the oxidative stress. The aim of this study was to investigate the effect of cigarette smoking on the erythrocyte antioxidative enzyme activities and the plasma ‎concentration of their cofactors. ‎Sixty eight healthy men were enrolled, 32 of whom had never smoked and 36 had smoked at least 10 cigarettes per day for ‎at least one year. Hemolysate superoxide dismutase (Cu-Zn SOD, glutathione peroxidase (GSH-Px and ‎catalase (CAT activities were measured using spectrophotometer. Plasma copper, zinc and selenium concentrations were determined ‎using atomic absorption spectrophotometer. Plasma iron concentration was determined by colorimetric ‎method. We found that erythrocyte Cu-Zn SOD activity was significantly higher in tobacco smokers ‎compared with non-smokers (1294 ± 206.7 U/gHb in smokers vs. 1121.6 ± 237.8 U/gHb in non-‎smokers, P < 0.01. While plasma selenium concentration was significantly lower in tobacco ‎smokers (62.7±14.8 μg/L in smokers vs. 92.1 ± 17.5 μg/L in non-smokers, P < 0.01, there were no significant ‎differences in erythrocyte GSH-Px and CAT activities and plasma copper, zinc and iron concentrations between the two groups. ‎It seems that cigarette smoking can alter antioxidative enzymes activity and plasma concentration of some trace elements.

  6. Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation.

    Science.gov (United States)

    Balamurugan, Appakalai N; Green, Michael L; Breite, Andrew G; Loganathan, Gopalakrishnan; Wilhelm, Joshua J; Tweed, Benjamin; Vargova, Lenka; Lockridge, Amber; Kuriti, Manikya; Hughes, Michael G; Williams, Stuart K; Hering, Bernhard J; Dwulet, Francis E; McCarthy, Robert C

    2016-01-01

    Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

  7. Enzyme clusters during the metamorphic period of Ambystoma mexicanum: role of thyroid hormone

    NARCIS (Netherlands)

    Lamers, W. H.; Mooren, P. G.; de Graaf, A.

    1982-01-01

    Enzyme activities and DNA content have been measure in axolotl liver during the metamorphic period (4-8 months after spawning). Three different types of enzyme activity profiles were observed. In the type I profile (carbamoyl-phosphate synthase, arginase, ornithine transcarbamoylase, and glutamate

  8. A new versatile microarray-based method for high-throughput screening of carbohydrate-active enzymes

    DEFF Research Database (Denmark)

    Vidal Melgosa, Silvia; Pedersen, Henriette Lodberg; Schückel, Julia

    2015-01-01

    Carbohydrate-active enzymes have multiple biological roles and industrial applications. Advances in genome and transcriptome sequencing, together with associated bioinformatic tools have identified vast numbers of putative carbohydrate degrading and modifying enzymes including glycoside hydrolases...... that the technique can be used to analyse both endo-acting and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and lytic polysaccharide monooxygenases. We demonstrate the potential of the technique by identifying the substrate specificities of purified un-characterised enzymes...

  9. Thioredoxin 1 in Prostate Tissue Is Associated with Gleason Score, Erythrocyte Antioxidant Enzyme Activity, and Dietary Antioxidants

    Directory of Open Access Journals (Sweden)

    Terrence M. Vance

    2015-01-01

    Full Text Available Background. Prostate cancer is the most common noncutaneous cancer and second leading cause of cancer-related mortality in men in the US. Growing evidence suggests that oxidative stress is involved in prostate cancer. Methods. In this study, thioredoxin 1 (Trx 1, an enzyme and subcellular indicator of redox status, was measured in prostate biopsy tissue from 55 men from the North Carolina-Louisiana Prostate Cancer Project. A pathologist blindly scored levels of Trx 1. The association between Trx 1 and the Gleason score, erythrocyte antioxidant enzyme activity, and dietary antioxidant intake was determined using Fisher’s exact test. Results. Trx 1 levels in benign prostate tissue in men with incident prostate cancer were positively associated with the Gleason score (P=0.01 and inversely associated with dietary antioxidant intake (P=0.03. In prostate cancer tissue, Trx 1 levels were associated with erythrocyte glutathione peroxidase activity (P=0.01. No association was found for other erythrocyte enzymes. Greater Gleason score of malignant tissue corresponds to a greater difference in Trx 1 levels between malignant and benign tissue (P=0.04. Conclusion. These results suggest that the redox status of prostate tissue is associated with prostate cancer grade and both endogenous and exogenous antioxidants.

  10. Effect of high temperature on grain filling period, yield, amylose content and activity of starch biosynthesis enzymes in endosperm of basmati rice.

    Science.gov (United States)

    Ahmed, Nisar; Tetlow, Ian J; Nawaz, Sehar; Iqbal, Ahsan; Mubin, Muhammad; Nawaz ul Rehman, Muhammad Shah; Butt, Aisha; Lightfoot, David A; Maekawa, Masahiko

    2015-08-30

    High temperature during grain filling affects yield, starch amylose content and activity of starch biosynthesis enzymes in basmati rice. To investigate the physiological mechanisms underpinning the effects of high temperature on rice grain, basmati rice was grown under two temperature conditions - 32 and 22 °C - during grain filling. High temperature decreased the grain filling period from 32 to 26 days, reducing yield by 6%, and caused a reduction in total starch (3.1%) and amylose content (22%). Measurable activities of key enzymes involved in sucrose to starch conversion, sucrose synthase, ADP-glucose pyrophosphorylase, starch phosphorylase and soluble starch synthase in endosperms developed at 32 °C were lower than those at 22 °C compared with similar ripening stage on an endosperm basis. In particular, granule-bound starch synthase (GBSS) activity was significantly lower than corresponding activity in endosperms developing at 22 °C during all developmental stages analyzed. Results suggest changes in amylose/amylopectin ratio observed in plants grown at 32 °C was attributable to a reduction in activity of GBSS, the sole enzyme responsible for amylose biosynthesis. © 2014 Society of Chemical Industry.

  11. Effect of norfloxacin and moxifloxacin on melanin synthesis and antioxidant enzymes activity in normal human melanocytes.

    Science.gov (United States)

    Beberok, Artur; Wrześniok, Dorota; Otręba, Michał; Miliński, Maciej; Rok, Jakub; Buszman, Ewa

    2015-03-01

    Fluoroquinolone antibiotics provide broad-spectrum coverage for a number of infectious diseases, including respiratory as well as urinary tract infections. One of the important adverse effects of these drugs is phototoxicity which introduces a serious limitation to their use. To gain insight the molecular mechanisms underlying the fluoroquinolones-induced phototoxic side effects, the impact of two fluoroquinolone derivatives with different phototoxic potential, norfloxacin and moxifloxacin, on melanogenesis and antioxidant enzymes activity in normal human melanocytes HEMa-LP was determined. Both drugs induced concentration-dependent loss in melanocytes viability. The value of EC50 for these drugs was found to be 0.5 mM. Norfloxacin and moxifloxacin suppressed melanin biosynthesis; antibiotics were shown to inhibit cellular tyrosinase activity and to reduce melanin content in melanocytes. When comparing the both analyzed fluoroquinolones, it was observed that norfloxacin possesses greater inhibitory effect on tyrosinase activity in melanocytes than moxifloxacin. The extent of oxidative stress in cells was assessed by measuring the activity of antioxidant enzymes: SOD, CAT, and GPx. It was observed that norfloxacin caused higher depletion of antioxidant status in melanocytes when compared with moxifloxacin. The obtained results give a new insight into the mechanisms of fluoroquinolones toxicity directed to pigmented tissues. Moreover, the presented differences in modulation of biochemical processes in melanocytes may be an explanation for various phototoxic activities of the analyzed fluoroquinolone derivatives in vivo.

  12. Diel changes in stream periphyton extracellular enzyme activity throughout community development on inert and organic substrates

    Science.gov (United States)

    Rier, S. T.; Francoeur, S. N.; Kuehn, K. A.

    2005-05-01

    We tested the hypothesis that algal photosynthesis in stream periphyton communities would influence the activities of extracellular enzymes produced by associated heterotrophic bacteria and fungi to acquire organic compounds and inorganic nutrients. We approached this question by looking for diurnal variation in activities of four extracellular enzymes in periphyton communities that were grown on either inert (glass fiber filters) or organic (leaves) substrata that there were incubated in stream-side channels that were either open to full sun or shaded. Substrata were subsampled for β-glucosidase, alkaline phosphotase, leucine-aminopeptidase, and phenol oxidase activities at 3-5 hr. intervals over two consecutive diurnal cycles that were repeated at an early and later stage of periphyton community development. Activities of all enzymes displayed diurnal periodicity but the strength of the diurnal effects depended largely on the substrate type and stage of community development. The most consistent diurnal change was observed with phenol oxidase activity with significantly greater (p<0.05) activities being observed in during the day for both stages of community development and for both substrate types. It is likely that oxygen produced by algal photosynthesis is driving the activity of this oxidative enzyme and that algae might indirectly influence the decomposition of phenolic compounds.

  13. Effect of sprint cycle training on activities of antioxidant enzymes in human skeletal muscle

    DEFF Research Database (Denmark)

    Hellsten, Ylva; Apple, F. S.; Sjödin, B.

    1996-01-01

    (P anaerobic capacity in the trained muscle. The present study demonstrates that intermittent sprint cycle training that induces an enhanced capacity for anaerobic energy generation also improves......The effect of intermittent sprint cycle training on the level of muscle antioxidant enzyme protection was investigated. Resting muscle biopsies, obtained before and after 6 wk of training and 3, 24, and 72 h after the final session of an additional 1 wk of more frequent training, were analyzed...... for activities of the antioxidant enzymes glutathione peroxidase (GPX), glutathione reductase (GR), and superoxide dismutase (SOD). Activities of several muscle metabolic enzymes were determined to assess the effectiveness of the training. After the first 6-wk training period, no change in GPX, GR, or SOD...

  14. Variation in pH optima of hydrolytic enzyme activities in tropical rain forest soils.

    Science.gov (United States)

    Turner, Benjamin L

    2010-10-01

    Extracellular enzymes synthesized by soil microbes play a central role in the biogeochemical cycling of nutrients in the environment. The pH optima of eight hydrolytic enzymes involved in the cycles of carbon, nitrogen, phosphorus, and sulfur, were assessed in a series of tropical forest soils of contrasting pH values from the Republic of Panama. Assays were conducted using 4-methylumbelliferone-linked fluorogenic substrates in modified universal buffer. Optimum pH values differed markedly among enzymes and soils. Enzymes were grouped into three classes based on their pH optima: (i) enzymes with acidic pH optima that were consistent among soils (cellobiohydrolase, β-xylanase, and arylsulfatase), (ii) enzymes with acidic pH optima that varied systematically with soil pH, with the most acidic pH optima in the most acidic soils (α-glucosidase, β-glucosidase, and N-acetyl-β-glucosaminidase), and (iii) enzymes with an optimum pH in either the acid range or the alkaline range depending on soil pH (phosphomonoesterase and phosphodiesterase). The optimum pH values of phosphomonoesterase were consistent among soils, being 4 to 5 for acid phosphomonoesterase and 10 to 11 for alkaline phosphomonoesterase. In contrast, the optimum pH for phosphodiesterase activity varied systematically with soil pH, with the most acidic pH optima (3.0) in the most acidic soils and the most alkaline pH optima (pH 10) in near-neutral soils. Arylsulfatase activity had a very acidic optimum pH in all soils (pH ≤3.0) irrespective of soil pH. The differences in pH optima may be linked to the origins of the enzymes and/or the degree of stabilization on solid surfaces. The results have important implications for the interpretation of hydrolytic enzyme assays using fluorogenic substrates.

  15. Effects of heavy metal pollution on enzyme activities in railway cut slope soils.

    Science.gov (United States)

    Meng, Xiaoyi; Ai, Yingwei; Li, Ruirui; Zhang, Wenjuan

    2018-03-07

    Railway transportation is an important transportation mode. However, railway transportation causes heavy metal pollution in surrounding soils. Heavy metal pollution has a serious negative impact on the natural environment, including a decrease of enzyme activities in soil and degradation of sensitive ecosystems. Some studies investigated the heavy metal pollution at railway stations or certain transportation hubs. However, the pollution accumulated in artificial cut slope soil all along the rails is still questioned. The interest on non-point source pollution from railways is increasing in an effort to protect the soil quality along the line. In this study, we studied spatial distributions of heavy metals and five enzyme activities, i.e., urease (UA), saccharase (SAC), protease (PRO), catalase (CAT), and polyphenol oxidase (POA) in the soil, and the correlation among them beside three different railways in Sichuan Province, China, as well. Soil samples were respectively collected from 5, 10, 25, 50, 100, and 150 m away from the rails (depth of 0-8 cm). Results showed that Mn, Cd, Cu, and Zn were influenced by railway transportation in different degrees while Pb was not. Heavy metal pollution was due to the abrasion of the gravel bed as well as the tracks and freight transportation which caused more heavy metal pollution than passenger transportation. Enzymatic activities were significantly negatively correlated with heavy metals in soils, especially Zn and Cu. Finally, it is proposed that combined use of PRO and POA activities could be an indicator of the heavy metal pollution in cut slope soils. The protective measures aimed at heavy metal pollution caused by railway transportation in cut slope soils are urgent.

  16. The effect of aluminium on enzyme activities in two wheat cultivars ...

    African Journals Online (AJOL)

    The effect of aluminium on enzyme activities in two wheat cultivars. ... African Journal of Biotechnology ... and Maroon (Al-tolerant) were grown on hydroponic solution (non modified Hoagland solution) containing AlCl3 (0-100-200-300 M).

  17. Enzyme activities at different stages of plant biomass decomposition in three species of fungus-growing termites

    DEFF Research Database (Denmark)

    da Costa, Rafael R.; Hu, Haofu; Pilgaard, Bo

    2018-01-01

    contributing to the success of the termites as the main plant decomposers in the Old World. Here we evaluate which plant polymers are decomposed and which enzymes are active during the decomposition process in two major genera of fungus-growing termites. We find a diversity of active enzymes at different...... stages of decomposition and a consistent decrease in plant components during the decomposition process. Furthermore, our findings are consistent with the hypothesis that termites transport enzymes from the older mature parts of the fungus comb through young worker guts to freshly inoculated plant...... substrate. However, preliminary fungal RNAseq analyses suggest that this likely transport is supplemented with enzymes produced in situ Our findings support that the maintenance of an external fungus comb, inoculated with an optimal mix of plant material, fungal spores, and enzymes, is likely the key...

  18. Analysis of soil microbial community structure and enzyme activities associated with negative effects of pseudostellaria heterophylla consecutive monoculture on yield

    International Nuclear Information System (INIS)

    Lin, S.; Lin, W.X.

    2015-01-01

    Pseudostellaria heterophylla is an important medicinal plant in China. However, cultivation of P. heterophylla using consecutive monoculture results in significant reductions in yield and quality. In this study, terminal-restriction fragment length polymorphism (T-RFLP) analysis and measurement of soil enzyme activities were used to investigate the regulation of soil micro-ecology to identify ways to overcome the negative effects of P. heterophylla consecutive monoculture. T-RFLP analysis showed that rice/P. heterophylla (RP) and bean/P. heterophylla (BP) crop rotation systems increased the number and diversity of microbial groups in P. heterophylla rhizosphere soil. In particular, the RP and BP crop rotations increased the number and abundance of beneficial bacterial species compared with two-year consecutive monoculture of P. heterophylla. The presence of these beneficial bacteria was positively correlated with soil enzyme activities which increased in rhizosphere soils of the RP and BP crop rotation systems. The results indicated that crop rotation systems could increase activities of key soil enzymes and beneficial microbial groups and improve soil health. This study could provide a theoretical basis to resolve the problems associated with P. heterophylla consecutive monoculture. (author)

  19. Effects of Heat Shock on Photosynthetic Properties, Antioxidant Enzyme Activity, and Downy Mildew of Cucumber (Cucumis sativus L..

    Directory of Open Access Journals (Sweden)

    Xiaotao Ding

    Full Text Available Heat shock is considered an abiotic stress for plant growth, but the effects of heat shock on physiological responses of cucumber plant leaves with and without downy mildew disease are still not clear. In this study, cucumber seedlings were exposed to heat shock in greenhouses, and the responses of photosynthetic properties, carbohydrate metabolism, antioxidant enzyme activity, osmolytes, and disease severity index of leaves with or without the downy mildew disease were measured. Results showed that heat shock significantly decreased the net photosynthetic rate, actual photochemical efficiency, photochemical quenching coefficient, and starch content. Heat shock caused an increase in the stomatal conductance, transpiration rate, antioxidant enzyme activities, total soluble sugar content, sucrose content, soluble protein content and proline content for both healthy leaves and downy mildew infected leaves. These results demonstrate that heat shock activated the transpiration pathway to protect the photosystem from damage due to excess energy in cucumber leaves. Potential resistance mechanisms of plants exposed to heat stress may involve higher osmotic regulation capacity related to an increase of total accumulations of soluble sugar, proline and soluble protein, as well as higher antioxidant enzymes activity in stressed leaves. Heat shock reduced downy mildew disease severity index by more than 50%, and clearly alleviated downy mildew development in the greenhouses. These findings indicate that cucumber may have a complex physiological change to resist short-term heat shock, and suppress the development of the downy mildew disease.

  20. Boosting the oxidase mimicking activity of nanoceria by fluoride capping: rivaling protein enzymes and ultrasensitive F- detection

    Science.gov (United States)

    Liu, Biwu; Huang, Zhicheng; Liu, Juewen

    2016-07-01

    Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement.Nanomaterial-based enzyme mimics (nanozymes) are currently a new forefront of chemical research. However, the application of nanozymes is limited by their low catalytic activity and low turnover numbers. Cerium dioxide nanoparticles (nanoceria) are among the few with oxidase activity. Herein, we report an interesting finding addressing their limitations. The oxidase activity of nanoceria is improved by over 100-fold by fluoride capping, making it more close to real oxidases. The turnover number reached 700 in 15 min, drastically improved from ~15 turnovers for the naked particles. The mechanism is attributed to surface charge modulation and facilitated electron transfer by F- capping based on ζ-potential and free radical measurements. Ultrasensitive sensing of fluoride was achieved with a detection limit of 0.64 μM F- in water and in toothpastes, while no other tested anions can achieve the activity enhancement. Electronic supplementary information (ESI) available: Methods, TMB oxidation kinetics and control experiments. See DOI: 10.1039/c6nr02730j

  1. Identification of 5'-adenylylimidodiphosphate-hydrolyzing enzyme activity in rabbit taste bud cells using X-ray microanalysis

    International Nuclear Information System (INIS)

    Asanuma, N.

    1990-01-01

    X-ray microanalysis has been used to characterize the enzyme activity hydrolyzing the ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) in taste bud cells. Rabbit foliate papillae fixed with paraformaldehyde and glutaraldehyde were incubated cytochemically with AMP-PNP as the substrate and lead ion as capture agent. The reaction product which appeared on the microvilli of taste bud cells was examined using an energy dispersive X-ray microanalyzer connected to an analytical electron microscope. The X-ray spectrum thus obtained was compared with that obtained from the product obtained from the demonstration of ATPase activity. Comparison of the phosphorus/lead ratios in the two products showed that twice as much phosphorus was released from an AMP-PNP molecule by the activity in question compared with that released from an ATP molecule by ATPase activity. This indicates that the enzyme hydrolyzes AMP-PNP into AMP and imidodiphosphate and that the enzyme is adenylate cyclase or ATP pyrophosphohydrolase, which possesses a similar hydrolytic property, but not ATPase or alkaline phosphatase, which hydrolyzes AMP-PNP into ADP-NH2 and orthophosphate. This paper provides an example of the use of X-ray microanalysis as a tool for enzyme distinction. The method is applicable to a variety of enzymes and tissues

  2. Serum prolidase enzyme activity in obese subjects and its relationship with oxidative stress markers.

    Science.gov (United States)

    Aslan, Mehmet; Duzenli, Ufuk; Esen, Ramazan; Soyoral, Yasemin Usul

    2017-10-01

    The relationship between increased serum enzyme activity of prolidase and increased rate of collagen turnover in the arterial wall has been asserted in previous studies. Collagen reflects much of the strength to the connective tissue involved in the arterial wall. Atherosclerosis is very common vessel disease and oxidative stress plays a pivotal role in the etiopathogenesis. Our objective was to examine the serum enzyme activity of prolidase and its possible relationships with oxidative stress parameters in obese subjects. Our present study was conducted 27 obese subjects and 26 age-matched healthy control subjects. The serum enzyme activity of prolidase in all study population was evaluated spectrophotometrically. Oxidative stress levels in obese subjects were analyzed with total antioxidant capacity (TAC) and total oxidant status (TOS) as well as oxidative stress index (OSI). Obese subjects have higher serum TOS and OSI indicators as well as prolidase activity than those in control subjects (for all; pstress levels in obese subjects. The significantly correlation between increased oxidative stress and increased prolidase activity may play a pivotal role in etiopathogenesis of atherosclerotic cardiovascular diseases in obese subjects. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Tumour Microenvironments Induce Expression of Urokinase Plasminogen Activator Receptor (uPAR) and Concomitant Activation of Gelatinolytic Enzymes

    Science.gov (United States)

    Magnussen, Synnøve; Hadler-Olsen, Elin; Latysheva, Nadezhda; Pirila, Emma; Steigen, Sonja E.; Hanes, Robert; Salo, Tuula; Winberg, Jan-Olof; Uhlin-Hansen, Lars; Svineng, Gunbjørg

    2014-01-01

    Background The urokinase plasminogen activator receptor (uPAR) is associated with poor prognosis in oral squamous cell carcinoma (OSCC), and increased expression of uPAR is often found at the invasive tumour front. The aim of the current study was to elucidate the role of uPAR in invasion and metastasis of OSCC, and the effects of various tumour microenvironments in these processes. Furthermore, we wanted to study whether the cells’ expression level of uPAR affected the activity of gelatinolytic enzymes. Methods The Plaur gene was both overexpressed and knocked-down in the murine OSCC cell line AT84. Tongue and skin tumours were established in syngeneic mice, and cells were also studied in an ex vivo leiomyoma invasion model. Soluble factors derived from leiomyoma tissue, as well as purified extracellular matrix (ECM) proteins, were assessed for their ability to affect uPAR expression, glycosylation and cleavage. Activity of gelatinolytic enzymes in the tissues were assessed by in situ zymography. Results We found that increased levels of uPAR did not induce tumour invasion or metastasis. However, cells expressing low endogenous levels of uPAR in vitro up-regulated uPAR expression both in tongue, skin and leiomyoma tissue. Various ECM proteins had no effect on uPAR expression, while soluble factors originating from the leiomyoma tissue increased both the expression and glycosylation of uPAR, and possibly also affected the proteolytic processing of uPAR. Tumours with high levels of uPAR, as well as cells invading leiomyoma tissue with up-regulated uPAR expression, all displayed enhanced activity of gelatinolytic enzymes. Conclusions Although high levels of uPAR are not sufficient to induce invasion and metastasis, the activity of gelatinolytic enzymes was increased. Furthermore, several tumour microenvironments have the capacity to induce up-regulation of uPAR expression, and soluble factors in the tumour microenvironment may have an important role in the

  4. Tumour microenvironments induce expression of urokinase plasminogen activator receptor (uPAR and concomitant activation of gelatinolytic enzymes.

    Directory of Open Access Journals (Sweden)

    Synnøve Magnussen

    Full Text Available The urokinase plasminogen activator receptor (uPAR is associated with poor prognosis in oral squamous cell carcinoma (OSCC, and increased expression of uPAR is often found at the invasive tumour front. The aim of the current study was to elucidate the role of uPAR in invasion and metastasis of OSCC, and the effects of various tumour microenvironments in these processes. Furthermore, we wanted to study whether the cells' expression level of uPAR affected the activity of gelatinolytic enzymes.The Plaur gene was both overexpressed and knocked-down in the murine OSCC cell line AT84. Tongue and skin tumours were established in syngeneic mice, and cells were also studied in an ex vivo leiomyoma invasion model. Soluble factors derived from leiomyoma tissue, as well as purified extracellular matrix (ECM proteins, were assessed for their ability to affect uPAR expression, glycosylation and cleavage. Activity of gelatinolytic enzymes in the tissues were assessed by in situ zymography.We found that increased levels of uPAR did not induce tumour invasion or metastasis. However, cells expressing low endogenous levels of uPAR in vitro up-regulated uPAR expression both in tongue, skin and leiomyoma tissue. Various ECM proteins had no effect on uPAR expression, while soluble factors originating from the leiomyoma tissue increased both the expression and glycosylation of uPAR, and possibly also affected the proteolytic processing of uPAR. Tumours with high levels of uPAR, as well as cells invading leiomyoma tissue with up-regulated uPAR expression, all displayed enhanced activity of gelatinolytic enzymes.Although high levels of uPAR are not sufficient to induce invasion and metastasis, the activity of gelatinolytic enzymes was increased. Furthermore, several tumour microenvironments have the capacity to induce up-regulation of uPAR expression, and soluble factors in the tumour microenvironment may have an important role in the regulation of posttranslational

  5. Biological energy from the igneous rock enhances cell growth and enzyme activity

    International Nuclear Information System (INIS)

    Lin Y.-L.; Kuo, H.-S; Chen, C.-T.; Kuo, S.-C.

    2000-01-01

    Some effects from natural resources might be ignored and unused by humans. Environmental hormesis could be a phenomena necessary to bio-organism existence on earth. Since 1919, radiation and some heavy metal hormesis from the environment were proved in various reports. In this study, igneous rock with very low radioactivity and high ferrous activity was measured by multichannel analyzer and inductively coupled plasma analyzer. The water treated by igneous rock, both directly soaked or indirectly in contact, induced increased activities of glucose oxidase, catalase, peroxidase, and superoxide dismutase. It also increased cell growth of SC-M1, HCT-15, Raji, and fibroblast cell lines. The water after treatment of igneous rock had no change in pH values, but displayed decreased conductivity values. We assume that the igneous rock could transfer energy to water to change the molecular structure or conformation of water cluster, or by radiation hormesis effect could then induce increased enzyme activity and cell growth. It is also possible that the energy from rock may combine radiation hormesis with other transferable biological energy forms to change water cluster conformation

  6. Ultra-performance liquid chromatography-tandem mass spectrometry-based multiplex enzyme assay for six enzymes associated with hereditary hemolytic anemia.

    Science.gov (United States)

    Park, Chul Min; Lee, Kyunghoon; Jun, Sun-Hee; Song, Sang Hoon; Song, Junghan

    2017-08-15

    Deficiencies in erythrocyte metabolic enzymes are associated with hereditary hemolytic anemia. Here, we report the development of a novel multiplex enzyme assay for six major enzymes, namely glucose-6-phosphate dehydrogenase, pyruvate kinase, pyrimidine 5'-nucleotidase, hexokinase, triosephosphate isomerase, and adenosine deaminase, deficiencies in which are implicated in erythrocyte enzymopathies. To overcome the drawbacks of traditional spectrophotometric enzyme assays, the present assay was based on ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The products of the six enzymes were directly measured by using ion pairing UPLC-MS/MS, and the precision, linearity, ion suppression, optimal sample amounts, and incubation times were evaluated. Eighty-three normal individuals and 13 patients with suspected enzymopathy were analyzed. The UPLC running time was within 5min. No ion suppression was observed at the retention time for the products or internal standards. We selected an optimal dilution factor and incubation time for each enzyme system. The intra- and inter-assay imprecision values (CVs) were 2.5-12.1% and 2.9-14.3%, respectively. The linearity of each system was good, with R 2 values >0.97. Patient samples showed consistently lower enzyme activities than those from normal individuals. The present ion paring UPLC-MS/MS assay enables facile and reproducible multiplex evaluation of the activity of enzymes implicated in enzymopathy-associated hemolytic anemia. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Measurement of Enzyme Kinetics by Use of a Blood Glucometer: Hydrolysis of Sucrose and Lactose

    Science.gov (United States)

    Heinzerling, Peter; Schrader, Frank; Schanze, Sascha

    2012-01-01

    An alternative analytical method for measuring the kinetic parameters of the enzymes invertase and lactase is described. Invertase hydrolyzes sucrose to glucose and fructose and lactase hydrolyzes lactose to glucose and galactose. In most enzyme kinetics studies, photometric methods or test strips are used to quantify the derivates of the…

  8. Soil zymography - A novel technique for mapping enzyme activity in the rhizosphere

    Science.gov (United States)

    Spohn, Marie

    2014-05-01

    The effect plant roots on microbial activity in soil at the millimeter scale is poorly understood. One reason for this is that spatially explicit methods for the study of microbial activity in soil are limited. Here we present a quantitative in situ technique for mapping the distribution of exoenzymes in soil along with some results about the effects of roots on exoenzyme activity in soil. In the first study we showed that both acid and alkaline phosphatase activity were up to 5.4-times larger in the rhizosphere of Lupinus albus than in the bulk soil. While acid phosphatase activity (produced by roots and microorganisms) was closely associated with roots, alkaline phosphatase activity (produced only by microorganisms) was more widely distributed, leading to a 2.5-times larger area of activity of alkaline than of acid phosphatase. These results indicate a spatial differentiation of different ecophysiological groups of organic phosphorus mineralizing organisms in the rhizosphere which might alleviate a potential competition for phosphorus between them. In a second study cellulase, chitinase and phosphatase activities were analyzed in the presence of living Lupinus polyphyllus roots and dead/dying roots (in the same soils 10, 20 and 30 days after cutting the L. polyphyllus shoots). The activity of all three enzymes was 9.0 to 13.9-times higher at the living roots compared to the bulk soil. Microhotspots of cellulase, chitinase and phosphatase activity in the soil were found up to 60 mm away from the living roots. 10 days after shoot cutting, the areas of high activities of cellulase and phosphatase activity were extend up to 55 mm away from the next root, while the extension of the area of chitinase activity did not change significantly. At the root, cellulase and chitinase activity increased first at the root tips after shoot cutting and showed maximal activity 20 days after shoot cutting. The number and activity of microhotspots of chitinase activity was maximal 10

  9. Phosphoenolpyruvate-dependent protein kinase enzyme I of Streptococcus faecalis: purification and properties of the enzyme and characterization of its active center

    International Nuclear Information System (INIS)

    Alpert, C.A.; Frank, R.; Stueber, K.D.; Deutscher, J.; Hengstenberg, W.

    1985-01-01

    Enzyme I, the phosphoenolpyruvate:protein phosphotransferase (EC 2.7.3.9), which is part of the bacterial phosphoenolpyruvate-(PEP) dependent phosphotransferase system, has been purified from Streptococcus faecalis by using a large-scale preparation. Size exclusion chromatography revealed a molecular weight of 140,000. On sodium dodecyl sulfate gels, enzyme I gave one band with a molecular weight of 70,000, indicating that enzyme I consists of two identical subunits. The first 59 amino acids of the amino-terminal part of the protein have been sequenced. It showed some similarities with enzyme I of Salmonella typhimurium. The active center of enzyme I has also been determined. After phosphorylation with [ 32 P]PEP, the enzyme was cleaved by using different proteases. Labeled peptides were isolated by high-performance liquid chromatography on a reversed-phase column. The amino acid composition or amino acid sequence of the peptides has been determined. The largest labeled peptide was obtained with Lys-C protease and had the following sequence: -Ala-Phe-Val-Thr-Asp-Ile-Gly- Gly-Arg-Thr-Ser-His*-Ser-Ala-Ile-Met-Ala-Arg-Ser-Leu-Glu-Ile-Pro-Ala- Ile-Val-Gly-Thr-Lys-. It has previously been shown that the phosphoryl group is bound to the N-3 position of a histidyl residue in phosphorylated enzyme I. The single His in position 12 of the above peptide must therefore carry the phosphoryl group

  10. Atrazine degradation and enzyme activities in an agricultural soil under two tillage systems.

    Science.gov (United States)

    Mahía, Jorge; Martín, Angela; Carballas, Tarsy; Díaz-Raviña, Montserrat

    2007-05-25

    The content of atrazine and its metabolites (hydroxyatrazine, deethylatrazine and deisopropylatrazine) as well as the activities of two soil enzymes (urease and beta-glucosidase) were evaluated in an acid agricultural soil, located in a temperate humid zone (Galicia, NW Spain), with an annual ryegrass-maize rotation under conventional tillage (CT) and no tillage (NT). Samples were collected during two consecutive years from the arable layer at two depths (0-5 cm and 5-20 cm) and different times after atrazine application. Hydroxyatrazine and deisopropylatrazine were the main metabolites resulting from atrazine degradation in the acid soil studied, the highest levels being detected in the surface layer of the NT treatment. A residual effect of atrazine was observed since hydroxyatrazine was detected in the arable layer (0-5 cm, 5-20 cm) even one year after the herbicide application. Soil enzyme activities in the upper 5 cm layer under NT were consistently higher than those in the same layer under CT. Urease and beta-glucosidase activities decreased with depth in the profile under NT but they did not show any differences between the two depths for the plots under CT. For both tillage systems enzyme activities also reflected temporal changes during the maize cultivation; however, no consistent effect of the herbicide application was observed.

  11. Acrolein inhibits NADH-linked mitochondrial enzyme activity: implications for Alzheimer's disease.

    Science.gov (United States)

    Pocernich, Chava B; Butterfield, D Allan

    2003-01-01

    In Alzheimer's disease (AD) brain increased lipid peroxidation and decreased energy utilization are found. Mitochondria membranes contain a significant amount of arachidonic and linoleic acids, precursors of lipid peroxidation products, 4-hydroxynonenal (HNE) and 2-propen-1-al (acrolein), that are extremely reactive. Both alkenals are increased in AD brain. In this study, we examined the effects of nanomolar levels of acrolein on the activities of pyruvate dehydrogenase (PDH) and Alpha-ketoglutarate dehydrogenase (KGDH), both reduced nicotinamide adenine dinucleotide (NADH)-linked mitochondrial enzymes. Acrolein decreased PDH and KGDH activities significantly in a dose-dependent manner. Using high performance liquid chromatography coupled to mass spectrometry (HPLC-MS), acrolein was found to bind lipoic acid, a component in both the PDH and KGDH complexes, most likely explaining the loss of enzyme activity. Acrolein also interacted with oxidized nicotinamide adenine dinucleotide (NAD(+)) in such a way as to decrease the production of NADH. Acrolein, which is increased in AD brain, may be partially responsible for the dysfunction of mitochondria and loss of energy found in AD brain by inhibition of PDH and KGDH activities, potentially contributing to the neurodegeneration in this disorder.

  12. Acetate Activation in Methanosaeta thermophila: Characterization of the Key Enzymes Pyrophosphatase and Acetyl-CoA Synthetase

    Directory of Open Access Journals (Sweden)

    Stefanie Berger

    2012-01-01

    Full Text Available The thermophilic methanogen Methanosaeta thermophila uses acetate as sole substrate for methanogenesis. It was proposed that the acetate activation reaction that is needed to feed acetate into the methanogenic pathway requires the hydrolysis of two ATP, whereas the acetate activation reaction in Methanosarcina sp. is known to require only one ATP. As these organisms live at the thermodynamic limit that sustains life, the acetate activation reaction in Mt. thermophila seems too costly and was thus reevaluated. It was found that of the putative acetate activation enzymes one gene encoding an AMP-forming acetyl-CoA synthetase was highly expressed. The corresponding enzyme was purified and characterized in detail. It catalyzed the ATP-dependent formation of acetyl-CoA, AMP, and pyrophosphate (PPi and was only moderately inhibited by PPi. The breakdown of PPi was performed by a soluble pyrophosphatase. This enzyme was also purified and characterized. The pyrophosphatase hydrolyzed the major part of PPi (KM=0.27±0.05 mM that was produced in the acetate activation reaction. Activity was not inhibited by nucleotides or PPi. However, it cannot be excluded that other PPi-dependent enzymes take advantage of the remaining PPi and contribute to the energy balance of the cell.

  13. Circadian variation in metabolite and enzyme activities in the femoral and thoracic muscles of adult variegated grasshoppers, Zonocerus variegatus (Linnaeus, 1758 (Orthoptera: Pyrgomorphidae

    Directory of Open Access Journals (Sweden)

    Ademolu Kehinde Olutoyin

    2018-03-01

    Full Text Available The African variegated grasshopper, Zonocerus variegatus, exhibits daily variations in its feeding and destructive activities. A study to investigate circadian variation in metabolites (lipids, protein, glucose, K+, Ca2+, Mg2+, Cl− concentrations and enzymes (lipase, amylase, proteinase, α-glucosidase activities in the femoral and thoracic muscles of adult Z. variegatus was carried out by collecting samples at 06:00, 12:00, 18:00 and 24:00 hrs GMT. The four enzymes were present throughout the day in both thoracic and femoral muscles but at varying levels. Significantly (p<0.05 higher enzymes activities were measured during the day (between 06:00 and 18:00 hours GMT (except proteinase than at night (24:00 hrs. Organic substances in the two tissues were present in significantly higher concentrations during the day than at night. Similarly, significantly more inorganic substances were recorded in the afternoon (12:00-18:00 hrs than at night in both femoral and thoracic muscles. It can thus be concluded that locomotor activities in Z. variegatus reach a peak during the day.

  14. Correction: Mesoporous titania thin films as efficient enzyme carriers for paraoxon determination/detoxification: effects of enzyme binding and pore hierarchy on the biocatalyst activity and reusability.

    Science.gov (United States)

    Frančič, N; Bellino, M G; Soler-Illia, G J A A; Lobnik, A

    2016-07-07

    Correction for 'Mesoporous titania thin films as efficient enzyme carriers for paraoxon determination/detoxification: effects of enzyme binding and pore hierarchy on the biocatalyst activity and reusability' by N. Frančičet al., Analyst, 2014, 139, 3127-3136.

  15. Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT).

    Science.gov (United States)

    Francis, R J; Mather, S J; Chester, K; Sharma, S K; Bhatia, J; Pedley, R B; Waibel, R; Green, A J; Begent, R H J

    2004-08-01

    MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99mTc-carbonyl [99mTc(H2O)3(CO)3]+ (abbreviated to TcCO) mediated labelling of 99mTc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins.

  16. Radiolabelling of glycosylated MFE-23::CPG2 fusion protein (MFECP1) with 99mTc for quantitation of tumour antibody-enzyme localisation in antibody-directed enzyme pro-drug therapy (ADEPT)

    International Nuclear Information System (INIS)

    Francis, R.J.; Chester, K.; Sharma, S.K.; Bhatia, J.; Pedley, R.B.; Green, A.J.; Begent, R.H.J.; Mather, S.J.; Waibel, R.

    2004-01-01

    MFECP1 is a glycosylated recombinant fusion protein composed of MFE-23, a high-affinity anti-carcinoembryonic antigen (CEA) single chain Fv (scFv), fused to the enzyme carboxypeptidase G2 (CPG2), and has been constructed for use in antibody-directed enzyme pro-drug therapy (ADEPT). Radiolabelling of glycosylated MFECP1 with technetium-99m was developed for the purpose of determining tumour localisation of MFECP1 in a phase I ADEPT clinical study. The method used was 99m Tc-carbonyl [ 99m Tc(H 2 O) 3 (CO) 3 ] + (abbreviated to TcCO) mediated labelling of 99m Tc to the hexahistidine (His) tag of MFECP1. MFECP1 fusion protein was labelled with TcCO under a variety of conditions, and this was shown to be a relatively simple and robust method. Tissue biodistribution was assessed in a CEA-expressing LS174T (human colon carcinoma) nude mouse xenograft model. Tissues were taken at 1, 4 and 6 h for assessment of distribution of radioactivity and for measurement of CPG2 enzyme levels. The amount of radioactivity retained by the tumour proved to be an accurate estimation of actual measured enzyme activity, indicating that this radiolabelling method does not appear to damage the antibody-antigen binding or the enzyme activity of MFECP1. However, correlation between CPG2 enzyme activity and measured radioactivity in liver, spleen and kidney was poor, indicating retention of radioactivity in non-tumour sites but loss of enzyme activity. The high retention of technetium radioisotope in normal tissues may limit the clinical applicability of this radiolabelling method for MFECP1; however, these results suggest that this technique does have applicability for measuring the biodistribution of His-tagged recombinant proteins. (orig.)

  17. Prenatal ethanol exposure alters steroidogenic enzyme activity in newborn rat testes.

    Science.gov (United States)

    Kelce, W R; Rudeen, P K; Ganjam, V K

    1989-10-01

    We have examined the in utero effects of ethanol exposure on testicular steroidogenesis in newborn male pups. Pregnant Sprague-Dawley rats were fed a liquid ethanol diet (35% ethanol-derived calories), a pair-fed isocaloric liquid diet, or a standard laboratory rat chow and water diet beginning on Day 12 of gestation and continuing through parturition. Although there were no significant differences in the enzymatic activity of 5-ene-3 beta-hydroxysteroid dehydrogenase/isomerase or C17,20-lyase, the enzymatic activity of 17 alpha-hydroxylase was significantly (p less than 0.01) reduced (i.e., approximately 36%) in the ethanol-exposed pups compared to those from the pair-fed and chow treatment groups. This lesion in testicular steroidogenic enzyme activity in newborn male pups exposed to alcohol in utero was transient as 17 alpha-hydroxylase activity from the ethanol-exposed animals returned to control levels by postnatal Day 20 and remained at control levels through adulthood (postnatal Day 60). These data suggest that the suppression of the perinatal testosterone surge in male rats exposed to alcohol in utero and the associated long term demasculinizing effects of prenatal ethanol exposure might be the result of reduced testicular steroidogenic enzyme activity in the perinatal animal.

  18. Studies on the effects of radiation on enzyme activity and chromosome in mammals (Mus musuculus)

    International Nuclear Information System (INIS)

    Kim, J.B.; Lee, K.S.; Kim, Y.J.

    1982-01-01

    From the results of many researches in radiation biology, it is well known that the radiation induces gene mutation, aberration of chromosome which is a carrier of genes and the increase or decrease of enzyme activities in living organisms. However, the frequency of chromosomal aberration or the degree of enzyme activities according to the animal's age when they are irradiated with radiation and time pass after irradiation are known a little if any. From these viewpoints, the research on the frequencies of chromosomal aberrations in bone marrow cells and the degree of activities of glucose-6-phosphate dehydrogenase in liver, kidney and brain, and isocitrate dehydrogense in kidney and brain of mouse has been carried out according to the mice age when they are irradiated with 200 rad of whole body irradiation. The chromosomes and enzyme activities were observed at 24 hours, 48 hours and 4 days to 90 days after irradiation. (Author)

  19. Antioxidant enzymes activity in embryogenic and non-embryogenic tissues in Sugarcane

    International Nuclear Information System (INIS)

    Marina Medeiros de Araujo Silva; Ulisses, Claudia; Lacerda E Medeiros, Maria Jaislanny; Cavalcante Granja, Manuela Maria; Willadino, Lilia; Camara, Terezinha

    2014-01-01

    The objective of this work was to induce direct somatic embryogenesis from segments of immature leaves of the RB872552 variety of sugarcane and to correlate this morphogenic event with oxidative stress. Two previously described protocols were utilized for the induction of somatic embryogenesis in sugarcane with different supplementations of the culture medium and different incubation conditions. For the conversion of embryos into plants was used ms medium without phytoregulators. Histological analyses and activity of antioxidant enzymes were also conducted for the embryogenic and non-embryogenic tissues. The formation of somatic embryos was obtained in 81 % of the explants with the combination of regulators 2,4-D (2,4-dichlorophenoxyacetic acid)and BAP (6-benzylaminopurine) when incubated under 16 h photoperiod. With regards to the antioxidant enzymes, there was increased activity of peroxidase and an increase in the soluble protein content in embryogenic tissues, whereas lower activities of polyphenol oxidase and catalase appeared in these tissues compared to nonembryogenic tissues. It could be inferred that oxidative stress plays an important role in the induction of somatic embryogenesis in sugarcane.

  20. Simplified methods for in vivo measurement of acetylcholinesterase activity in rodent brain

    International Nuclear Information System (INIS)

    Kilbourn, Michael R.; Sherman, Phillip S.; Snyder, Scott E.

    1999-01-01

    Simplified methods for in vivo studies of acetylcholinesterase (AChE) activity in rodent brain were evaluated using N-[ 11 C]methylpiperidinyl propionate ([ 11 C]PMP) as an enzyme substrate. Regional mouse brain distributions were determined at 1 min (representing initial brain uptake) and 30 min (representing trapped product) after intravenous [ 11 C]PMP administration. Single time point tissue concentrations (percent injected dose/gram at 30 min), tissue concentration ratios (striatum/cerebellum and striatum/cortex ratios at 30 min), and regional tissue retention fractions (defined as percent injected dose 30 min/percent injected dose 1 min) were evaluated as measures of AChE enzymatic activity in mouse brain. Studies were carried out in control animals and after dosing with phenserine, a selective centrally active AChE inhibitor; neostigmine, a peripheral cholinesterase inhibitor; and a combination of the two drugs. In control and phenserine-treated animals, absolute tissue concentrations and regional retention fractions provide good measures of dose-dependent inhibition of brain AChE; tissue concentration ratios, however, provide erroneous conclusions. Peripheral inhibition of cholinesterases, which changes the blood pharmacokinetics of the radiotracer, diminishes the sensitivity of all measures to detect changes in central inhibition of the enzyme. We conclude that certain simple measures of AChE hydrolysis rates for [ 11 C]PMP are suitable for studies where alterations of the peripheral blood metabolism of the tracer are kept to a minimum

  1. Real-time investigation of human topoisomerase I reaction kinetics using an optical sensor: a fast method for drug screening and determination of active enzyme concentrations.

    Science.gov (United States)

    Kristoffersen, Emil L; Jørgensen, Line A; Franch, Oskar; Etzerodt, Michael; Frøhlich, Rikke; Bjergbæk, Lotte; Stougaard, Magnus; Ho, Yi-Ping; Knudsen, Birgitta R

    2015-06-07

    Human DNA topoisomerase I (hTopI) is a nuclear enzyme that catalyzes relaxation of super helical tension that arises in the genome during essential DNA metabolic processes. This is accomplished through a common reaction mechanism shared among the type IB topoisomerase enzymes, including eukaryotic and poxvirus topoisomerase I. The mechanism of hTopI is specifically targeted in cancer treatment using camptothecin derivatives. These drugs convert the hTopI activity into a cellular poison, and hence the cytotoxic effects of camptothecin derivatives correlate with the hTopI activity. Therefore, fast and reliable techniques for high throughput measurements of hTopI activity are of high clinical interest. Here we demonstrate potential applications of a fluorophore-quencher based DNA sensor designed for measurement of hTopI cleavage-ligation activities, which are the catalytic steps affected by camptothecin. The kinetic analysis of the hTopI reaction with the DNA sensor exhibits a characteristic burst profile. This is the result of a two-step ping-pong reaction mechanism, where a fast first reaction, the one creating the signal, is followed by a slower second reaction necessary for completion of the catalytic cycle. Hence, the burst profile holds information about two reactions in the enzymatic mechanism. Moreover, it allows the amount of active enzyme in the reaction to be determined. The presented results pave the way for future high throughput drug screening and the potential of measuring active hTopI concentrations in clinical samples for individualized treatment.

  2. Antioxidant and Angiotensin-Converting Enzyme Inhibitory Activity of Eucalyptus camaldulensis and Litsea glaucescens Infusions Fermented with Kombucha Consortium

    OpenAIRE

    Gamboa-Gómez, Claudia I.; González-Laredo, Rubén F.; Gallegos-Infante, José Alberto; Pérez, MŞ del Mar Larrosa; Moreno-Jiménez, Martha R.; Flores-Rueda, Ana G.; Rocha-Guzmán, Nuria E.

    2016-01-01

    Physicochemical properties, consumer acceptance, antioxidant and angiotensin-converting enzyme (ACE) inhibitory activities of infusions and fermented beverages of Eucalyptus camaldulensis and Litsea glaucescens were compared. Among physicochemical parameters, only the pH of fermented beverages decreased compared with the unfermented infusions. No relevant changes were reported in consumer preference between infusions and fermented beverages. Phenolic profi le measured by UPLC MS/MS analysis d...

  3. Assaying Oxidative Coupling Activity of CYP450 Enzymes.

    Science.gov (United States)

    Agarwal, Vinayak

    2018-01-01

    Cytochrome P450 (CYP450) enzymes are ubiquitous catalysts in natural product biosynthetic schemes where they catalyze numerous different transformations using radical intermediates. In this protocol, we describe procedures to assay the activity of a marine bacterial CYP450 enzyme Bmp7 which catalyzes the oxidative radical coupling of polyhalogenated aromatic substrates. The broad substrate tolerance of Bmp7, together with rearrangements of the aryl radical intermediates leads to a large number of products to be generated by the enzymatic action of Bmp7. The complexity of the product pool generated by Bmp7 thus presents an analytical challenge for structural elucidation. To address this challenge, we describe mass spectrometry-based procedures to provide structural insights into aryl crosslinked products generated by Bmp7, which can complement subsequent spectroscopic experiments. Using the procedures described here, for the first time, we show that Bmp7 can efficiently accept polychlorinated aryl substrates, in addition to the physiological polybrominated substrates for the biosynthesis of polyhalogenated marine natural products. © 2018 Elsevier Inc. All rights reserved.

  4. Ultrasound in Enzyme Activation and Inactivation

    Science.gov (United States)

    Mawson, Raymond; Gamage, Mala; Terefe, Netsanet Shiferaw; Knoerzer, Kai

    As discussed in previous chapters, most effects due to ultrasound arise from cavitation events, in particular, collapsing cavitation bubbles. These collapsing bubbles generate very high localized temperatures and pressure shockwaves along with micro-streaming that is associated with high shear forces. These effects can be used to accelerate the transport of substrates and reaction products to and from enzymes, and to enhance mass transfer in enzyme reactor systems, and thus improve efficiency. However, the high velocity streaming, together with the formation of hydroxy radicals and heat generation during collapsing of bubbles, may also potentially affect the biocatalyst stability, and this can be a limiting factor in combined ultrasound/enzymatic applications. Typically, enzymes can be readily denatured by slight changes in environmental conditions, including temperature, pressure, shear stress, pH and ionic strength.

  5. Influence of protoplast fusion between two Trichoderma spp. on extracellular enzymes production and antagonistic activity.

    Science.gov (United States)

    Hassan, Mohamed M

    2014-11-02

    Biological control plays a crucial role in grapevine pathogens disease management. The cell-wall degrading enzymes chitinase, cellulase and β-glucanase have been suggested to be essential for the mycoparasitism activity of Trichoderma species against grapevine fungal pathogens. In order to develop a useful strain as a single source of these vital enzymes, it was intended to incorporate the characteristics of two parental fungicides tolerant mutants of Trichoderma belonging to the high chitinase producing species T. harzianum and the high cellulase producing species T. viride , by fusing their protoplasts. The phylogeny of the parental strains was carried out using a sequence of the 5.8S-ITS region. The BLAST of the obtained sequence identified these isolates as T. harzianum and T. viride . Protoplasts were isolated using lysing enzymes and were fused using polyethylene glycol. The fused protoplasts have been regenerated on protoplast regeneration minimal medium supplemented with two selective fungicides. Among the 40 fast growing fusants, 17 fusants were selected based on their enhanced growth on selective media for further studies. The fusant strains were growing 60%-70% faster than the parents up to third generation. All the 17 selected fusants exhibited morphological variations. Some fusant strains displayed threefold increased chitinase enzyme activity and twofold increase in β-glucanase enzyme activity compared to the parent strains. Most fusants showed powerful antagonistic activity against Macrophomin aphaseolina , Pythium ultimum and Sclerotium rolfsii pathogens. Fusant number 15 showed the highest inhibition percentage (92.8%) against M. phaseolina and P. ultimum, while fusant number 9 showed the highest inhibition percentage (98.2%) against the growth of S. rolfsii. A hyphal intertwining and degradation phenomenon was observed by scanning electron microscope. The Trichoderma antagonistic effect against pathogenic fungal mycelia was due to the

  6. Hydrolytic enzyme activities in shiitake mushroom (Lentinula edodes) strains cultivated on coffee pulp.

    Science.gov (United States)

    Mata, Gerardo; Salmones, Dulce; Pérez-Merlo, Rosalía

    Hydrolytic enzyme production (cellulases, laminarinases and xylanases) was studied in cultures of Lentinula edodes on sterilized coffee pulp. Samples of substrate colonized by mycelia were taken after 7, 14, 21, 28 and 35 days of incubation at 25°C (W1 to W5) and during the fruiting period at different stages: formation of primordia (PF), first harvest (H) and one week after the first harvest (PH). The enzymatic activity was lower during the early mycelial growth and showed higher levels during the formation and development of fruiting bodies. During the reproductive stage of the fungus, the samples were subjected to a soaking treatment; however, it was not possible to relate this soaking treatment to the increase in enzyme production. The levels of enzymatic activity suggest that secretion of the studied enzymes does not influence the adaptability of the strains to the substrate. Copyright © 2016 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

  7. Effect of water quality and confounding factors on digestive enzyme activities in Gammarus fossarum.

    Science.gov (United States)

    Charron, L; Geffard, O; Chaumot, A; Coulaud, R; Queau, H; Geffard, A; Dedourge-Geffard, O

    2013-12-01

    The feeding activity and subsequent assimilation of the products resulting from food digestion allow organisms to obtain energy for growth, maintenance and reproduction. Among these biological parameters, we studied digestive enzymes (amylase, cellulase and trypsin) in Gammarus fossarum to assess the impact of contaminants on their access to energy resources. However, to enable objective assessment of a toxic effect of decreased water quality on an organisms' digestive capacity, it is necessary to establish reference values based on its natural variability as a function of changing biotic and abiotic factors. To limit the confounding influence of biotic factors, a caging approach with calibrated male organisms from the same population was used. This study applied an in situ deployment at 23 sites of the Rhone basin rivers, complemented by a laboratory experiment assessing the influence of two abiotic factors (temperature and conductivity). The results showed a small effect of conductivity on cellulase activity and a significant effect of temperature on digestive enzyme activity but only at the lowest temperature (7 °C). The experimental conditions allowed us to define an environmental reference value for digestive enzyme activities to select sites where the quality of the water impacted the digestive capacity of the organisms. In addition to the feeding rate, this study showed the relevance of digestive enzymes as biomarkers to be used as an early warning tool to reflect organisms' health and the chemical quality of aquatic ecosystems.

  8. Modeling the effects of tree species and incubation temperature on soil's extracellular enzyme activity in 78-year-old tree plantations

    Directory of Open Access Journals (Sweden)

    X. Zhou

    2017-12-01

    Full Text Available Forest plantations have been widely used as an effective measure for increasing soil carbon (C, and nitrogen (N stocks and soil enzyme activities play a key role in soil C and N losses during decomposition of soil organic matter. However, few studies have been carried out to elucidate the mechanisms behind the differences in soil C and N cycling by different tree species in response to climate warming. Here, we measured the responses of soil's extracellular enzyme activity (EEA to a gradient of temperatures using incubation methods in 78-year-old forest plantations with different tree species. Based on a soil enzyme kinetics model, we established a new statistical model to investigate the effects of temperature and tree species on soil EEA. In addition, we established a tree species–enzyme–C∕N model to investigate how temperature and tree species influence soil C∕N contents over time without considering plant C inputs. These extracellular enzymes included C acquisition enzymes (β-glucosidase, BG, N acquisition enzymes (N-acetylglucosaminidase, NAG; leucine aminopeptidase, LAP and phosphorus acquisition enzymes (acid phosphatases. The results showed that incubation temperature and tree species significantly influenced all soil EEA and Eucalyptus had 1.01–2.86 times higher soil EEA than coniferous tree species. Modeling showed that Eucalyptus had larger soil C losses but had 0.99–2.38 times longer soil C residence time than the coniferous tree species over time. The differences in the residual soil C and N contents between Eucalyptus and coniferous tree species, as well as between slash pine (Pinus elliottii Engelm. var. elliottii and hoop pine (Araucaria cunninghamii Ait., increase with time. On the other hand, the modeling results help explain why exotic slash pine can grow faster, as it has 1.22–1.38 times longer residual soil N residence time for LAP, which mediate soil N cycling in the long term, than native

  9. Suite of Activity-Based Probes for Cellulose-Degrading Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Chauvigne-Hines, Lacie M.; Anderson, Lindsey N.; Weaver, Holly M.; Brown, Joseph N.; Koech, Phillip K.; Nicora, Carrie D.; Hofstad, Beth A.; Smith, Richard D.; Wilkins, Michael J.; Callister, Stephen J.; Wright, Aaron T.

    2012-12-19

    Microbial glycoside hydrolases play a dominant role in the biochemical conversion of cellulosic biomass to high-value biofuels. Anaerobic cellulolytic bacteria are capable of producing multicomplex catalytic subunits containing cell-adherent cellulases, hemicellulases, xylanases, and other glycoside hydrolases to facilitate the degradation of highly recalcitrant cellulose and other related plant cell wall polysaccharides. Clostridium thermocellum is a cellulosome producing bacterium that couples rapid reproduction rates to highly efficient degradation of crystalline cellulose. Herein, we have developed and applied a suite of difluoromethylphenyl aglycone, N-halogenated glycosylamine, and 2-deoxy-2-fluoroglycoside activity-based protein profiling (ABPP) probes to the direct labeling of the C. thermocellum cellulosomal secretome. These activity-based probes (ABPs) were synthesized with alkynes to harness the utility and multimodal possibilities of click chemistry, and to increase enzyme active site inclusion for LC-MS analysis. We directly analyzed ABP-labeled and unlabeled global MS data, revealing ABP selectivity for glycoside hydrolase (GH) enzymes in addition to a large collection of integral cellulosome-containing proteins. By identifying reactivity and selectivity profiles for each ABP, we demonstrate our ability to widely profile the functional cellulose degrading machinery of the bacterium. Derivatization of the ABPs, including reactive groups, acetylation of the glycoside binding groups, and mono- and disaccharide binding groups, resulted in considerable variability in protein labeling. Our probe suite is applicable to aerobic and anaerobic cellulose degrading systems, and facilitates a greater understanding of the organismal role associated within biofuel development.

  10. Exploring functionally related enzymes using radially distributed properties of active sites around the reacting points of bound ligands

    Directory of Open Access Journals (Sweden)

    Ueno Keisuke

    2012-04-01

    Full Text Available Abstract Background Structural genomics approaches, particularly those solving the 3D structures of many proteins with unknown functions, have increased the desire for structure-based function predictions. However, prediction of enzyme function is difficult because one member of a superfamily may catalyze a different reaction than other members, whereas members of different superfamilies can catalyze the same reaction. In addition, conformational changes, mutations or the absence of a particular catalytic residue can prevent inference of the mechanism by which catalytic residues stabilize and promote the elementary reaction. A major hurdle for alignment-based methods for prediction of function is the absence (despite its importance of a measure of similarity of the physicochemical properties of catalytic sites. To solve this problem, the physicochemical features radially distributed around catalytic sites should be considered in addition to structural and sequence similarities. Results We showed that radial distribution functions (RDFs, which are associated with the local structural and physicochemical properties of catalytic active sites, are capable of clustering oxidoreductases and transferases by function. The catalytic sites of these enzymes were also characterized using the RDFs. The RDFs provided a measure of the similarity among the catalytic sites, detecting conformational changes caused by mutation of catalytic residues. Furthermore, the RDFs reinforced the classification of enzyme functions based on conventional sequence and structural alignments. Conclusions Our results demonstrate that the application of RDFs provides advantages in the functional classification of enzymes by providing information about catalytic sites.

  11. 21 CFR 862.1090 - Angiotensin converting enzyme (A.C.E.) test system.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Angiotensin converting enzyme (A.C.E.) test system... Test Systems § 862.1090 Angiotensin converting enzyme (A.C.E.) test system. (a) Identification. An angiotensin converting enzyme (A.C.E.) test system is a device intended to measure the activity of angiotensin...

  12. Andrographolide powder treatment as antifeedant decreased digestive enzyme activity from Plutella xylostella (L.) larvae midgut

    Science.gov (United States)

    Madihah, Malini, Desak Made; Roviani, Hana; Rani, Nessa Vidya; Hermawan, Wawan

    2018-02-01

    Andrographolide, an active compound of Andrographis paniculata, has shown antifeedant activity against Plutella xylostella larvae by disrupting the midgut histological structures. This study aims to determine the activity of andrographolide in crystallized powder form against several digestive enzymes from the midgut of 4th instar P. xylostella larvae. The concentrations used were 0 (control), 1000, 1600, 2500, 4000 and 6500 ppm with four replications each. No-choice antifeedant test with leaf disc method is used in a bioassay for 24 hours. The midgut was dissected from 2nd until 6th segment of 4th instar larvae and was homogenized in iced-buffer solution. Furthermore, larvae's midgut samples were centrifuged at 10,000 rpm, 4°C for 20 min and the supernatant is used as enzyme source. The results showed that andrographolide significantly reduces the amylase, invertase, protease and trypsin activity, as well as total protein concentration compared with control (p<0.05) in a dose-dependent manner. This study provides information about the mode of action of andrographolide in inhibiting feed activity by the reduced digestive enzyme activity of 4th instar P. xylostella larvae.

  13. Comparison of anti-oxidant enzymes activity and levels of zinc and selenium in sperm and seminal plasma between fertile and idiopathic infertile men

    Directory of Open Access Journals (Sweden)

    Hadi Kharazi

    2010-12-01

    Full Text Available Background: Reactive oxygen species (ROS-induced lipidperoxidation can lead to dysfunction of sperm and thereby, infertility may be occurred. So, always there is a balance between amount of ROS and anti-oxidant molecules in semen. Anti-oxidant enzymes of sperm; superoxide dismutase (SOD, glutathione peroxidase (GPX, catalse and zinc and selenium can protect it from destructive effects of ROS. Hence, the present study was designed to compare the activities of these enzymes and trace elements between fertile and idiopathic infertile men.Methods: Semen specimens were collected from 30 infertile men with proven infertility by an urologist, and 30 fertile men as control donors, with age range between 20-40 years old. Semen analysis was conducted by CASA method. Atomic absorption method was used for measuring of zinc and selenium concentration. Activity assays of SOD and GPX were performed by Randox Kits. Aebi method also was applied for evaluation of catalase activity.Results: There was no difference between the activities of enzymes in fertile men and infertile ones. Also, it wasn't seen any difference in the selenium and zinc levels of seminal plasma. There was no relationship between evaluated items with sperm parameters. Only, in asthenoteratospermic individuals negative correlations were found between GPX and sperm motility, selenium and sperm morphology. Also, in these individuals ,there was a positive correlation between SOD and catalse activity.Conclusion: Measuring activities of SOD, GPx, and catalase and the contents of zinc and selenium of seminal plasma do not appear to be suitable tools for determining the fertility potential of sperm.

  14. Effect of high-intensity intermittent swimming training on fatty acid oxidation enzyme activity in rat skeletal muscle.

    Science.gov (United States)

    Terada, Shin; Tabata, Izumi; Higuchi, Mitsuru

    2004-02-01

    We previously reported that high-intensity exercise training significantly increased citrate synthase (CS) activity, a marker of oxidative enzyme, in rat skeletal muscle to a level equaling that attained after low-intensity prolonged exercise training (Terada et al., J Appl Physiol 90: 2019-2024, 2001). Since mitochondrial oxidative enzymes and fatty acid oxidation (FAO) enzymes are often increased simultaneously, we assessed the effect of high-intensity intermittent swimming training on FAO enzyme activity in rat skeletal muscle. Male Sprague-Dawley rats (3 to 4 weeks old) were assigned to a 10-day period of high-intensity intermittent exercise training (HIT), low-intensity prolonged exercise training (LIT), or sedentary control conditions. In the HIT group, the rats repeated fourteen 20 s swimming sessions with a weight equivalent to 14-16% of their body weight. Between the exercise sessions, a 10 s pause was allowed. Rats in the LIT group swam 6 h/day in two 3 h sessions separated by 45 min of rest. CS activity in the triceps muscle of rats in the HIT and LIT groups was significantly higher than that in the control rats by 36 and 39%, respectively. Furthermore, 3-beta hydroxyacyl-CoA dehydrogenase (HAD) activity, an important enzyme in the FAO pathway in skeletal muscle, was higher in the two training groups than in the control rats (HIT: 100%, LIT: 88%). No significant difference in HAD activity was observed between the two training groups. In conclusion, the present investigation demonstrated that high-intensity intermittent swimming training elevated FAO enzyme activity in rat skeletal muscle to a level similar to that attained after 6 h of low-intensity prolonged swimming exercise training.

  15. Changes in Soil Enzyme Activities and Microbial Biomass after Revegetation in the Three Gorges Reservoir, China

    Directory of Open Access Journals (Sweden)

    Qingshui Ren

    2018-05-01

    Full Text Available Soil enzymes and microbes are central to the decomposition of plant and microbial detritus, and play important roles in carbon, nitrogen, and phosphorus biogeochemistry cycling at the ecosystem level. In the present study, we characterized the soil enzyme activity and microbial biomass in revegetated (with Taxodium distichum (L. Rich. and Cynodon dactylon (L. Pers. versus unplanted soil in the riparian zone of the Three Gorges Dam Reservoir (TGDR, in order to quantify the effect of revegetation on the edaphic microenvironment after water flooding in situ. After revegetation, the soil physical and chemical properties in revegetated soil showed significant differences to those in unplanted soil. The microbial biomass carbon and phosphorus in soils of T. distichum were significantly higher than those in C. dactylon and unplanted soils, respectively. The microbial biomass nitrogen in revegetated T. distichum and C. dactylon soils was significantly increased by 273% and 203%, respectively. The enzyme activities of T. distichum and C. dactylon soils displayed no significant difference between each other, but exhibited a great increase compared to those of the unplanted soil. Elements ratio (except C/N (S did not vary significantly between T. distichum and C. dactylon soils; meanwhile, a strong community-level elemental homeostasis in the revegetated soils was found. The correlation analyses demonstrated that only microbial biomass carbon and phosphorus had a significantly positive relationship with soil enzyme activities. After revegetation, both soil enzyme activities and microbial biomasses were relatively stable in the T. distichum and C. dactylon soils, with the wooded soil being more superior. The higher enzyme activities and microbial biomasses demonstrate the C, N, and P cycling and the maintenance of soil quality in the riparian zone of the TGDR.

  16. Using Carbohydrate Interaction Assays to Reveal Novel Binding Sites in Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Cockburn, Darrell; Wilkens, Casper; Dilokpimol, Adiphol

    2016-01-01

    Carbohydrate active enzymes often contain auxiliary binding sites located either on independent domains termed carbohydrate binding modules (CBMs) or as so-called surface binding sites (SBSs) on the catalytic module at a certain distance from the active site. The SBSs are usually critical...

  17. Quantitative Structure-Activity Relationship Modeling Coupled with Molecular Docking Analysis in Screening of Angiotensin I-Converting Enzyme Inhibitory Peptides from Qula Casein Hydrolysates Obtained by Two-Enzyme Combination Hydrolysis.

    Science.gov (United States)

    Lin, Kai; Zhang, Lanwei; Han, Xue; Meng, Zhaoxu; Zhang, Jianming; Wu, Yifan; Cheng, Dayou

    2018-03-28

    In this study, Qula casein derived from yak milk casein was hydrolyzed using a two-enzyme combination approach, and high angiotensin I-converting enzyme (ACE) inhibitory activity peptides were screened by quantitative structure-activity relationship (QSAR) modeling integrated with molecular docking analysis. Hydrolysates (casein presents an excellent source to produce ACE inhibitory peptides.

  18. Subtropical urban turfs: Carbon and nitrogen pools and the role of enzyme activity.

    Science.gov (United States)

    Kong, Ling; Chu, L M

    2018-03-01

    Urban grasslands not only provide a recreational venue for urban residents, but also sequester organic carbon in vegetation and soils through photosynthesis, and release carbon dioxide through respiration, which largely contribute to carbon storage and fluxes at regional and global scales. We investigated organic carbon and nitrogen pools in subtropical turfs and found that dissolved organic carbon (DOC) and dissolved organic nitrogen (DON) were regulated by several factors including microbial activity which is indicated by soil enzymatic activity. We observed a vertical variation and different temporal patterns in both soil DOC, DON and enzyme activities, which decreased significantly with increasing soil depths. We further found that concentration of soil DON was linked with turf age. There were correlations between grass biomass and soil properties, and soil enzyme activities. In particular, soil bulk density was significantly correlated with soil moisture and soil organic carbon (SOC). In addition, DOC correlated significantly with DON. Significant negative correlations were also observed between soil total dissolved nitrogen (TDN) and grass biomass of Axonopus compressus and Zoysia matrella. Specifically, grass biomass was significantly correlated with the soil activity of urease and β-glucosidase. Soil NO 3 -N concentration also showed negative correlations with the activity of both β-glucosidase and protease but there were no significant correlations between cellulase and soil properties or grass biomass. Our study demonstrated a relationship between soil C and N dynamics and soil enzymes that could be modulated to enhance SOC pools through management and maintenance practices. Copyright © 2017. Published by Elsevier B.V.

  19. Quality of Water Content, Diastase Enzyme Activity and Hidroximetilfurfural (HMF in Rubber and Rambutan Honey

    Directory of Open Access Journals (Sweden)

    Sulis Setio Toto Harjo

    2017-03-01

    Full Text Available The purpose of this research was to determine the water content, diastase enzyme activity and HMF of the rubber and rambutan honey. The method was a laboratory experiments with statistical analysis unpaired student t-test by two treatments and fifteen replications. The variable of this research were water content, diastase enzyme activity and HMF. The results of rubber and rambutan honey showed that there were significant difference effect (P0.05 that is 11 DN and there is a highly significant difference (P<0.01 on the HMF content of 17.23±0.54 mg/kg and 7.61±0.23 mg/kg. Rubber and rambutan honey have good quality based on the water content, diastase enzyme activity and HMF. It was concluded that the rubber and rambutan honey used were of good quality because it has met the requirements of SNI.

  20. Activation of PAF-synthesizing enzymes in rat brain stem slices after LTP induction in the medial vestibular nuclei.

    Science.gov (United States)

    Francescangeli, Ermelinda; Grassi, Silvarosa; Pettorossi, Vito E; Goracci, Gianfrancesco

    2002-11-01

    LysoPAF acetyltransferase (lysoPAF-AT) and PAF-synthesizing phosphocholinetransferase (PAF-PCT) are the two enzymes which catalyze the final reactions for the synthesis of PAF. Their activities, assayed in the homogenate of rat brain stem slices and under their optimal conditions, increased 5 min after high frequency stimulation of vestibular afferents, inducing LTP in the medial vestibular nuclei. The activity of phosphatidylcholine-synthesizing phosphocholinetransferase, was not affected. Sixty minutes from the induction of LTP, PAF-PCT activity, but not that of lysoPAF-AT, was still significantly higher with respect to 5 min test stimulated control. We used AP-5 to verify whether this increase was strictly dependent upon LTP induction, which requires NMDA receptor activation. In AP-5 treated slices, lysoPAF-acetyltransferase and PAF-synthesizing phosphocholinetransferase activities increased, but they were reduced after high frequency stimulation under AP-5. In conclusion, we have demonstrated that the activities of PAF-synthesizing enzymes are activated soon after the induction of LTP and that this effect is linked to the activation of NMDA-receptors. We suggest that the enzyme activation by AP-5, preventing LTP, might be due to glutamate enhancement but, in neurons showing LTP and under normal conditions, the activation of potentiation mechanisms is critical for the enhancement of enzyme activities.

  1. Measuring Intracellular Enzyme Concentrations: Assessing the Effect of Oxidative Stress on the Amount of Glyoxalase I

    Science.gov (United States)

    Miranda, Hugo Vicente; Ferreira, Antonio E. N.; Quintas, Alexandre; Cordeiro, Carlos; Freire, Ana Ponces

    2008-01-01

    Enzymology is one of the fundamental areas of biochemistry and involves the study of the structure, kinetics, and regulation of enzyme activity. Research in this area is often conducted with purified enzymes and extrapolated to "in vivo" conditions. The specificity constant, k[subscript S], is the ratio between k[subscript cat] (the catalytic…

  2. Quantum mechanical design of enzyme active sites.

    Science.gov (United States)

    Zhang, Xiyun; DeChancie, Jason; Gunaydin, Hakan; Chowdry, Arnab B; Clemente, Fernando R; Smith, Adam J T; Handel, T M; Houk, K N

    2008-02-01

    The design of active sites has been carried out using quantum mechanical calculations to predict the rate-determining transition state of a desired reaction in presence of the optimal arrangement of catalytic functional groups (theozyme). Eleven versatile reaction targets were chosen, including hydrolysis, dehydration, isomerization, aldol, and Diels-Alder reactions. For each of the targets, the predicted mechanism and the rate-determining transition state (TS) of the uncatalyzed reaction in water is presented. For the rate-determining TS, a catalytic site was designed using naturalistic catalytic units followed by an estimation of the rate acceleration provided by a reoptimization of the catalytic site. Finally, the geometries of the sites were compared to the X-ray structures of related natural enzymes. Recent advances in computational algorithms and power, coupled with successes in computational protein design, have provided a powerful context for undertaking such an endeavor. We propose that theozymes are excellent candidates to serve as the active site models for design processes.

  3. Mini Review: Basic Physiology and Factors Influencing Exogenous Enzymes Activity in the Porcine Gastrointestinal Tract

    DEFF Research Database (Denmark)

    Strube, Mikael Lenz; Meyer, Anne S.; Boye, Mette

    2013-01-01

    activity during intestinal transit are few, it is known that the enzymes, being protein molecules, can be negatively affected by the gastrointestinal proteolytic enzymes and the low pH in the stomach ventricle. In this review, the pH-values, endogenous proteases and other factors native to the digestive......The addition of exogenous enzymes to pig feed is used to enhance general nutrient availability and thus increase daily weight gain per feed unit. The enzymes used are mainly beta-glucanase (EC 3.2.1.4) and xylanase (EC 3.2.1.8) and phytase (EC 3.1.3.8). Although in vivo data assessing feed enzyme...... tract of the adult pig and the piglet are discussed in relation to the stability of exogenous feed enzymes. Development of more consistent assessment methods which acknowledge such factors is warranted both in vitro and in vivo for proper evaluation and prediction of the efficiency of exogenous enzymes...

  4. Analysis of metallotionein expression and antioxidant enzyme activities in Meretrix meretrix larvae under sublethal cadmium exposure

    Energy Technology Data Exchange (ETDEWEB)

    Wang Qing [Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7th Nanhai Road, Qingdao 266071 (China); Graduate University, Chinese Academy of Sciences, Beijing 100049 (China); Wang Xiaomei [Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071 (China); Wang Xiaoyu [Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7th Nanhai Road, Qingdao 266071 (China); Engineering Research Center of Seawater Utilization Technology, Ministry of Education, Hebei University of Technology, Tianjin 300130 (China); Yang Hongsheng, E-mail: hshyang@ms.qdio.ac.cn [Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, 7th Nanhai Road, Qingdao 266071 (China); Liu Baozhong, E-mail: bzliu@ms.qdio.ac.cn [Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071 (China)

    2010-11-15

    To investigate the possible role of metallothioneins (MTs) and antioxidant enzymes in cadmium (Cd) tolerance in Meretrix meretrix larvae, a new MT (designated MmMT) gene was identified and cloned from M. meretrix. The full-length cDNA of MmMT consisted of an open reading frame (ORF) of 231 bp encoding a protein of 76 amino acids, with 21 cysteine residues and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys at the C-terminus. The deduced amino acid sequence of MmMT showed about 57-84% identity with previously published MT sequences of mussels and oysters. Real-time PCR was used to analyze the expression level of MmMT mRNA at different M. meretrix larval stages under Cd exposure (25 {mu}g L{sup -1}). Results showed that Cd could induce the expression of MmMT mRNA in the larvae, and the expression level increased 5.04-fold and 3.99-fold in D-shaped larvae and pediveligers, respectively. Immunolocalization of MmMT in the stressed larvae revealed that MmMT was synthesized in almost all of the soft parts at the trochophore and postlarva stages, whereas it was only synthesized in the velum and epidermis at the D-shaped larva and pediveliger stages. The activities of three antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), also were measured in larvae at different developmental stages. Increased enzymatic activities were detected mainly in D-shaped larvae and pediveligers under Cd stress, suggesting that these enzymes respond synchronously with MT. Our results indicate that MmMT and antioxidant enzymes played important roles in counteracting Cd stress in M. meretrix larvae.

  5. Analysis of metallotionein expression and antioxidant enzyme activities in Meretrix meretrix larvae under sublethal cadmium exposure

    International Nuclear Information System (INIS)

    Wang Qing; Wang Xiaomei; Wang Xiaoyu; Yang Hongsheng; Liu Baozhong

    2010-01-01

    To investigate the possible role of metallothioneins (MTs) and antioxidant enzymes in cadmium (Cd) tolerance in Meretrix meretrix larvae, a new MT (designated MmMT) gene was identified and cloned from M. meretrix. The full-length cDNA of MmMT consisted of an open reading frame (ORF) of 231 bp encoding a protein of 76 amino acids, with 21 cysteine residues and a conserved structural pattern Cys-x-Cys-x(3)-Cys-Tyr-Gly-x(3)-Cys-x-Cys-x(3)-Cys-x-Cys-Lys at the C-terminus. The deduced amino acid sequence of MmMT showed about 57-84% identity with previously published MT sequences of mussels and oysters. Real-time PCR was used to analyze the expression level of MmMT mRNA at different M. meretrix larval stages under Cd exposure (25 μg L -1 ). Results showed that Cd could induce the expression of MmMT mRNA in the larvae, and the expression level increased 5.04-fold and 3.99-fold in D-shaped larvae and pediveligers, respectively. Immunolocalization of MmMT in the stressed larvae revealed that MmMT was synthesized in almost all of the soft parts at the trochophore and postlarva stages, whereas it was only synthesized in the velum and epidermis at the D-shaped larva and pediveliger stages. The activities of three antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx), also were measured in larvae at different developmental stages. Increased enzymatic activities were detected mainly in D-shaped larvae and pediveligers under Cd stress, suggesting that these enzymes respond synchronously with MT. Our results indicate that MmMT and antioxidant enzymes played important roles in counteracting Cd stress in M. meretrix larvae.

  6. Acute effects of cadmium on osmoregulation of the freshwater teleost Prochilodus lineatus: Enzymes activity and plasma ions

    Energy Technology Data Exchange (ETDEWEB)

    Silva, Alexandre O.F. da [Departamento de Ciências Fisiológicas, Laboratório de Ecofisiologia Animal, UEL, Londrina (Brazil); Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, UEL, Londrina (Brazil); Centro de Ciências Humanas e da Educação, UENP, Jacarezinho (Brazil); Martinez, Cláudia B.R., E-mail: cbueno@uel.br [Departamento de Ciências Fisiológicas, Laboratório de Ecofisiologia Animal, UEL, Londrina (Brazil); Programa Multicêntrico de Pós-graduação em Ciências Fisiológicas, UEL, Londrina (Brazil)

    2014-11-15

    Highlights: • Cd effects were evaluated on plasma ions and ATPases of Prochilodus lineatus. • Fish were exposed to 1 and 10 μg Cd L{sup −1} for 24 and 96 h. • Na{sup +}/K{sup +}-ATPase and carbonic anhydrase decreased in gills and kidney after Cd exposure. • Gill Ca{sup 2+}-ATPase and plasma Ca{sup 2+} decreased after Cd exposure. • Cd concentrations set by Brazilian guidelines affect Ca{sup 2+} regulation of P. lineatus. - Abstract: Cadmium (Cd) is a trace element that is very toxic to fish. It is commonly found in surface waters contaminated with industrial effluents. When dissolved in water, Cd can rapidly cause physiological changes in the gills and kidneys of freshwater fish. The objective of this study was to evaluate the acute effects of Cd on the osmoregulation of the Neotropical fish Prochilodus lineatus. Juvenile fish were exposed to Cd at two concentrations [1 (Cd1) and 10 (Cd10) μg L{sup −1}] for 24 and 96 h. The effects of Cd were evaluated through the analysis of ions (Na{sup +}, K{sup +}, Ca{sup 2+}, and Cl{sup −}) and plasma osmolality, and by measuring the activities of enzymes involved in osmoregulation obtained from the gills and kidney. Fish exposed to Cd for 24 and 96 h showed a decrease in Na{sup +}/K{sup +}-ATPase activity in the gills and kidney. The activity of carbonic anhydrase decreased in the gills after 24 h and in both tissues after 96 h of Cd exposure. The gill Ca{sup 2+}-ATPase activity also decreased with Cd exposure, with a concomitant drop in the plasma concentration of Ca{sup 2+}. Despite the hypocalcemia, there were no changes in the concentration of the ions Na{sup +}, K{sup +}, and Cl{sup −} or in plasma osmolality. Among the enzymes involved in ion transport, H{sup +}-ATPase was the only enzyme that showed increased activity in gills, whereas its activity in kidney remained unchanged. The results of this study demonstrate that waterborne Cd at the maximum concentrations set by Brazilian guidelines for

  7. Acute effects of cadmium on osmoregulation of the freshwater teleost Prochilodus lineatus: Enzymes activity and plasma ions

    International Nuclear Information System (INIS)

    Silva, Alexandre O.F. da; Martinez, Cláudia B.R.

    2014-01-01

    Highlights: • Cd effects were evaluated on plasma ions and ATPases of Prochilodus lineatus. • Fish were exposed to 1 and 10 μg Cd L −1 for 24 and 96 h. • Na + /K + -ATPase and carbonic anhydrase decreased in gills and kidney after Cd exposure. • Gill Ca 2+ -ATPase and plasma Ca 2+ decreased after Cd exposure. • Cd concentrations set by Brazilian guidelines affect Ca 2+ regulation of P. lineatus. - Abstract: Cadmium (Cd) is a trace element that is very toxic to fish. It is commonly found in surface waters contaminated with industrial effluents. When dissolved in water, Cd can rapidly cause physiological changes in the gills and kidneys of freshwater fish. The objective of this study was to evaluate the acute effects of Cd on the osmoregulation of the Neotropical fish Prochilodus lineatus. Juvenile fish were exposed to Cd at two concentrations [1 (Cd1) and 10 (Cd10) μg L −1 ] for 24 and 96 h. The effects of Cd were evaluated through the analysis of ions (Na + , K + , Ca 2+ , and Cl − ) and plasma osmolality, and by measuring the activities of enzymes involved in osmoregulation obtained from the gills and kidney. Fish exposed to Cd for 24 and 96 h showed a decrease in Na + /K + -ATPase activity in the gills and kidney. The activity of carbonic anhydrase decreased in the gills after 24 h and in both tissues after 96 h of Cd exposure. The gill Ca 2+ -ATPase activity also decreased with Cd exposure, with a concomitant drop in the plasma concentration of Ca 2+ . Despite the hypocalcemia, there were no changes in the concentration of the ions Na + , K + , and Cl − or in plasma osmolality. Among the enzymes involved in ion transport, H + -ATPase was the only enzyme that showed increased activity in gills, whereas its activity in kidney remained unchanged. The results of this study demonstrate that waterborne Cd at the maximum concentrations set by Brazilian guidelines for freshwater affects the gills and kidney functions of P. lineatus. Acute exposure to

  8. Reliability of soluble IL-2 receptor measurements obtained with enzyme-linked immunosorbent assay

    International Nuclear Information System (INIS)

    Akiyama, Mitoshi; Takaishi, Masatoshi; Murakami, Yoshie; Ueda, Ryuzo; Yamakido, Michio; Tsubokura, Tokuo.

    1989-09-01

    Using an enzyme-linked immunosorbent assay (ELISA), human soluble interleukin-2 receptors (IL-2R) were measured in the serum of patients with various autoimmune system diseases. To study the sensitivity and specificity of the assay, soluble IL-2Rs were measured in the culture supernatants and in the cell extracts of peripheral blood mononuclear cells activated with phytohemagglutinin (PHA), purified protein derivative of tuberculin, and allogeneic lymphocytes, as well as in the serum of patients with various collagen diseases. The results correlated well with reports from other laboratories. For example, when stimulated by PHA, the greatest amount of soluble IL-2Rs was produced at the fastest rate. In addition, soluble IL-2R levels in the serum of collagen disease patients were significantly higher than those in healthy persons, who themselves exhibited low levels of detectable soluble IL-2Rs. It is hoped that reliable ELISA measurements of soluble IL-2Rs in the serum of atomic bomb survivors will assist in the interpretation of data collected during the work described in RP 2-87, a study of autoimmunity and autoimmune diseases in the Adult Health Study. (author)

  9. Fluvoxamine alters the activity of energy metabolism enzymes in the brain

    Directory of Open Access Journals (Sweden)

    Gabriela K. Ferreira

    2014-09-01

    Full Text Available Objective: Several studies support the hypothesis that metabolism impairment is involved in the pathophysiology of depression and that some antidepressants act by modulating brain energy metabolism. Thus, we evaluated the activity of Krebs cycle enzymes, the mitochondrial respiratory chain, and creatine kinase in the brain of rats subjected to prolonged administration of fluvoxamine. Methods: Wistar rats received daily administration of fluvoxamine in saline (10, 30, and 60 mg/kg for 14 days. Twelve hours after the last administration, rats were killed by decapitation and the prefrontal cortex, cerebral cortex, hippocampus, striatum, and cerebellum were rapidly isolated. Results: The activities of citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV were decreased after prolonged administration of fluvoxamine in rats. However, the activities of complex II, succinate dehydrogenase, and creatine kinase were increased. Conclusions: Alterations in activity of energy metabolism enzymes were observed in most brain areas analyzed. Thus, we suggest that the decrease in citrate synthase, malate dehydrogenase, and complexes I, II-III, and IV can be related to adverse effects of pharmacotherapy, but long-term molecular adaptations cannot be ruled out. In addition, we demonstrated that these changes varied according to brain structure or biochemical analysis and were not dose-dependent.

  10. Warming increases hotspot areas of enzyme activity and shortens the duration of hot moments in the detritusphere

    Science.gov (United States)

    Ma, Xiaomin; Razavi, Bahar S.; Holz, Maire; Blagodatskaya, Evgenia; Kuzyakov, Yakov

    2017-04-01

    Temperature effects on enzyme kinetics and on the spatial distribution of microbial hotspots are important because of their potential feedback to climate change. We used direct zymography to study the spatial distributions of enzymes responsible for P (phosphatase), C (cellobiohydrolase) and N (leucine-aminopeptidase) cycles in the rhizosphere (living roots of maize) and detritusphere (7 and 14 days after cutting shoots). Soil zymography was coupled with enzyme kinetics to test temperature effects (10, 20, 30 and 40 °C) on the dynamics and localization of these three enzymes in the detritusphere. Total hotspot areas of enzyme activity were 1.9-7.9 times larger and their extension was broader in the detritusphere compared to rhizosphere. From 10 to 30 °C, the hotspot areas enlarged by a factor of 2-24 and Vmax increased by 1.5-6.6 times; both, however, decreased at 40 °C. For the first time, we found a close positive correlation between Vmax and the areas of enzyme activity hotspots, indicating that maximum reaction rate is coupled with hotspot formation. The substrate turnover time at 30 °C were 1.7-6.7-fold faster than at 10 °C. The Km of cellobiohydrolase and phosphatase significantly increased at 30 and 40 °C, indicating high enzyme conformational flexibility, or isoenzyme production at warm temperatures. We conclude that soil warming (at least up to 30°C) increases hotspot areas of enzyme activity and the maximum reaction rate (Vmax) in the detritusphere. This, in turn, leads to faster substrate exhaustion and shortens the duration of hot moments.

  11. Changes of enzyme activities in lens after glaucoma trabecular resection

    Directory of Open Access Journals (Sweden)

    Jian-Ping Wang

    2013-08-01

    Full Text Available AIM: To observe the change of lens antioxidant enzyme activity after glaucoma trabecular resection. METHODS: Thirty-two eyes of sixteen New-Zealand rabbits(2.2-2.4kgwere divided into two groups. The left eyes of rabbits underwent standard glaucoma trabecular resection were treatment group, and the normal right eyes served as controls. Transparency of lenses was monitored by a slit-lamp biomicroscopy before and after glaucoma trabecular resection. The morphology of lens cells was observed under the light microscope.The activities of Na+-K+-ATPase,catalase(CAT, glutathion peroxidase(GSH-px, glutathione reductase(GR, superoxide dismutase(SODand content of malondialdehyde(MDAin lenses were detected six months after trabecular resection. RESULTS: Lenses were clear in both treatment group and normal control group during the six months after operation. The morphology and structure of lens cells were normal under the light microscope in both operation group and normal group. The activity of lens cells antioxidant enzyme activity were significantly decreased in operation group compared with control group, Na+-K+-ATPase declined by 20.97%, CAT declined by 16.36%, SOD declined by 4.46%, GR declined by 4.85%, GSH-px declined by 10.02%, and MDA increased by 16.31%. CONCLUSION: Glaucoma trabecular resection can induce the change of Na+-K+-ATPase, CAT, GSH-px, GR, SOD and MDA in lens of rabbit. Glaucoma filtration surgery for the occurrence of cataract development mechanism has important guiding significance.

  12. Approaching a Conceptual Understanding of Enzyme Kinetics and Inhibition: Development of an Active Learning Inquiry Activity for Prehealth and Nonscience Majors

    Science.gov (United States)

    House, Chloe; Meades, Glen; Linenberger, Kimberly J.

    2016-01-01

    Presented is a guided inquiry activity designed to be conducted with prenursing students using an analogous system to help develop a conceptual understanding of factors impacting enzyme kinetics and the various types of enzyme inhibition. Pre- and postconceptual understanding evaluations and effectiveness of implementation surveys were given to…

  13. Enzymes in Commercial Cellulase Preparations Bind Differently to Dioxane Extracted Lignins

    Energy Technology Data Exchange (ETDEWEB)

    Yarbrough, John M.; Mittal, Ashutosh; Katahira, Rui; Mansfield, Elisabeth; Taylor, Larry E.; Decker, Stephen R.; Himmel, Michael E.; Vinzant, Todd

    2017-04-24

    Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. To evaluate lignin binding affinities of different enzyme activities in various commercial cellulase formulations in order to determine if enzyme losses due to lignin binding can be modulated by using different enzymes of the same activity We used water:dioxane (1:9) to extract lignin from pretreated corn stover. Commercial cellulases were incubated with lignin and the unbound supernatants were evaluated for individual enzyme loss by SDS=PAGE and these were correlated with activity loss using various pNP-sugar substrates. Colorimetric assays for general glycosyl hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native systems demonstrated low binding of endo- and exo-cellulases, high binding of xylanase, and moderate ..beta..-glucosidase binding. Engineered cellulase mixtures exhibited low binding of exo-cellulases, very strong binding of endocellulases and ..beta..- glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of ..beta..-glucosidase activities. Bound and unbound activities were correlated with general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated with binding of ..beta..-glucosidase activity. While ..beta..-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated with xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between the three classes of cellulases preparations indicate that it is certainly possible to alter the binding of specific

  14. Allosteric regulation of epigenetic modifying enzymes.

    Science.gov (United States)

    Zucconi, Beth E; Cole, Philip A

    2017-08-01

    Epigenetic enzymes including histone modifying enzymes are key regulators of gene expression in normal and disease processes. Many drug development strategies to target histone modifying enzymes have focused on ligands that bind to enzyme active sites, but allosteric pockets offer potentially attractive opportunities for therapeutic development. Recent biochemical studies have revealed roles for small molecule and peptide ligands binding outside of the active sites in modulating the catalytic activities of histone modifying enzymes. Here we highlight several examples of allosteric regulation of epigenetic enzymes and discuss the biological significance of these findings. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. [Hydrogen production and enzyme activity of acidophilic strain X-29 at different C/N ratio].

    Science.gov (United States)

    Li, Qiu-bo; Xing, De-feng; Ren, Nan-qi; Zhao, Li-hua; Song, Ye-ying

    2006-04-01

    Some fermentative bacteria can produce hydrogen by utilizing carbohydrate and other kinds of organic compounds as substrates. Hydrogen production was also determined by both the limiting of growth and related enzyme activity in energy metabolism. Carbon and nitrogen are needed for the growth and metabolism of microorganisms. In addition, the carbon/nitrogen (C/N) ratio can influence the material metabolized and the energy produced. In order to improve the hydrogen production efficiency of the bacteria, we analyzed the effect of different C/N ratios on hydrogen production and the related enzyme activities in the acidophilic strain X-29 using batch test. The results indicate that the differences in the metabolism level and enzyme activity are obvious at different C/N ratios. Although the difference in liquid fermentative products produced per unit of biomass is not obvious, hydrogen production is enhanced at a specifically determined ratio. At a C/N ratio of 14 the accumulative hydrogen yield of strain X-29 reaches the maximum, 2210.9 mL/g. At different C/N ratios, the expression of hydrogenase activity vary; the activity of hydrogenase decrease quickly after reaching a maximum along with the fermentation process, but the time of expression is short. The activity of alcohol dehydrogenase (ADH) tend to stabilize after reaching a peak along with the fermentation process, the difference in expression activity is little, and the expression period is long at different C/N ratios. At a C/N ratio of 14 hydrogenase and ADH reach the maximum 2.88 micromol x (min x mg)(-1) and 33.2 micromol x (min x mg)(-1), respectively. It is shown that the C/N ratio has an important effect on enhancing hydrogen production and enzyme activity.

  16. Prescribed burning effects on soil enzyme activity in a southern Ohio hardwood forest: A landscape-scale analysis

    Science.gov (United States)

    Ralph E. J. Boerner; Kelly L. M. Decker; Elaine K. Sutherland

    2000-01-01

    We assessed the effect of a single, dormant season prescribed fire on soil enzyme activity in oak-hickory (Quercus-Carya) forests in southern Ohio, USA. Four enzymes specific for different C sources were chosen for monitoring: acid phosphatase, beta-glucosidase, chitinase and phenol oxidase. Postfire acid phosphatase activity was generally reduced by burning and...

  17. Skeletal Muscle Fibre-Specific Knockout of p53 Does Not Reduce Mitochondrial Content or Enzyme Activity

    Directory of Open Access Journals (Sweden)

    Ben Stocks

    2017-12-01

    Full Text Available Tumour protein 53 (p53 has been implicated in the regulation of mitochondrial biogenesis in skeletal muscle, with whole-body p53 knockout mice displaying impairments in basal mitochondrial content, respiratory capacity, and enzyme activity. This study aimed to determine the effect of skeletal muscle-specific loss of p53 on mitochondrial content and enzyme activity. Mitochondrial protein content, enzyme activity and mRNA profiles were assessed in skeletal muscle of 8-week-old male muscle fibre-specific p53 knockout mice (p53 mKO and floxed littermate controls (WT under basal conditions. p53 mKO and WT mice displayed similar content of electron transport chain proteins I-V and citrate synthase enzyme activity in skeletal muscle. In addition, the content of proteins regulating mitochondrial morphology (MFN2, mitofillin, OPA1, DRP1, FIS1, fatty acid metabolism (β-HAD, ACADM, ACADL, ACADVL, carbohydrate metabolism (HKII, PDH, energy sensing (AMPKα2, AMPKβ2, and gene transcription (NRF1, PGC-1α, and TFAM were comparable in p53 mKO and WT mice (p > 0.05. Furthermore, p53 mKO mice exhibited normal mRNA profiles of targeted mitochondrial, metabolic and transcriptional proteins (p > 0.05. Thus, it appears that p53 expression in skeletal muscle fibres is not required to develop or maintain mitochondrial protein content or enzyme function in skeletal muscle under basal conditions.

  18. [Diversity and enzyme-producing activity of culturable halophilic bacteria in Daishan Saltern of East China].

    Science.gov (United States)

    Yang, Dan-Dan; Li, Qian; Huang, Jing-Jing; Chen, Min

    2012-11-01

    Soil and saline water samples were collected from the Daishan Saltern of East China, and the halophilic bacteria were isolated and cultured by using selective media, aimed to investigate the diversity and enzyme-producing activity of culturable halophilic bacteria in saltern environment. A total of 181 strains were isolated by culture-dependent method. Specific primers were used to amplify the 16S rRNA gene of bacteria and archaea. The operation taxonomy units (OTUs) were determined by ARDRA method, and the representative strain of each OTU was sequenced. The phylogenetic position of all the isolated strains was determined by 16S rRNA sequencing. The results showed that the isolated 181 strains displayed 21 operational taxonomic units (OTUs), of which, 12 OTUs belonged to halophilic bacteria, and the others belonged to halophilic archaea. Phylogenetic analysis indicated that there were 7 genera presented among the halophilic bacteria group, and 4 genera presented among the halophilic archaea group. The dominant halophilic strains were of Halomonas and Haloarcula, with 46.8% in halophilic bacteria and 49.1% in halophilic archaea group, respectively. Enzyme-producing analysis indicated that most strains displayed enzyme-producing activity, including the activities of producing amylase, proteinase and lipase, and the dominant strains capable of enzyme-producing were of Haloarcula. Our results showed that in the environment of Daishan Saltern, there existed a higher diversity of halophilic bacteria, being a source sink for screening enzyme-producing bacterial strains.

  19. Differences in forage-acquisition and fungal enzyme activity contribute to niche segregation in Panamanian leaf-cutting ants.

    Directory of Open Access Journals (Sweden)

    Pepijn W Kooij

    Full Text Available The genera Atta and Acromyrmex are often grouped as leaf-cutting ants for pest management assessments and ecological surveys, although their mature colony sizes and foraging niches may differ substantially. Few studies have addressed such interspecific differences at the same site, which prompted us to conduct a comparative study across six sympatric leaf-cutting ant species in Central Panama. We show that foraging rates during the transition between dry and wet season differ about 60 fold between genera, but are relatively constant across species within genera. These differences appear to match overall differences in colony size, especially when Atta workers that return to their nests without leaves are assumed to carry liquid food. We confirm that Panamanian Atta specialize primarily on tree-leaves whereas Acromyrmex focus on collecting flowers and herbal leaves and that species within genera are similar in these overall foraging strategies. Species within genera tended to be spaced out over the three habitat categories that we distinguished (forest, forest edge, open grassland, but each of these habitats normally had only a single predominant Atta and Acromyrmex species. We measured activities of twelve fungus garden decomposition enzymes, belonging to the amylases, cellulases, hemicellulases, pectinases and proteinases, and show that average enzyme activity per unit of fungal mass in Atta gardens is lower than in Acromyrmex gardens. Expression profiles of fungal enzymes in Atta also appeared to be more specialized than in Acromyrmex, possibly reflecting variation in forage material. Our results suggest that species- and genus-level identities of leaf-cutting ants and habitat-specific foraging profiles may give predictable differences in the expression of fungal genes coding for decomposition enzymes.

  20. Characterization of biotransformation enzyme activities in primary rat proximal tubular cells

    NARCIS (Netherlands)

    Schaaf, G.; de Groene, E.M.; Maas, R.; Commandeur, J.N.M.; Fink-Gremmels, J.

    2001-01-01

    The proximal tubule is a frequent target for nephrotoxic compounds due to it's ability to transport and accumulate xenobiotics and their metabolites, as well as by the presence of an organ-selective set of biotransformation enzymes. The aim of the present study was to characterize the activities of

  1. Accurate and reproducible measurements of RhoA activation in small samples of primary cells.

    Science.gov (United States)

    Nini, Lylia; Dagnino, Lina

    2010-03-01

    Rho GTPase activation is essential in a wide variety of cellular processes. Measurement of Rho GTPase activation is difficult with limited material, such as tissues or primary cells that exhibit stringent culture requirements for growth and survival. We defined parameters to accurately and reproducibly measure RhoA activation (i.e., RhoA-GTP) in cultured primary keratinocytes in response to serum and growth factor stimulation using enzyme-linked immunosorbent assay (ELISA)-based G-LISA assays. We also established conditions that minimize RhoA-GTP in unstimulated cells without affecting viability, allowing accurate measurements of RhoA activation on stimulation or induction of exogenous GTPase expression. Copyright 2009 Elsevier Inc. All rights reserved.

  2. Partial Characterization of α-Galactosidic Activity from the Antarctic Bacterial Isolate, . LX-20 as a Potential Feed Enzyme Source

    Directory of Open Access Journals (Sweden)

    Inkyung Park

    2012-06-01

    Full Text Available An Antarctic bacterial isolate displaying extracellular α-galactosidic activity was named Paenibacillus sp. LX-20 based on 16S rRNA gene sequence analysis. Optimal activity for the LX-20 α-galactosidase occurred at pH 6.0–6.5 and 45°C. The enzyme immobilized on the smart polymer Eudragit L-100 retained 70% of its original activity after incubation for 30 min at 50°C, while the free enzyme retained 58% of activity. The enzyme had relatively high specificity for α-D-galactosides such as p-nitrophenyl-α-galactopyranoside, melibiose, raffinose and stachyose, and was resistant to some proteases such as trypsin, pancreatin and pronase. Enzyme activity was almost completely inhibited by Ag+, Hg2+, Cu2+, and sodium dodecyl sulfate, but activity was not affected by β-mercaptoethanol or EDTA. LX-20 α-galactosidase may be potentially useful as an additive for soybean processing in the feed industry.

  3. Simplified methods for in vivo measurement of acetylcholinesterase activity in rodent brain

    Energy Technology Data Exchange (ETDEWEB)

    Kilbourn, Michael R. E-mail: mkilbour@umich.edu; Sherman, Phillip S.; Snyder, Scott E

    1999-07-01

    Simplified methods for in vivo studies of acetylcholinesterase (AChE) activity in rodent brain were evaluated using N-[{sup 11}C]methylpiperidinyl propionate ([{sup 11}C]PMP) as an enzyme substrate. Regional mouse brain distributions were determined at 1 min (representing initial brain uptake) and 30 min (representing trapped product) after intravenous [{sup 11}C]PMP administration. Single time point tissue concentrations (percent injected dose/gram at 30 min), tissue concentration ratios (striatum/cerebellum and striatum/cortex ratios at 30 min), and regional tissue retention fractions (defined as percent injected dose 30 min/percent injected dose 1 min) were evaluated as measures of AChE enzymatic activity in mouse brain. Studies were carried out in control animals and after dosing with phenserine, a selective centrally active AChE inhibitor; neostigmine, a peripheral cholinesterase inhibitor; and a combination of the two drugs. In control and phenserine-treated animals, absolute tissue concentrations and regional retention fractions provide good measures of dose-dependent inhibition of brain AChE; tissue concentration ratios, however, provide erroneous conclusions. Peripheral inhibition of cholinesterases, which changes the blood pharmacokinetics of the radiotracer, diminishes the sensitivity of all measures to detect changes in central inhibition of the enzyme. We conclude that certain simple measures of AChE hydrolysis rates for [{sup 11}C]PMP are suitable for studies where alterations of the peripheral blood metabolism of the tracer are kept to a minimum.

  4. Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF

    International Nuclear Information System (INIS)

    Park, Kiyoung; Li, Ning; Kwak, Yeonju; Srnec, Martin

    2017-01-01

    Binuclear non-heme iron enzymes activate O 2 for diverse chemistries that include oxygenation of organic substrates and hydrogen atom abstraction. This process often involves the formation of peroxo-bridged biferric intermediates, only some of which can perform electrophilic reactions. To elucidate the geometric and electronic structural requirements to activate peroxo reactivity, the active peroxo intermediate in 4-aminobenzoate N-oxygenase (AurF) has been characterized spectroscopically and computationally. A magnetic circular dichroism study of reduced AurF shows that its electronic and geometric structures are poised to react rapidly with O 2 . Nuclear resonance vibrational spectroscopic definition of the peroxo intermediate formed in this reaction shows that the active intermediate has a protonated peroxo bridge. Density functional theory computations on the structure established here show that the protonation activates peroxide for electrophilic/single-electron-transfer reactivity. As a result, this activation of peroxide by protonation is likely also relevant to the reactive peroxo intermediates in other binuclear non-heme iron enzymes.

  5. Zymography methods for visualizing hydrolytic enzymes

    OpenAIRE

    Vandooren, Jennifer; Geurts, Nathalie; Martens, Erik; Van den Steen, Philippe E.; Opdenakker, Ghislain

    2013-01-01

    Zymography is a technique for studying hydrolytic enzymes on the basis of substrate degradation. It is a powerful., but often misinterpreted, tool. yielding information on potential. hydrolytic activities, enzyme forms and the locations of active enzymes. In this Review, zymography techniques are compared in terms of advantages, limitations and interpretations. With in gel zymography, enzyme forms are visualized according to their molecular weights. Proteolytic activities are localized in tis...

  6. Loss in photosynthesis during senescence is accompanied by an increase in the activity of β-galactosidase in leaves of Arabidopsis thaliana: modulation of the enzyme activity by water stress.

    Science.gov (United States)

    Pandey, Jitendra Kumar; Dash, Sidhartha Kumar; Biswal, Basanti

    2017-07-01

    The precise nature of the developmental modulation of the activity of cell wall hydrolases that breakdown the wall polysaccharides to maintain cellular sugar homeostasis under sugar starvation environment still remains unclear. In this work, the activity of β-galactosidase (EC 3.2.1.23), a cell-wall-bound enzyme known to degrade the wall polysaccharides, has been demonstrated to remarkably enhance during senescence-induced loss in photosynthesis in Arabidopsis thaliana. The enhancement in the enzyme activity reaches a peak at the terminal phase of senescence when the rate of photosynthesis is at its minimum. Although the precise nature of chemistry of the interface between the decline in photosynthesis and enhancement in the activity of the enzyme could not be fully resolved, the enhancement in its activity in dark and its suppression in light or with exogenous sugars may indicate the involvement of loss of photosynthetic production of sugars as a key factor that initiates and stimulates the activity of the enzyme. The hydrolase possibly participates in the catabolic network of cell wall polysaccharides to produce sugars for execution of energy-dependant senescence program in the background of loss of photosynthesis. Drought stress experienced by the senescing leaves accelerates the decline in photosynthesis with further stimulation in the activity of the enzyme. The stress recovery of photosynthesis and suppression of the enzyme activity on withdrawal of stress support the proposition of photosynthetic modulation of the cell-wall-bound enzyme activity.

  7. Regulation of sucrose metabolism in higher plants: localization and regulation of activity of key enzymes

    Science.gov (United States)

    Winter, H.; Huber, S. C.; Brown, C. S. (Principal Investigator)

    2000-01-01

    Sucrose (Suc) plays a central role in plant growth and development. It is a major end product of photosynthesis and functions as a primary transport sugar and in some cases as a direct or indirect regulator of gene expression. Research during the last 2 decades has identified the pathways involved and which enzymes contribute to the control of flux. Availability of metabolites for Suc synthesis and 'demand' for products of sucrose degradation are important factors, but this review specifically focuses on the biosynthetic enzyme sucrose-phosphate synthase (SPS), and the degradative enzymes, sucrose synthase (SuSy), and the invertases. Recent progress has included the cloning of genes encoding these enzymes and the elucidation of posttranslational regulatory mechanisms. Protein phosphorylation is emerging as an important mechanism controlling SPS activity in response to various environmental and endogenous signals. In terms of Suc degradation, invertase-catalyzed hydrolysis generally has been associated with cell expansion, whereas SuSy-catalyzed metabolism has been linked with biosynthetic processes (e.g., cell wall or storage products). Recent results indicate that SuSy may be localized in multiple cellular compartments: (1) as a soluble enzyme in the cytosol (as traditionally assumed); (2) associated with the plasma membrane; and (3) associated with the actin cytoskeleton. Phosphorylation of SuSy has been shown to occur and may be one of the factors controlling localization of the enzyme. The purpose of this review is to summarize some of the recent developments relating to regulation of activity and localization of key enzymes involved in sucrose metabolism in plants.

  8. Change of various enzyme activities of koji and sterilization of koiji by γ-radiation

    International Nuclear Information System (INIS)

    Iwano, Kimio; Mikami, Shigeaki; Oishi, Atsushi; Shiinoki, Satoshi

    1987-01-01

    Sterilization and changes of various enzyme activities of koji by gamma irradiation were investigated. A dose of 1 Mrad gamma irradiation was effective for the sterilization of koji. Various enzymes of koji were inactivated about 10-30% by the irradiation, while no influence was observed for shochu fermentation. There seemed to be no influence for qualities of sake and shochu by the irradiation. (author)

  9. Evaluation of the Activities of Antioxidant Enzyme and Lysosomal Enzymes of the Longissimus dorsi Muscle from Hanwoo (Korean Cattle) in Various Freezing Conditions

    OpenAIRE

    Kang, Sun Moon; Kang, Geunho; Seong, Pil-Nam; Park, Beomyoung; Kim, Donghun; Cho, Soohyun

    2014-01-01

    This study was conducted to evaluate the activities of antioxidant enzyme (glutathione peroxidase (GSH-Px)) and lysosomal enzymes (alpha-glucopyranosidase (AGP) and beta-N-acetyl-glucosaminidase (BNAG)) of the longissimus dorsi (LD) muscle from Hanwoo (Korean cattle) in three freezing conditions. Following freezing at -20, -60, and -196? (liquid nitrogen), LD samples (48 h post-slaughter) were treated as follows: 1) freezing for 14 d, 2) 1 to 4 freeze-thaw cycles (2 d of freezing in each cycl...

  10. Abalone Protein Hydrolysates: Preparation, Angiotensin I Converting Enzyme Inhibition and Cellular Antioxidant Activity.

    Science.gov (United States)

    Park, Soo Yeon; Je, Jae-Young; Hwang, Joung-Youl; Ahn, Chang-Bum

    2015-09-01

    Abalone protein was hydrolyzed by enzymatic hydrolysis and the optimal enzyme/substrate (E/S) ratios were determined. Abalone protein hydrolysates (APH) produced by Protamex at E/S ratio of 1:100 showed angiotensin I converting enzyme inhibitory activity with IC50 of 0.46 mg/mL, and APH obtained by Flavourzyme at E/S ratio of 1:100 possessed the oxygen radical absorbance capacity value of 457.6 μM trolox equivalent/mg sample. Flavourzyme abalone protein hydrolysates (FAPH) also exhibited H2O2 scavenging activity with IC50 of 0.48 mg/mL and Fe(2+) chelating activity with IC50 of 2.26 mg/mL as well as high reducing power. FAPH significantly (P<0.05) protected H2O2-induced hepatic cell damage in cultured hepatocytes, and the cell viability was restored to 90.27% in the presence of FAPH. FAPH exhibited 46.20% intracellular ROS scavenging activity and 57.89% lipid peroxidation inhibition activity in cultured hepatocytes. Overall, APH may be useful as an ingredient for functional foods.

  11. Imbalance between pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in acute respiratory distress syndrome

    NARCIS (Netherlands)

    Wösten-van Asperen, Roelie M.; Bos, Albert P.; Bem, Reinout A.; Dierdorp, Barbara S.; Dekker, Tamara; van Goor, Harry; Kamilic, Jelena; van der Loos, Chris M.; van den Berg, Elske; Bruijn, Martijn; van Woensel, Job B.; Lutter, René

    2013-01-01

    Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts angiotensin

  12. Imbalance between pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in acute respiratory distress syndrome

    NARCIS (Netherlands)

    Wosten-van Asperen, Roelie M.; Bos, Albert; Bem, Reinout A.; Dierdorp, Barbara S.; Dekker, Tamara; van Goor, Harry; Kamilic, Jelena; van der Loos, Chris M.; van den Berg, Elske; Bruijn, Martijn; van Woensel, Job B.; Lutter, Rene

    2013-01-01

    Objective: Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts

  13. Effect of “Tai Chi” exercise on antioxidant enzymes activities and ...

    African Journals Online (AJOL)

    ... exercise that increases physical strength and relax the mind. Materials and Methods: The study investigates effect of “tai chi” exercise on antioxidant enzymes activities and immunity function in participants. These participants were randomly divided into two groups: “tai chi” exercise group (n=25) and control group (n=25).

  14. Fungal Community and Ligninolytic Enzyme Activities in Quercus deserticola Trel. Litter from Forest Fragments with Increasing Levels of Disturbance

    Directory of Open Access Journals (Sweden)

    Jesús A. Rosales-Castillo

    2017-12-01

    Full Text Available Litter fungal communities and their ligninolytic enzyme activities (laccase, Mn-peroxidase, and lignin-peroxidase play a vital role in forest biogeochemical cycles by breaking down plant cell wall polymers, including recalcitrant lignin. However, litter fungal communities and ligninolytic enzyme activities have rarely been studied in Neotropical, non-coniferous forests. Here, we found no significant differences in litter ligninolytic enzyme activities from well preserved, moderately disturbed, and heavily disturbed Quercus deserticola Trel. forests in central Mexico. However, we did find seasonal effects on enzyme activities: during the dry season, we observed lower laccase, and increased Mn-peroxidase and lignin-peroxidase activities, and in the rainy season, Mn-peroxidase and lignin-peroxidase activities were lower, while laccase activity peaked. Fungal diversity (Shannon-Weaver and Simpson indices based on ITS-rDNA analyses decreased with increased disturbance, and principal component analysis showed that litter fungal communities are structured differently between forest types. White-rot Polyporales and Auriculariales only occurred in the well preserved forest, and a high number of Ascomycota were shared between forests. While the degree of forest disturbance significantly affected the litter fungal community structure, the ligninolytic enzyme activities remained unaffected, suggesting functional redundancy and a possible role of generalist Ascomycota taxa in litter delignification. Forest conservation and restoration strategies must account for leaf litter and its associated fungal community.

  15. Effect of hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion tissues

    Directory of Open Access Journals (Sweden)

    Lushchak V.I.

    1998-01-01

    Full Text Available The effect of hypoxia on the levels of glycogen, glucose and lactate as well as the activities and binding of glycolytic and associated enzymes to subcellular structures was studied in brain, liver and white muscle of the teleost fish, Scorpaena porcus. Hypoxia exposure decreased glucose levels in liver from 2.53 to 1.70 µmol/g wet weight and in muscle led to its increase from 3.64 to 25.1 µmol/g wet weight. Maximal activities of several enzymes in brain were increased by hypoxia: hexokinase by 23%, phosphoglucoisomerase by 47% and phosphofructokinase (PFK by 56%. However, activities of other enzymes in brain as well as enzymes in liver and white muscle were largely unchanged or decreased during experimental hypoxia. Glycolytic enzymes in all three tissues were partitioned between soluble and particulate-bound forms. In several cases, the percentage of bound enzymes was reduced during hypoxia; bound aldolase in brain was reduced from 36.4 to 30.3% whereas glucose-6-phosphate dehydrogenase fell from 55.7 to 28.7% bound. In muscle PFK was reduced from 57.4 to 41.7% bound. Oppositely, the proportion of bound aldolase and triosephosphate isomerase increased in hypoxic muscle. Phosphoglucomutase did not appear to occur in a bound form in liver and bound phosphoglucomutase disappeared in muscle during hypoxia exposure. Anoxia exposure also led to the disappearance of bound fructose-1,6-bisphosphatase in liver, whereas a bound fraction of this enzyme appeared in white muscle of anoxic animals. The possible function of reversible binding of glycolytic enzymes to subcellular structures as a regulatory mechanism of carbohydrate metabolism is discussed.

  16. Gelatin in situ zymography on fixed, paraffin-embedded tissue: zinc and ethanol fixation preserve enzyme activity.

    Science.gov (United States)

    Hadler-Olsen, Elin; Kanapathippillai, Premasany; Berg, Eli; Svineng, Gunbjørg; Winberg, Jan-Olof; Uhlin-Hansen, Lars

    2010-01-01

    In situ zymography is a method for the detection and localization of enzymatic activity in tissue sections. This method is used with frozen sections because routine fixation of tissue in neutral-buffered formalin inhibits enzyme activity. However, frozen sections present with poor tissue morphology, making precise localization of enzymatic activity difficult to determine. Ethanol- and zinc-buffered fixative (ZBF) are known to preserve both morphological and functional properties of the tissue well, but it has not previously been shown that these fixatives preserve enzyme activity. In the present study, we show that in situ zymography can be performed on ethanol- and ZBF-fixed paraffin-embedded tissue. Compared with snap-frozen tissue, ethanol- and ZBF-fixed tissue showed stronger signals and superior morphology, allowing for a much more precise detection of gelatinolytic activity. Gelatinolytic enzymes could also be extracted from both ethanol- and ZBF-fixed tissue. The yield, as analyzed by SDS-PAGE gelatin zymography and Western blotting, was influenced by the composition of the extraction buffer, but was generally lower than that obtained from unfixed tissue.

  17. Effect of fluorozis on the erythrocyte antioxidant enzyme activity levels

    International Nuclear Information System (INIS)

    Akdogan, M.; YiImaz, D.; Yontem, M.; Kalei, S.; Kilic, I.

    2011-01-01

    While the flourine level of (drinking) water was higher than normal ranges in the center of Isparta region before 1995 year, this problematic situation is solved in later years. (However) the individuals who are staying in Yenice district are still expose to high levels of fluorine because of the usage of Andik spring water (3.8 mg/L flour level) as drinking water. In this study we aimed to investigate the harmful effect of floride on human erythrocytes via antioxidant defence system and lipid peroxidation. Therefore, we studied the activities of erythrocyte antioxidant enzymes such as Superoxide Dismutase (SOD), Glutathione Peroxidase (GSH-Px) and Catalase (CAT), and the level of erythrocyte Glutathione (GSH), thiobarbituric acid reactive substance (TBARS) and the level of urine floride in high floride exposed people (children, adult and elderly). The activities of SOD, GSH-Px and CAT and the level of GSH, TBARS and urine floride were higher in 3.8 mg/L floride exposed children (Group II) than 0.8 mg/L floride exposed control children (Group I) (p 0.05). The activities of SOD, GSH-Px and CAT were lower and the levels of TBARS and urine floride were higher in 3.8 mg/L floride exposed elderly people (Group VI) than 0.8 mg/L floride exposed control elderly people (Group V) (p 0.05). As a result we thought that increased SOD, GSH-Px and CAT activities in floride exposed children and adult people, decreased activities of these enzymes in floride exposed elderly people, and increased TBARS in all groups may indicate floride caused oxidative damage in erythrocytes. (author)

  18. Site- and horizon-specific patterns of microbial community structure and enzyme activities in permafrost-affected soils of Greenland

    Science.gov (United States)

    Gittel, Antje; Bárta, Jiří; Kohoutová, Iva; Schnecker, Jörg; Wild, Birgit; Čapek, Petr; Kaiser, Christina; Torsvik, Vigdis L.; Richter, Andreas; Schleper, Christa; Urich, Tim

    2014-01-01

    Permafrost-affected soils in the Northern latitudes store huge amounts of organic carbon (OC) that is prone to microbial degradation and subsequent release of greenhouse gasses to the atmosphere. In Greenland, the consequences of permafrost thaw have only recently been addressed, and predictions on its impact on the carbon budget are thus still highly uncertain. However, the fate of OC is not only determined by abiotic factors, but closely tied to microbial activity. We investigated eight soil profiles in northeast Greenland comprising two sites with typical tundra vegetation and one wet fen site. We assessed microbial community structure and diversity (SSU rRNA gene tag sequencing, quantification of bacteria, archaea and fungi), and measured hydrolytic and oxidative enzyme activities. Sampling site and thus abiotic factors had a significant impact on microbial community structure, diversity and activity, the wet fen site exhibiting higher potential enzyme activities and presumably being a hot spot for anaerobic degradation processes such as fermentation and methanogenesis. Lowest fungal to bacterial ratios were found in topsoils that had been relocated by cryoturbation (“buried topsoils”), resulting from a decrease in fungal abundance compared to recent (“unburied”) topsoils. Actinobacteria (in particular Intrasporangiaceae) accounted for a major fraction of the microbial community in buried topsoils, but were only of minor abundance in all other soil horizons. It was indicated that the distribution pattern of Actinobacteria and a variety of other bacterial classes was related to the activity of phenol oxidases and peroxidases supporting the hypothesis that bacteria might resume the role of fungi in oxidative enzyme production and degradation of phenolic and other complex substrates in these soils. Our study sheds light on the highly diverse, but poorly-studied communities in permafrost-affected soils in Greenland and their role in OC degradation. PMID

  19. Toward single enzyme analysis in a droplet-based micro and nanofluidic system

    NARCIS (Netherlands)

    Arayanarakool, Rerngchai

    2012-01-01

    In this thesis, we have demonstrated the application of micro- and nanofluidic devices to generate an array of aqueous droplets in oil phase for single-enzyme encapsulation and activity measurement. We chose droplet-based microfluidics for this purpose of monitoring single-enzyme reactions since the

  20. Acetylcholinesterase immobilization and characterization, and comparison of the activity of the porous silicon-immobilized enzyme with its free counterpart.

    Science.gov (United States)

    Saleem, Muhammad; Rafiq, Muhammad; Seo, Sung-Yum; Lee, Ki Hwan

    2016-02-02

    A successful prescription is presented for acetylcholinesterase physically adsorbed on to a mesoporous silicon surface, with a promising hydrolytic response towards acetylthiocholine iodide. The catalytic behaviour of the immobilized enzyme was assessed by spectrophotometric bioassay using neostigmine methyl sulfate as a standard acetycholinesterase inhibitor. The surface modification was studied through field emission SEM, Fourier transform IR spectroscopy, energy-dispersive X-ray spectroscopy, cathode luminescence and X-ray photoelectron spectroscopy analysis, photoluminescence measurement and spectrophotometric bioassay. The porous silicon-immobilized enzyme not only yielded greater enzyme stability, but also significantly improved the native photoluminescence at room temperature of the bare porous silicon architecture. The results indicated the promising catalytic behaviour of immobilized enzyme compared with that of its free counterpart, with a greater stability, and that it aided reusability and easy separation from the reaction mixture. The porous silicon-immobilized enzyme was found to retain 50% of its activity, promising thermal stability up to 90°C, reusability for up to three cycles, pH stability over a broad pH of 4-9 and a shelf-life of 44 days, with an optimal hydrolytic response towards acetylthiocholine iodide at variable drug concentrations. On the basis of these findings, it was believed that the porous silicon-immobilized enzyme could be exploited as a reusable biocatalyst and for screening of acetylcholinesterase inhibitors from crude plant extracts and synthesized organic compounds. Moreover, the immobilized enzyme could offer a great deal as a viable biocatalyst in bioprocessing for the chemical and pharmaceutical industries, and bioremediation to enhance productivity and robustness. © 2016 Authors.

  1. Bacterial community composition and extracellular enzyme activity in temperate streambed sediment during drying and rewetting.

    Directory of Open Access Journals (Sweden)

    Elisabeth Pohlon

    Full Text Available Droughts are among the most important disturbance events for stream ecosystems; they not only affect stream hydrology but also the stream biota. Although desiccation of streams is common in Mediterranean regions, phases of dryness in headwaters have been observed more often and for longer periods in extended temperate regions, including Central Europe, reflecting global climate change and enhanced water withdrawal. The effects of desiccation and rewetting on the bacterial community composition and extracellular enzyme activity, a key process in the carbon flow of streams and rivers, were investigated in a typical Central European stream, the Breitenbach (Hesse, Germany. Wet streambed sediment is an important habitat in streams. It was sampled and exposed in the laboratory to different drying scenarios (fast, intermediate, slow for 13 weeks, followed by rewetting of the sediment from the fast drying scenario via a sediment core perfusion technique for 2 weeks. Bacterial community structure was analyzed using CARD-FISH and TGGE, and extracellular enzyme activity was assessed using fluorogenic model substrates. During desiccation the bacterial community composition shifted toward composition in soil, exhibiting increasing proportions of Actinobacteria and Alphaproteobacteria and decreasing proportions of Bacteroidetes and Betaproteobacteria. Simultaneously the activities of extracellular enzymes decreased, most pronounced with aminopeptidases and less pronounced with enzymes involved in the degradation of polymeric carbohydrates. After rewetting, the general ecosystem functioning, with respect to extracellular enzyme activity, recovered after 10 to 14 days. However, the bacterial community composition had not yet achieved its original composition as in unaffected sediments within this time. Thus, whether the bacterial community eventually recovers completely after these events remains unknown. Perhaps this community undergoes permanent changes

  2. Immobilization of the Enzyme Glucose Oxidase on Both Bulk and Porous SiO2 Surfaces

    Directory of Open Access Journals (Sweden)

    Fulvia Sinatra

    2008-09-01

    Full Text Available Silicon dioxide surfaces, both bulk and porous, were used to anchor the enzyme glucose oxidase. The immobilization protocol was optimized and the samples characterized using X-ray Photoelectron Spectroscopy, Energy Dispersive X-rays coupled to scanning electron microscopy and enzymatic activity measurements. We show that a uniform layer was obtained by activating the oxide before immobilization. X-ray Photoelectron Spectroscopy measurements carried out on bulk oxide showed that the silicon substrate signal was fully screened after the enzyme deposition showing the absence of uncovered surface regions. The enzyme presence was detected monitoring both the C 1s and N 1s signals. Finally, enzymatic activity measurements confirmed that the glucose oxidase activity was preserved after immobilization and maintained after three months of shelf life if the sample was properly stored. The importance of using porous silicon oxide to maximize the surface area was also evidenced.

  3. Global characterization of in vivo enzyme catalytic rates and their correspondence to in vitro kcat measurements

    Science.gov (United States)

    Davidi, Dan; Noor, Elad; Liebermeister, Wolfram; Bar-Even, Arren; Flamholz, Avi; Tummler, Katja; Barenholz, Uri; Goldenfeld, Miki; Shlomi, Tomer; Milo, Ron

    2016-01-01

    Turnover numbers, also known as kcat values, are fundamental properties of enzymes. However, kcat data are scarce and measured in vitro, thus may not faithfully represent the in vivo situation. A basic question that awaits elucidation is: how representative are kcat values for the maximal catalytic rates of enzymes in vivo? Here, we harness omics data to calculate kmaxvivo, the observed maximal catalytic rate of an enzyme inside cells. Comparison with kcat values from Escherichia coli, yields a correlation of r2= 0.62 in log scale (p enzymes and the backward flux dictated by thermodynamics, we further refine the correspondence between kmaxvivo and kcat values. The approach we present here characterizes the quantitative relationship between enzymatic catalysis in vitro and in vivo and offers a high-throughput method for extracting enzyme kinetic constants from omics data. PMID:26951675

  4. Activation of Pseudomonas aeruginosa elastase in Pseudomonas putida by triggering dissociation of the propeptide-enzyme complex

    NARCIS (Netherlands)

    Braun, P; Bitter, W; Tommassen, J

    2000-01-01

    The propeptide of Pseudomonas aeruginosa elastase functions both as an intramolecular chaperone required for the folding of the enzyme and as an inhibitor that prevents activity of the enzyme before its secretion into the extracellular medium. Since expression of the lasB gene, which encodes

  5. Surface binding sites in carbohydrate active enzymes: An emerging picture of structural and functional diversity

    DEFF Research Database (Denmark)

    Svensson, Birte; Cockburn, Darrell

    2013-01-01

    is not universal and is in fact rare among some families of enzymes. In some cases an alternative to possessing a CBM is for the enzyme to bind to the substrate at a site on the catalytic domain, but away from the active site. Such a site is termed a surface (or secondary) binding site (SBS). SBSs have been...

  6. Protein stability and enzyme activity at extreme biological temperatures

    International Nuclear Information System (INIS)

    Feller, Georges

    2010-01-01

    Psychrophilic microorganisms thrive in permanently cold environments, even at subzero temperatures. To maintain metabolic rates compatible with sustained life, they have improved the dynamics of their protein structures, thereby enabling appropriate molecular motions required for biological activity at low temperatures. As a consequence of this structural flexibility, psychrophilic proteins are unstable and heat-labile. In the upper range of biological temperatures, thermophiles and hyperthermophiles grow at temperatures > 100 0 C and synthesize ultra-stable proteins. However, thermophilic enzymes are nearly inactive at room temperature as a result of their compactness and rigidity. At the molecular level, both types of extremophilic proteins have adapted the same structural factors, but in opposite directions, to address either activity at low temperatures or stability in hot environments. A model based on folding funnels is proposed accounting for the stability-activity relationships in extremophilic proteins. (topical review)

  7. Effects of manufactured nano-copper on copper uptake, bioaccumulation and enzyme activities in cowpea grown on soil substrate.

    Science.gov (United States)

    Ogunkunle, Clement O; Jimoh, Mahboob A; Asogwa, Nnaemeka T; Viswanathan, K; Vishwakarma, Vinita; Fatoba, Paul O

    2018-07-15

    Increased use of nanoparticles-based products in agriculture portends important implications for agriculture. Therefore, the impact of nano-copper particles (nano-Cu for 65 days. Results indicated significant (Pnano-Cu levels compared to control, and bioaccumulation increased in seeds by at least 250%. Response of antioxidant enzymes to both nano-Cu types was concentration-dependent. Activity of APX and GR was enhanced in leaves and roots in response to both nano-Cu treatments in similar patterns compared to control. Both nano-Cu increased CAT activity in roots while SOD activity reduced in both leaves and roots. This shows that response of antioxidant enzymes to nano-Cu toxicity was organ-specific in cowpea. Malondialdehyde, a measure of lipid peroxidation, increased at 500 -1000 mg/kg of 25 nm nano-Cu in leaves by average of 8.4%, and 60-80 nm nano-Cu in root by 52.8%, showing particle-size and organ-dependent toxicity of nano-Cu. In conclusion, exposure of cowpea to nano-Cu treatments increased both the uptake and bioaccumulation of Cu, and also promoted the activity of APX and GR in root and leaf tissues of cowpea. Therefore, APX- and GR-activity level could be a useful predictive biomarker of nano-Cu toxicity in cowpea. Copyright © 2018 Elsevier Inc. All rights reserved.

  8. Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by 14C putrescine method

    International Nuclear Information System (INIS)

    Fogel, W.A.; Bieganski, T.; Wozniak, J.; Maslinski, C.

    1978-01-01

    The Δ 1 pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi 14 C putrescine test (1961, Archs Biochem. Biophys., vol.95, 242), has been investigated in several tissue homogenates. When guinea pig liver homogenate was used as a source of enzyme in the presence of aldehyde dehydrogenase inhibitors chlorate hydrate and acetaldehyde the level of formation Δ 1 pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced Δ 1 pyrroline formation, but to a lesser degree. In other tissues, with very high initial diamine oxidase activity (rat intestine, dog kidney) or with very low diamine oxidase activity (guinea pig skin, dog liver) the influence of these inhibitors was only slight. Pyrazole, an inhibitor of alcohol dehydrogenase exerted only a small effect on Δ 1 pyrroline formation. All aldehyde-metabolizing enzymes inhibitors, except pyrazole, were without effect on purified pea seddling and hog kidney diamine oxidases. The use of aldehyde-metabolizing enzymes inhibitors may help to reveal the real values of diamine oxidase activity, when tissues homogenates are used as a source of enzyme. (author)

  9. Circulating angiotensin-converting enzyme 2 activity in patients with chronic kidney disease without previous history of cardiovascular disease.

    Science.gov (United States)

    Anguiano, Lidia; Riera, Marta; Pascual, Julio; Valdivielso, José Manuel; Barrios, Clara; Betriu, Angels; Mojal, Sergi; Fernández, Elvira; Soler, María José

    2015-07-01

    Patients with cardiovascular (CV) disease have an increased circulating angiotensin-converting enzyme 2 (ACE2) activity, but there is little information about changes in ACE2 in chronic kidney disease (CKD) patients without history of CV disease. We examined circulating ACE2 activity in CKD patients at stages 3-5 (CKD3-5) and in dialysis (CKD5D) without any history of CV disease. Circulating ACE2 activity was measured in human ethylenediamine-tetraacetic acid (EDTA)-plasma samples from the NEFRONA study (n = 2572): control group (CONT) (n = 568), CKD3-5 (n = 1458) and CKD5D (n = 546). Different clinical and analytical variables such as gender; age; history of diabetes mellitus (DM), dyslipidemia and hypertension; glycaemic, renal, lipid and anaemia profiles; vitamin D analogues treatment and antihypertensive treatments (angiotensin-converting enzyme inhibitor and angiotensin receptor blockade) were analysed. Circulating ACE2 and ACE activities were measured using modified fluorimetric assay for EDTA-plasma samples, where zinc chloride was added to recover enzymatic activity. In CKD3-5 and CKD5D, significant decrease in circulating ACE2 activity was observed when compared with CONT, but no differences were found between CKD3-5 and CKD5 when performing paired case-control studies. By multivariate linear regression analysis, male gender and advanced age were identified as independent predictors of ACE2 activity in all groups. Diabetes was identified as independent predictor of ACE2 activity in CKD3-5. Significant increase in the activity of circulating ACE was found in CKD3-5 and CKD5D when compared with CONT and in CKD5D when compared with CKD3-5. By multiple regression analysis, female gender and younger age were identified as independent predictors of ACE activity in CONT and CKD3-5. Diabetes was also identified as an independent predictor of ACE activity in CKD3-5 patients. Circulating ACE2 and ACE activities can be measured in human EDTA-plasma samples with zinc

  10. Digestive enzymes of some earthworms.

    Science.gov (United States)

    Mishra, P C; Dash, M C

    1980-10-15

    4 species of tropical earthworms differed with regard to enzyme activity. The maximum activity of protease and of cellulase occurred in the posterior region of the gut of the earthworms. On the average Octochaetona surensis shows maximum activity and Drawida calebi shows minimum activity for all the enzymes studied.

  11. Definition of regional dependence of activity antioxidative enzymes means of the dispersive analysis

    Directory of Open Access Journals (Sweden)

    Anatoly T. Bykov

    2011-05-01

    Full Text Available In article application of the dispersive analysis for an estimation of dependence of activity antioxidative enzymes from region of constant residing, age, sex and the disease diagnosis is considered.

  12. Soil enzyme dynamics in chlorpyrifos-treated soils under the influence of earthworms.

    Science.gov (United States)

    Sanchez-Hernandez, Juan C; Notario Del Pino, J; Capowiez, Yvan; Mazzia, Christophe; Rault, Magali

    2018-01-15

    Earthworms contribute, directly and indirectly, to contaminant biodegradation. However, most of bioremediation studies using these annelids focus on pollutant dissipation, thus disregarding the health status of the organism implied in bioremediation as well as the recovery of indicators of soil quality. A microcosm study was performed using Lumbricus terrestris to determine whether earthworm density (2 or 4individuals/kg wet soil) and the time of exposure (1, 2, 6, 12, and 18wk) could affect chlorpyrifos persistence in soil initially treated with 20mg active ingredientkg -1 wet soil. Additionally, selected earthworm biomarkers and soil enzyme activities were measured as indicators of earthworm health and soil quality, respectively. After an 18-wk incubation period, no earthworm was killed by the pesticide, but clear signs of severe intoxication were detected, i.e., 90% inhibition in muscle acetylcholinesterase and carboxylesterase (CbE) activities. Unexpectedly, the earthworm density had no significant impact on chlorpyrifos dissipation rate, for which the measured half-life ranged between 30.3d (control soils) and 44.5d (low earthworm density) or 36.7d (high earthworm density). The dynamic response of several soil enzymes to chlorpyrifos exposure was examined calculating the geometric mean and the treated-soil quality index, which are common enzyme-based indexes of microbial functional diversity. Both indexes showed a significant and linear increase of the global enzyme response after 6wk of chlorpyrifos treatment in the presence of earthworms. Examination of individual enzymes revealed that soil CbE activity could decrease chlorpyrifos-oxon impact upon the rest of enzyme activities. Although L. terrestris was found not to accelerate chlorpyrifos dissipation, a significant increase in the activity of soil enzyme activities was achieved compared with earthworm-free, chlorpyrifos-treated soils. Therefore, the inoculation of organophosphorus-contaminated soils with L

  13. ACTIVITY OF SUPEROXIDE DISMUTASE ENZYME IN YEAST SACCHAROMYCES CEREVISIAE

    Directory of Open Access Journals (Sweden)

    Blažena Lavová

    2014-02-01

    Full Text Available Reactive oxygen species (ROS with reactive nitrogen species (RNS are known to play dual role in biological systems, they can be harmful or beneficial to living systems. ROS can be important mediators of damage to cell structures, including proteins, lipids and nucleic acids termed as oxidative stress. The antioxidant enzymes protect the organism against the oxidative damage caused by active oxygen forms. The role of superoxide dismutase (SOD is to accelerate the dismutation of the toxic superoxide radical, produced during oxidative energy processes, to hydrogen peroxide and molecular oxygen. In this study, SOD activity of three yeast strains Saccharomyces cerevisiae was determined. It was found that SOD activity was the highest (23.7 U.mg-1 protein in strain 612 after 28 hours of cultivation. The lowest SOD activity from all tested strains was found after 56 hours of cultivation of strain Gyöng (0.7 U.mg-1 protein.

  14. THE COORDINATION COMPOUNDS OF COBALT (II, III WITH DITHIOCARBAMIC ACID DERIVATIVES — MODIFICATORS OF HYDROLYTIC ENZYMES ACTIVITY

    Directory of Open Access Journals (Sweden)

    L. D. Varbanets

    2013-02-01

    Full Text Available Chloride, bromide and isothiocyanate complexes of cobalt(II with N-substituted thiocarbamoyl-N?-pentamethylenesulfenamides (1–(12, and also complexes of cobalt(II, Ш with derivatives of morpholine-4-carbodithioic acid (13–(18 have been used as modificators of enzymes of hydrolytic action — Bacillus thurin-giensis ІМВ В-7324 peptidases, Bacillus subtilis 147 and Aspergillus flavus var. oryzae 80428 amylases, Eupenicillium erubescens 248 and Cryptococcus albidus 1001 rhamnosidases. It was shown that cobalt (II, Ш compounds influence differently on the activity of enzymes tested, exerted both inhibitory and stimulatory action. It gives a possibility to expect that manifestation of activity by complex molecule depends on ligand and anion presence — Cl–, Br– or NCS–. The high activating action of cobalt(II complexes with N-substituted thiocarbamoyl-N?-pentamethylenesulphenamides (1–(12 on elastase and fibrinolytic activity of peptidases compared to tris(4-morpholinecarbodithioatocobalt(ІІІ (14 and products of its interaction with halogens (15–(17, causes inhibitory effect that is probably due to presence of a weekly S–N link, which is easy subjected to homolytic breaking. The studies of influences of cobalt(II complexes on activity of C. аlbidus and E. еrubescens ?-Lrhamnosidases showed, that majority of compounds inhibits of its activity, at that the most inhibitory effect exerts to C. аlbidus enzyme.To sum up, it is possible to state that character of influence of cobalt(II complexes with N-substituted thiocarbamoyl-N?-pentamethylenesulphenamides, and also cobalt(II, Ш complexes with derivatives of morpholine-4-carbodithioic acid varies depending on both strain producer and enzyme tested. The difference in complex effects on enzymes tested are due to peculiarities of building and functional groups of their active centers, which are also responsible for binding with modificators.

  15. Enzyme active site mimics based on TriAzaCyclophane (TAC)-scaffolded peptides and amino acid residues

    NARCIS (Netherlands)

    Albada, H.B.

    2009-01-01

    This thesis describes the scope and limitations of the application of TriAzaCyclophane (TAC)-scaffolded peptides or amino acid residues as enzyme active site mimics, as ligands in asymmetric catalysis and as hydrolysis catalysts attached to vancomycin. For the mimicry of functional group enzymes, of

  16. Laccase Enzymes in Inocula Pleurotus spp

    Directory of Open Access Journals (Sweden)

    Nora García-Oduardo

    2017-01-01

    Full Text Available The cultivation of edible and medicinal mushrooms Pleurotus has been aimed at promoting alternative management for agricultural products. This basidiomicete has been the subject of numerous studies because of its fruiting body constitutes a food, being a producer of enzymes with industrial interest and for its ability of biotransformation of lignocellulosic substrates. Pleurotus inocula in the established technology for growing edible and medicinal mushrooms in the CEBI Research- Production Plant were performed using sorghum or wheat. However, it is possible to expand the possibilities with other substrates. In this paper, the results of laccase enzymes production in inocula prepared with sorghum, corn and coffee pulp with two strains Pleurotus ostreatus CCEBI 3021 and Pleurotus ostreatus CCEBI 3024 are presented. The period of preparation of seed reaches 15-21 days, the measurements of laccase activity were performed in periods of seven days. Extraction of crude enzyme was performed in aqueous phase, the determination of the laccase enzyme activity, using guaiacol as substrate. The results obtained in this work with studies in previous work using sorghum as inocula are compared. It is found that higher yields are obtained laccase in coffee pulp. This study contributes to the theoretical knowledge and to provide alternatives for securing the production process of the plant.

  17. Insights into catalytic activity of industrial enzyme Co-nitrile hydratase. Docking studies of nitriles and amides.

    Science.gov (United States)

    Peplowski, Lukasz; Kubiak, Karina; Nowak, Wieslaw

    2007-07-01

    Nitrile hydratase (NHase) is an enzyme containing non-corrin Co3+ in the non-standard active site. NHases from Pseudonocardia thermophila JCM 3095 catalyse hydration of nitriles to corresponding amides. The efficiency of the enzyme is 100 times higher for aliphatic nitriles then aromatic ones. In order to understand better this selectivity dockings of a series of aliphatic and aromatic nitriles and related amides into a model protein based on an X-ray structure were performed. Substantial differences in binding modes were observed, showing better conformational freedom of aliphatic compounds. Distinct interactions with postranslationally modified cysteines present in the active site of the enzyme were observed. Modeling shows that water molecule activated by a metal ion may easily directly attack the docked acrylonitrile to transform this molecule into acryloamide. Thus docking studies provide support for one of the reaction mechanisms discussed in the literature.

  18. Effect of Probiotic Bacillus megaterium PTB 1.4 on the Population of Intestinal Microflora, Digestive Enzyme Activity and the Growth of Catfish (Clarias sp.

    Directory of Open Access Journals (Sweden)

    Wahyu Afrilasari

    2016-10-01

    Full Text Available This study aimed to analyze the effect of Bacillus megaterium PTB 1.4 on the population of intestinal microflora, digestive enzyme activity, and the growth of catfish. Gnotobiotic and normal fish were used. Treatment using gnotobiotic was divided into gnoto (with feed and 100 μg/mL rifampicin and gnotoplus (with feed, 100 μg/mL rifampicin, and 1% probiotic; whereas treatment using normal fish was divided into normalplus (with feed and 1% probiotic and normal (only feed. The amount of bacteria on gastrointestinal tract was measured 30 days after treatments using the total plate count method. The results indicated no significant difference in bacterial growth between gnotobiotic and normal fish. The total amount of probiotic bacteria with normalplus treatment was significantly different with gnotoplus. The activity of protease and amylase enzymes, and specific growth rate in normalplus treatment were significantly higher (p < 0.05 than other treatments. Bacillus megaterium PTB 1.4 increased the activity of digestive enzymes and the growth of catfish.

  19. Response of hydrolytic enzyme activities and nitrogen mineralization to fertilizer and organic matter application in subtropical paddy soils

    Science.gov (United States)

    Kader, Mohammed Abdul; Yeasmin, Sabina; Akter, Masuda; Sleutel, Steven

    2016-04-01

    Driving controllers of nitrogen (N) mineralization in paddy soils, especially under anaerobic soil conditions, remain elusive. The influence of exogenous organic matter (OM) and fertilizer application on the activities of five relevant enzymes (β-glucosaminidase, β-glucosidase, L-glutaminase, urease and arylamidase) was measured in two long-term field experiments. One 18-years field experiment was established on a weathered terrace soil with a rice-wheat crop rotation at the Bangabandhu Sheikh Mujibur Rahman Agricultural University (BSMRAU) having five OM treatments combined with two mineral N fertilizer levels. Another 30-years experiment was established on a young floodplain soil with rice-rice crop rotation at the Bangladesh Agricultural University (BAU) having eight mineral fertilizer treatments combined with organic manure. At BSMRAU, N fertilizer and OM amendments significantly increased all enzyme activities, suggesting them to be primarily determined by substrate availability. At BAU, non-responsiveness of β-glucosidase activity suggested little effect of the studied fertilizer and OM amendments on general soil microbial activity. Notwithstanding probably equal microbial demand for N, β-glucosaminidase and L-glutaminase activities differed significantly among the treatments (P>0.05) and followed strikingly opposite trends and correlations with soil organic N mineralization. So enzymatic pathways to acquire N differed by treatment at BAU, indicating differences in soil N quality and bio-availability. L-glutaminase activity was significantly positively correlated to the aerobic and anaerobic N mineralization rates at both field experiments. Combined with negative correlations between β-glucosaminidase activity and N mineralization rates, it appears that terminal amino acid NH2 hydrolysis was a rate-limiting step for soil N mineralization at BAU. Future investigations with joint quantification of polyphenol accumulation and binding of N, alongside an

  20. Screening and characterization of thermo-active enzymes of biotechnological interest produced by thermophilic Bacillus isolated from hot springs in Tunisia.

    Science.gov (United States)

    Thebti, Wajdi; Riahi, Yosra; Gharsalli, Rawand; Belhadj, Omrane

    2016-01-01

    As part of the contribution to the global efforts in research of thermostable enzymes being of industrial interest, we focus on the isolation of thermophilic bacteria from Tunisian hot springs. Among the collection of 161 strains of thermophilic Bacillus isolated from different samples of thermal water in Tunisia, 20% are capable of growing at 100°C and the rest grow at 70°C or above. Preliminary activity tests on media supplemented with enzyme-substrates confirmed that 35 strains produced amylases, 37 - proteases, 43 - cellulases, 31 - xylanases and 37 - mannanases. The study of the effect of temperature on enzyme activity led to determination of the optimal temperatures of activities that vary between 60 and 100°C. Several enzymes were active at high temperatures (80, 90 and 100°C) and kept their activity even at 110°C. Several isolated strains producing enzymes with high optimal temperatures of activity were described for the first time in this study. Both strains B62 and B120 are producers of amylase, protease, cellulase, xylanase, and mannanase. The sequencing of 16S DNA identified isolated strains as Geobacillus kaustophillus, Aeribacillus pallidus, Geobacillus galactosidasus and Geobacillus toebii.

  1. Measure of enzymatic activity coincident with 2450 MHz microwave exposure

    Energy Technology Data Exchange (ETDEWEB)

    Ward, T R; Allis, J W; Elder, J A

    1975-09-01

    Enzyme preparations were exposed to microwave radiation at 2450 MHz and enzymatic activity was simultaneously monitored spectrophotometrically with a crossed-beam exposure detection system. Enzymes studied were glucose 6-phosphate dehydrogenase from human red blood cells and yeast, adenylate kinase from rat liver mitochondria and rabbit muscle, and rat liver microsomal NADPH cytochrome c reductase. No difference was found between the specific activity at 25/sup 0/C of unirradiated controls and enzyme preparations irradiated at an absorbed dose rate of 42 W/kg.

  2. The Effects of Ibogaine on Uterine Smooth Muscle Contractions: Relation to the Activity of Antioxidant Enzymes

    Directory of Open Access Journals (Sweden)

    Zorana Oreščanin-Dušić

    2018-01-01

    Full Text Available Ibogaine is an indole alkaloid originally extracted from the root bark of the African rainforest shrub Tabernanthe iboga. It has been explored as a treatment for substance abuse because it interrupts drug addiction and relieves withdrawal symptoms. However, it has been shown that ibogaine treatment leads to a sharp and transient fall in cellular ATP level followed by an increase of cellular respiration and ROS production. Since contractile tissues are sensitive to changes in the levels of ATP and ROS, here we investigated an ibogaine-mediated link between altered redox homeostasis and uterine contractile activity. We found that low concentrations of ibogaine stimulated contractile activity in spontaneously active uteri, but incremental increase of doses inhibited it. Inhibitory concentrations of ibogaine led to decreased SOD1 and elevated GSH-Px activity, but doses that completely inhibited contractions increased CAT activity. Western blot analyses showed that changes in enzyme activities were not due to elevated enzyme protein concentrations but posttranslational modifications. Changes in antioxidant enzyme activities point to a vast concentration-dependent increase in H2O2 level. Knowing that extracellular ATP stimulates isolated uterus contractility, while H2O2 has an inhibitory effect, this concentration-dependent stimulation/inhibition could be linked to ibogaine-related alterations in ATP level and redox homeostasis.

  3. Changes in element accumulation, phenolic metabolism, and antioxidative enzyme activities in the red-skin roots of Panax ginseng.

    Science.gov (United States)

    Zhou, Ying; Yang, Zhenming; Gao, Lingling; Liu, Wen; Liu, Rongkun; Zhao, Junting; You, Jiangfeng

    2017-07-01

    Red-skin root disease has seriously decreased the quality and production of Panax ginseng (ginseng). To explore the disease's origin, comparative analysis was performed in different parts of the plant, particularly the epidermis, cortex, and/or fibrous roots of 5-yr-old healthy and diseased red-skin ginseng. The inorganic element composition, phenolic compound concentration, reactive oxidation system, antioxidant concentrations such as ascorbate and glutathione, activities of enzymes related to phenolic metabolism and oxidation, and antioxidative system particularly the ascorbate-glutathione cycle were examined using conventional methods. Aluminum (Al), iron (Fe), magnesium, and phosphorus were increased, whereas manganese was unchanged and calcium was decreased in the epidermis and fibrous root of red-skin ginseng, which also contained higher levels of phenolic compounds, higher activities of the phenolic compound-synthesizing enzyme phenylalanine ammonia-lyase and the phenolic compound oxidation-related enzymes guaiacol peroxidase and polyphenoloxidase. As the substrate of guaiacol peroxidase, higher levels of H 2 O 2 and correspondingly higher activities of superoxide dismutase and catalase were found in red-skin ginseng. Increased levels of ascorbate and glutathione; increased activities of l-galactose 1-dehydrogenase, ascorbate peroxidase, ascorbic acid oxidase, and glutathione reductase; and lower activities of dehydroascorbate reductase, monodehydroascorbate reductase, and glutathione peroxidase were found in red-skin ginseng. Glutathione- S -transferase activity remained constant. Hence, higher element accumulation, particularly Al and Fe, activated multiple enzymes related to accumulation of phenolic compounds and their oxidation. This might contribute to red-skin symptoms in ginseng. It is proposed that antioxidant and antioxidative enzymes, especially those involved in ascorbate-glutathione cycles, are activated to protect against phenolic compound

  4. Bioactive properties of commercialised pomegranate (Punica granatum) juice: antioxidant, antiproliferative and enzyme inhibiting activities.

    Science.gov (United States)

    Les, Francisco; Prieto, Jose M; Arbonés-Mainar, Jose Miguel; Valero, Marta Sofía; López, Víctor

    2015-06-01

    Pomegranate juice and related products have long been used either in traditional medicine or as nutritional supplements claiming beneficial effects. Although there are several studies on this food plant, only a few studies have been performed with pomegranate juice or marketed products. The aim of this work is to evaluate the antioxidant effects of pomegranate juice on cellular models using hydrogen peroxide as an oxidizing agent or DPPH and superoxide radicals in cell free systems. The antiproliferative effects of the juice were measured on HeLa and PC-3 cells by the MTT assay and pharmacologically relevant enzymes (cyclooxygenases, xanthine oxidase, acetylcholinesterase and monoamine oxidase A) were selected for enzymatic inhibition assays. Pomegranate juice showed significant protective effects against hydrogen peroxide induced toxicity in the Artemia salina and HepG2 models; these effects may be attributed to radical scavenging properties of pomegranate as the juice was able to reduce DPPH and superoxide radicals. Moderate antiproliferative activities in HeLa and PC-3 cancer cells were observed. However, pomegranate juice was also able to inhibit COX-2 and MAO-A enzymes. This study reveals some mechanisms by which pomegranate juice may have interesting and beneficial effects in human health.

  5. Ultrasound-assisted extraction and characterization of hydrolytic and oxidative enzymes produced by solid state fermentation.

    Science.gov (United States)

    Szabo, Orsolya Erzsebet; Csiszar, Emilia; Toth, Karolina; Szakacs, George; Koczka, Bela

    2015-01-01

    Ligninolytic and hydrolytic enzymes were produced with six selected fungi on flax substrate by solid state fermentation (SSF). The extracellular enzyme production of the organisms in two SSF media was evaluated by measuring the soluble protein concentration and the filter paper, endoxylanase, 1,4-β-d-glucosidase, 1,4-β-d-endoglucanase, polygalacturonase, lignin peroxidase, manganese peroxidase and laccase activities of the clear culture solutions produced by conventional extraction from the SSF materials. The SSF material of the best enzyme producer (Trichoderma virens TUB F-498) was further investigated to enhance the enzyme recovery by low frequency ultrasound treatment. Performance of both the original and ultrasound macerated crude enzyme mixtures was evaluated in degradation of the colored lignin-containing and waxy materials of raw linen fabric. Results proved that sonication (at 40%, 60% and 80% amplitudes, for 60min) did not result in reduction in the filter paper, lignin peroxidase and laccase activities of the crude enzyme solution, but has a significant positive effect on the efficiency of enzyme extraction from the SSF material. Depending on the parameters of sonication, the enzyme activities in the extracts obtained can be increased up to 129-413% of the original activities measured in the control extracts recovered by a common magnetic stirrer. Sonication also has an effect on both the enzymatic removal of the lignin-containing color materials and hydrophobic surface layer from the raw linen. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Enzymes in biogenesis of plant cell wall polysaccharides. Enzyme characterization using tracer techniques

    International Nuclear Information System (INIS)

    Dickinson, D.B.

    1975-01-01

    Enzymes and metabolic pathways, by which starch and cell wall polysaccharides are formed, were investigated in order to learn how these processes are regulated and to identify the enzymatic regulatory mechanisms involved. Germinating lily pollen was used for studies of cell wall formation, and pollen and maize endosperm for studies of starch biosynthesis. Hexokinase being the first step in conversion of hexoses to starch, wall polysaccharides and respiratory substrates, maize endosperm enzyme was assayed by its conversion of 14 C-hexose to 14 C-hexose-6-P, and rapid separation of the two labelled compounds on anion-exchange paper. This enzyme did not appear to be under tight regulation by feed-back inhibition or activation, nor to be severely inhibited by glucose-6-P or activated by citrate. ADP-glucose pyrophosphorylase and other pyrophosphorylases were assayed radiochemically with 14 C-glucose-1-P (forward direction) or 32-PPsub(i) (reverse direction). They showed that the maize endosperm enzyme was activated by the glycolytic intermediates fructose-6-P and 3-phosphoglycerate, and that low levels of the enzyme were present in the high sucrose-low starch mutant named shrunken-2. Under optimal in-vitro assay conditions, the pollen enzyme reacted four times faster than the observed in-vivo rate of starch accumulation. Biogenesis of plant cell wall polysaccharides requires the conversion of hexose phosphates to various sugar nucleotides and utilization of the latter by the appropriate polysaccharide synthetases. Lily pollen possesses a β-1,3-glucan synthetase which is activated up to six-fold by β-linked oligosaccharides. Hence, the in-vivo activity of this enzyme may be modulated by such effector molecules

  7. Effect of tillage systems and permanent groundcover intercropped with orange trees on soil enzyme activities

    Directory of Open Access Journals (Sweden)

    Elcio Liborio Balota

    2011-04-01

    Full Text Available The objective of this study was to evaluate the effect of different soil tillage systems and groundcover crops intercropped with orange trees on soil enzyme activities. The experiment was performed in an Ultisol soil in northwestern Paraná State. Two soil tillage systems were evaluated [conventional tillage (CT across the entire area and strip tillage (ST with a 2-m strip width] in combination with various groundcover vegetation management systems. Soil samples were collected after five years of experimental management at a depth of 0-15 cm under the tree canopy and in the inter-row space in the following treatments: (1 CT-Calopogonium mucunoides; (2 CT-Arachis pintoi; (3 CT-Bahiagrass; (4 CT-Brachiaria humidicola; and (5 ST-B. humidicola. The soil tillage systems and groundcover crops influenced the soil enzyme activities both under the tree canopy and in the inter-row space. The cultivation of B. humidicola provided higher amylase, arylsulfatase, acid phosphatase and alkaline phosphatase than other groundcover species. Strip tillage increased enzyme activities compared to the conventional tillage system.

  8. Enzyme inhibition by iminosugars

    DEFF Research Database (Denmark)

    López, Óscar; Qing, Feng-Ling; Pedersen, Christian Marcus

    2013-01-01

    Imino- and azasugar glycosidase inhibitors display pH dependant inhibition reflecting that both the inhibitor and the enzyme active site have groups that change protonation state with pH. With the enzyme having two acidic groups and the inhibitor one basic group, enzyme-inhibitor complexes...

  9. Enzymes with activity toward Xyloglucan

    NARCIS (Netherlands)

    Vincken, J.P.

    2003-01-01

    Xyloglucans are plant cell wall polysaccharides, which belong to the hemicellulose class. Here the structural variations of xyloglucans will be reviewed. Subsequently, the anchoring of xyloglucan in the plant cell wall will be discussed. Enzymes involved in degradation or modification of xyloglucan

  10. Enzyme immunoassay for measurement of murine plasminogen activator inhibitor-1, employing a specific antibody produced by the DNA vaccine method.

    Science.gov (United States)

    Yamada, Takayuki; Takagi, Akira; Takeshita, Kyosuke; Yamamoto, Koji; Ito, Masafumi; Matsushita, Tadashi; Murate, Takashi; Saito, Hidehiko; Kojima, Tetsuhito

    2003-01-01

    We developed a sensitive immunoassay to determine the concentration of mouse plasminogen activator inhibitor-1. The assay was a non-competitive sandwich enzyme-linked immunosorbent assay (ELISA) based on the production of a specific polyclonal antibody against mouse plasminogen activator inhibitor type-1 (PAI-1) used both as a trapping and detecting antibody. This antibody was raised in a rabbit by direct introduction of the expression vector plasmid DNA encoding mouse PAI-1, instead of conventional immunization with the purified protein. The standard curve was constructed with a recombinant glutathione S-transferase (GST)-mouse PAI-1 fusion protein (GST-mPAI-1) and dose-response of the assay was linear for GST-mPAI-1 between 6.25 and 100 pM. In order to assess the consistency of the assay, we measured PAI-1 antigen in normal mouse pooled plasma several times. We found that the intra-assay and inter-assay coefficients of variation (CV) were 4.8% and 9.2%, respectively, indicating that the ELISA would be sufficiently repeatable and reproducible. In this assay, lipopolysaccharide (LPS)-injected mice showed substantially higher levels (22-fold) of plasma PAI-1 antigen than did control mice (12.5+/-2.4 vs. 0.58+/-0.16 nM), similar to results reported elsewhere. Taken together, the DNA vaccine method is extremely useful for preparing specific antibodies against mouse PAI-1, which can be utilized to establish the ELISA and analyze the profile of PAI-1 distributions in mice under various conditions. This approach might also be useful for immunological investigation of other coagulation factors and related proteins.

  11. Activity of peroxisomal enzymes and intracellular distribution of catalase in Zellweger syndrome

    NARCIS (Netherlands)

    Schrakamp, G.; Bosch, H. van den; Roest, B.; Kos, M.; Meijer, A.J.; Heymans, H.S.A.; Tegelaers, W.H.H.; Schutgens, R.B.H.; Tager, J.M.; Wanders, R.J.A.

    1984-01-01

    The activity of peroxisomal enzymes was studied in human liver and cultured human skin fibroblasts in relation to the finding (Goldfischer, S. et al. (1973) Science 182, 62–64) that morphologically distinct peroxisomes are not detectable in patients with the cerebro-hepato-renal (Zellweger)

  12. Activity of peroxisomal enzymes and intracellular distribution of catalase in Zellweger syndrome

    NARCIS (Netherlands)

    Wanders, R. J.; Kos, M.; Roest, B.; Meijer, A. J.; Schrakamp, G.; Heymans, H. S.; Tegelaers, W. H.; van den Bosch, H.; Schutgens, R. B.; Tager, J. M.

    1984-01-01

    The activity of peroxisomal enzymes was studied in human liver and cultured human skin fibroblasts in relation to the finding (Goldfischer, S. et al. (1973) Science 182, 62-64) that morphologically distinct peroxisomes are not detectable in patients with the cerebro-hepato-renal (Zellweger)

  13. Seed reserve utilization and hydrolytic enzyme activities in germinating seeds of sweet corn

    International Nuclear Information System (INIS)

    Cheng, X.; Xiong, F.; Wang, C.; He, S.; Zhou, Y.

    2018-01-01

    In this study, two sh2 sweet corn cultivars (i.e., the initial seed dry weight for FT018 and TB010 was 0.16+-0.02 g/grain and 0.09+-0.01 g/grain, respectively) were used to determine the physiological characteristics of seed reserve utilization in germination. The data implied that the weight of mobilized seed reserve (WMSR) and seed reserve utilization efficiency (SRUE) increased with seed germination. FT018 exhibited higher SRUE than TB010 due to its sufficient energy production for growth. Sugar (sucrose and fructose) contents were at different levels in the germinating seed of sh2 sweet corn. The protein content and number of protein species were highest in the early stage of germination. Enzyme activity in the germinating seed indicated that enzymes for starch and sugar hydrolysis were important and that enzyme activities significantly differed at each germination stage and between the cultivars under dark conditions. Succinate dehydrogenase, sucrose synthase, and glucose-6-phosphate dehydrogenase accumulated in the late germination stage. Thus, appropriate efforts should be focused on improving the seed reserve utilization in sweet corn by identifying the physiological mechanism of germinating seed. (author)

  14. Warming Effects on Enzyme Activities are Predominant in Sub-surface Soils of an Arctic Tundra Ecosystem over 6-Year Field Manipulation

    Science.gov (United States)

    Kang, H.; Seo, J.; Kim, M.; Jung, J. Y.; Lee, Y. K.

    2017-12-01

    Arctic tundra ecosystems are of great importance because they store a large amount of carbon as un-decomposed organic matter. Global climate change is expected to affect enzyme activities and heterotrophic respiration in Arctic soils, which may accelerate greenhouse gas (GHG) emission through positive biological feedbacks. Unlike laboratory-based incubation experiments, field measurements often show different warming effects on decomposition of organic carbon and releases of GHGs. In the present study, we conducted a field-based warming experiment in Cambridge Bay, Canada (69°07'48″N, 105°03'36″W) by employing passive chambers during growing seasons over 6 years. A suite of enzyme activities (ß-glucosidase, cellobiohydrolase, N-acetylglucosaminidase, leucine aminopeptidase and phenol oxidase), microbial community structure (NGS), microbial abundances (gene copy numbers of bacteria and fungi), and soil chemical properties have been monitored in two depths (0-5 cm and 5-10 cm) of tundra soils, which were exposed to four different treatments (`control', `warming-only', `water-addition only', and both `warming and water-addition'). Phenol oxidase activity increased substantially, and bacterial community structure and abundance changed in the early stage (after 1 year's warming manipulation), but these changes disappeared afterwards. Most hydrolases were enhanced in surface soils by `water-addition only' over the period. However, the long-term effects of warming appeared in sub-surface soils where both `warming only' and `warming and water addition' increased hydrolase activities. Overall results of this study indicate that the warming effects on enzyme activities in surface soils are only short-term (phenol oxidase) or masked by water-limitation (hydrolases). However, hydrolases activities in sub-surface soils are more strongly enhanced than surface soils by warming, probably due to the lack of water limitation. Meanwhile, negative correlations between hydrolase

  15. Mining anaerobic digester consortia metagenomes for secreted carbohydrate active enzymes

    DEFF Research Database (Denmark)

    Wilkens, Casper; Busk, Peter Kamp; Pilgaard, Bo

    thermophilic and mesophilic ADs a wide variety of carbohydrate active enzyme functions were discovered in the metagenomic sequencing of the microbial consortia. The most dominating type of glycoside hydrolases were β-glucosidases (up to 27%), α-amylases (up to 10%), α-glucosidases (up to 8%), α......, and food wastes (Alvarado et al., 2014). The processes and the roles of the microorganisms that are involved in biomass conversion and methane production in ADs are still not fully understood. We are investigating thermophilic and mesophilic ADs that use wastewater surplus sludge for methane production...... was done with the Peptide Pattern Recognition (PPR) program (Busk and Lange, 2013), which is a novel non-alignment based approach that can predict function of e.g. CAZymes. PPR identifies a set of short conserved sequences, which can be used as a finger print when mining genomes for novel enzymes. In both...

  16. Covalent Immobilization of Cellulase Using Magnetic Poly(ionic liquid) Support: Improvement of the Enzyme Activity and Stability.

    Science.gov (United States)

    Hosseini, Seyed Hassan; Hosseini, Seyedeh Ameneh; Zohreh, Nasrin; Yaghoubi, Mahshid; Pourjavadi, Ali

    2018-01-31

    A magnetic nanocomposite was prepared by entrapment of Fe 3 O 4 nanoparticles into the cross-linked ionic liquid/epoxy type polymer. The resulting support was used for covalent immobilization of cellulase through the reaction with epoxy groups. The ionic surface of the support improved the adsorption of enzyme, and a large amount of enzyme (106.1 mg/g) was loaded onto the support surface. The effect of the presence of ionic monomer and covalent binding of enzyme was also investigated. The structure of support was characterized by various instruments such as FT-IR, TGA, VSM, XRD, TEM, SEM, and DLS. The activity and stability of immobilized cellulase were investigated in the prepared support. The results showed that the ionic surface and covalent binding of enzyme onto the support improved the activity, thermal stability, and reusability of cellulase compared to free cellulase.

  17. Phlorotannins from Alaskan Seaweed Inhibit Carbolytic Enzyme Activity

    Science.gov (United States)

    Kellogg, Joshua; Grace, Mary H.; Lila, Mary Ann

    2014-01-01

    Global incidence of type 2 diabetes has escalated over the past few decades, necessitating a continued search for natural sources of enzyme inhibitors to offset postprandial hyperglycemia. The objective of this study was to evaluate coastal Alaskan seaweed inhibition of α-glucosidase and α-amylase, two carbolytic enzymes involved in serum glucose regulation. Of the six species initially screened, the brown seaweeds Fucus distichus and Alaria marginata possessed the strongest inhibitory effects. F. distichus fractions were potent mixed-mode inhibitors of α-glucosidase and α-amylase, with IC50 values of 0.89 and 13.9 μg/mL, respectively; significantly more efficacious than the pharmaceutical acarbose (IC50 of 112.0 and 137.8 μg/mL, respectively). The activity of F. distichus fractions was associated with phlorotannin oligomers. Normal-phase liquid chromatography-mass spectrometry (NPLC-MS) was employed to characterize individual oligomers. Accurate masses and fragmentation patterns confirmed the presence of fucophloroethol structures with degrees of polymerization from 3 to 18 monomer units. These findings suggest that coastal Alaskan seaweeds are sources of α-glucosidase and α-amylase inhibitory phlorotannins, and thus have potential to limit the release of sugar from carbohydrates and thus alleviate postprandial hyperglycemia. PMID:25341030

  18. Phlorotannins from Alaskan Seaweed Inhibit Carbolytic Enzyme Activity

    Directory of Open Access Journals (Sweden)

    Joshua Kellogg

    2014-10-01

    Full Text Available Global incidence of type 2 diabetes has escalated over the past few decades, necessitating a continued search for natural sources of enzyme inhibitors to offset postprandial hyperglycemia. The objective of this study was to evaluate coastal Alaskan seaweed inhibition of α-glucosidase and α-amylase, two carbolytic enzymes involved in serum glucose regulation. Of the six species initially screened, the brown seaweeds Fucus distichus and Alaria marginata possessed the strongest inhibitory effects. F. distichus fractions were potent mixed-mode inhibitors of α-glucosidase and α-amylase, with IC50 values of 0.89 and 13.9 μg/mL, respectively; significantly more efficacious than the pharmaceutical acarbose (IC50 of 112.0 and 137.8 μg/mL, respectively. The activity of F. distichus fractions was associated with phlorotannin oligomers. Normal-phase liquid chromatography-mass spectrometry (NPLC-MS was employed to characterize individual oligomers. Accurate masses and fragmentation patterns confirmed the presence of fucophloroethol structures with degrees of polymerization from 3 to 18 monomer units. These findings suggest that coastal Alaskan seaweeds are sources of α-glucosidase and α-amylase inhibitory phlorotannins, and thus have potential to limit the release of sugar from carbohydrates and thus alleviate postprandial hyperglycemia.

  19. Soil enzyme activities in Pinus tabuliformis (Carriere) plantations in northern China

    Science.gov (United States)

    Weiwei Wang; Deborah Page-Dumroese; Ruiheng Lv; Chen Xiao; Guolei Li; Yong Liu

    2016-01-01

    Changes in forest stand structure may alter the activity of invertase, urease, catalase and phenol oxidase after thinning Pinus tabuliformis (Carriére) plantations in Yanqing County of Beijing, China. We examined changes in these soil enzymes as influenced by time since thinning (24, 32, and 40 years since thinning) for 3 seasons (spring, summer and autumn)...

  20. Cell in situ zymography: an in vitro cytotechnology for localization of enzyme activity in cell culture.

    Science.gov (United States)

    Chhabra, Aastha; Jaiswal, Astha; Malhotra, Umang; Kohli, Shrey; Rani, Vibha

    2012-09-01

    In situ zymography is a unique technique for detection and localization of enzyme-substrate interactions majorly in histological sections. Substrate with quenched fluorogenic molecule is incorporated in gel over which tissue sections are mounted and then incubated in buffer. The enzymatic activity is observed in the form of fluorescent signal. With the advancements in the field of biological research, use of in vitro cell culture has become very popular and holds great significance in multiple fields including inflammation, cancer, stem cell biology and the still emerging 3-D cell cultures. The information on analysis of enzymatic activity in cell lines is inadequate presently. We propose a single-step methodology that is simple, sensitive, cost-effective, and functional to perform and study the 'in position' activity of enzyme on substrate for in vitro cell cultures. Quantification of enzymatic activity to carry out comparative studies on cells has also been illustrated. This technique can be applied to a variety of enzyme classes including proteases, amylases, xylanases, and cellulases in cell cultures.

  1. A plasmonic nanosensor for lipase activity based on enzyme-controlled gold nanoparticles growth in situ

    Science.gov (United States)

    Tang, Yan; Zhang, Wei; Liu, Jia; Zhang, Lei; Huang, Wei; Huo, Fengwei; Tian, Danbi

    2015-03-01

    A plasmonic nanosensor for lipase activity was developed based on one-pot nanoparticle growth. Tween 80 was selected not only as the substrate for lipase recognition but also as the reducing and stabilizing agent for the sensor fabrication. The different molecular groups in Tween 80 could have different roles in the fabrication procedure; the H2O2 produced by the autoxidation of the ethylene oxide subunits in Tween 80 could reduce the AuCl4- ions to Au atoms, meanwhile, the lipase could hydrolyze its carboxyl ester bond, which could, in turn, control the rate of nucleation of the gold nanoparticles (AuNPs) and tailor the localized surface plasmon resonance (LSPR) of the AuNP transducers. The color changes, which depend on the absence or presence of the lipase, could be used to sense the lipase activity. A linear response ranging from 0.025 to 4 mg mL-1 and a detection limit of the lipase as low as 3.47 μg mL-1 were achieved. This strategy circumvents the problems encountered by general enzyme assays that require sophisticated instruments and complicated assembling steps. The methodology can benefit the assays of heterogeneous-catalyzed enzymes.A plasmonic nanosensor for lipase activity was developed based on one-pot nanoparticle growth. Tween 80 was selected not only as the substrate for lipase recognition but also as the reducing and stabilizing agent for the sensor fabrication. The different molecular groups in Tween 80 could have different roles in the fabrication procedure; the H2O2 produced by the autoxidation of the ethylene oxide subunits in Tween 80 could reduce the AuCl4- ions to Au atoms, meanwhile, the lipase could hydrolyze its carboxyl ester bond, which could, in turn, control the rate of nucleation of the gold nanoparticles (AuNPs) and tailor the localized surface plasmon resonance (LSPR) of the AuNP transducers. The color changes, which depend on the absence or presence of the lipase, could be used to sense the lipase activity. A linear response

  2. The effect of water temperature on food transit time and digestive enzymes activity in Caspian kutum (Rutilus kutum larvae

    Directory of Open Access Journals (Sweden)

    Nahid Ghysvandi

    2014-07-01

    Full Text Available The present study investigates the effects of water temperature on digestive enzymes activity and food transit time in Caspian kutum (Rutilus kutum larvae. Caspian kutum larvae (532 ± 0.05 and 543 ± 0.02 mg were divided into two groups with three replicates and reared at different water temperature i.e. 25.6 ± 0.4°C (T1 and 18.4 ± 0.1°C (T2. At the end of the experiment, sampling of intestine was performed at 0, 1, 3, 5, 8, 16, 24 and 30 h after feeding from each treatment. In T2, food was observed until 24 h after feeding and the intestine was empty 29 h after feeding, while in T1 19 h after feeding the intestine was empty. Digestive enzymes activities were higher in T2 treatment. The peaks of trypsin and alkaline phosphatase enzymes activity were found 8 h after feeding in T1, while occurred 16 h after feeding in T2. The highest chymotrypsin and alpha-amylase enzymes activity were observed 5 and 8h after feeding in T1 and T2, respectively. These results confirmed remarkable effects of temperature on food transit time and digestive enzymes activity of Caspian kutum.

  3. Mitochondrial oxidative enzyme activity in individual fibre types in hypo- and hyperthyroid rat skeletal muscles.

    Science.gov (United States)

    Johnson, M A; Turnbull, D M

    1984-04-01

    Quantitative cytochemical and biochemical techniques have been used in combination to study the response of mitochondrial oxidative enzymes in individual muscle fibre types to hypo- and hyperthyroidism. Hypothyroidism resulted in decreased activity of succinate dehydrogenase (SDH), L-glycerol-3-phosphate dehydrogenase (L-GPDH), and D-3-hydroxybutyrate dehydrogenase (D-HBDH) in all fibre types of both slow-twitch soleus and fast-twitch extensor digitorum longus (e.d.l.) muscles. In hyperthyroidism, only L-GPDH activity increased in e.d.l. but more marked increases were seen in soleus muscles, which also showed increased SDH activity. In addition to these alterations in the enzyme activity in individual fibre types the metabolic profile of the muscle is further modified by the hormone-induced interconversion of slow- to fast-twitch fibres and vice versa.

  4. Bacillus subtilis BY-kinase PtkA controls enzyme activity and localization of its protein substrates

    DEFF Research Database (Denmark)

    Jers, Carsten; Pedersen, Malene Mejer; Paspaliari, Dafni Katerina

    2010-01-01

    -phosphorylated proteins in B. subtilis. We found that the majority of these proteins could be phosphorylated by PtkA in vitro. Among these new substrates, single-stranded DNA exonuclease YorK, and aspartate semialdehyde dehydrogenase Asd were activated by PtkA-dependent phosphorylation. Because enzyme activity......A was dramatically altered in Delta ptkA background. Our results confirm that PtkA can control enzyme activity of its substrates in some cases, but also reveal a new mode of action for PtkA, namely ensuring correct cellular localization of its targets.......P>Bacillus subtilis BY-kinase PtkA was previously shown to phosphorylate, and thereby regulate the activity of two classes of protein substrates: UDP-glucose dehydrogenases and single-stranded DNA-binding proteins. Our recent phosphoproteome study identified nine new tyrosine...

  5. A rapid non invasive L-DOPA-¹³C breath test for optimally suppressing extracerebral AADC enzyme activity - toward individualizing carbidopa therapy in Parkinson’s disease.

    Science.gov (United States)

    Modak, Anil; Durso, Raymon; Josephs, Ephraim; Rosen, David

    2012-01-01

    Peripheral carbidopa (CD) levels directly impact on central dopamine (DA) production in Parkinson disease (PD) through extracerebral inhibition of dopa decarboxylase (AADC) resulting in an increase in levodopa (LD) bioavailability. Recent data suggests that higher CD doses than those presently used in PD treatment may result in improved clinical response. Optimizing CD doses in individual patients may, therefore, result in ideal individualized treatment. A single center, randomized, double-blind study was carried out recruiting 5 Parkinson’s disease (PD) patients already on LD/CD and 1 treatment näve PD patient using stable isotope labeled LD-1-¹³C as a substrate for a noninvasive breath test to evaluate individual AADC enzyme activity. Each patient was studied five times, receiving 200 mg LD-¹³C at each visit along with one of five randomized CD doses (0, 25, 50, 100 and 200 mg). The metabolite ¹³CO₂ in breath was measured for evaluating AADC enzyme activity and plasma metabolite levels for LD-¹³C and homovanillic acid (HVA) were measured for 4 hours. HVA in plasma and ¹³CO₂ in breath are metabolic products of LD. We found a significant positive correlation of ¹³CO₂ DOB AUC0-240 with serum HVA AUC0-240 following the oral dose of LD-1-¹³C for all 5 doses of CD (r² = 0.9378). With increasing inhibition of AADC enzyme activity with CD, we observed an increase in the plasma concentration of LD.We found an inverse correlation of the 13CO2 DOB AUC with serum LD-¹³C AUC. Our studies indicate the optimal dose of CD for maximal suppression of AADC enzyme activity can be determined for each individual from ¹³CO₂ generation in breath. The LD-breath test can be a useful noninvasive diagnostic tool for evaluation of AADC enzyme activity using the biomarker ¹³CO₂ in breath, a first step in personalizing CD doses for PD patients.

  6. Magnetically responsive enzyme powders

    Energy Technology Data Exchange (ETDEWEB)

    Pospiskova, Kristyna, E-mail: kristyna.pospiskova@upol.cz [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Safarik, Ivo, E-mail: ivosaf@yahoo.com [Regional Centre of Advanced Technologies and Materials, Palacky University, Slechtitelu 11, 783 71 Olomouc (Czech Republic); Department of Nanobiotechnology, Institute of Nanobiology and Structural Biology of GCRC, Na Sadkach 7, 370 05 Ceske Budejovice (Czech Republic)

    2015-04-15

    Powdered enzymes were transformed into their insoluble magnetic derivatives retaining their catalytic activity. Enzyme powders (e.g., trypsin and lipase) were suspended in various liquid media not allowing their solubilization (e.g., saturated ammonium sulfate and highly concentrated polyethylene glycol solutions, ethanol, methanol, 2-propanol) and subsequently cross-linked with glutaraldehyde. Magnetic modification was successfully performed at low temperature in a freezer (−20 °C) using magnetic iron oxides nano- and microparticles prepared by microwave-assisted synthesis from ferrous sulfate. Magnetized cross-linked enzyme powders were stable at least for two months in water suspension without leakage of fixed magnetic particles. Operational stability of magnetically responsive enzymes during eight repeated reaction cycles was generally without loss of enzyme activity. Separation of magnetically modified cross-linked powdered enzymes from reaction mixtures was significantly simplified due to their magnetic properties. - Highlights: • Cross-linked enzyme powders were prepared in various liquid media. • Insoluble enzymes were magnetized using iron oxides particles. • Magnetic iron oxides particles were prepared by microwave-assisted synthesis. • Magnetic modification was performed under low (freezing) temperature. • Cross-linked powdered trypsin and lipase can be used repeatedly for reaction.

  7. Biosynthesis of quinoxaline antibiotics: Purification and characterization of the quinoxaline-2-carboxylic acid activating enzyme from Streptomyces triostinicus

    International Nuclear Information System (INIS)

    Glund, K.; Schlumbohm, W.; Bapat, M.; Keller, U.

    1990-01-01

    A quinoxaline-2-carboxylic acid activating enzyme was purified to homogeneity from triostin-producing Streptomyces triostinicus. It could also be purified from quinomycin-producing Streptomyces echinatus. Triostins and quinomycins are peptide lactones that contain quinoxaline-2-carboxylic acid as chromophoric moiety. The enzyme catalyzes the ATP-pyrophosphate exchange reaction dependent on quinoxaline-2-carboxylic acid and the formation of the corresponding adenylate. Besides quinoxaline-2-carboxylic acid, the enzyme also catalyzes the formation of adenylates from quinoline-2-carboxylic acid and thieno[3,2-b]pyridine-5-carboxylic acid. No adenylates were seen from quinoline-3-carboxylic acid, quinoline-4-carboxylic acid, pyridine-2-carboxylic acid, and 2-pyrazinecarboxylic acid. Previous work revealed that quinoline-2-carboxylic acid and thieno[3,2-b]pyridine-5-carboxylic acid became efficiently incorporated into the corresponding quinoxaline antibiotic analogues in vivo. Together with the data described here, this suggests that the enzyme is part of the quinoxaline antibiotics synthesizing enzyme system. The enzyme displays a native molecular weight of 42,000, whereas in its denatured form it is a polypeptide of Mr 52,000-53,000. It resembles in its behavior actinomycin synthetase I, the chromophore activating enzyme involved in actinomycin biosynthesis

  8. Determining soil enzyme activities for the assessment of fungi and citric acid-assisted phytoextraction under cadmium and lead contamination.

    Science.gov (United States)

    Mao, Liang; Tang, Dong; Feng, Haiwei; Gao, Yang; Zhou, Pei; Xu, Lurong; Wang, Lumei

    2015-12-01

    Microorganism or chelate-assisted phytoextraction is an effective remediation tool for heavy metal polluted soil, but investigations into its impact on soil microbial activity are rarely reported. Consequently, cadmium (Cd)- and lead (Pb)-resistant fungi and citric acid (CA) were introduced to enhance phytoextraction by Solanum nigrum L. under varied Cd and Pb pollution levels in a greenhouse pot experiment. We then determined accumulation of Cd and Pb in S. nigrum and the soil enzyme activities of dehydrogenase, phosphatase, urease, catalase, sucrase, and amylase. Detrended canonical correspondence analysis (DCCA) was applied to assess the interactions between remediation strategies and soil enzyme activities. Results indicated that the addition of fungi, CA, or their combination enhanced the root biomass of S. nigrum, especially at the high-pollution level. The combined treatment of CA and fungi enhanced accumulation of Cd about 22-47 % and of Pb about 13-105 % in S. nigrum compared with the phytoextraction alone. However, S. nigrum was not shown to be a hyperaccumulator for Pb. Most enzyme activities were enhanced after remediation. The DCCA ordination graph showed increasing enzyme activity improvement by remediation in the order of phosphatase, amylase, catalase, dehydrogenase, and urease. Responses of soil enzyme activities were similar for both the addition of fungi and that of CA. In summary, results suggest that fungi and CA-assisted phytoextraction is a promising approach to restoring heavy metal polluted soil.

  9. Increased angiotensin-converting enzyme activity in the left ventricle after infarction

    Directory of Open Access Journals (Sweden)

    V.C.W. Busatto

    1997-05-01

    Full Text Available An increase in angiotensin-converting enzyme (ACE activity has been observed in the heart after myocardial infarction (MI. Since most studies have been conducted in chronically infarcted individuals exhibiting variable degrees of heart failure, the present study was designed to determine ACE activity in an earlier phase of MI, before heart failure development. MI was produced in 3-month old male Wistar rats by ligation of the anterior branches of the left coronary artery, control rats underwent sham surgery and the animals were studied 7 or 15 days later. Hemodynamic data obtained for the anesthetized animals showed normal values of arterial blood pressure and of end-diastolic pressure in the right and left ventricular cavities of MI rats. Right and left ventricular (RV, LV muscle and scar tissue homogenates were prepared to determine ACE activity in vitro by measuring the velocity of His-Leu release from the synthetic substrate Hyp-His-Leu. ACE activity was corrected to the tissue wet weight and is reported as nmol His-Leu g-1 min-1. No significant change in ACE activity in the RV homogenates was demonstrable. A small nonsignificant increase of ACE activity (11 ± 9%; P0.05 was observed 7 days after MI in the surviving left ventricular muscle. Two weeks after surgery, however, ACE activity was 46 ± 11% (P<0.05 higher in infarcted rats compared to sham-operated rats. The highest ACE activity was demonstrable in the scar tissue homogenate. In rats studied two weeks after surgery, ACE activity in the LV muscle increased from 105 ± 7 nmol His-Leu g-1 min-1 in control hearts to 153 ± 11 nmol His-Leu g-1 min-1 (P<0.05 in the remaining LV muscle of MI rats and to 1051 ± 208 nmol His-Leu g-1 min-1 (P<0.001 in the fibrous scar. These data indicate that ACE activity increased in the heart after infarction before heart failure was demonstrable by hemodynamic measurements. Since the blood vessels of the scar drain to the remaining LV myocardium, the

  10. Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica: A Simple and Effective Method for Enzyme Stabilization

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Moon Il; Kim, Jungbae; Lee, Jinwoo; Jia, Hongfei; Na, Hyon Bin; Youn, Jongkyu; Kwak, Ja Hun; Dohnalkova, Alice; Grate, Jay W.; Wang, Ping; Hyeon, Taeghwan; Park, Hyun-Gyu; Chang, Ho Nam

    2007-02-01

    alpha-chymotrypsin (CT) and lipase (LP) were immobilized in hierarchically-ordered mesocellular mesoporous silica (HMMS) in a simple but effective way for the enzyme stabilization, which was achieved by the enzyme adsorption followed by glutaraldehyde (GA) crosslinking. This resulted in the formation of nanometer scale crosslinked enzyme aggregates (CLEAs) entrapped in the mesocellular pores of HMMS (37 nm), which did not leach out of HMMS through narrow mesoporous channels (13 nm). CLEA of alpha-chymotrypsin (CLEA-CT) in HMMS showed a high enzyme loading capacity and significantly increased enzyme stability. No activity decrease of CLEA-CT was observed for two weeks under even rigorously shaking condition, while adsorbed CT in HMMS and free CT showed a rapid inactivation due to the enzyme leaching and presumably autolysis, respectively. With the CLEA-CT in HMMS, however, there was no tryptic digestion observed suggesting that the CLEA-CT is not susceptible to autolysis. Moreover, CLEA of lipase (CLEA-LP) in HMMS retained 30% specific activity of free lipase with greatly enhanced stability. This work demonstrates that HMMS can be efficiently employed as host materials for enzyme immobilization leading to highly enhanced stability of the immobilized enzymes with high enzyme loading and activity.

  11. Effects of chilled storage and cryopreservation on sperm characteristics, antioxidant enzyme activities, and lipid peroxidation in Pacific cod Gadus microcephalus

    Science.gov (United States)

    Wang, Xueying; Shi, Xuehui; Liu, Yifan; Yu, Daode; Guan, Shuguang; Liu, Qinghua; Li, Jun

    2016-07-01

    The present study evaluated the effects of chilled storage and cryopreservation on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod Gadus macrocephalus. Sperm motility and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (Gr), and lipid peroxidation (measured via malondialdehyde (MDA) content) were determined after the milt was stored at 4°C for 12 h, cryopreserved without cryoprotectant in 12% propylene glycol (PG), cryopreserved in 12% PG+0.1 mol/L trehalose, or cryopreserved in 12% PG spermatozoa but centrifuged to decant the supernatant prior to cryopreservation (only sperm cells were cryopreserved). After chilled storage or cryopreservation, the SOD, CAT and GPx activities were reduced in sperm cells and increased in seminal plasma in almost all treatments; sperm motility parameters were also decreased. However, the addition of trehalose into the cryoprotectant could significantly improve the postthaw sperm quality as revealed by the sperm average path velocity. This improvement might be attributed to the function of trehalose in scavenging reactive oxygen species. Chilled storage and cryopreservation had significant effects on sperm motion characteristics, antioxidant enzyme activities, and lipid peroxidation in the Pacific cod.

  12. Positron emitter labeled enzyme inhibitors

    International Nuclear Information System (INIS)

    Fowler, J.S.; MacGregor, R.R.; Wolf, A.P.; Langstrom, B.

    1990-01-01

    This invention involves a new strategy for imagining and mapping enzyme activity in the living human and animal body using positron emitter-labeled suicide enzyme inactivators or inhibitors which become covalently bound to the enzyme as a result of enzymatic catalysis. Two such suicide inactivators for monoamine oxidase have been labeled with carbon-11 and used to map the enzyme subtypes in the living human and animal body using PET. By using positron emission tomography to image the distribution of radioactivity produced by the body penetrating radiation emitted by carbon-11, a map of functionally active monoamine oxidase activity is obtained. Clorgyline and L-deprenyl are suicide enzyme inhibitors and irreversibly inhibit monoamine oxidase. When these inhibitors are labeled with carbon-11 they provide selective probes for monoamine oxidase localization and reactivity in vivo using positron emission tomography

  13. Activities of xenobiotic metabolizing enzymes in rat placenta and liver in vitro

    NARCIS (Netherlands)

    Fabian, Eric; Wang, Xinyi; Engel, Franziska; Li, Hequn; Landsiedel, Robert; Ravenzwaay, van Bennard

    2016-01-01

    In order to assess whether the placental metabolism of xenobiotic compounds should be taken into consideration for physiologically-based toxicokinetic (PBTK) modelling, the activities of seven phase I and phase II enzymes have been quantified in the 18-day placenta of untreated Wistar rats. To

  14. pH-sensitive pHluorins as a molecular sensor for in situ monitoring of enzyme-catalyzed prodrug activation.

    Science.gov (United States)

    Liu, Hui; Cao, Xiaodan; Wang, Ping; Ma, Xingyuan

    2017-07-01

    This work examines the feasibility of using a pH-sensitive fluorescent protein as a molecular reporter for enzyme-catalyzed prodrug activation reaction. Specifically, a ratiometric pHluorins was examined for detection of the activity of horseradish peroxidase (HRP) for the activation of indole-3-acetic acid. The pHluorins and HRP were conjugated chemically, forming a biocatalyst with a self-reporting function. Results showed that the characteristic fluorescence intensity ratio of the conjugate shifted from 1.47 to 1.40 corresponding to the progress of the prodrug activation reaction. The effectiveness of applying the conjugate for inhibition of the growth of Bcap-37 cells was also demonstrated simultaneously with reaction monitoring. The results reveal a very promising approach to realizing in situ monitoring of enzyme activities based on pH shifting for enzyme-based prodrug therapy applications. © 2016 International Union of Biochemistry and Molecular Biology, Inc.

  15. Influence of exogenous lead pollution on enzyme activities and organic matter degradation in the surface of river sediment.

    Science.gov (United States)

    Huang, Danlian; Xu, Juanjuan; Zeng, Guangming; Lai, Cui; Yuan, Xingzhong; Luo, Xiangying; Wang, Cong; Xu, Piao; Huang, Chao

    2015-08-01

    As lead is one of the most hazardous heavy metals in river ecosystem, the influence of exogenous lead pollution on enzyme activities and organic matter degradation in the surface of river sediment with high moisture content were studied at laboratory scale. The dynamic changes of urease, catalase, protease activities, organic matter content, and exchangeable or ethylenediaminetetraacetic acid (EDTA)-extractable Pb concentration in sediment were monitored during different levels of exogenous lead infiltrating into sediment. At the early stage of incubation, the activities of catalase and protease were inhibited, whereas the urease activities were enhanced with different levels of exogenous lead. Organic matter content in polluted sediment with exogenous lead was lower than control and correlated with enzyme activities. In addition, the effects of lead on the three enzyme activities were strongly time-dependent and catalase activities showed lower significant difference (P < 0.05) than urease and protease. Correlations between catalase activities and EDTA-extractable Pb in the experiment were significantly negative. The present findings will improve the understandings about the ecotoxicological mechanisms in sediment.

  16. Acute Exercise Increases Plasma Total Antioxidant Status and Antioxidant Enzyme Activities in Untrained Men

    Directory of Open Access Journals (Sweden)

    C. Berzosa

    2011-01-01

    Full Text Available Antioxidant defences are essential for cellular redox regulation. Since free-radical production may be enhanced by physical activity, herein, we evaluated the effect of acute exercise on total antioxidant status (TAS and the plasma activities of catalase, glutathione reductase, glutathione peroxidase, and superoxide dismutase and its possible relation to oxidative stress resulting from exercise. Healthy untrained male subjects (=34 performed three cycloergometric tests, including maximal and submaximal episodes. Venous blood samples were collected before and immediately after each different exercise. TAS and enzyme activities were assessed by spectrophotometry. An increase of the antioxidant enzyme activities in plasma was detected after both maximal and submaximal exercise periods. Moreover, under our experimental conditions, exercise also led to an augmentation of TAS levels. These findings are consistent with the idea that acute exercise may play a beneficial role because of its ability to increase antioxidant defense mechanisms through a redox sensitive pathway.

  17. Effect of allyl isothiocyanate on ultra-structure and the activities of four enzymes in adult Sitophilus zeamais.

    Science.gov (United States)

    Wu, Hua; Liu, Xue-ru; Yu, Dong-dong; Zhang, Xing; Feng, Jun-tao

    2014-02-01

    Rarefaction and vacuolization of the mitochondrial matrix of AITC-treated (allyl isothiocyanate-treated) adult Sitophilus zeamais were evident according to the ultra-structural by TEM. Four important enzymes in adult S. zeamais were further studied after fumigation treatment with allyl isothiocyanate (AITC) extracted from Armoracia rusticana roots and shoots. The enzymes were glutathione S-transferase (GST), catalase (CAT), cytochrome c oxidase, and acetylcholinesterase (AChE). The results indicated that the activities of the four enzymes were strongly time and dose depended. With prolonged exposure time, treatment with 0.74μg/mL AITC inhibited the activities of cytochrome c oxidase, AChE, and CAT, but induced the activity of GST. The activities of cytochrome c oxidase, AChE, and CAT were remarkably induced at a low AITC dosage (0.25μg/mL), but were restrained with increased AITC dosage. The activity of GST was inhibited at a low AITC dosage (0.5μg/mL), but was induced at a high AITC dosage (1.5μg/mL). According to the results of TEM, toxic symptoms and enzymes activities, it suggested that mitochondrial maybe the one site of action of AITC against the adult S. zeamais and it also suggested that cytochrome c oxidase maybe one target protein of AITC against the adult S. zeamais, which need to further confirmed by protein function tested. Copyright © 2014 Elsevier Inc. All rights reserved.

  18. Influence of altered precipitation pattern on greenhouse gas emissions and soil enzyme activities in Pannonian soils

    Science.gov (United States)

    Forstner, Stefan Johannes; Michel, Kerstin; Berthold, Helene; Baumgarten, Andreas; Wanek, Wolfgang; Zechmeister-Boltenstern, Sophie; Kitzler, Barbara

    2013-04-01

    water only once in two weeks (D=dry). Both groups received same water totals for each soil. At the end of each two week drying period, greenhouse gas fluxes were measured via an open-chamber-system (CO2, NO) and a closed-chamber-approach (CH4, N2O, CO2). Additional cylinders were harvested destructively to quantify inorganic N forms, microbial biomass C, N and extracellular enzyme activity (Cellulase, Xylanase, Protease, Phenoloxidase, Peroxidase). We hypothesize that after rewetting (1) rates of greenhouse gas fluxes will generally increase, as well as (2) extracellular enzyme activity indicating enhanced microbial activity. However, response may be different for gases and enzymes involved in the C and N cycle, respectively, as drying/rewetting stress may uncouple microbial mediated biogeochemical cycles. Results will be presented at the EGU General Assembly. Reference: Schimel, J., Balser, T.C., and Wallenstein, M. (2007). Microbial stress-response physiology and its implications for ecosystem function. Ecology 88, 1386-1394.

  19. Functional Trade-Offs in Promiscuous Enzymes Cannot Be Explained by Intrinsic Mutational Robustness of the Native Activity.

    Directory of Open Access Journals (Sweden)

    Miriam Kaltenbach

    2016-10-01

    Full Text Available The extent to which an emerging new function trades off with the original function is a key characteristic of the dynamics of enzyme evolution. Various cases of laboratory evolution have unveiled a characteristic trend; a large increase in a new, promiscuous activity is often accompanied by only a mild reduction of the native, original activity. A model that associates weak trade-offs with "evolvability" was put forward, which proposed that enzymes possess mutational robustness in the native activity and plasticity in promiscuous activities. This would enable the acquisition of a new function without compromising the original one, reducing the benefit of early gene duplication and therefore the selection pressure thereon. Yet, to date, no experimental study has examined this hypothesis directly. Here, we investigate the causes of weak trade-offs by systematically characterizing adaptive mutations that occurred in two cases of evolutionary transitions in enzyme function: (1 from phosphotriesterase to arylesterase, and (2 from atrazine chlorohydrolase to melamine deaminase. Mutational analyses in various genetic backgrounds revealed that, in contrast to the prevailing model, the native activity is less robust to mutations than the promiscuous activity. For example, in phosphotriesterase, the deleterious effect of individual mutations on the native phosphotriesterase activity is much larger than their positive effect on the promiscuous arylesterase activity. Our observations suggest a revision of the established model: weak trade-offs are not caused by an intrinsic robustness of the native activity and plasticity of the promiscuous activity. We propose that upon strong adaptive pressure for the new activity without selection against the original one, selected mutations will lead to the largest possible increases in the new function, but whether and to what extent they decrease the old function is irrelevant, creating a bias towards initially weak

  20. Mutual synergy between catalase and peroxidase activities of the bifunctional enzyme KatG is facilitated by electron hole-hopping within the enzyme.

    Science.gov (United States)

    Njuma, Olive J; Davis, Ian; Ndontsa, Elizabeth N; Krewall, Jessica R; Liu, Aimin; Goodwin, Douglas C

    2017-11-10

    KatG is a bifunctional, heme-dependent enzyme in the front-line defense of numerous bacterial and fungal pathogens against H 2 O 2 -induced oxidative damage from host immune responses. Contrary to the expectation that catalase and peroxidase activities should be mutually antagonistic, peroxidatic electron donors (PxEDs) enhance KatG catalase activity. Here, we establish the mechanism of synergistic cooperation between these activities. We show that at low pH values KatG can fully convert H 2 O 2 to O 2 and H 2 O only if a PxED is present in the reaction mixture. Stopped-flow spectroscopy results indicated rapid initial rates of H 2 O 2 disproportionation slowing concomitantly with the accumulation of ferryl-like heme states. These states very slowly returned to resting ( i.e. ferric) enzyme, indicating that they represented catalase-inactive intermediates. We also show that an active-site tryptophan, Trp-321, participates in off-pathway electron transfer. A W321F variant in which the proximal tryptophan was replaced with a non-oxidizable phenylalanine exhibited higher catalase activity and less accumulation of off-pathway heme intermediates. Finally, rapid freeze-quench EPR experiments indicated that both WT and W321F KatG produce the same methionine-tyrosine-tryptophan (MYW) cofactor radical intermediate at the earliest reaction time points and that Trp-321 is the preferred site of off-catalase protein oxidation in the native enzyme. Of note, PxEDs did not affect the formation of the MYW cofactor radical but could reduce non-productive protein-based radical species that accumulate during reaction with H 2 O 2 Our results suggest that catalase-inactive intermediates accumulate because of off-mechanism oxidation, primarily of Trp-321, and PxEDs stimulate KatG catalase activity by preventing the accumulation of inactive intermediates. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.