WorldWideScience

Sample records for high enzyme activity

  1. 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

  2. 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.

  3. Styrene Oxide Isomerase of Rhodococcus opacus 1CP, a Highly Stable and Considerably Active Enzyme

    Science.gov (United States)

    Gröning, Janosch A. D.; Tischler, Dirk; Kaschabek, Stefan R.; Schlömann, Michael

    2012-01-01

    Styrene oxide isomerase (SOI) is involved in peripheral styrene catabolism of bacteria and converts styrene oxide to phenylacetaldehyde. Here, we report on the identification, enrichment, and biochemical characterization of a novel representative from the actinobacterium Rhodococcus opacus 1CP. The enzyme, which is strongly induced during growth on styrene, was shown to be membrane integrated, and a convenient procedure was developed to highly enrich the protein in active form from the wild-type host. A specific activity of about 370 U mg−1 represents the highest activity reported for this enzyme class so far. This, in combination with a wide pH and temperature tolerance, the independence from cofactors, and the ability to convert a spectrum of substituted styrene oxides, makes a biocatalytic application imaginable. First, semipreparative conversions were performed from which up to 760 μmol of the pure phenylacetaldehyde could be obtained from 130 U of enriched SOI. Product concentrations of up to 76 mM were achieved. However, due to the high chemical reactivity of the aldehyde function, SOI was shown to be the subject of an irreversible product inhibition. A half-life of 15 min was determined at a phenylacetaldehyde concentration of about 55 mM, indicating substantial limitations of applicability and the need to modify the process. PMID:22504818

  4. Computationally optimized deimmunization libraries yield highly mutated enzymes with low immunogenicity and enhanced activity.

    Science.gov (United States)

    Salvat, Regina S; Verma, Deeptak; Parker, Andrew S; Kirsch, Jack R; Brooks, Seth A; Bailey-Kellogg, Chris; Griswold, Karl E

    2017-06-27

    Therapeutic proteins of wide-ranging function hold great promise for treating disease, but immune surveillance of these macromolecules can drive an antidrug immune response that compromises efficacy and even undermines safety. To eliminate widespread T-cell epitopes in any biotherapeutic and thereby mitigate this key source of detrimental immune recognition, we developed a Pareto optimal deimmunization library design algorithm that optimizes protein libraries to account for the simultaneous effects of combinations of mutations on both molecular function and epitope content. Active variants identified by high-throughput screening are thus inherently likely to be deimmunized. Functional screening of an optimized 10-site library (1,536 variants) of P99 β-lactamase (P99βL), a component of ADEPT cancer therapies, revealed that the population possessed high overall fitness, and comprehensive analysis of peptide-MHC II immunoreactivity showed the population possessed lower average immunogenic potential than the wild-type enzyme. Although similar functional screening of an optimized 30-site library (2.15 × 10 9 variants) revealed reduced population-wide fitness, numerous individual variants were found to have activity and stability better than the wild type despite bearing 13 or more deimmunizing mutations per enzyme. The immunogenic potential of one highly active and stable 14-mutation variant was assessed further using ex vivo cellular immunoassays, and the variant was found to silence T-cell activation in seven of the eight blood donors who responded strongly to wild-type P99βL. In summary, our multiobjective library-design process readily identified large and mutually compatible sets of epitope-deleting mutations and produced highly active but aggressively deimmunized constructs in only one round of library screening.

  5. 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

  6. Functional Metagenomics: Construction and High-Throughput Screening of Fosmid Libraries for Discovery of Novel Carbohydrate-Active Enzymes.

    Science.gov (United States)

    Ufarté, Lisa; Bozonnet, Sophie; Laville, Elisabeth; Cecchini, Davide A; Pizzut-Serin, Sandra; Jacquiod, Samuel; Demanèche, Sandrine; Simonet, Pascal; Franqueville, Laure; Veronese, Gabrielle Potocki

    2016-01-01

    Activity-based metagenomics is one of the most efficient approaches to boost the discovery of novel biocatalysts from the huge reservoir of uncultivated bacteria. In this chapter, we describe a highly generic procedure of metagenomic library construction and high-throughput screening for carbohydrate-active enzymes. Applicable to any bacterial ecosystem, it enables the swift identification of functional enzymes that are highly efficient, alone or acting in synergy, to break down polysaccharides and oligosaccharides.

  7. 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.

  8. 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

  9. Effect of high hydrostatic pressure on mycelial development, spore viability and enzyme activity of Penicillium Roqueforti.

    Science.gov (United States)

    Martínez-Rodríguez, Yamile; Acosta-Muñiz, Carlos; Olivas, Guadalupe I; Guerrero-Beltrán, José; Rodrigo-Aliaga, Dolores; Mujica-Paz, Hugo; Welti-Chanes, Jorge; Sepulveda, David R

    2014-01-03

    This study investigated the effect of high hydrostatic pressure treatments on mycelial development, spore viability, and total proteolytic and lipolytic activity of Penicillium roqueforti PV-LYO 10 D. Fungus growing in liquid medium was pressure-treated at 300, 400, and 500 MPa for 10 min at 20°C following seven days of incubation at 25°C and analyzed periodically up to day 9 after treatments to evaluate the effect on fungal growth. Mycelial mass of P. roqueforti was significantly affected at all pressure treatments evaluated, being 15.48%, 22.28%, 30.03%, and 12.53% lower than controls on day 1, 3, 6, and 9 after 300 MPa treatment, respectively. In a similar way, at 400 and 500 MPa, mycelial mass was 31.08% and 60.34% lower than controls one day after treatments and 49.74% and 80.85% lower on day 9, respectively. The viability of P. roqueforti spores decreased by 36.53% at 300 MPa, and complete inactivation took place at ≥400 MPa from an initial count of 7 log cfu/mL. Total proteolytic activity was not significantly affected at 300 MPa but was reduced by 18.22% at 400 MPa and by 43.18% at 500 MPa. Total lipolytic activity also decreased as the intensity of the pressure treatments increased. 21.69%, 39.12%, and 56.26% activity reductions were observed when treatments of 300, 400 and 500 MPa were applied, respectively. The results from this study show that pressure treatments are able to control growth, inactivate spores, and alter enzyme activity of P. roqueforti, which could be of interest in extending the shelf-life of blue-veined cheeses and other food products. © 2013.

  10. High Pressure Homogenization of Porcine Pepsin Protease: Effects on Enzyme Activity, Stability, Milk Coagulation Profile and Gel Development

    Science.gov (United States)

    Leite Júnior, Bruno Ricardo de Castro; Tribst, Alline Artigiani Lima; Cristianini, Marcelo

    2015-01-01

    This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G’ value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese. PMID:25938823

  11. 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.

  12. Highly active engineered-enzyme oriented monolayers: formation, characterization and sensing applications

    Directory of Open Access Journals (Sweden)

    Patolsky Fernando

    2011-06-01

    Full Text Available Abstract Background The interest in introducing ecologically-clean, and efficient enzymes into modern industry has been growing steadily. However, difficulties associated with controlling their orientation, and maintaining their selectivity and reactivity is still a significant obstacle. We have developed precise immobilization of biomolecules, while retaining their native functionality, and report a new, fast, easy, and reliable procedure of protein immobilization, with the use of Adenylate kinase as a model system. Methods Self-assembled monolayers of hexane-1,6-dithiol were formed on gold surfaces. The monolayers were characterized by contact-angle measurements, Elman-reagent reaction, QCM, and XPS. A specifically designed, mutated Adenylate kinase, where cysteine was inserted at the 75 residue, and the cysteine at residue 77 was replaced by serine, was used for attachment to the SAM surface via spontaneously formed disulfide (S-S bonds. QCM, and XPS were used for characterization of the immobilized protein layer. Curve fitting in XPS measurements used a Gaussian-Lorentzian function. Results and Discussion Water contact angle (65-70°, as well as all characterization techniques used, confirmed the formation of self-assembled monolayer with surface SH groups. X-ray photoelectron spectroscopy showed clearly the two types of sulfur atom, one attached to the gold (triolate and the other (SH/S-S at the ω-position for the hexane-1,6-dithiol SAMs. The formation of a protein monolayer was confirmed using XPS, and QCM, where the QCM-determined amount of protein on the surface was in agreement with a model that considered the surface area of a single protein molecule. Enzymatic activity tests of the immobilized protein confirmed that there is no change in enzymatic functionality, and reveal activity ~100 times that expected for the same amount of protein in solution. Conclusions To the best of our knowledge, immobilization of a protein by the method

  13. 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.

  14. High titers of autoantibodies to glutamate decarboxylase in Type 1 Diabetes Patients: Epitope Analysis and Inhibition of Enzyme Activity

    Science.gov (United States)

    Hampe, Christiane S.; Maitland, Murray E.; Gilliam, Lisa K.; Thi Phan, Thanh-H.; Sweet, Ian R.; Radtke, Jared R.; Bota, Vasile; Ransom, Bruce R.; Hirsch, Irl B.

    2014-01-01

    Objective Autoantibodies to glutamate decarboxylase (GAD65Ab) are found in patients with autoimmune neurological disorders and patients with type 1 diabetes. The correct diagnosis of GAD65Ab-associated neurological disorders is often delayed by the variability of symptoms and a lack of diagnostic markers. We hypothesize that the frequency of neurological disorders with high GAD65Ab titers is significantly higher than currently recognized. Methods We analyzed GAD65Ab titer, inhibition of GAD65 enzyme activity, and pattern of GAD65Ab epitopes in a cohort of type 1 diabetes patients (n=100) and correlated our findings with neurological symptoms and diseases. Results Fourty-three percent (43/100) of the patients had detectable GAD65Ab titers (median=400 U/ml, range: 142–250,000U/ml). The GAD65Ab titers in 10 type 1 diabetes patients exceeded the 90th percentile of the cohort (2,000–250,000 U/ml). Sera of these 10 patients were analyzed for their GAD65Ab epitope specificity and their ability to inhibit GAD65 enzyme activity in vitro. GAD65Ab of five patients inhibited the enzyme activity significantly (by 34–55%). Three of these patients complained of muscle stiffness and pain, which was documented in two of these patients. Conclusions Based on our findings we suggest that neurological disorders with high GAD65Ab titers are more frequent in type 1 diabetes patients than currently recognized. PMID:23512385

  15. High activity antioxidant enzymes protect flying-fox haemoglobin against damage: an evolutionary adaptation for flight?

    Science.gov (United States)

    Reinke, N B; O'Brien, G M

    2006-11-01

    Flying-foxes are better able to defend haemoglobin against autoxidation than non-volant mammals such as sheep. When challenged with the common physiological oxidant, hydrogen peroxide, haemolysates of flying-fox red blood cells (RBC) were far less susceptible to methaemoglobin formation than sheep. Challenge with 1-acetyl-2-phenylhydrazine (APH) caused only half as much methaemoglobin formation in flying-fox as in ovine haemolysates. When intact cells were challenged with phenazine methosulfate (PMS), flying-fox RBC partially reversed the oxidant damage, and reduced methaemoglobin from 40 to 20% over 2 h incubation, while ovine methaemoglobin remained at 40%. This reflected flying-fox cells' capacity to replenish GSH fast enough that it did not deplete beyond 50%, while ovine RBC GSH was depleted to around 20%. The greater capacity of flying-foxes to defend haemoglobin against oxidant damage may be explained in part by antioxidant enzymes catalase, superoxide dismutase and cytochrome-b ( 5 ) reductase having two- to four-fold higher activity than in sheep (P foxes.

  16. Application of high hydrostatic pressure for increasing activity and stability of enzymes.

    Science.gov (United States)

    Mozhaev, V V; Lange, R; Kudryashova, E V; Balny, C

    1996-10-20

    Elevated hydrostatic pressure has been used to increase catalytic activity and thermal stability of alpha-chymotrypsin (CT). For an anilide substrate, characterized by a negative value of the reaction activation volume (DeltaV( not equal)), an increase in pressure at 20 degrees C results in an exponential acceleration of the hydrolysis rate catalyzed by CT reaching a 6.5-fold increase in activity at 4700 atm (4.7 kbar). Due to a strong temperature dependence of DeltaV( not equal), the acceleration effect of high pressure becomes more pronounced at high temperatures. For example, at 50 degrees C, under a pressure of 3.6 kbar, CT shows activity which is more than 30 times higher than the activity at normal conditions (20 degrees C, 1 atm). At pressures of higher than 3.6 kbar, the enzymatic activity is decreased due to a pressure-induced denaturation.Elevated hydrostatic pressure is also efficient for increasing stability of CT against thermal denaturation. For example, at 55 degrees C, CT is almost instantaneously inactivated at atmospheric pressure, whereas under a pressure of 1.8 kbar CT retains its anilide-hydrolyzing activity during several dozen minutes. Additional stabilization can be achieved in the presence of glycerol, which is most effective for protection of CT at an intermediate concentration of 40% (v/v). There has been observed an additivity in stabilization effects of high pressure and glycerol: thermal inactivation of pressure-stabilized CT can be decelerated in a supplementary manner by addition of 40% (v/v) glycerol. The protection effect of glycerol on the catalytic activity and stability of CT becomes especially pronounced when both extreme factors of temperature and pressure reach critical values. For example, at approximately 55 degrees C and 4.7 kbar, enzymatic activity of CT in the presence of 40% (v/v) glycerol is severalfold higher than in aqueous buffer.The results of this study are discussed in terms of the hypotheses which explain the

  17. 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...

  18. 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.

  19. 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.

  20. Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

    Science.gov (United States)

    Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor

    2015-11-01

    Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

  1. 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...

  2. 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.

  3. 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

  4. [Dynamic changes of soil microbial populations and enzyme activities in super-high yielding summer maize farmland soil].

    Science.gov (United States)

    Hou, Peng; Wang, Yong-jun; Wang, Kong-jun; Yang, Jin-sheng; Li, Deng-hai; Dong, Shu-ting; Liu, Jing-guo

    2008-08-01

    To reveal the characteristics of the dynamic changes of soil microbial populations and enzyme activities in super-high yielding ( > 15,000 kg x hm(-2)) summer maize farmland soil, a comparative study was conducted in the experimental fields in National Maize Engineering Research Center (Shandong). On the fields with an annual yield of >15,000 kg x hm(-2) in continuous three years, a plot with the yield of 20 322 kg x hm(-2) (HF) was chosen to make comparison with the conventional farmland (CF) whose maize yield was 8920. 1 kg x hm(-2). The numbers of bacteria, fungi, and actinomycetes as well as the activities of urease and invertase in 0-20 cm soil layer were determined. The results showed that in the growth period of maize, the numbers of bacteria, fungi, and actinomycetes in the two farmland soils increased first and declined then. At the later growth stages of maize, the numbers of soil microbes, especially those of bacteria and actinomycetes, were lower in HF than those in CF. At harvest stage, the ratio of the number of soil bacteria to fungi (B/ F) in HF was 2.03 times higher than that at sowing stage, and 3.02 times higher than that in CF. The B/F in CF had less difference at harvest and sowing stages. The soil urease activity in HF was significantly lower than that in CF at jointing stage, and the invertase activity in HF decreased rapidly after blooming stage, being significantly lower than that in CF.

  5. 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),.

  6. 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.

  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. 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.

  9. Method of preparing highly active and thermostable preparations of liver uridin-kinase usable for enzymic synthesis of radioactive nucleoside-5'-phosphates

    International Nuclear Information System (INIS)

    Cihak, A.; Vesely, J.

    1975-01-01

    A method is described of preparing a high-activity uridine kinase for the enzymic synthesis of radioactive nucleoside-5m-phosphates of the pyrimidine series. The preparation is separated from male rat liver after intraperitoneal application of 5'-azacytidine. Examples are given showing detailed procedures for the conversion of uridine and 6-azauridine to the corresponding 5'-phosphates. (L.K.)

  10. 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.

  11. 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...

  12. 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.

  13. Influence of high temperature and ethanol on thermostable lignocellulolytic enzymes

    DEFF Research Database (Denmark)

    Skovgaard, Pernille Anastasia; Jørgensen, Henning

    2013-01-01

    the influence of temperature and ethanol on enzyme activity and stability in the distillation step, where most enzymes are inactivated due to high temperatures. Two enzyme mixtures, a mesophilic and a thermostable mixture, were exposed to typical process conditions [temperatures from 55 to 65 °C and up to 5...... % ethanol (w/v)] followed by specific enzyme activity analyses and SDS-PAGE. The thermostable and mesophilic mixture remained active at up to 65 and 55 °C, respectively. When the enzyme mixtures reached their maximum temperature limit, ethanol had a remarkable influence on enzyme activity, e.g., the more...... ethanol, the faster the inactivation. The reason could be the hydrophobic interaction of ethanol on the tertiary structure of the enzyme protein. The thermostable mixture was more tolerant to temperature and ethanol and could therefore be a potential candidate for recycling after distillation....

  14. 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.

  15. 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

    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...... hydrolysis in order to determine the relative temperature responses of the initial and terminal steps in microbial remineralization of carbon. The temperature optimum of sulfate reduction, 21degreesC, was considerably lower than previous reports of sulfate reduction in marine sediments, but is consistent...... with recent studies of psychrophilic sulfate reducers isolated from Svalbard sediments. A calculation of potential carbon flow into the microbial food chain demonstrated that the activity of just one type of polysaccharide-hydrolyzing enzyme could in theory supply 21 to 100% of the carbon consumed via sulfate...

  16. 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.

  17. Functional Comparison for Lipid Metabolism and Intestinal and Fecal Microflora Enzyme Activities between Low Molecular Weight Chitosan and Chitosan Oligosaccharide in High-Fat-Diet-Fed Rats.

    Science.gov (United States)

    Chiu, Chen-Yuan; Feng, Shih-An; Liu, Shing-Hwa; Chiang, Meng-Tsan

    2017-07-24

    The present study investigated and compared the regulatory effects on the lipid-related metabolism and intestinal disaccharidase/fecal bacterial enzyme activities between low molecular weight chitosan and chitosan oligosaccharide in high-fat-diet-fed rats. Diet supplementation of low molecular weight chitosan showed greater efficiency than chitosan oligosaccharide in suppressing the increased weights in body and in liver and adipose tissues of high-fat-diet-fed rats. Supplementation of low molecular weight chitosan also showed a greater improvement than chitosan oligosaccharide in imbalance of plasma, hepatic, and fecal lipid profiles, and intestinal disaccharidase activities in high-fat-diet-fed rats. Moreover, both low molecular weight chitosan and chitosan oligosaccharide significantly decreased the fecal microflora mucinase and β-glucuronidase activities in high-fat-diet-fed rats. These results suggest that low molecular weight chitosan exerts a greater positive improvement than chitosan oligosaccharide in lipid metabolism and intestinal disaccharidase activity in high-fat-diet-induced obese rats.

  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. 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

  20. Highly efficient enzyme encapsulation in a protein nanocage: towards enzyme catalysis in a cellular nanocompartment mimic

    Science.gov (United States)

    Schoonen, Lise; Nolte, Roeland J. M.; van Hest, Jan C. M.

    2016-07-01

    The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions.The study of enzyme behavior in small nanocompartments is crucial for the understanding of biocatalytic processes in the cellular environment. We have developed an enzymatic conjugation strategy to attach a model enzyme to the interior of a cowpea chlorotic mottle virus capsid. It is shown that with this methodology high encapsulation efficiencies can be achieved. Additionally, we demonstrate that the encapsulation does not affect the enzyme performance in terms of a decreased activity or a hampered substrate diffusion. Finally, it is shown that the encapsulated enzymes are protected against proteases. We believe that our strategy can be used to study enzyme kinetics in an environment that approaches physiological conditions. Electronic supplementary information (ESI) available: Experimental procedures for the cloning, expression, and purification of all proteins, as well as supplementary figures and calculations. See DOI: 10.1039/c6nr04181g

  1. Modification of enzymes by use of high-pressure homogenization.

    Science.gov (United States)

    Dos Santos Aguilar, Jessika Gonçalves; Cristianini, Marcelo; Sato, Helia Harumi

    2018-07-01

    High-pressure is an emerging and relatively new technology that can modify various molecules. High-pressure homogenization (HPH) has been used in several studies on protein modification, especially in enzymes used or found in food, from animal, plant or microbial resources. According to the literature, the enzymatic activity can be modulated under pressure causing inactivation, stabilization or activation of the enzymes, which, depending on the point of view could be very useful. Homogenization can generate changes in the structure of the enzyme modifying various chemical bonds (mainly weak bonds) causing different denaturation levels and, consequently, affecting the catalytic activity. This review aims to describe the various alterations due to HPH treatment in enzymes, to show the influence of high-pressure on proteins and to report the HPH effects on the enzymatic activity of different enzymes employed in the food industry and research. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. 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...

  3. 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.

  4. 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.

  5. High content of endogenous cytokinins stimulates activity of enzymes and proteins involved in stress response in Nicotiana tabacum

    Czech Academy of Sciences Publication Activity Database

    Synková, Helena; Semorádová, Šárka; Burketová, Lenka

    2004-01-01

    Roč. 79, č. 2 (2004), s. 169-179 ISSN 0167-6857 R&D Projects: GA ČR GA206/01/1061; GA ČR GA206/03/0310 Grant - others:Grantová agentura Univerzity Karlovy(CZ) 134/2001/B-Bio/PrF; Grantová agentura Univerzity Karlovy(CZ) Z5038910 Institutional research plan: CEZ:AV0Z5038910 Keywords : antioxidant enzymes * enzymes of intermediary metabolism * ex vitro Subject RIV: ED - Physiology Impact factor: 1.028, year: 2004

  6. 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.

  7. 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

  8. Enhancement of catalytic activity of enzymes by heating in anhydrous organic solvents: 3D structure of a modified serine proteinase at high resolution.

    Science.gov (United States)

    Sharma, S; Tyagi, R; Gupta, M N; Singh, T P

    2001-01-01

    For the first time, it is demonstrated that exposure of an enzyme to anhydrous organic solvents at optimized high temperature enhances its catalytic power through local changes at the binding region. Six enzymes, namely, proteinase K, wheat germ acid phosphatase, alpha-amylase, beta-glucosidase, chymotrypsin and trypsin were exposed to acetonitrile at 70 degrees C for three hr. The activities of these enzymes were found to be considerably enhanced. In order to understand the basis of this change in the activity of these enzymes, proteinase K was analyzed in detail using X-ray diffraction method. The overall structure of the enzyme was found to be similar to the native structure in aqueous environment. The hydrogen bonding system of the catalytic triad remained intact after the treatment. However, the water structure in the substrate binding site underwent some rearrangement as some of the water molecules were either displaced or completely absent. The most striking observation concerning the water structure was the complete deletion of the water molecule which occupied the position at the so-called oxyanion hole in the active site of the native enzyme. Three acetonitrile molecules were found in the present structure. All the acetonitrile molecules were located in the recognition site. Interlinked through water molecules, the sites occupied by acetonitrile molecules were independent of water molecules. The acetonitrile molecules are involved in extensive interactions with the protein atoms. The methyl group of one of the acetonitrile molecules (CCN1) interacts simultaneously with the hydrophobic side chains of Leu 96, Ile 107 and Leu 133. The development of such a hydrophobic environment at the recognition site introduced a striking conformation change in Ile 107 by rotating its side chain about C alpha-C beta bond by 180 degrees to bring about the delta-methyl group within the range of attractive van der Waals interactions with the methyl group of CCN1. A similar

  9. 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

  10. Fabrication of highly electro catalytic active layer of multi walled carbon nanotube/enzyme for Pt-free dye sensitized solar cells

    Energy Technology Data Exchange (ETDEWEB)

    Arbab, Alvira Ayoub, E-mail: alvira_arbab@yahoo.com [Department of Organic and Nano Engineering, Hanyang University, Seoul 133-791 (Korea, Republic of); Sun, Kyung Chul, E-mail: hytec@hanyang.ac.kr [Department of Fuel cells and hydrogen technology, Hanyang University, Seoul 133-791 (Korea, Republic of); Sahito, Iftikhar Ali, E-mail: iftikhar.sahito@faculty.muet.edu.pk [Department of Organic and Nano Engineering, Hanyang University, Seoul 133-791 (Korea, Republic of); Qadir, Muhammad Bilal, E-mail: bilal_ntu81@hotmail.com [Department of Organic and Nano Engineering, Hanyang University, Seoul 133-791 (Korea, Republic of); Jeong, Sung Hoon, E-mail: shjeong@hanyang.ac.kr [Department of Organic and Nano Engineering, Hanyang University, Seoul 133-791 (Korea, Republic of)

    2015-09-15

    Graphical abstract: - Highlights: • We prepared three different types of enzyme dispersed multiwall carbon nanotube (E-MWCNT) layer for application in Pt-free dye sensitized solar cell (DSSCs). • E-MWCNT catalysts exhibited an extremely good electro-catalytic activity (ECA), compared with the conventional catalyst, when synthesized with lipase enzyme. • E-MWCNT as counter electrode exhibits a high power conversion efficiency (PCE) of 7.5%, which can be compared to 8% efficiency of Pt catalyst. - Abstract: Highly dispersed conductive suspensions of multi walled carbon nanotubes (MWCNT) can have intrinsic electrical and electrochemical characteristics, which make them useful candidate for platinum (Pt)-free, dye sensitized solar cells (DSSCs). High energy conversion efficiency of 7.52% is demonstrated in DSSCs, based on enzyme dispersed MWCNT (E-MWCNT) layer deposited on fluorine doped tin oxide (FTO) glass. The E-MWCNT layer shows a pivotal role as platform to reduce large amount of iodide species via electro catalytically active layer, fabricated by facile tape casting under air drying technique. The E-MWCNT layer with large surface area, high mechanical adhesion, and good interconnectivity is derived from an appropriate enzyme dispersion, which provides not only enhanced interaction sites for the electrolyte/counter electrode interface but also improved electron transport mechanism. The surface morphology and structural characterization were investigated using field emission-scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), x-ray photoelectron spectroscopy (XPS), Raman spectroscopy and electronic microscopy techniques. Electro catalytic activity (ECA) and electrochemical properties of E-MWCNT counter electrode (CE) were investigated using cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) measurements. The high power conversion efficiency (PCE) of E-MWCNT CE is associated with the low charge transfer

  11. Fabrication of highly electro catalytic active layer of multi walled carbon nanotube/enzyme for Pt-free dye sensitized solar cells

    International Nuclear Information System (INIS)

    Arbab, Alvira Ayoub; Sun, Kyung Chul; Sahito, Iftikhar Ali; Qadir, Muhammad Bilal; Jeong, Sung Hoon

    2015-01-01

    Graphical abstract: - Highlights: • We prepared three different types of enzyme dispersed multiwall carbon nanotube (E-MWCNT) layer for application in Pt-free dye sensitized solar cell (DSSCs). • E-MWCNT catalysts exhibited an extremely good electro-catalytic activity (ECA), compared with the conventional catalyst, when synthesized with lipase enzyme. • E-MWCNT as counter electrode exhibits a high power conversion efficiency (PCE) of 7.5%, which can be compared to 8% efficiency of Pt catalyst. - Abstract: Highly dispersed conductive suspensions of multi walled carbon nanotubes (MWCNT) can have intrinsic electrical and electrochemical characteristics, which make them useful candidate for platinum (Pt)-free, dye sensitized solar cells (DSSCs). High energy conversion efficiency of 7.52% is demonstrated in DSSCs, based on enzyme dispersed MWCNT (E-MWCNT) layer deposited on fluorine doped tin oxide (FTO) glass. The E-MWCNT layer shows a pivotal role as platform to reduce large amount of iodide species via electro catalytically active layer, fabricated by facile tape casting under air drying technique. The E-MWCNT layer with large surface area, high mechanical adhesion, and good interconnectivity is derived from an appropriate enzyme dispersion, which provides not only enhanced interaction sites for the electrolyte/counter electrode interface but also improved electron transport mechanism. The surface morphology and structural characterization were investigated using field emission-scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), x-ray photoelectron spectroscopy (XPS), Raman spectroscopy and electronic microscopy techniques. Electro catalytic activity (ECA) and electrochemical properties of E-MWCNT counter electrode (CE) were investigated using cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS) measurements. The high power conversion efficiency (PCE) of E-MWCNT CE is associated with the low charge transfer

  12. Influence of high glycine diets on the activity of glycine-catabolizing enzymes and on glycine catabolism in rats

    International Nuclear Information System (INIS)

    Petzke, K.J.; Albrecht, V.; Przybilski, H.

    1986-01-01

    Male albino rats were adapted to isocaloric purified diets that differed mainly in their glycine and casein contents. Controls received a 30% casein diet. In experimental diets gelatin or gelatin hydrolysate was substituted for half of the 30% casein. An additional group was fed a glycine-supplemented diet, which corresponded in glycine level to the gelatin diet but in which the protein level was nearly the same as that of the casein control diet. Another group received a 15% casein diet. Rat liver glycine cleavage system, serine hydroxymethyltransferase and serine dehydratase activities were measured. 14 CO 2 production from the catabolism of 14 C-labeled glycine was measured in vivo and in vitro (from isolated hepatocytes). Serine dehydratase and glycine cleavage system activities were higher in animals fed 30% casein diets than in those fed 15% casein diets. Serine hydroxymethyltransferase activity of the cytosolic and mitochondrial fractions was highest when a high glycine diet (glycine administered as pure, protein bound in gelatin or peptide bound in gelatin hydrolysate) was fed. 14 CO 2 formation from [1- 14 C]- and [2- 14 C]glycine both in vivo and in isolated hepatocytes was higher when a high glycine diet was fed than when a casein diet was fed. These results suggest that glycine catabolism is dependent on and adaptable to the glycine content of the diet. Serine hydroxymethyltransferase appears to play a major role in the regulation of glycine degradation via serine and pyruvate

  13. Method of preparing uridine-diphospho-/sup 14/C-D-glucose of high molar activity and radiochemical purity by enzyme synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Farkas, V; Biely, P; Koelbl, J

    1980-06-15

    A new method is described of enzyme synthesis of uridine-diphospho-/sup 14/C-D-glucose of high molar activity. After conversion of the initial /sup 14/C-D-glucose, the accompanying sugar phosphates and nucleoside-5'-phosphates are selectively hydrolyzed by the action of alkaline phosphatase in the presence of phenolphthalein as an indicator. Uridine-diphospho-/sup 14/C-D-glucose is separated by paper chromatography from the reaction mixture thus modified. Synthesis yields range within 75% and 85% relative to the starting /sup 14/C-D-glucose.

  14. Antimutagenic activity of oxidase enzymes

    International Nuclear Information System (INIS)

    Agabeili, R.A.

    1986-01-01

    By means of a cytogenetic analysis of chromosomal aberrations in plant cells (Welsh onion, wheat) it was found that the cofactors nicotinamide adenine phosphate (NAD), nicotinamide adenine dinucleotide phosphate (NADPH), and riboflavin possess antimutagenic activity

  15. 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)

  16. The expression and activity of antioxidant enzymes in the liver of rats exposed to high-fructose diet in the period from weaning to adulthood.

    Science.gov (United States)

    Glban, Alhadi M; Vasiljević, Ana; Veličković, Nataša; Nikolić-Kokić, Aleksandra; Blagojević, Duško; Matić, Gordana; Nestorov, Jelena

    2015-08-30

    Increased fructose consumption correlates with rising prevalence of various metabolic disorders, some of which were linked to oxidative stress. The relationship between fructose consumption and oxidative stress is complex and effects of a fructose-rich diet on the young population have not been fully elucidated. The aim of this study was to investigate whether high-fructose diet applied in the period from weaning to adulthood induces oxidative stress in the liver, thus contributing to induction or aggravation of metabolic disturbances in later adulthood. To that end we examined the effects of high-fructose diet on expression and activity of antioxidant enzymes, markers of lipid peroxidation and protein damage in the liver as the main fructose metabolizing tissue. High-fructose diet increased only SOD2 (mitochondrial manganese superoxide dismutase) activity, with no effect on other antioxidant enzymes, lipid peroxidation or accumulation of damaged proteins in the liver. The results show that fructose-induced metabolic disturbances could not be attributed to oxidative stress, at least not at young age. The absence of oxidative stress in the liver observed herein implies that young organisms are capable of maintaining redox homeostasis when challenged by fructose-derived energy overload. © 2014 Society of Chemical Industry.

  17. 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

  18. Inhibition of angiotensin-1-converting enzyme activity by two varieties of ginger (Zingiber officinale) in rats fed a high cholesterol diet.

    Science.gov (United States)

    Akinyemi, Ayodele Jacob; Ademiluyi, Adedayo Oluwaseun; Oboh, Ganiyu

    2014-03-01

    Angiotensin-1-converting enzyme (ACE) inhibitors are widely used in the treatment of cardiovascular diseases. This study sought to investigate the inhibitory effect of two varieties of ginger (Zingiber officinale) commonly consumed in Nigeria on ACE activity in rats fed a high cholesterol diet. The inhibition of ACE activity of two varieties of ginger (Z. officinale) was investigated in a high cholesterol (2%) diet fed to rats for 3 days. Feeding high cholesterol diets to rats caused a significant (Pginger varieties. Rats that were fed 4% white ginger had the greatest inhibitory effect as compared with a control diet. Furthermore, there was a significant (Pginger (either 2% or 4%) caused a significant (Pginger had the greatest reduction as compared with control diet. In conclusion, both ginger varieties exhibited anti-hypercholesterolemic properties in a high cholesterol diet fed to rats. This activity of the gingers may be attributed to its ACE inhibitory activity. However, white ginger inhibited ACE better in a high cholesterol diet fed to rats than red ginger. Therefore, both gingers could serve as good functional foods/nutraceuticals in the management/treatment of hypertension and other cardiovascular diseases.

  19. 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.

  20. Structure of a Highly Active Cephalopod S-crystallin Mutant: New Molecular Evidence for Evolution from an Active Enzyme into Lens-Refractive Protein.

    Science.gov (United States)

    Tan, Wei-Hung; Cheng, Shu-Chun; Liu, Yu-Tung; Wu, Cheng-Guo; Lin, Min-Han; Chen, Chiao-Che; Lin, Chao-Hsiung; Chou, Chi-Yuan

    2016-08-08

    Crystallins are found widely in animal lenses and have important functions due to their refractive properties. In the coleoid cephalopods, a lens with a graded refractive index provides good vision and is required for survival. Cephalopod S-crystallin is thought to have evolved from glutathione S-transferase (GST) with various homologs differentially expressed in the lens. However, there is no direct structural information that helps to delineate the mechanisms by which S-crystallin could have evolved. Here we report the structural and biochemical characterization of novel S-crystallin-glutathione complex. The 2.35-Å crystal structure of a S-crystallin mutant from Octopus vulgaris reveals an active-site architecture that is different from that of GST. S-crystallin has a preference for glutathione binding, although almost lost its GST enzymatic activity. We've also identified four historical mutations that are able to produce a "GST-like" S-crystallin that has regained activity. This protein recapitulates the evolution of S-crystallin from GST. Protein stability studies suggest that S-crystallin is stabilized by glutathione binding to prevent its aggregation; this contrasts with GST-σ, which do not possess this protection. We suggest that a tradeoff between enzyme activity and the stability of the lens protein might have been one of the major driving force behind lens evolution.

  1. 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.

  2. 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 ...

  3. Two enzymes catalyze vitamin K 2,3-epoxide reductase activity in mouse: VKORC1 is highly expressed in exocrine tissues while VKORC1L1 is highly expressed in brain.

    Science.gov (United States)

    Caspers, Michael; Czogalla, Katrin J; Liphardt, Kerstin; Müller, Jens; Westhofen, Philipp; Watzka, Matthias; Oldenburg, Johannes

    2015-05-01

    VKORC1 and VKORC1L1 are enzymes that both catalyze the reduction of vitamin K2,3-epoxide via vitamin K quinone to vitamin K hydroquinone. VKORC1 is the key enzyme of the classical vitamin K cycle by which vitamin K-dependent (VKD) proteins are γ-carboxylated by the hepatic γ-glutamyl carboxylase (GGCX). In contrast, the VKORC1 paralog enzyme, VKORC1L1, is chiefly responsible for antioxidative function by reduction of vitamin K to prevent damage by intracellular reactive oxygen species. To investigate tissue-specific vitamin K 2,3-epoxide reductase (VKOR) function of both enzymes, we quantified mRNA levels for VKORC1, VKORC1L1, GGCX, and NQO1 and measured VKOR enzymatic activities in 29 different mouse tissues. VKORC1 and GGCX are highly expressed in liver, lung and exocrine tissues including mammary gland, salivary gland and prostate suggesting important extrahepatic roles for the vitamin K cycle. Interestingly, VKORC1L1 showed highest transcription levels in brain. Due to the absence of detectable NQO1 transcription in liver, we assume this enzyme has no bypass function with respect to activation of VKD coagulation proteins. Our data strongly suggest diverse functions for the vitamin K cycle in extrahepatic biological pathways. Copyright © 2015. Published by Elsevier Ltd.

  4. Characterization of the cloned full-length and a truncated human target of rapamycin: Activity, specificity, and enzyme inhibition as studied by a high capacity assay

    International Nuclear Information System (INIS)

    Toral-Barza, Lourdes; Zhang Weiguo; Lamison, Craig; LaRocque, James; Gibbons, James; Yu, Ker

    2005-01-01

    The mammalian target of rapamycin (mTOR/TOR) is implicated in cancer and other human disorders and thus an important target for therapeutic intervention. To study human TOR in vitro, we have produced in large scale both the full-length TOR (289 kDa) and a truncated TOR (132 kDa) from HEK293 cells. Both enzymes demonstrated a robust and specific catalytic activity towards the physiological substrate proteins, p70 S6 ribosomal protein kinase 1 (p70S6K1) and eIF4E binding protein 1 (4EBP1), as measured by phosphor-specific antibodies in Western blotting. We developed a high capacity dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA) for analysis of kinetic parameters. The Michaelis constant (K m ) values of TOR for ATP and the His6-S6K substrate were shown to be 50 and 0.8 μM, respectively. Dose-response and inhibition mechanisms of several known inhibitors, the rapamycin-FKBP12 complex, wortmannin and LY294002, were also studied in DELFIA. Our data indicate that TOR exhibits kinetic features of those shared by traditional serine/threonine kinases and demonstrate the feasibility for TOR enzyme screen in searching for new inhibitors

  5. 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.

  6. High-yield production of biologically active recombinant protein in shake flask culture by combination of enzyme-based glucose delivery and increased oxygen transfer

    Directory of Open Access Journals (Sweden)

    Ukkonen Kaisa

    2011-12-01

    Full Text Available Abstract This report describes the combined use of an enzyme-based glucose release system (EnBase® and high-aeration shake flask (Ultra Yield Flask™. The benefit of this combination is demonstrated by over 100-fold improvement in the active yield of recombinant alcohol dehydrogenase expressed in E. coli. Compared to Terrific Broth and ZYM-5052 autoinduction medium, the EnBase system improved yield mainly through increased productivity per cell. Four-fold increase in oxygen transfer by the Ultra Yield Flask contributed to higher cell density with EnBase but not with the other tested media, and consequently the product yield per ml of EnBase culture was further improved.

  7. Development and Application of a New Microarray- Based Method for High-Throughput Screening of Carbohydrate Active Enzymes

    DEFF Research Database (Denmark)

    Vidal Melgosa, Silvia

    single defined polysaccharides, mixtures of defined polysaccharides and complex biomass extracts. Furthermore, the capacity of the technique to analyse endo- and exo-acting glycoside hydrolases, polysaccharide lyases, carbohydrate esterases and lytic polysaccharide monooxygenases has been confirmed...... will contribute to both the discovery of CAZymes and the empirical characterisation of their activities, thus aiding their industrial utilisation and biological understanding...

  8. Spectrophotometric Enzyme Assays for High-Throughput Screening

    Directory of Open Access Journals (Sweden)

    Jean-Louis Reymond

    2004-01-01

    Full Text Available This paper reviews high-throughput screening enzyme assays developed in our laboratory over the last ten years. These enzyme assays were initially developed for the purpose of discovering catalytic antibodies by screening cell culture supernatants, but have proved generally useful for testing enzyme activities. Examples include TLC-based screening using acridone-labeled substrates, fluorogenic assays based on the β-elimination of umbelliferone or nitrophenol, and indirect assays such as the back-titration method with adrenaline and the copper-calcein fluorescence assay for aminoacids.

  9. 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.

  10. 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.

  11. A maize gene encoding an NADPH binding enzyme highly homologous to isoflavone reductases is activated in response to sulfur starvation.

    Science.gov (United States)

    Petrucco, S; Bolchi, A; Foroni, C; Percudani, R; Rossi, G L; Ottonello, S

    1996-01-01

    we isolated a novel gene that is selectively induced both in roots and shoots in response to sulfur starvation. This gene encodes a cytosolic, monomeric protein of 33 kD that selectively binds NADPH. The predicted polypeptide is highly homologous ( > 70%) to leguminous isoflavone reductases (IFRs), but the maize protein (IRL for isoflavone reductase-like) belongs to a novel family of proteins present in a variety of plants. Anti-IRL antibodies specifically recognize IFR polypeptides, yet the maize protein is unable to use various isoflavonoids as substrates. IRL expression is correlated closely to glutathione availability: it is persistently induced in seedlings whose glutathione content is about fourfold lower than controls, and it is down-regulated rapidly when control levels of glutathione are restored. This glutathione-dependent regulation indicates that maize IRL may play a crucial role in the establishment of a thiol-independent response to oxidative stress under glutathione shortage conditions.

  12. 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.

  13. High-pressure tolerance of earthworm fibrinolytic and digestive enzymes.

    Science.gov (United States)

    Akazawa, Shin-Ichi; Tokuyama, Haruka; Sato, Shunsuke; Watanabe, Toshinori; Shida, Yosuke; Ogasawara, Wataru

    2018-02-01

    Earthworms contain several digestive and therapeutic enzymes that are beneficial to our health and useful for biomass utilization. Specifically, earthworms contain potent fibrinolytic enzymes called lumbrokinases, which are highly stable even at room temperature and remain active in dried earthworm powder. However, the high-temperature sterilization method leads to the inactivation of enzymes. Therefore, we investigated the effect of high-pressure treatment (HPT) (from 0.1 MPa to 500 MPa at 25°C and 50°C) on the enzymatic activity of lumbrokinase (LK), α-amylase (AMY), endoglucanase (EG), β-glucosidase (BGL), and lipase (LP) of the earthworm Eisenia fetida, Waki strain, and its sterilization ability in producing dietary supplement. LK showed thermo- and high-pressure tolerance. In addition, HPT may have resulted in pressure-induced stabilization and activation of LK. Although AMY activity was maintained up to 400 MPa at 25°C, the apparent activity decreased slightly at 50°C with HPT. EG showed almost the same pattern as AMY. However, it is possible that the effects of temperature and pressure compensated each other under 100 MPa at 50°C. BGL was shown to be a pressure- and temperature-sensitive enzyme, and LP showed a thermo- and high-pressure tolerance. The slight decrease in apparent activity occurred under 200 MPa at both temperatures. Furthermore, the low-temperature and pressure treatment completely sterilized the samples. These results provide a basis for the development of a novel earthworm dietary supplement with fibrinolytic and digestive activity and of high-pressure-tolerant enzymes to be used for biomass pretreatment. Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

  14. Carbohydrate-active enzymes from the zygomycete fungus Rhizopus oryzae: a highly specialized approach to carbohydrate degradation depicted at genome level

    Directory of Open Access Journals (Sweden)

    Henrissat Bernard

    2011-01-01

    Full Text Available Abstract Background Rhizopus oryzae is a zygomycete filamentous fungus, well-known as a saprobe ubiquitous in soil and as a pathogenic/spoilage fungus, causing Rhizopus rot and mucomycoses. Results Carbohydrate Active enzyme (CAZy annotation of the R. oryzae identified, in contrast to other filamentous fungi, a low number of glycoside hydrolases (GHs and a high number of glycosyl transferases (GTs and carbohydrate esterases (CEs. A detailed analysis of CAZy families, supported by growth data, demonstrates highly specialized plant and fungal cell wall degrading abilities distinct from ascomycetes and basidiomycetes. The specific genomic and growth features for degradation of easily digestible plant cell wall mono- and polysaccharides (starch, galactomannan, unbranched pectin, hexose sugars, chitin, chitosan, β-1,3-glucan and fungal cell wall fractions suggest specific adaptations of R. oryzae to its environment. Conclusions CAZy analyses of the genome of the zygomycete fungus R. oryzae and comparison to ascomycetes and basidiomycete species revealed how evolution has shaped its genetic content with respect to carbohydrate degradation, after divergence from the Ascomycota and Basidiomycota.

  15. 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.

  16. 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...

  17. 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

  18. 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.

  19. 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.

  20. 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

  1. 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...

  2. 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.

  3. 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- ...

  4. 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)

  5. 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.

  6. Stabilization of red fruit-based smoothies by high-pressure processing. Part A. Effects on microbial growth, enzyme activity, antioxidant capacity and physical stability.

    Science.gov (United States)

    Hurtado, Adriana; Guàrdia, Maria Dolors; Picouet, Pierre; Jofré, Anna; Ros, José María; Bañón, Sancho

    2017-02-01

    Non-thermal pasteurization by high-pressure processing (HPP) is increasingly replacing thermal processing (TP) to maintain the properties of fresh fruit products. However, most of the research on HPP-fruit products only partially addresses fruit-pressure interaction, which limits its practical interest. The objective of this study was to assess the use of a mild HPP treatment to stabilize red fruit-based smoothies (microbial, enzymatic, oxidative and physical stability). HPP (350 MPa/10 °C/5 min) was slightly less effective than TP (85 °C/7 min) in inactivating microbes (mesophilic and psychrophilic bacteria, coliforms, yeasts and moulds) in smoothies kept at 4 °C for up to 28 days. The main limitation of using HPP was its low efficacy in inactivating oxidative (polyphenol oxidase and peroxidase) and hydrolytic (pectin methyl esterase) enzymes. Data on antioxidant status, colour parameters, browning index, transmittance, turbidity and viscosity confirmed that the HPP-smoothies have a greater tendency towards oxidation and clarification, which might lead to undesirable sensory and nutritional changes (see Part B). The microbial quality of smoothies was adequately controlled by mild HPP treatment without affecting their physical-chemical characteristics; however, oxidative and hydrolytic enzymes are highly pressure-resistant, which suggests that additional strategies should be used to stabilize smoothies. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  7. 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...

  8. Quantification of trypsin with a radioimmunoassay in herring larvae (Clupea harengus L.) compared with a highly sensitive fluorescence technique to determine tryptic enzyme activity

    International Nuclear Information System (INIS)

    Ueberschaer, B.; Pedersen, B.H.; Hjelmeland, K.

    1993-01-01

    Enzymatic activity and quantity of the protease trypsin were measured in individual herring larvae (Clupea harengus L.). The enzymatic activity assay was done by a fluorescence technique, and a radioimmunoassay was used for quantification of trypsin. The results are compared and the differences between the techniques discussed. Both methods have similar results, as high or low values in trypsin quantity were reflected in high or low values of tryptic activity. Quantity and activity were linearly and positively correlated, but small differences between methods were found at the lowest detection limits. Both techniques reflect the high variability between individual larvae. (orig.)

  9. 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

  10. 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)

  11. 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.

  12. Inhibitory Activities of Zygophyllum album: A Natural Weight-Lowering Plant on Key Enzymes in High-Fat Diet-Fed Rats

    Science.gov (United States)

    Mnafgui, Kais; Hamden, Khaled; Ben Salah, Hichem; Kchaou, Mouna; Nasri, Mbarek; Slama, Sadok; Derbali, Fatma; Allouche, Noureddine; Elfeki, Abdelfattah

    2012-01-01

    Obesity is a serious health problem that increased risk for many complications, including diabetes and cardiovascular disease. The results showed EZA, which found rich in flavonoids and phenolic compounds, exhibited an inhibitory activity on pancreatic lipase in vitro with IC50 of 91.07 μg/mL. In vivo administration of this extract to HFD-rats lowered body weight and serum leptin level; and inhibited lipase activity of obese rats by 37% leading to notable decrease of T-Ch, TGs and LDL-c levels accompanied with an increase in HDL-c concentration in serum and liver of EZA treated HFD-rats. Moreover, the findings revealed that EZA helped to protect liver tissue from the appearance of fatty cysts. Interestingly, supplementation of EZA modulated key enzyme related to hypertension such as ACE by 36% in serum of HFD animals and improve some of serum electrolytes such as Na+, K+, Cl−, Ca2+ and Mg2+. Moreover, EZA significantly protected the liver-kidney function by reverted back near to normal the values of the liver-kidney dysfunction indices AST&ALT, ALP, CPK and GGT activities, decreased T-Bili, creat, urea and uric acid rates. In conclusion, these results showed a strong antihypelipidemic effect of EZA which can delay the occurrence of dislipidemia and hypertension. PMID:23258993

  13. 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.

  14. Antioxidant Enzyme Activities of some Brassica Species

    Directory of Open Access Journals (Sweden)

    Rodica SOARE

    2017-11-01

    Full Text Available This paper set out to comparatively study five species: white cabbage (Brassica oleracea L. var. capitata alba Alef., red cabbage (Brassica oleracea L. var. capitata f. rubra Alef., Kale (Brassica oleracea L. var. Acephala, cauliflower (Brassica oleracea var. botrytis and broccoli (Brassica oleracea var. cymosa in order to identify those with high enzymatic and antioxidant activities. The enzymatic activity of superoxide dismutase (SOD, catalase (CAT and soluble peroxidase (POX as well as the antioxidant activity against 2.2’-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS radical cation were determined. Total superoxide dismutase activity was measured spectrophotometrically based on inhibition in the photochemical reduction of nitroblue tetrazolium. Total soluble peroxidase was assayed by measuring the increase in A436 due to the guaiacol oxidation and the catalase activity was assayed through the colorimetric method. The capacity of extracts to scavenge the ABTS radical cation was assessed colorimetric using Trolox as a standard. The obtained results show that studied enzymatic activities and the antioxidant activity against ABTS vary depending on the analyzed species. So, among the studied Brassicaceae species, it emphasize red cabbage with the highest enzymatic activity (CAT 22.54 mM H2O2/min/g and POX 187.2 mM ΔA/1min/1g f.w. and kale with highest antioxidant activity, of 767 μmol TE/100g f.w. The results of this study recommendintroducing the studied varieties in diet due to the rich antioxidant properties.

  15. 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.

  16. 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.

  17. Enzyme

    Science.gov (United States)

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  18. 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

  19. 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,…

  20. 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

  1. 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.

  2. 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.

  3. 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...

  4. 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.

  5. 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

  6. 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 ...

  7. 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 ...

  8. 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 ...

  9. 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

  10. 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)

  11. 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)

  12. 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.

  13. 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.

  14. 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.

  15. 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.

  16. 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 ...

  17. 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.

  18. 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)

  19. Carbon-cycle effects of differences in soil moisture and soil extracellular enzyme activity at sites representing different land-use histories in high-elevation Ecuadorian páramo landscapes

    Science.gov (United States)

    McKnight, J.; Harden, C. P.; Schaeffer, S. M.

    2016-12-01

    Ecuadorian páramo grasslands are important regional soil carbon sinks. In the páramo of the Mazar Wildlife Reserve, differences in soil carbon content among different types of land use may reflect changes in soil carbon-acquisition related microbial enzyme activity after land cover and soil moisture are altered; however, this hypothesis has not been tested explicitly for Ecuadorian páramos. This study used a fluorescence enzyme assay to assess the activities of four different extracellular enzymes representing carbon acquisition: α-glucosidase, β-glucosidase, β-D-cellulobiohydrolase, and β-xylosidase in Andean páramo soils. Acquisition activities were also measured for nitrogen (N-acetyl-β-glucosidase and leucine aminopeptidase) and phosphorus (phosphatase) to assess stoichiometric differences between land-uses, which can affect soil microbial activity related to carbon acquisition. Soils were analyzed from four land uses: native forest, grass páramo, recently burned grass páramo, and non-native pine plantation. Carbon acquisition activity was highest at the pine site (678 nmol h-1 g-1) and lowest at the recently burned páramo site (252 nmol h-1 g-1), indicating the lowest and highest available soil carbon, respectively. Carbon-acquisition EE activity was significantly higher at the grass páramo site (595 nmol h-1 g-1) than at the recently burned páramo and native forest sites. At the grass páramo site, a history of burning as a management strategy and high carbon-acquisition EE activity could indicate the presence of pyrogenic soil organic matter, which is more resistant to microbial decomposition. Soils at the native forest and both grassland sites were phosphorus limited, and soil at the pine site had higher nitrogen-acquisition activity, indicative of a shift to nitrogen-limited soil stoichiometric conditions. To our knowledge these are the first data reported for soil extracellular enzyme activities for Ecuadorian páramos.

  20. 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...

  1. Pepsinogens and pepsins from largemouth bass, Micropterus salmoides: purification and characterization with special reference to high proteolytic activities of bass enzymes.

    Science.gov (United States)

    Miura, Yoko; Kageyama, Takashi; Moriyama, Akihiko

    2015-05-01

    Six pepsinogens were purified from the gastric mucosa of largemouth bass (Micropterus salmoides) by DEAE-Sephacel chromatography, Sephadex G-100 gel filtration, and Mono Q FPLC. The potential specific activities of two major pepsinogens, PG1-1 and PG2-2, against hemoglobin were 51 and 118 units/mg protein, respectively. The activity of pepsin 2-2 was the highest among the pepsins reported to date; this might be linked to the strongly carnivorous diet of the largemouth bass. The molecular masses of PG1-1 and PG2-2 were 39.0 and 41.0 kDa, respectively. The N-terminal amino acid sequences of PG1-1 and PG2-2 were LVQVPLEVGQTAREYLE- and LVRLPLIVGKTARQALLE-, respectively, showing similarities with those of fish type-A pepsinogens. The optimal pHs for hemoglobin-digestive activity of pepsins 1-1 and 2-2 were around 1.5 and 2.0, respectively, though both pepsins retained considerable activity at pHs over 3.5. They showed maximal activity around 50 and 40 °C, respectively. They were inhibited by pepstatin similarly to porcine pepsin A. The cleavage specificities clarified with oxidized insulin B chain were shown to be restricted to a few bonds consisting of hydrophobic/aromatic residues, such as the Leu(15)-Tyr(16), Phe(24)-Phe(25) and Phe(25)-Tyr(26) bonds. When hemoglobin was used as a substrate, the kcat/Km value of bass pepsin 2-2 was 4.6- to 36.8-fold larger than those of other fish pepsins. In the case of substance P, an ideal pepsin substrate mimic, the kcat/Km values were about 200-fold larger than those of porcine pepsin A, supporting the high activity of the bass pepsin. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. 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.

  3. Combined pressure and cosolvent effects on enzyme activity - a high-pressure stopped-flow kinetic study on α-chymotrypsin.

    Science.gov (United States)

    Luong, Trung Quan; Winter, Roland

    2015-09-21

    We investigated the combined effects of cosolvents and pressure on the hydrolysis of a model peptide catalysed by α-chymotrypsin. The enzymatic activity was measured in the pressure range from 0.1 to 200 MPa using a high-pressure stopped-flow systems with 10 ms time resolution. A kosmotropic (trimethalymine-N-oxide, TMAO) and chaotropic (urea) cosolvent and mixtures thereof were used as cosolvents. High pressure enhances the hydrolysis rate as a consequence of a negative activation volume, ΔV(#), which, depending on the cosolvent system, amounts to -2 to -4 mL mol(-1). A more negative activation volume can be explained by a smaller compression of the ES complex relative to the transition state. Kinetic constants, such as kcat and the Michaelis constant KM, were determined for all solution conditions as a function of pressure. With increasing pressure, kcat increases by about 35% and its pressure dependence by a factor of 1.9 upon addition of 2 M urea, whereas 1 M TMAO has no significant effect on kcat and its pressure dependence. Similarly, KM increases upon addition of urea 6-fold. Addition of TMAO compensates the urea-effect on kcat and KM to some extent. The maximum rate of the enzymatic reaction increases with increasing pressure in all solutions except in the TMAO : urea 1 : 2 mixture, where, remarkably, pressure is found to have no effect on the rate of the enzymatic reaction anymore. Our data clearly show that compatible solutes can easily override deleterious effects of harsh environmental conditions, such as high hydrostatic pressures in the 100 MPa range, which is the maximum pressure encountered in the deep biosphere on Earth.

  4. 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.

  5. 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.

  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. 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.

  8. Chimeric enzymes with improved cellulase activities

    Science.gov (United States)

    Xu, Qi; Baker, John O; Himmel, Michael E

    2015-03-31

    Nucleic acid molecules encoding chimeric cellulase polypeptides that exhibit improved cellulase activities are disclosed herein. The chimeric cellulase polypeptides encoded by these nucleic acids and methods to produce the cellulases are also described, along with methods of using chimeric cellulases for the conversion of cellulose to sugars such as glucose.

  9. Alginate Immobilization of Metabolic Enzymes (AIME) for High ...

    Science.gov (United States)

    Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput screening (HTS) assays to assess chemical perturbations of molecular and cellular endpoints. A key criticism of using HTS assays for toxicity assessment is the lack of xenobiotic metabolism (XM) which precludes both metabolic detoxification as well as bioactivation of chemicals tested in vitro thereby mischaracterizing the potential risk posed by these chemicals. To address this deficiency, we have developed an extracellular platform to retrofit existing HTS assays with XM activity. This platform utilizes the S9 fraction of liver homogenate encapsulated in an alginate gel network which reduces the cytotoxicity caused by direct addition of S9 to cells in culture. Alginate microspheres containing encapsulated human liver S9 were cross-linked to solid supports extending from a 96-well plate lid and were assayed using a pro-luciferin substrate specific for CYP3A4 (IPA). We demonstrate that S9 was successfully encapsulated and remained enzymatically active post-encapsulation with 5-10X the CYP3A4 activity as compared to 1 µg solubilized human liver S9. Ketoconazole, a known inhibitor of human CYP3A4, inhibited CYP3A4 activity in a concentration-dependent manner (IC50: 0.27 µM) and inhibiti

  10. Enzyme inhibitory activity of selected Philippine plants

    International Nuclear Information System (INIS)

    Sasotona, Joseph S.; Hernandez, Christine C.

    2015-01-01

    In the Philippines, the number one cause of death are cardiovascular diseases. Diseases linked with inflammation are proliferating. This research aims to identify plant extracts that have potential activity of cholesterol-lowering, anti-hypertension, anti-gout, anti-inflammatory and fat blocker agents. Although there are commercially available drugs to treat the aforementioned illnesses, these medicine have adverse side-effects, aside from the fact that they are expensive. The results of this study will serve as added knowledge to contribute to the development of cheaper, more readily available, and effective alternative medicine. 100 plant extracts from different areas in the Philippines have been tested for potential inhibitory activity against Hydroxymethylglutaryl-coenzyme A (HMG-CoA), Lipoxygenase, and Xanthine Oxidase. The plant samples were labeled with codes and distributed to laboratories for blind testing. The effective concentration of the samples tested for Xanthine oxidase is 100 ppm. Samples number 9, 11, 14, 29, 43, 46, and 50 have shown significant inhibitory activity at 78.7%, 78.4%, 70%, 89.2%, 79%, 67.4%, and 67.5% respectively. Samples tested for Lipoxygenase inhibition were set at 33ppm. Samples number 2, 37, 901, 1202, and 1204 have shown significant inhibitory activity at 66, 84.9%, 88.55%, 93.3%, and 84.7% respectively. For HMG-CoA inhibition, the effective concentration of the samples used was 100 ppm. Samples number 1 and 10 showed significant inhibitory activity at 90.1% and 81.8% respectively. (author)

  11. 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.

  12. 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

  13. Contemplation of wheat genotypes for enhanced antioxidant enzyme activity

    International Nuclear Information System (INIS)

    Nasim, S.; Shabbir, G.; Ilyas, M.

    2017-01-01

    Wheat (Triticum aestivum L.) is leading cereal crop in Pakistan but its yield is highly affected due to various abiotic factors especially drought stress, which affects the metabolism of plants. The present study was conducted at Pir Mehr Ali Shah Arid Agriculture University Rawalpindi, using thirty three genotypes during 2011 to investigate the response of anti oxidative enzymes. Seedlings were subjected to stress condition with 30 % PEG 6000 solution along with control (irrigated with water) under in vitro conditions. The experiment was conducted in pots following Complete Randomized Design in Laboratory. Results revealed that under control conditions the maximum values for Guaiacol peroxidase were found in Punjab-96 and Auqab-2000 (2.523), for superoxide in C-273 (0.294), for ascorbate peroxide in PAK-81 (2.523) and for catalase in Kohsar-95 (0.487). Under moisture stress condition the maximum value for Guaiacol peroxidase were recorded for Kohsar-95 (2.699), for superoxide in Kohsar-95 (1.259), for ascorbate peroxide in Pak-81, SA-75, Mexipak-65 and PARI-73 (3.000) and for catalase in Mexipak-65 (0.640). The genotypes which showed higher antioxidant enzyme activity under drought stress have the ability to perform better under adverse soil moisture condition. Such potential genotypes can be utilized in the future breeding programs and also in improving the wheat varieties against drought stress. (author)

  14. DUOX enzyme activity promotes AKT signalling in prostate cancer cells.

    Science.gov (United States)

    Pettigrew, Christopher A; Clerkin, John S; Cotter, Thomas G

    2012-12-01

    Reactive oxygen species (ROS) and oxidative stress are related to tumour progression, and high levels of ROS have been observed in prostate tumours compared to normal prostate. ROS can positively influence AKT signalling and thereby promote cell survival. The aim of this project was to establish whether the ROS generated in prostate cancer cells positively regulate AKT signalling and enable resistance to apoptotic stimuli. In PC3 cells, dual oxidase (DUOX) enzymes actively generate ROS, which inactivate phosphatases, thereby maintaining AKT phosphorylation. Inhibition of DUOX by diphenylene iodium (DPI), intracellular calcium chelation and small-interfering RNA (siRNA) resulted in lower ROS levels, lower AKT and glycogen synthase kinase 3β (GSK3β) phosphorylation, as well as reduced cell viability and increased susceptibility to apoptosis stimulating fragment (FAS) induced apoptosis. This report shows that ROS levels in PC3 cells are constitutively maintained by DUOX enzymes, and these ROS positively regulate AKT signalling through inactivating phosphatases, leading to increased resistance to apoptosis.

  15. 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....

  16. 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

  17. 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

  18. 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...

  19. 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.

  20. 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.

  1. Highly efficient Fenton and enzyme-mimetic activities of NH2-MIL-88B(Fe) metal organic framework for methylene blue degradation.

    Science.gov (United States)

    He, Jianchuan; Zhang, Yao; Zhang, Xiaodan; Huang, Yuming

    2018-03-26

    Here, we show that NH 2 -MIL-88B(Fe) can be used as a peroxidase-like catalyst for Fenton-like degradation of methylene blue (MB) in water. The iron-based NH 2 -MIL-88B(Fe) metal organic framework (MOF) was synthesized by a facile and rapid microwave heating method. It was characterized by scanning electron microscopy, Fourier transform infrared spectroscopy, powder X-ray diffraction, and the Brunauer-Emmett-Teller method. The NH 2 -MIL-88B(Fe) MOF possesses intrinsic oxidase-like and peroxidase-like activities. The reaction parameters that affect MB degradation were investigated, including the solution pH, NH 2 -MIL-88B(Fe) MOF and H 2 O 2 concentrations, and temperature. The results show that the NH 2 -MIL-88B(Fe) MOF exhibits a wide working pH range (pH 3.0-11.0), temperature tolerance, and good recyclability for MB removal. Under the optimal conditions, complete removal of MB was achieved within 45 min. In addition, removal of MB was above 80% after five cycles, showing the good recyclability of NH 2 -MIL-88B(Fe). The NH 2 -MIL-88B(Fe) MOF has the features of easy preparation, high efficiency, and good recyclability for MB removal in a wide pH range. Electron spin resonance and fluorescence probe results suggest the involvement of hydroxyl radicals in MB degradation. These findings provide new insight into the application of high-efficient MOF-based Fenton-like catalysts for water purification.

  2. Antioxidant enzymes activities in obese Tunisian children

    Directory of Open Access Journals (Sweden)

    Sfar Sonia

    2013-01-01

    Full Text Available Abstract Background The oxidant stress, expected to increase in obese adults, has an important role in the pathogenesis of many diseases. It results when free radical formation is greatly increased or protective antioxidant mechanisms are compromised. The main objective of this study is to evaluate the antioxidant response to obesity-related stress in healthy children. Methods A hundred and six healthy children (54 obese and 52 controls, aged 6–12 years old, participated in this study. The collected data included anthropometric measures, blood pressure, fasting glucose, total cholesterol, triglycerides and enzymatic antioxidants (Superoxide dismutase: SOD, Catalase: CAT and Glutathione peroxidase: GPx. Results The first step antioxidant response, estimated by the SOD activity, was significantly higher in obese children compared with normal-weight controls (p  Conclusions The obesity-related increase of the oxidant stress can be observed even in the childhood period. In addition to the complications of an increased BMI, obesity itself can be considered as an independent risk factor of free radical production resulting in an increased antioxidant response.

  3. The pecan nut (Carya illinoinensis) and its oil and polyphenolic fractions differentially modulate lipid metabolism and the antioxidant enzyme activities in rats fed high-fat diets.

    Science.gov (United States)

    Domínguez-Avila, Jesús A; Alvarez-Parrilla, Emilio; López-Díaz, José A; Maldonado-Mendoza, Ignacio E; Gómez-García, María Del Consuelo; de la Rosa, Laura A

    2015-02-01

    Tree nuts such as pecans (Carya illinoinensis) contain mostly oil but are also a source of polyphenols. Nut consumption has been linked to a reduction in serum lipid levels and oxidative stress. These effects have been attributed to the oil while overlooking the potential contribution of the polyphenols. Because the evidence regarding each fraction's bioactivity is scarce, we administered high-fat (HF) diets to male Wistar rats, supplementing them with pecan oil (HF+PO), pecan polyphenols (HF+PP) or whole pecans (HF+WP), and analysed the effects of each fraction. The HF diet increased the serum leptin and total cholesterol (TC) with respect to the control levels. The HF+WP diet prevented hyperleptinemia and decreased the TC compared with the control. The HF+WP diet upregulated the hepatic expression of apolipoprotein B and LDL receptor mRNAs with respect to the HF levels. The HF+PO diet reduced the level of triacylglycerols compared with the control. The HF+PP diet stimulated the hepatic expression of liver X receptor alpha mRNA. The HF+WP diet increased the activities of hepatic catalase, glutathione peroxidase and glutathione S transferase compared with the control, and decreased the degree of lipid peroxidation compared with the HF diet. The most bioactive diet was the WP diet. Copyright © 2014 Elsevier Ltd. All rights reserved.

  4. 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.

  5. Effects of Fertilization on Tomato Growth and Soil Enzyme Activity

    Science.gov (United States)

    Mu, Zhen; Hu, Xue-Feng; Cheng, Chang; Luo, Zhi-qing

    2015-04-01

    66 days after base fertilizer application, but maintained the high level for a long time. In short, the application of organic manure, especially the fermenting manure, is more beneficial to maintain high levels of soil enzyme activities and biodiversity. (2) The tomato yield treated with the CF, CM, FM and CK was 50055 kg/ha, 37814 kg/ha, 36965 kg/ha and 29937 kg/ha, respectively. The yield increasing rates of the CF, CM and FM were 67.2%, 26.3% and 23.5%, respectively. The application of chemical fertilizer could raise the tomato yield more effectively. The use of organic manure, especially the fermenting manure, however, could improve the fruit quality more effectively, especially increase soluble sugar and vitamin C contents and reduce nitrate content in tomato fruit significantly. The application of biological fermenting manure is beneficial to promote the recycling agriculture in China. It could also be used in the organic farming promisingly.

  6. 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.

  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. 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.

  9. 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.

  10. 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.

  11. 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.

  12. High-throughput screening of carbohydrate-degrading enzymes using novel insoluble chromogenic substrate assay kits

    DEFF Research Database (Denmark)

    Schückel, Julia; Kracun, Stjepan Kresimir; Willats, William George Tycho

    2016-01-01

    for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay...... CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used...... for screening the activities of enzymes, enzyme cocktails, and broths....

  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. 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

  15. 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

  16. Enzyme free cloning for high throughput gene cloning and expression

    NARCIS (Netherlands)

    de Jong, R.N.; Daniëls, M.; Kaptein, R.; Folkers, G.E.

    2006-01-01

    Structural and functional genomics initiatives significantly improved cloning methods over the past few years. Although recombinational cloning is highly efficient, its costs urged us to search for an alternative high throughput (HTP) cloning method. We implemented a modified Enzyme Free Cloning

  17. 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....

  18. 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

  19. Enzymic hydrolysis of xylans. I. A high xylanase and beta-xylosidase producing strain of Aspergillus niger

    Energy Technology Data Exchange (ETDEWEB)

    Conrad, D.

    1981-01-01

    Aspergillus niger, strain 110.42 (CBS) was selected as a producer of high xylanolytic activities. The time course of xylanase and beta-xylosidase production as well as the effect of pH and temperature on the activity of these enzymes were studied. High-performance liquid chromatography analysis of the enzymic degradation of arabinoxylan showed a nearly complete conversion to pentose sugars. Aspects of using crude xylanase preparations for enzymic saccharification of xylans are discussed.

  20. 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.

  1. 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 ...

  2. 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.

  3. 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.

  4. 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...

  5. Inhibition of raw starch digestion by one glucoamylase preparation from black Aspergillus at high enzyme concentration

    Energy Technology Data Exchange (ETDEWEB)

    Saka, B C; Veda, S

    1981-09-01

    Raw starch digestion by glucoamylase I (Ab. G-I) preparation from black Aspergillus was inhibited significantly at relatively high concentration of the enzyme. The properties of this enzyme were studied together with those of another glucoamylase I (Nor. G-I), also from black Aspergillus. The two glucoamylases do not differ so much in their physico-chemical properties such as molecular weight, pH and thermal stability, pH and temperature optimum, substrate specificity, debranching activity, isoelectric point etc. The adsorption rate of both enzymes on raw starch increased by the increase of enzyme concentration. The raw starch digestion rate by adsorbed Ab. G-I, however, was decreased with the increase of concentration of enzyme whereas the same was increased in case of Nor. G-I. The inhibitory effect was weaker at 60 deg. Celcius or above. (Refs. 11).

  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. 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.

  8. 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.

  9. 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.

  10. 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

  11. 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.

  12. 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.

  13. 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

    Full Text Available Introduction Corn has been recently used for producing ethanol fuel in the major corn-producing countries such as the US and Brazil. Recent diversion of corn for biofuel production along with the increased world's demand for this feedstuff has resulted in unprecedented rise in feed cost for poultry worldwide. Alternative grains such as wheat and barley can be successfully replaced for corn in poultry diets. These cereal grains can locally grow in many parts of the world as they have remarkably lower water requirement than corn. Wheat and barley are generally used as major sources of energy in poultry diets. Though the major components of these grains are starch and proteins, they have considerable content of non-starch polysaccharides (NSPs, derived from the cell walls (Olukosi et al. 2007; Mirzaie et al. 2012. NSPs are generally considered as anti-nutritional factors (Jamroz et al. 2002. The content and structure of NSP polymers vary between different grains, which consequently affect their nutritive value (Olukosi et al. 2007.Wheat and barley are generally used as major sources of energy in poultry diets. The major components of these grains are starch and proteins, they have considerable content of non-starch polysaccharides (NSPs, derived from the cell walls. NSPs are generally considered as anti-nutritional factors. The content and structure of NSP polymers vary between different grains, which consequently affect their nutritive value. The major part of NSPs in barley comprises polymers of (1→3 (1→4-β- glucans which could impede growth factors and consequently carcass quality through lowering the rate and amount of available nutrients in the mucosal surface of the intestinal. Materials and Methods This experiment was conducted to evaluate the effect of corn and barley based diets supplemented with multi-enzyme on growth, carcass, pancreas enzyme activity and physiological characteristics of broilers. A total number of 375 one day old

  14. 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.

  15. 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.

  16. 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)

  17. 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

  18. 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.

  19. 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

  20. 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.

  1. 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.

  2. Efeito da alta pressão hidrostática na atividade de enzimas da polpa de açaí High hydrostatic pressure effect on enzyme activity of açai pulp

    Directory of Open Access Journals (Sweden)

    Ellen Mayra da Silva Menezes

    2008-12-01

    case, High Hydrostatic Pressure (HHP use can be an alternative to traditional processing techniques for enzyme activation or inactivation. Peroxidases (POD and Polyphenoloxidases (PPO are the main enzymes responsible for undesirable changes in the original characteristics of vegetable products, but using HHP to inactivate them, it is possible to avoid enzymatic browning maintaining sensorial properties. Aiming to evaluate HHP effect on POD and PPO, present in the açaí pulp, the variables pressure, temperature, and time were adopted. Enzyme activities, expressed in percentages, indicate a more stable POD, achieving percentages close to 100% (control. The lowest activities were at 90.74%, and when applied 300 and 500 MPa, 25 °C for 5 minutes treatments, the activities increased (112.34 and 132.98%, respectively. PPO activity rose up to 83.03% in relation to the to control even though inactivation was present in most of the processes (35 °C/5 minutes at 300 and 500 MPa, 53.25, and 53.75%, respectively. Under the experimental conditions, HHP proved effective for the partial inactivation of açaí pulp oxidases.

  3. Engineering of GlcNAc-1-Phosphotransferase for Production of Highly Phosphorylated Lysosomal Enzymes for Enzyme Replacement Therapy.

    Science.gov (United States)

    Liu, Lin; Lee, Wang-Sik; Doray, Balraj; Kornfeld, Stuart

    2017-06-16

    Several lysosomal enzymes currently used for enzyme replacement therapy in patients with lysosomal storage diseases contain very low levels of mannose 6-phosphate, limiting their uptake via mannose 6-phosphate receptors on the surface of the deficient cells. These enzymes are produced at high levels by mammalian cells and depend on endogenous GlcNAc-1-phosphotransferase α/β precursor to phosphorylate the mannose residues on their glycan chains. We show that co-expression of an engineered truncated GlcNAc-1-phosphotransferase α/β precursor and the lysosomal enzyme of interest in the producing cells resulted in markedly increased phosphorylation and cellular uptake of the secreted lysosomal enzyme. This method also results in the production of highly phosphorylated acid β-glucocerebrosidase, a lysosomal enzyme that normally has just trace amounts of this modification.

  4. 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...

  5. Antioxidant enzyme activities, plasma hormone levels and serum metabolites of finishing broiler chickens reared under high ambient temperature and fed lemon and orange peel extracts and Curcuma xanthorrhiza essential oil.

    Science.gov (United States)

    Akbarian, A; Golian, A; Kermanshahi, H; De Smet, S; Michiels, J

    2015-02-01

    The negative effects of high ambient temperature during some months of the year on poultry production have been of great concern in many countries. Dietary modifications are among the most practical ways to alleviate the effects of high temperature. Possible effects of dietary supplementation with 200 or 400 mg/kg feed of lemon peel extract (LPE), orange peel extract (OPE) and Curcuma xanthorrhiza essential oil (CXEO) under hot conditions (34 °C with 50% relative humidity for 5 h daily starting from day 28 until day 38 of age) on blood antioxidant enzyme activities, biochemical parameters and antibody titres of broiler chickens were investigated. All extracts are rich in phenolic compounds and highly available. Compared to control, supplementation with OPE at 400 mg/kg and CXEO significantly increased erythrocyte glutathione peroxidase and superoxide dismutase activity, plasma growth hormone concentrations and serum phosphorus, total protein and chloride concentrations and decreased serum low-density lipoprotein and cholesterol concentrations in chickens at 38 days of age. Regarding antibody titres, CXEO supplementation at 400 mg/kg caused a significant increase in bronchitis antibody titres. Supplementation with LPE and OPE gave more inconsistent results. Most interesting, 400 mg/kg LPE significantly increased 3,5,3'-triiodothyronine and GH concentration as compared to the control. In conclusion, the herbal extracts tested in this study, in particular CXEO at 400 mg/kg, may relieve some of the changes in blood composition induced by increased ambient temperatures. Journal of Animal Physiology and Animal Nutrition © 2014 Blackwell Verlag GmbH.

  6. 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

  7. 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.

  8. 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.

  9. Relationship between Estradiol and Antioxidant Enzymes Activity of Ischemic Stroke

    Directory of Open Access Journals (Sweden)

    Nasrin Sheikh

    2009-01-01

    Full Text Available Some evidence suggests the neuroprotection of estrogen provided by the antioxidant activity of this compound. The main objective of this study was to determine the level of estradiol and its correlation with the activity of antioxidant enzymes, total antioxidant status and ferritin from ischemic stroke subjects. The study population consisted of 30 patients with acute ischemic stroke and 30 controls. There was no significant difference between estradiol in stroke and control group. The activity of superoxide dismutase and level of ferritin was higher in stroke compared with control group (<.05, <.001, resp.. There was no significant correlation between estradiol and glutathione peroxidase, glutathione reductase, catalase, total antioxidant status, and ferritin in stroke and control groups. We observed inverse correlation between estradiol with superoxide dismutase in males of stroke patients (=−0.54, =.029. Our results supported that endogenous estradiol of elderly men and women of stroke or control group has no antioxidant activity.

  10. 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.

  11. 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.

  12. 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...

  13. 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.

  14. 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.

  15. Toward a generalized and high-throughput enzyme screening system based on artificial genetic circuits.

    Science.gov (United States)

    Choi, Su-Lim; Rha, Eugene; Lee, Sang Jun; Kim, Haseong; Kwon, Kilkoang; Jeong, Young-Su; Rhee, Young Ha; Song, Jae Jun; Kim, Hak-Sung; Lee, Seung-Goo

    2014-03-21

    Large-scale screening of enzyme libraries is essential for the development of cost-effective biological processes, which will be indispensable for the production of sustainable biobased chemicals. Here, we introduce a genetic circuit termed the Genetic Enzyme Screening System that is highly useful for high-throughput enzyme screening from diverse microbial metagenomes. The circuit consists of two AND logics. The first AND logic, the two inputs of which are the target enzyme and its substrate, is responsible for the accumulation of a phenol compound in cell. Then, the phenol compound and its inducible transcription factor, whose activation turns on the expression of a reporter gene, interact in the other logic gate. We confirmed that an individual cell harboring this genetic circuit can present approximately a 100-fold higher cellular fluorescence than the negative control and can be easily quantified by flow cytometry depending on the amounts of phenolic derivatives. The high sensitivity of the genetic circuit enables the rapid discovery of novel enzymes from metagenomic libraries, even for genes that show marginal activities in a host system. The crucial feature of this approach is that this single system can be used to screen a variety of enzymes that produce a phenol compound from respective synthetic phenyl-substrates, including cellulase, lipase, alkaline phosphatase, tyrosine phenol-lyase, and methyl parathion hydrolase. Consequently, the highly sensitive and quantitative nature of this genetic circuit along with flow cytometry techniques could provide a widely applicable toolkit for discovering and engineering novel enzymes at a single cell level.

  16. 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.

  17. 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.

  18. 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.

  19. 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)

  20. High-solids enrichment of thermophilic microbial communities and their enzymes on bioenergy feedstocks

    Energy Technology Data Exchange (ETDEWEB)

    Reddy, A. P.; Allgaier, M.; Singer, S.W.; Hazen, T.C.; Simmons, B.A.; Hugenholtz, P.; VanderGheynst, J.S.

    2011-04-01

    Thermophilic microbial communities that are active in a high-solids environment offer great potential for the discovery of industrially relevant enzymes that efficiently deconstruct bioenergy feedstocks. In this study, finished green waste compost was used as an inoculum source to enrich microbial communities and associated enzymes that hydrolyze cellulose and hemicellulose during thermophilic high-solids fermentation of the bioenergy feedstocks switchgrass and corn stover. Methods involving the disruption of enzyme and plant cell wall polysaccharide interactions were developed to recover xylanase and endoglucanase activity from deconstructed solids. Xylanase and endoglucanase activity increased by more than a factor of 5, upon four successive enrichments on switchgrass. Overall, the changes for switchgrass were more pronounced than for corn stover; solids reduction between the first and second enrichments increased by a factor of four for switchgrass while solids reduction remained relatively constant for corn stover. Amplicon pyrosequencing analysis of small-subunit ribosomal RNA genes recovered from enriched samples indicated rapid changes in the microbial communities between the first and second enrichment with the simplified communities achieved by the third enrichment. The results demonstrate a successful approach for enrichment of unique microbial communities and enzymes active in a thermophilic high-solids environment.

  1. 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.

  2. 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

  3. 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.

  4. 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.

  5. 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

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. Enzyme architecture: deconstruction of the enzyme-activating phosphodianion interactions of orotidine 5'-monophosphate decarboxylase.

    Science.gov (United States)

    Goldman, Lawrence M; Amyes, Tina L; Goryanova, Bogdana; Gerlt, John A; Richard, John P

    2014-07-16

    The mechanism for activation of orotidine 5'-monophosphate decarboxylase (OMPDC) by interactions of side chains from Gln215 and Try217 at a gripper loop and R235, adjacent to this loop, with the phosphodianion of OMP was probed by determining the kinetic parameters k(cat) and K(m) for all combinations of single, double, and triple Q215A, Y217F, and R235A mutations. The 12 kcal/mol intrinsic binding energy of the phosphodianion is shown to be equal to the sum of the binding energies of the side chains of R235 (6 kcal/mol), Q215 (2 kcal/mol), Y217 (2 kcal/mol), and hydrogen bonds to the G234 and R235 backbone amides (2 kcal/mol). Analysis of a triple mutant cube shows small (ca. 1 kcal/mol) interactions between phosphodianion gripper side chains, which are consistent with steric crowding of the side chains around the phosphodianion at wild-type OMPDC. These mutations result in the same change in the activation barrier to the OMPDC-catalyzed reactions of the whole substrate OMP and the substrate pieces (1-β-D-erythrofuranosyl)orotic acid (EO) and phosphite dianion. This shows that the transition states for these reactions are stabilized by similar interactions with the protein catalyst. The 12 kcal/mol intrinsic phosphodianion binding energy of OMP is divided between the 8 kcal/mol of binding energy, which is utilized to drive a thermodynamically unfavorable conformational change of the free enzyme, resulting in an increase in (k(cat))(obs) for OMPDC-catalyzed decarboxylation of OMP, and the 4 kcal/mol of binding energy, which is utilized to stabilize the Michaelis complex, resulting in a decrease in (K(m))(obs).

  11. 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...

  12. 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

  13. Versatile de novo enzyme activity in capsid proteins from an engineered M13 bacteriophage library.

    Science.gov (United States)

    Casey, John P; Barbero, Roberto J; Heldman, Nimrod; Belcher, Angela M

    2014-11-26

    Biocatalysis has grown rapidly in recent decades as a solution to the evolving demands of industrial chemical processes. Mounting environmental pressures and shifting supply chains underscore the need for novel chemical activities, while rapid biotechnological progress has greatly increased the utility of enzymatic methods. Enzymes, though capable of high catalytic efficiency and remarkable reaction selectivity, still suffer from relative instability, high costs of scaling, and functional inflexibility. Herein, we developed a biochemical platform for engineering de novo semisynthetic enzymes, functionally modular and widely stable, based on the M13 bacteriophage. The hydrolytic bacteriophage described in this paper catalyzes a range of carboxylic esters, is active from 25 to 80 °C, and demonstrates greater efficiency in DMSO than in water. The platform complements biocatalysts with characteristics of heterogeneous catalysis, yielding high-surface area, thermostable biochemical structures readily adaptable to reactions in myriad solvents. As the viral structure ensures semisynthetic enzymes remain linked to the genetic sequences responsible for catalysis, future work will tailor the biocatalysts to high-demand synthetic processes by evolving new activities, utilizing high-throughput screening technology and harnessing M13's multifunctionality.

  14. 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.

  15. 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.

  16. 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

  17. Expression and Enzyme Activity of Catalase in Chilo suppressalis (Lepidoptera: Crambidae) Is Responsive to Environmental Stresses.

    Science.gov (United States)

    Lu, Yanhui; Bai, Qi; Zheng, Xusong; Lu, Zhongxian

    2017-08-01

    Catalase (CAT) is an important antioxidant enzyme that protects organisms against oxidative stresses by eliminating hydrogen peroxide. In this study, we cloned and characterized a full-length cDNA of CAT from Chilo suppressalis (CsCAT) and examined the influence of environmental stresses on CsCAT expression and enzyme activity. The cDNA contains a 1659-bp open reading frame encoding a polypeptide of 553 amino acids most closely related (90.14%) to Papilio polytes catalases. The CsCAT was expressed in all developmental stages with the highest expression in the fat body, and the CsCAT enzyme activity closely mirrored its observed mRNA expression patterns. The CsCAT mRNA was up-regulated when the larvae were exposed to high temperature (≥30 °C), insecticides (abamectin and chlorantraniliprole), chemicals (H2O2, CHP, CdCl2, and CuSO4), and a dead-end trap plant (vetiver grass), and the CsCAT enzyme activity again mirrored the observed CsCAT expression patterns. These results suggest that up-regulation of CsCAT may enhance the defense response of C. suppressalis by weakening the effects of environmental stresses, and provide insight into the role of CsCAT during development of C. suppressalis. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Angiotensin-converting enzyme activity and cognitive impairment during hypoglycaemia in healthy humans

    DEFF Research Database (Denmark)

    Pedersen-Bjergaard, Ulrik; Thomsen, Carsten E; Høgenhaven, Hans

    2008-01-01

    INTRODUCTION: In type 1 diabetes increased risk of severe hypoglycaemia is associated with high angiotensin-converting enzyme (ACE) activity. We tested in healthy humans the hypothesis that this association is explained by the reduced ability of subjects with high ACE activity to maintain normal...... cognitive function during hypoglycaemia. METHODS: Sixteen healthy volunteers selected by either particularly high or low serum ACE activity were subjected to hypoglycaemia (plasma glucose 2.7 mmol/L). Cognitive function was assessed by choice reaction tests. RESULTS: Despite a similar hypoglycaemic stimulus...... in the two groups, only the group with high ACE activity showed significant deterioration in cognitive performance during hypoglycaemia. In the high ACE group mean reaction time (MRT) in the most complex choice reaction task was prolonged and error rate (ER) was increased in contrast to the low ACE group...

  19. Hierarchically Nanoporous Bioactive Glasses for High Efficiency Immobilization of Enzymes

    DEFF Research Database (Denmark)

    He, W.; Min, D.D.; Zhang, X.D.

    2014-01-01

    Bioactive glasses with hierarchical nanoporosity and structures have been heavily involved in immobilization of enzymes. Because of meticulous design and ingenious hierarchical nanostructuration of porosities from yeast cell biotemplates, hierarchically nanostructured porous bioactive glasses can...... and products of catalytic reactions can freely diffuse through open mesopores (2–40 nm). The formation mechanism of hierarchically structured porous bioactive glasses, the immobilization mechanism of enzyme and the catalysis mechanism of immobilized enzyme are then discussed. The novel nanostructure...

  20. The effect of angiotensin converting enzyme genotype on aerobic capacity following high intensity interval training

    OpenAIRE

    Goddard, N; Baker, M.D; Higgins, T; Cobbold, C

    2014-01-01

    Obesity increases the risk of developing type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD). Physical activity can reduce T2DM and CVD risk, and increase aerobic capacity, a significant predictor of all-cause mortality and morbidity. High intensity interval training (HIIT) produces similar improvements in aerobic capacity to continuous moderate exercise (CME). Different genotypes of angiotensin converting enzyme (ACE) have been implicated in improving aerobic capacity and theref...

  1. 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

  2. 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.

  3. 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.

  4. Physicochemical Properties and Enzymes Activity Studies in a ...

    African Journals Online (AJOL)

    Soil Physicochemical properties and enzyme concentration were evaluated in soil from a refined-oil contaminated community in Isiukwuato, Abia State three years after the spill. The soil enzymes examined were urease, lipase, oxidase, alkaline and acid phosphatases. Results show a significant (P< 0.05) decrease in the ...

  5. 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...

  6. 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...

  7. 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.

  8. 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

  9. 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.

  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. 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

    Rhizosphere and detritusphere are soil microsites with very high resource availability for microorganisms affecting their biomass, composition and functions. In the rhizosphere low molecular compounds occur with root exudates and low available polymeric compounds, as belowground plant senescence. In detritusphere the substrate for decomposition is mainly a polymeric material of low availability. We hypothesized that microorganisms adapted to contrasting quality and availability of substrates in the rhizosphere and detritusphere are strongly different in affinity of hydrolytic enzymes responsible for decomposition of organic compounds. According to common ecological principles easily available substrates are quickly consumed by microorganisms with enzymes of low substrate affinity (i.e. r-strategists). The slow-growing K-strategists with enzymes of high substrate affinity are better adapted for growth on substrates of low availability. Estimation of affinity of enzyme systems to the substrate is based on Michaelis-Menten kinetics, reflecting the dependency of decomposition rates on substrate amount. As enzymes-mediated reactions are substrate-dependent, we further hypothesized that the largest differences in hydrolytic activity between the rhizosphere and detritusphere occur at substrate saturation and that these differences are smoothed with increasing limitation of substrate. Affected by substrate limitation, microbial species follow a certain adaptation strategy. To achieve different depth gradients of substrate availability 12 plots on an agricultural field were established in the north-west of Göttingen, Germany: 1) 4 plots planted with maize, reflecting lower substrate availability with depth; 2) 4 unplanted plots with maize litter input (0.8 kg m-2 dry maize residues), corresponding to detritusphere; 3) 4 bare fallow plots as control. Maize litter was grubbed homogenously into the soil at the first 5 cm to ensure comparable conditions for the herbivore and

  12. 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.

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

    Directory of Open Access Journals (Sweden)

    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

  14. Antitumor properties and modulation of antioxidant enzymes' activity by Aloe vera leaf active principles isolated via supercritical carbon dioxide extraction.

    Science.gov (United States)

    El-Shemy, H A; Aboul-Soud, M A M; Nassr-Allah, A A; Aboul-Enein, K M; Kabash, A; Yagi, A

    2010-01-01

    The aim of this study was to evaluate the potential anticancer properties and modulatory effect of selected Aloe vera (A. vera) active principles on antioxidant enzyme activities. Thus, three anthraquinones (Namely: aloesin, aloe-emodin and barbaloin) were extracted from A. vera leaves by supercritical fluid extraction and subsequently purified by high performance liquid chromatography. Additionally, the N-terminal octapeptide derived from verectin, a biologically active 14 kDa glycoprotein present in A. vera, was also tested. In vivo, active principles exhibited significant prolongation of the life span of tumor-transplanted animals in the following order: barbaloin> octapeptide> aloesin > aloe-emodin. A. vera active principles exhibited significant inhibition on Ehrlich ascite carcinoma cell (EACC) number, when compared to positive control group, in the following order: barbaloin> aloe-emodin > octapeptide > aloesin. Moreover, in trypan blue cell viability assay, active principles showed a significant concentration-dependent cytotoxicity against acute myeloid leukemia (AML) and acute lymphocytes leukemia (ALL) cancerous cells. Furthermore, in MTT cell viability test, aloe-emodin was found to be active against two human colon cancer cell lines (i.e. DLD-1 and HT2), with IC(50) values of 8.94 and 10.78 microM, respectively. Treatments of human AML leukemic cells with active principles (100 microg ml(-1)) resulted in varying intensities of internucleosomal DNA fragmentation, hallmark of cells undergoing apoptosis, in the following order: aloe-emodin> aloesin> barbaloin> octapeptide. Intererstingly, treatment of EACC tumors with active principles resulted in a significant elevation activity of key antioxidant enzymes (SOD, GST, tGPx, and LDH). Our data suggest that the tested A. vera compounds may exert their chemo-preventive effect through modulating antioxidant and detoxification enzyme activity levels, as they are one of the indicators of tumorigenesis. These

  15. 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.

  16. 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.

  17. Response of broiler chickens to diets containing artificially dried high-moisture maize supplemented with microbial enzymes

    OpenAIRE

    Bhuiyan, M.M; Islam, A.F; Iji, P.A

    2010-01-01

    The effect of feeding high-moisture maize grains dried in the sun or artificially in a forced draught oven at 80, 90 or 100 ºC for 24 hours and supplemented with microbial enzymes (Avizyme 1502 and Phyzyme XP) on growth performance, visceral organs, tissue protein, enzyme activity and gut development was investigated in a broiler growth trial. Feed intake (FI) up to 21 days decreased as a results of oven drying of grains whereas supplementation with microbial enzymes increased FI compared to ...

  18. 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.

  19. 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.

  20. 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.

  1. Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content.

    Science.gov (United States)

    Lee, Do Kyung; Jang, Seok; Baek, Eun Hye; Kim, Mi Jin; Lee, Kyung Soon; Shin, Hea Soon; Chung, Myung Jun; Kim, Jin Eung; Lee, Kang Oh; Ha, Nam Joo

    2009-06-11

    Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as lower cholesterol. Although present in many foods, most trials have been in spreads or dairy products. Here we tested whether Bifidobacteria isolates could lower cholesterol, inhibit harmful enzyme activities, and control fecal water content. In vitro culture experiments were performed to evaluate the ability of Bifidobacterium spp. isolated from healthy Koreans (20 approximately 30 years old) to reduce cholesterol-levels in MRS broth containing polyoxyethanylcholesterol sebacate. Animal experiments were performed to investigate the effects on lowering cholesterol, inhibiting harmful enzyme activities, and controlling fecal water content. For animal studies, 0.2 ml of the selected strain cultures (108 approximately 109 CFU/ml) were orally administered to SD rats (fed a high-cholesterol diet) every day for 2 weeks. B. longum SPM1207 reduced serum total cholesterol and LDL levels significantly (p water content, and reduced body weight and harmful intestinal enzyme activities. Daily consumption of B. longum SPM1207 can help in managing mild to moderate hypercholesterolemia, with potential to improve human health by helping to prevent colon cancer and constipation.

  2. Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content

    Directory of Open Access Journals (Sweden)

    Chung Myung

    2009-06-01

    Full Text Available Abstract Background Lactic acid bacteria (LAB are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as lower cholesterol. Although present in many foods, most trials have been in spreads or dairy products. Here we tested whether Bifidobacteria isolates could lower cholesterol, inhibit harmful enzyme activities, and control fecal water content. Methods In vitro culture experiments were performed to evaluate the ability of Bifidobacterium spp. isolated from healthy Koreans (20~30 years old to reduce cholesterol-levels in MRS broth containing polyoxyethanylcholesterol sebacate. Animal experiments were performed to investigate the effects on lowering cholesterol, inhibiting harmful enzyme activities, and controlling fecal water content. For animal studies, 0.2 ml of the selected strain cultures (108~109 CFU/ml were orally administered to SD rats (fed a high-cholesterol diet every day for 2 weeks. Results B. longum SPM1207 reduced serum total cholesterol and LDL levels significantly (p B. longum SPM1207 also increased fecal LAB levels and fecal water content, and reduced body weight and harmful intestinal enzyme activities. Conclusion Daily consumption of B. longum SPM1207 can help in managing mild to moderate hypercholesterolemia, with potential to improve human health by helping to prevent colon cancer and constipation.

  3. Mining lipolytic enzymes in community DNA from high Andean soils using a targeted approach.

    Science.gov (United States)

    Borda-Molina, Daniel; Montaña, José Salvador; Zambrano, María Mercedes; Baena, Sandra

    2017-08-01

    Microbial enrichments cultures are a useful strategy to speed up the search for enzymes that can be employed in industrial processes. Lipases have gained special attention because they show unique properties such as: broad substrate specificity, enantio- and regio-selectivity and stability in organic solvents. A major goal is to identify novel lipolytic enzymes from microorganisms living in cold extreme environments such as high Andean soils, of relevance to our study being their capability be used in industrial processes. Paramo and glacier soils from the Nevados National Park in Colombia were sampled and microbial communities enriched through a fed-batch fermentation using olive oil as an inductor substrate. After 15 days of enrichment under aerobic conditions, total DNA was extracted. Subsequently, metagenomic libraries were constructed in the cosmid vector pWEB-TNC™. After functional screening, twenty and eighteen lipolytic clones were obtained from Paramo and Glacier soil enrichments, respectively. Based on lipid hydrolysis halo dimensions, the clone (Gla1) from a glacier enrichment was selected. A gene related to lipolytic activity was subcloned to evaluate enzyme properties. Phylogenetic analysis of the identified gene showed that the encoded lipase belongs to the family GDSL from a Ralstonia-like species. Interestingly, the secreted enzyme exhibited stability at high temperature and alkaline conditions, specifically the preferred conditions at 80 °C and pH 9.0. Thus, with the identification of an enzyme with non-expected properties, in this study is shown the potential of extreme cold environments to be explored for new catalytic molecules, using current molecular biology techniques, with applications in industrial processes, which demand stability under harsh conditions.

  4. 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.

  5. Effects of microcystins contamination on soil enzyme activities and microbial community in two typical lakeside soils.

    Science.gov (United States)

    Cao, Qing; Steinman, Alan D; Su, Xiaomei; Xie, Liqiang

    2017-12-01

    A 30-day indoor incubation experiment was conducted to investigate the effects of different concentrations of microcystin (1, 10, 100 and 1000 μg eq. MC-LR L -1 ) on soil enzyme activity, soil respiration, physiological profiles, potential nitrification, and microbial abundance (total bacteria, total fungi, ammonia-oxidizing bacteria and archaea) in two lakeside soils in China (Soil A from the lakeside of Lake Poyanghu at Jiujiang; Soil B from the lakeside of Lake Taihu at Suzhou). Of the enzymes tested, only phenol oxidase activity was negatively affected by microcystin application. In contrast, dehydrogenase activity was stimulated in the 1000 μg treatment, and a stimulatory effect also occurred with soil respiration in contaminated soil. The metabolic profiles of the microbial communities indicated that overall carbon metabolic activity in the soils treated with high microcystin concentrations was inhibited, and high concentrations of microcystin also led to different patterns of potential carbon utilization. High microcystin concentrations (100, 1000 μg eq. MC-LR L -1 in Soil A; 10, 100 1000 μg eq. MC-LR L -1 in Soil B) significantly decreased soil potential nitrification rate. Furthermore, the decrease in soil potential nitrification rate was positively correlated with the decrease of the amoA gene abundance, which corresponds to the ammonia-oxidizing bacterial community. We conclude that application of microcystin-enriched irrigation water can significantly impact soil microbial community structure and function. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Evaluation, partial characterization and purification of acetylcholine esterase enzyme and antiangiogenic activity from marine sponges

    Directory of Open Access Journals (Sweden)

    Maushmi Shailesh Kumar

    2014-11-01

    Full Text Available Objective: To test three marine sponges Halichondria glabrata Keller, 1891; Spirastrella pachyspira (S. pachyspira Levi, 1958 and Cliona lobata Hancock, 1849 for the presence of the acetylcholinesterase (AChE in both young and developed samples from western coastal area of India. S. pachyspira methanolic extract was selected for anti/pro angiogenic activity. Methods: They were evaluated for AChE activity using Ellman’s assay based on production of yellow colored 5-thio-2-nitrobenzoate. Purification of the enzyme was planned using ammonium sulphate precipitation and characterization by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Chorioallantoic membrane (ChAM assay model was used for angiogenic/ antiangiogenic testing. Results: All the three sponges showed good specific enzyme activity and S. pachyspira contained maximum specific enzyme activity. Sixty percent of ammonium sulphate precipitation of crude protein sample gave single band at 66 kDa corresponding to the true AChE. ChAM assay was performed at 62.5, 125.0 and 250.0 µg/mL. Dosage beyond 250 µg/mL extract showed toxic response with anti angiogenic activity at all the concentrations. Conclusions: AChE activity was detected in all samples. Extract showed good anti-angiogenic response at 62.5 µg/mL. Extract was highly toxic affecting microvasculature of ChAM as well as normal growth and development of the embryo at 500 µg/mL. With further characterization of bioactive compounds from the extract of S. pachyspira, the compounds can be developed for anti tumor activity.

  7. Enzymes activities involving bacterial cytochromes incorporated in clays

    International Nuclear Information System (INIS)

    Lojou, E.; Giudici-Orticoni, M.Th.; Bianco, P.

    2005-01-01

    With the development of bio electrochemistry, researches appeared on the enzymes immobilization at the surface of electrodes for the realization of bioreactors and bio sensors. One of the main challenges is the development of host matrix able to immobilize the protein material preserving its integrity. In this framework the authors developed graphite electrodes modified by clay films. These electrodes are examined for two enzyme reactions involving proteins of sulfate-reduction bacteria. Then in the framework of the hydrogen biological production and bioreactors for the environmental pollution de-pollution, the electrochemical behavior of the cytochrome c3 in two different clays deposed at the electrode is examined

  8. 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.

  9. 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

  10. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes.

    Science.gov (United States)

    Greenough, Lucia; Schermerhorn, Kelly M; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E; Gardner, Andrew F

    2016-01-29

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3'-5' exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Chemical Composition and Enzymes Inhibitory, Brine Shrimp Larvae Toxicity, Antimicrobial and Antioxidant Activities of Caloplaca biatorina

    Directory of Open Access Journals (Sweden)

    Tahereh Valadbeigi

    2016-10-01

    Full Text Available Background This study evaluated the brine shrimp larvae toxicity and enzymes inhibitory especially anti-diabetic potential of Caloplaca biatorina via in vitro inhibition of α-amylase and α-glucosidase using the methanol extracts. Also aldehyde oxidase and xanthine oxidase enzymes inhibitory, cytotoxicity, and antioxidant activities of the species were determined. Methods In this experimental study, different concentrations of the extracts (0.2, 5.0, 1 and 1.5 mg/mL were incubated with enzyme substrate solution and the percentage of enzyme inhibitory activity and IC50 was calculated. Folin- Ciocalteu reagent and aluminium chloride colorimetric methods were used to estimate total phenolic and flavonoid content of extracts. The toxicity of the extract was assessed using the brine shrimp lethality bioassay. The minimal inhibitory concentration (MIC and minimum bactericidal concentration (MBC were determined. High-performance liquid chromatography and Thin-layer chromatography analysis were evaluated. The data were analyzed by SPSS V.21 software. Results Parietin, Emodin, 1,8-Dihydroxy-3-(hydroxymethyl-6- methoxy-9.10-anthracenedione and Rhein were identified. The extract showed strong α-glucosidase, aldehyde oxidase and xanthine oxidase inhibitory activities with IC50 value of 17.12, 40.09 and 11.02 µg/mL respectively. Also methanol extract displayed the strongest DPPH radical scavenging and brine shrimp toxicity (IC50 = 91.11 properties. Conclusions The result obtained suggests that the C. biatorina extract can be classified as non-toxic. Also, it revealed the antioxidant and antidiabetic potential of the lichen.

  12. Discovering an Accessible Enzyme: Salivary [alpha]-Amylase--"Prima Digestio Fit in Ore"--A Didactic Approach for High School Students

    Science.gov (United States)

    Marini, Isabella

    2005-01-01

    Human salivary [alpha]-amylase is used in this experimental approach to introduce biology high school students to the concept of enzyme activity in a dynamic way. Through a series of five easy, rapid, and inexpensive laboratory experiments students learn what the activity of an enzyme consists of: first in a qualitative then in a semi-quantitative…

  13. 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...

  14. Decrease in Activities of Selected Rat Liver Enzymes following ...

    African Journals Online (AJOL)

    The effects of the chemical effluent from Soap and Detergent Industry on some rat liver enzymes were investigated. Chemical analyses of both the effluent and tap water which served as the control were carried out before various concentrations of the effluent (5%v/v, 25%v/v, 50%v/v and 100%v/v) were made. The effluent ...

  15. Assessment of some Hepatic Enzyme activities in adult rabbits ...

    African Journals Online (AJOL)

    Therapeutic potentials of Garcinia kola (G. kola) have been extensively documented and several researches have asserted its protective uniqueness against liver disorders/diseases. It is the aim of this study to assess the level of some enzyme involved in liver cellular integrity in rabbits chronically fed G. kola. To achieve this ...

  16. 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

  17. The hormetic effect of cadmium on the activity of antioxidant enzymes in the earthworm Eisenia fetida

    International Nuclear Information System (INIS)

    Zhang Yan; Shen Guoqing; Yu Yueshu; Zhu Hongling

    2009-01-01

    The hormetic dose-response relationships induced by environmental toxic agents are often characterized by low-dose stimulation and high-dose inhibition. Confirmation of the general phenomenon of hormesis may have significant implications for ecological risk assessment, although the mechanisms that underlie hormesis remain an enigma. In this study, a model-based approach for describing a dose-response relationship incorporating the hormetic effect was applied to the detection and estimation of the hormetic effect of cadmium (Cd) on the activity of antioxidant enzymes in the earthworm Eisenia fetida. The results showed that Cd at low concentrations induced an increase in the activity of catalase and superoxide dismutase (SOD), but high concentrations inhibited the enzymes, and this was reflected in an inverted U-shaped curve. The maximum hormetic magnitude of SOD activity was higher than that of catalase. The presence of hormesis induced by cadmium in the earthworm may be related to activation of adaptive pathways. - A model-based approach and careful preliminary experiments are needed for detecting and estimating the hormetic effect.

  18. The hormetic effect of cadmium on the activity of antioxidant enzymes in the earthworm Eisenia fetida

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Yan [Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240 (China); Shen Guoqing, E-mail: gqsh@sjtu.edu.c [Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240 (China); Yu Yueshu; Zhu Hongling [Key Laboratory of Urban Agriculture (South), Ministry of Agriculture, School of Agriculture and Biology, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240 (China)

    2009-11-15

    The hormetic dose-response relationships induced by environmental toxic agents are often characterized by low-dose stimulation and high-dose inhibition. Confirmation of the general phenomenon of hormesis may have significant implications for ecological risk assessment, although the mechanisms that underlie hormesis remain an enigma. In this study, a model-based approach for describing a dose-response relationship incorporating the hormetic effect was applied to the detection and estimation of the hormetic effect of cadmium (Cd) on the activity of antioxidant enzymes in the earthworm Eisenia fetida. The results showed that Cd at low concentrations induced an increase in the activity of catalase and superoxide dismutase (SOD), but high concentrations inhibited the enzymes, and this was reflected in an inverted U-shaped curve. The maximum hormetic magnitude of SOD activity was higher than that of catalase. The presence of hormesis induced by cadmium in the earthworm may be related to activation of adaptive pathways. - A model-based approach and careful preliminary experiments are needed for detecting and estimating the hormetic effect.

  19. 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

  20. Effect of high dietary copper on growth, antioxidant and lipid metabolism enzymes of juvenile larger yellow croaker Larimichthys croceus

    Directory of Open Access Journals (Sweden)

    Fanxing Meng

    2016-05-01

    Full Text Available A study was carried out to test the responses of juvenile larger yellow croaker Larimichthys croceus to high Cu intake. Experimental diets were formulated containing three levels of Cu: low Cu (3.67 mg/kg, middle Cu (13.65 mg/kg and high Cu (25.78 mg/kg, and each diet were fed to large yellow croaker in triplicate for 10 weeks. Final body weight, weight gain and feed intake were the lowest in high Cu group, but hepatosomatic index was the highest; Cu concentrations in the whole-body, muscle and liver of fish fed low Cu diet was the lowest; Liver superoxide dismutase, catalase and glutathione peroxidase activities in fish fed high Cu diet were lower than those in fish fed other diets; The higher content of liver thiobarbituric acid reactive substance content was found in high Cu group, followed by middle Cu group, and the lowest in low Cu group; Liver 6-phosphogluconate dehydrogenase, glucose-6-phosphate dehydrogenase, malic enzyme, isocitrate dehydrogenase and fatty acid synthase activities were the lowest in high Cu group, but lipoprotein lipase activity was the highest. This study indicated that high copper intake reduced growth of juvenile larger yellow croaker, inhibited activities of antioxidant enzymes and lipid synthetases, and led to energy mobilization. Keywords: Larger yellow croaker, Copper, Antioxidant enzyme, Lipid metabolism enzyme

  1. 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.

  2. Continuous microwave pasteurization of a vegetable smoothie improves its physical quality and hinders detrimental enzyme activity.

    Science.gov (United States)

    Arjmandi, Mitra; Otón, Mariano; Artés, Francisco; Artés-Hernández, Francisco; Gómez, Perla A; Aguayo, Encarna

    2017-01-01

    The effect of a pasteurization treatment at 90 ± 2 ℃ for 35 s provided by continuous microwave under different doses (low power/long time and high power/short time) or conventional pasteurization on the quality of orange-colored smoothies and their changes throughout 45 days of storage at 5 ℃ was investigated. A better color retention of the microwave pasteurization- treated smoothie using high power/short time than in conventionally processed sample was evidenced by the stability of the hue angle. The continuous microwave heating increased the viscosity of the smoothie more than the conventional pasteurization in comparison with non-treated samples. Lower residual enzyme activities from peroxidase, pectin methylesterase and polygalacturonase were obtained under microwave heating, specifically due to the use of higher power/shorter time. For this kind of smoothie, polygalacturonase was the more thermo-resistant enzyme and could be used as an indicator of pasteurization efficiency. The use of a continuous semi-industrial microwave using higher power and shorter time, such as 1600 W/206 s and 3600 W/93 s, resulted in better quality smoothies and greater enzyme reduction than conventional thermal treatment. © The Author(s) 2016.

  3. Angiotensin converting enzyme 2 activity and human atrial fibrillation: increased plasma angiotensin converting enzyme 2 activity is associated with atrial fibrillation and more advanced left atrial structural remodelling.

    Science.gov (United States)

    Walters, Tomos E; Kalman, Jonathan M; Patel, Sheila K; Mearns, Megan; Velkoska, Elena; Burrell, Louise M

    2017-08-01

    Angiotensin converting enzyme 2 (ACE2) is an integral membrane protein whose main action is to degrade angiotensin II. Plasma ACE2 activity is increased in various cardiovascular diseases. We aimed to determine the relationship between plasma ACE2 activity and human atrial fibrillation (AF), and in particular its relationship to left atrial (LA) structural remodelling. One hundred and three participants from a tertiary arrhythmia centre, including 58 with paroxysmal AF (PAF), 20 with persistent AF (PersAF), and 25 controls, underwent clinical evaluation, echocardiographic analysis, and measurement of plasma ACE2 activity. A subgroup of 20 participants underwent invasive LA electroanatomic mapping. Plasma ACE2 activity levels were increased in AF [control 13.3 (9.5-22.3) pmol/min/mL; PAF 16.9 (9.7-27.3) pmol/min/mL; PersAF 22.8 (13.7-33.4) pmol/min/mL, P = 0.006]. Elevated plasma ACE2 was associated with older age, male gender, hypertension and vascular disease, elevated left ventricular (LV) mass, impaired LV diastolic function and advanced atrial disease (P < 0.05 for all). Independent predictors of elevated plasma ACE2 activity were AF (P = 0.04) and vascular disease (P < 0.01). There was a significant relationship between elevated ACE2 activity and low mean LA bipolar voltage (adjusted R2 = 0.22, P = 0.03), a high proportion of complex fractionated electrograms (R2 = 0.32, P = 0.009) and a long LA activation time (R2 = 0.20, P = 0.04). Plasma ACE2 activity is elevated in human AF. Both AF and vascular disease predict elevated plasma ACE2 activity, and elevated plasma ACE2 is significantly associated with more advanced LA structural remodelling. Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2016. For permissions please email: journals.permissions@oup.com.

  4. 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.

  5. 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.

  6. 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...

  7. Prediction of severe hypoglycaemia by angiotensin-converting enzyme activity and genotype in type 1 diabetes

    DEFF Research Database (Denmark)

    Pedersen-Bjerggaard, U.; Agerholm-Larsen, B.; Pramming, S.

    2003-01-01

    AIMS/HYPOTHESIS: We have previously shown a strong relationship between high angiotensin-converting enzyme (ACE) activity, presence of the deletion (D) allele of the ACEgene and recall of severe hypoglycaemic events in patients with Type 1 diabetes. This study was carried out to assess...... this relationship prospectively. METHODS: We followed 171 adult outpatients with Type 1 diabetes in a one-year observational study with the recording of severe hypoglycaemia. Participants were characterised by serum ACE activity and ACE genotype and not treated with ACE inhibitors or angiotensin II receptor...... antagonists. RESULTS: There was a positive relationship between serum ACE activity and rate of severe hypoglycaemia with a 2.7 times higher rate in the fourth quartile of ACE activity compared to the first quartile (p=0.0007). A similar relationship was observed for the subset of episodes with coma (2.9 times...

  8. Molecular Basis for Enzymatic Sulfite Oxidation -- HOW THREE CONSERVED ACTIVE SITE RESIDUES SHAPE ENZYME ACTIVITY

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, Susan; Rapson, Trevor; Johnson-Winters, Kayunta; Astashkin, Andrei; Enemark, John; Kappler, Ulrike

    2008-11-10

    Sulfite dehydrogenases (SDHs) catalyze the oxidation and detoxification of sulfite to sulfate, a reaction critical to all forms of life. Sulfite-oxidizing enzymes contain three conserved active site amino acids (Arg-55, His-57, and Tyr-236) that are crucial for catalytic competency. Here we have studied the kinetic and structural effects of two novel and one previously reported substitution (R55M, H57A, Y236F) in these residues on SDH catalysis. Both Arg-55 and His-57 were found to have key roles in substrate binding. An R55M substitution increased Km(sulfite)(app) by 2-3 orders of magnitude, whereas His-57 was required for maintaining a high substrate affinity at low pH when the imidazole ring is fully protonated. This effect may be mediated by interactions of His-57 with Arg-55 that stabilize the position of the Arg-55 side chain or, alternatively, may reflect changes in the protonation state of sulfite. Unlike what is seen for SDHWT and SDHY236F, the catalytic turnover rates of SDHR55M and SDHH57A are relatively insensitive to pH (~;;60 and 200 s-1, respectively). On the structural level, striking kinetic effects appeared to correlate with disorder (in SDHH57A and SDHY236F) or absence of Arg-55 (SDHR55M), suggesting that Arg-55 and the hydrogen bonding interactions it engages in are crucial for substrate binding and catalysis. The structure of SDHR55M has sulfate bound at the active site, a fact that coincides with a significant increase in the inhibitory effect of sulfate in SDHR55M. Thus, Arg-55 also appears to be involved in enabling discrimination between the substrate and product in SDH.

  9. 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.

  10. 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.

  11. Altered activity of heme biosynthesis pathway enzymes in individuals chronically exposed to arsenic in Mexico

    Energy Technology Data Exchange (ETDEWEB)

    Hernandez-Zavala, A.; Del Razo, L.M.; Garcia-Vargas, G.G.; Aguilar, C.; Borja, V.H.; Albores, A.; Cebrian, M.E. [CINVESTAV-IPN, Mexico (Mexico). Dept. de Farmacologia y Toxicologica

    1999-03-01

    Our objective was to evaluate the activities of some enzymes of the heme biosynthesis pathway and their relationship with the profile of urinary porphyrin excretion in individuals exposed chronically to arsenic (As) via drinking water in Region Lagunera, Mexico. We selected 17 individuals from each village studied: Benito Juarez, which has current exposure to 0.3 mg As/l; Santa Ana, where individuals have been exposed for more than 35 years to 0.4 mg As/l, but due to changes in the water supply (in 1992) exposure was reduced to its current level (0.1 mg As/l), and Nazareno, with 0.014 mg As/l. Average arsenic concentrations in urine were 2058, 398, and 88 {mu}g As/g creatinine, respectively. The more evident alterations in heme metabolism observed in the highly exposed individuals were: (1) small but significant increases in porphobilinogen deaminase (PBG-D) and uroporphyrinogen decarboxylase (URO-D) activities in peripheral blood erythrocytes; (2) increases in the urinary excretion of total porphyrins, mainly due to coproporphyrin III (COPROIII) and uroporphyrin III (UROIII); and (3) increases in the COPRO/URO and COPROIII/COPROI ratios. No significant changes were observed in uroporphyrinogen III synthetase (UROIII-S) activity. The direct relationships between enzyme activities and urinary porphyrins, suggest that the increased porphyrin excretion was related to PBG-D, whereas the increased URO-D activity would enhance coproporphyrin synthesis and excretion at the expense of uroporphyrin. None of the human studies available have reported the marked porphyric response and enzyme inhibition observed in rodents. In conclusion, chronic As exposure alters human heme metabolism; however the severity of the effects appears to depend on characteristics of exposure not yet fully characterized. (orig.) With 1 fig., 3 tabs., 20 refs.

  12. 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....

  13. 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 .

  14. 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.

  15. Zinc affects differently growth, photosynthesis, antioxidant enzyme activities and phytochelatin synthase expression of four marine diatoms.

    Science.gov (United States)

    Nguyen-Deroche, Thi Le Nhung; Caruso, Aurore; Le, Thi Trung; Bui, Trang Viet; Schoefs, Benoît; Tremblin, Gérard; Morant-Manceau, Annick

    2012-01-01

    Zinc-supplementation (20 μM) effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase), and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa). Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses.

  16. Zinc Affects Differently Growth, Photosynthesis, Antioxidant Enzyme Activities and Phytochelatin Synthase Expression of Four Marine Diatoms

    Directory of Open Access Journals (Sweden)

    Thi Le Nhung Nguyen-Deroche

    2012-01-01

    Full Text Available Zinc-supplementation (20 μM effects on growth, photosynthesis, antioxidant enzyme activities (superoxide dismutase, ascorbate peroxidase, catalase, and the expression of phytochelatin synthase gene were investigated in four marine diatoms (Amphora acutiuscula, Nitzschia palea, Amphora coffeaeformis and Entomoneis paludosa. Zn-supplementation reduced the maximum cell density. A linear relationship was found between the evolution of gross photosynthesis and total chlorophyll content. The Zn treatment decreased the electron transport rate except in A. coffeaeformis and in E. paludosa at high irradiance. A linear relationship was found between the efficiency of light to evolve oxygen and the size of the light-harvesting antenna. The external carbonic anhydrase activity was stimulated in Zn-supplemented E. paludosa but was not correlated with an increase of photosynthesis. The total activity of the antioxidant enzymes did not display any clear increase except in ascorbate peroxidase activity in N. palea. The phytochelatin synthase gene was identified in the four diatoms, but its expression was only revealed in N. palea, without a clear difference between control and Zn-supplemented cells. Among the four species, A. paludosa was the most sensitive and A. coffeaeformis, the most tolerant. A. acutiuscula seemed to be under metal starvation, whereas, to survive, only N. palea developed several stress responses.

  17. 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.

  18. 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.)

  19. 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.)

  20. 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

  1. 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...

  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. Polyphenols isolated from Acacia mearnsii bark with anti-inflammatory and carbolytic enzyme inhibitory activities

    Institute of Scientific and Technical Information of China (English)

    XIONG Jia; GRACE Mary H; ESPOSITO Debora; KOMARNYTSKY Slavko; WANG Fei; LILA Mary Ann

    2017-01-01

    The present study was designed to characterize the polyphenols isolated from Acacia mearnsii bark crude extract (B) and fractions (B1-B7) obtained by high-speed counter-current chromatography (HSCCC) and evaluate their anti-inflammatory and carbolytic enzymes (α-glucosidase and α-amylase) inhibitory activities.Fractions B4,B5,B6,B7 (total phenolics 850.3,983.0,843.9,and 572.5 mg·g-1,respectively;proanthocyanidins 75.7,90.5,95.0,and 44.8 mg·g-1,respectively) showed significant activities against reactive oxygen species (ROS),nitric oxide (NO) production,and expression of pro-inflammatory genes interleukin-lβ (IL-1β) and inducible nitric oxide synthase (iNOS) in a lipopolysaccharide (LPS)-stimulated mouse macrophage cell line RAW 264.7.All the extracts suppressed α-glucosidase and α-amylase activities,two primary enzymes responsible for carbohydrate digestion.A.mearnsii bark samples possessed significantly stronger inhibitory effects against α-glucosidase enzyme (IC50 of 0.4-1.4 tg·mL-1) than the pharmaceutical acarbose (IC50 141.8 μg·mL-1).B6 and B7 (IC5017.6 and 11.7 μg·mL-1,respectively) exhibited α-amylase inhibitory activity as efficacious as acarbose (IC50 15.4 μg·mL-1).Moreover,B extract,at 25 μg·mL-l,significantly decreased the non-mitochondrial oxidative burst that is often associated with inflammatory response in human monocytic macrophages.

  4. Direct Ionic Regulation of the Activity of Myo-Inositol Biosynthesis Enzymes in Mozambique Tilapia.

    Directory of Open Access Journals (Sweden)

    Fernando D Villarreal

    Full Text Available Myo-inositol (Ins is a major compatible osmolyte in many cells, including those of Mozambique tilapia (Oreochromis mossambicus. Ins biosynthesis is highly up-regulated in tilapia and other euryhaline fish exposed to hyperosmotic stress. In this study, enzymatic regulation of two enzymes of Ins biosynthesis, Ins phosphate synthase (MIPS and inositol monophosphatase (IMPase, by direct ionic effects is analyzed. Specific MIPS and IMPase isoforms from Mozambique tilapia (MIPS-160 and IMPase 1 were selected based on experimental, phylogenetic, and structural evidence supporting their role for Ins biosynthesis during hyperosmotic stress. Recombinant tilapia IMPase 1 and MIPS-160 activity was assayed in vitro at ionic conditions that mimic changes in the intracellular milieu during hyperosmotic stress. The in vitro activities of MIPS-160 and IMPase 1 are highest at alkaline pH of 8.8. IMPase 1 catalytic efficiency is strongly increased during hyperosmolality (particularly for the substrate D-Ins-3-phosphate, Ins-3P, mainly as a result of [Na+] elevation. Furthermore, the substrate-specificity of IMPase 1 towards D-Ins-1-phosphate (Ins-1P is lower than towards Ins-3P. Because MIPS catalysis results in Ins-3P this results represents additional evidence for IMPase 1 being the isoform that mediates Ins biosynthesis in tilapia. Our data collectively demonstrate that the Ins biosynthesis enzymes are activated under ionic conditions that cells are exposed to during hypertonicity, resulting in Ins accumulation, which, in turn, results in restoration of intracellular ion homeostasis. We propose that the unique and direct ionic regulation of the activities of Ins biosynthesis enzymes represents an efficient biochemical feedback loop for regulation of intracellular physiological ion homeostasis during hyperosmotic stress.

  5. Direct Ionic Regulation of the Activity of Myo-Inositol Biosynthesis Enzymes in Mozambique Tilapia.

    Science.gov (United States)

    Villarreal, Fernando D; Kültz, Dietmar

    2015-01-01

    Myo-inositol (Ins) is a major compatible osmolyte in many cells, including those of Mozambique tilapia (Oreochromis mossambicus). Ins biosynthesis is highly up-regulated in tilapia and other euryhaline fish exposed to hyperosmotic stress. In this study, enzymatic regulation of two enzymes of Ins biosynthesis, Ins phosphate synthase (MIPS) and inositol monophosphatase (IMPase), by direct ionic effects is analyzed. Specific MIPS and IMPase isoforms from Mozambique tilapia (MIPS-160 and IMPase 1) were selected based on experimental, phylogenetic, and structural evidence supporting their role for Ins biosynthesis during hyperosmotic stress. Recombinant tilapia IMPase 1 and MIPS-160 activity was assayed in vitro at ionic conditions that mimic changes in the intracellular milieu during hyperosmotic stress. The in vitro activities of MIPS-160 and IMPase 1 are highest at alkaline pH of 8.8. IMPase 1 catalytic efficiency is strongly increased during hyperosmolality (particularly for the substrate D-Ins-3-phosphate, Ins-3P), mainly as a result of [Na+] elevation. Furthermore, the substrate-specificity of IMPase 1 towards D-Ins-1-phosphate (Ins-1P) is lower than towards Ins-3P. Because MIPS catalysis results in Ins-3P this results represents additional evidence for IMPase 1 being the isoform that mediates Ins biosynthesis in tilapia. Our data collectively demonstrate that the Ins biosynthesis enzymes are activated under ionic conditions that cells are exposed to during hypertonicity, resulting in Ins accumulation, which, in turn, results in restoration of intracellular ion homeostasis. We propose that the unique and direct ionic regulation of the activities of Ins biosynthesis enzymes represents an efficient biochemical feedback loop for regulation of intracellular physiological ion homeostasis during hyperosmotic stress.

  6. Antioxidant and lipoxygenase activities of polyphenol extracts from oat brans treated with polysaccharide degrading enzymes

    Directory of Open Access Journals (Sweden)

    Nisita Ratnasari

    2017-07-01

    Full Text Available This study used polysaccharide degrading enzymes and protein precipitation to extract polyphenols from oats and to determine their bioactivity. Duplicate oat brans were treated with viscozyme (Vis, cellulase (Cel or no enzyme (control, CTL then, proteins were removed in one set (Vis1, Cel1, CTL1 and not in the other (Vis2, Cel2, CTL2. HPLC analyses showed that for cellulase treated brans, precipitation of proteins increased phenolic acids and avenanthramides by 14%. Meanwhile, a decreased of 67% and 20% respectively was found for viscozyme and control brans. The effect of protein precipitation on soluble polyphenols is therefore dependent of the carbohydrase, as proteins with different compositions will interact differently with other molecules. Radical scavenging data showed that Cel1 and Vis1 had higher quenching effects on ROO• radicals with activities of 22.1 ± 0.8 and 23.5 ± 1.2 μM Trolox Equivalents/g defatted brans. Meanwhile, CTL2 had the highest HO• radicals inhibition (49.4 ± 2.8% compared to 10.8–32.3% for others. Samples that highly inhibited lipoxygenase (LOX, an enzyme involved in lipid oxidation were Cel1 (23.4 ± 2.3% and CTL1 (18 ± 0.4%.

  7. Comparative evaluation of oxidative enzyme activities during adventitious rooting in the cuttings of grapevine rootstocks.

    Science.gov (United States)

    Kose, Cafer; Erdal, Serkan; Kaya, Ozkan; Atici, Okkeş

    2011-03-15

    This study investigated changes in peroxidase (POX) and polyphenol oxidase (PPO) activities through adventitious rooting in hardwood cuttings of grapevine rootstocks. Three grapevine rootstocks with different propensity to produce adventitious roots were selected: recalcitrant (Ramsey), non-recalcitrant (Rupestris du Lot) and intermediate (99R) cultivars. The averages of root number at 65 days were 96 in Lot, 76 in 99R and 30 in Ramsey. Both enzyme activities characteristically increased before adventitious rooting, regardless of rooting ability of the rootstocks, and then decreased. POX activity increased in Ramsey cuttings at 22 days, in Lot and 99R cuttings at 14 days after planting, and then decreased gradually until 51 days. The highest POX activity was determined in Ramsey rootstock with the highest rooting ability and the lowest activity was determined in the rootstocks with the lowest rooting ability. PPO activity gradually increased in Ramsey rootstock cuttings from 10 days to 22 days, in Lot and 99R cuttings at 14 days, and then decreased until 51 days. A significant correlation was identified between high POX activity and adventitious rooting capability in rootstocks, but the same result was not determined with PPO activity. A recalcitrant rooting variety cannot increase POX activity sufficiently before rooting. Therefore applications that could increase POX activity in stem cuttings during rooting may facilitate increased rooting in such rootstocks. Copyright © 2011 Society of Chemical Industry.

  8. 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.

  9. An enzyme-immunobinding assay for fast screening of expression of tissue plasminogen activator cDNA in E. coli

    International Nuclear Information System (INIS)

    Tang, J.C.T.; Li, S.H.

    1984-01-01

    Tissue plasminogen activator (TPA) has been isolated from normal human tissues and certain human cell lines in culture. The enzyme is a serine protease which converts an inactive zymogen, plasminogen to plasmin, and causes lysis of fibrin clots. The high affinity of TPA for fibrin indicates that it is a potential thrombolytic agent and is superior to urokinase-like plasminogen activators. Recently, TPA has been cloned and expressed in E. coli. Using TPA as a model protein, the authors report here the development of a direct, sensitive enzyme-immunoassay for the screening of a cDNA expression library using specific antibodies and peroxidase-labeled second antibody

  10. 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).

  11. 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

  12. Effect of different growth parameters on chitinase enzyme activity ...

    African Journals Online (AJOL)

    Optimization of culture conditions revealed that the enzyme production was maximum in pH 7.5 (107.4 ± 0.50 U/ml), temperature 35°C (103.15 ± 1.74 U/ml) when the carbon and the nitrogen sources used were CMC (106.0 ± 1.89 U/ml) and KNO3 (91.2 ± 1.51 U/ml), respectively. The total chitinase production for all optimum ...

  13. 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)

  14. Bromelain enzyme from pineapple: in vitro activity study under different micropropagation conditions.

    Science.gov (United States)

    Vilanova Neta, Jaci Lima; da Silva Lédo, Ana; Lima, Aloisio André Bonfim; Santana, José Carlos Curvelo; Leite, Nadjma Souza; Ruzene, Denise Santos; Silva, Daniel Pereira; de Souza, Roberto Rodrigues

    2012-09-01

    The aim of this work was to evaluate the activity of bromelain in pineapple plants (Ananas comosus var. Comosus), Pérola cultivar, produced in vitro in different culture conditions. This enzyme, besides its pharmacological effects, is also employed in food industries, such as breweries and meat processing. In this work, the enzymatic activity was evaluated in the tissues of leaves and stems of plants grown in culture medium without plant growth regulator. The most significant levels of bromelain were observed in leaf tissue after 4 months of culture in vitro in medium with a filter paper bridge, followed by medium gelled by the agar. The results of this study, regarding the different structures of the pineapple (leaves and stems) in vitro showed that the activity of bromelain varied depending on the culture conditions, the time and structure of which was quantified, ensuring a viable strategy in the production of seedlings with high levels of bromelain in subsequent phases of micropropagation.

  15. 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.

  16. 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

  17. 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

  18. 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.

  19. Enzyme controlled glucose auto-delivery for high cell density cultivations in microplates and shake flasks

    Directory of Open Access Journals (Sweden)

    Casteleijn Marco G

    2008-11-01

    potential of the system is that the microbial growth rate and oxygen consumption can be simply controlled by the amount (and principally also by the activity of the starch-degrading enzyme. This solves the problems of uncontrolled growth, oxygen limitation, and severe pH drop in shaken cultures. In parallel the method provides the basis for enhanced cell densities. The feasibility of the new method has been shown for 96-well plates and shake flasks and we believe that it can easily be adapted to different microwell and deepwell plate formats and shake flasks. Therefore EnBase will be a helpful tool especially in high throughput applications.

  20. 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

  1. Polychlorinated biphenyl (PCB) induction of CYP3A4 enzyme activity in healthy Faroese adults

    DEFF Research Database (Denmark)

    Petersen, Maria Skaalum; Halling, Jónrit; Damkier, Per

    2007-01-01

    The CYP3A4 enzyme is, along with other cytochrome P450 enzymes, involved in the metabolism of environmental pollutants and is highly inducible by these substances. A commercial polychlorinated biphenyl (PCB) mixture, 1,1,1,-trichloro-2-(o-chlorophenyl), 2-(p'-chlorophenyl)ethane (o,p'-DDT) and 1......,1,-dichloro-2,2-bis (p-chlorophenyl)ethene (p,p'-DDE) are known to induce CYP3A4 activity through activation of nuclear receptors, such as the pregnane X receptor. However, this induction of CYP3A4 has not yet been investigated in humans. Thus, the aim of the study was to determine the variability of the CYP3......A4 phenotype in regard to increased concentrations of PCBs and other persistent organohalogen pollutants (POPs) in healthy Faroese adults. In 310 randomly selected Faroese residents aged 18-60 years, the CYP3A4 activity was determined based on the urinary 6beta-hydroxycortisol/cortisol (6beta...

  2. Effects of isobutyrate supplementation on ruminal microflora, rumen enzyme activities and methane emissions in Simmental steers.

    Science.gov (United States)

    Wang, C; Liu, Q; Zhang, Y L; Pei, C X; Zhang, S L; Wang, Y X; Yang, W Z; Bai, Y S; Shi, Z G; Liu, X N

    2015-02-01

    The objective of this study was to evaluate the effects of isobutyrate supplementation on rumen microflora, enzyme activities and methane emissions in Simmental steers consuming a corn stover-based diet. Eight ruminally cannulated Simmental steers were used in a replicated 4 × 4 Latin square experiment. The treatments were control (without isobutyrate), low isobutyrate (LIB), moderate isobutyrate (MIB) and high isobutyrate (HIB) with 8.4, 16.8 and 25.2 g isobutyrate per steer per day respectively. Isobutyrate was hand-mixed into the concentrate portion. Diet consisted of 60% corn stover and 40% concentrate [dry matter (DM) basis]. Dry matter intake (averaged 9 kg/day) was restricted to a maximum of 90% of ad libitum intake. Population of total bacteria, cellulolytic bacteria and anaerobic fungi were linearly increased, whereas that of protozoa and total methanogens was linearly reduced with increasing isobutyrate supplementation. Real-time PCR quantification of population of Ruminococcus albus, Ruminococcus flavefaciens, Butyrivibrio fibrisolvens and Fibrobacter succinogenes was linearly increased with increasing isobutyrate supplementation. Activities of carboxymethyl cellulase, xylanase and β-glucosidase were linearly increased, whereas that of protease was linearly reduced. Methane production was linearly decreased with increasing isobutyrate supplementation. Effective degradabilities of cellulose and hemicellulose of corn stover were linearly increased, whereas that of crude protein in diet was linearly decreased with increasing isobutyrate supplementation. The present results indicate that isobutyrate supplemented improved microflora, rumen enzyme activities and methane emissions in steers. It was suggested that the isobutyrate stimulated the digestive micro-organisms or enzymes in a dose-dependent manner. In the experimental conditions of this trial, the optimum isobutyrate dose was approximately 16.8 g isobutyrate per steer per day. Journal of Animal

  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. 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.

  5. 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).

  6. 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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. Blood superoxiddismutase and catalase: enzymes activity under oxidative stress conditions

    OpenAIRE

    Каріна Леонідівна Шамелашвілі; Інга Володимирівна Леус; Тетяна Іванівна Сергієнко; Марина Вячеславівна Горіла; Наталія Іванівна Штеменко

    2015-01-01

    The activity of catalase and superoxide dismutase depends not only on the used compounds of rhenium, and also on their dimensional structure and form of applying. It is established that the cis- and trans-isomers of complex compounds of rhenium did countervailing effect on superoxide dismutase and catalase activities. Cis-isomers of Rhenium dycarboxylats agreed increased activity of superoxide dismutase and catalase. While under the action of trans-isomers, where increased activity of superox...

  12. 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.

  13. Recovery of active pathogenesis-related enzymes from the apoplast ...

    African Journals Online (AJOL)

    Overall protease activity intensity was higher in the symplast. Maximum symplast contamination of the apoplast was 2% as estimated by glucose 6-phosphate dehydrogenase activity, a biochemical marker for symplast. Accumulation of pathogenesis-related enzymatic activities in the apoplast of M. acuminata leaf tissue was ...

  14. Blood superoxiddismutase and catalase: enzymes activity under oxidative stress conditions

    Directory of Open Access Journals (Sweden)

    Каріна Леонідівна Шамелашвілі

    2015-05-01

    Full Text Available The activity of catalase and superoxide dismutase depends not only on the used compounds of rhenium, and also on their dimensional structure and form of applying. It is established that the cis- and trans-isomers of complex compounds of rhenium did countervailing effect on superoxide dismutase and catalase activities. Cis-isomers of Rhenium dycarboxylats agreed increased activity of superoxide dismutase and catalase. While under the action of trans-isomers, where increased activity of superoxide dismutase, catalase activity decreased

  15. 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.

  16. 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.

  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. 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.

  19. Structure of a Berberine Bridge Enzyme-Like Enzyme with an Active Site Specific to the Plant Family Brassicaceae

    DEFF Research Database (Denmark)

    Daniel, Bastian; Wallner, Silvia; Steiner, Barbara

    2016-01-01

    Berberine bridge enzyme-like (BBE-like) proteins form a multigene family (pfam 08031), which is present in plants, fungi and bacteria. They adopt the vanillyl alcohol-oxidase fold and predominantly show bi-covalent tethering of the FAD cofactor to a cysteine and histidine residue, respectively....... The Arabidopsis thaliana genome was recently shown to contain genes coding for 28 BBE-like proteins, while featuring four distinct active site compositions. We determined the structure of a member of the AtBBE-like protein family (termed AtBBE-like 28), which has an active site composition that has not been...... be exploited for catalysis. The structure also indicates a shift of the position of the isoalloxazine ring in comparison to other members of the BBE-like family. The dioxygen surrogate chloride was found near the C(4a) position of the isoalloxazine ring in the oxygen pocket, pointing to a rapid reoxidation...

  20. 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

  1. Developmental and hormone-induced changes of mitochondrial electron transport chain enzyme activities during the last instar larval development of maize stem borer, Chilo partellus (Lepidoptera: Crambidae).

    Science.gov (United States)

    VenkatRao, V; Chaitanya, R K; Naresh Kumar, D; Bramhaiah, M; Dutta-Gupta, A

    2016-12-01

    The energy demand for structural remodelling in holometabolous insects is met by cellular mitochondria. Developmental and hormone-induced changes in the mitochondrial respiratory activity during insect metamorphosis are not well documented. The present study investigates activities of enzymes of mitochondrial electron transport chain (ETC) namely, NADH:ubiquinone oxidoreductase or complex I, Succinate: ubiquinone oxidoreductase or complex II, Ubiquinol:ferricytochrome c oxidoreductase or complex III, cytochrome c oxidase or complex IV and F 1 F 0 ATPase (ATPase), during Chilo partellus development. Further, the effect of juvenile hormone (JH) analog, methoprene, and brain and corpora-allata-corpora-cardiaca (CC-CA) homogenates that represent neurohormones, on the ETC enzyme activities was monitored. The enzymatic activities increased from penultimate to last larval stage and thereafter declined during pupal development with an exception of ATPase which showed high enzyme activity during last larval and pupal stages compared to the penultimate stage. JH analog, methoprene differentially modulated ETC enzyme activities. It stimulated complex I and IV enzyme activities, but did not alter the activities of complex II, III and ATPase. On the other hand, brain homogenate declined the ATPase activity while the injected CC-CA homogenate stimulated complex I and IV enzyme activities. Cumulatively, the present study is the first to show that mitochondrial ETC enzyme system is under hormone control, particularly of JH and neurohormones during insect development. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. 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.

  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. Digestive enzyme activity and trophic behavior in two predator aquatic insects (Plecoptera, Perlidae): a comparative study.

    Science.gov (United States)

    López-Rodríguez, M J; Trenzado, C E; Tierno de Figueroa, J M; Sanz, A

    2012-05-01

    Plecoptera (Perlidae) are among the major macroinvertebrate predators in stream ecosystems and one of the insect families with lower tolerance to environmental alterations, being usually employed as bioindicators of high water ecological quality. The differences in the trophic roles of the coexisting species have been exclusively studied from their gut contents, while no data are available on the comparative digestive capacity. In the present paper, we make a comparative study of the activity of several digestive enzymes, namely proteases (at different pH), amylase, lipase, trypsin and chymotrypsin, in two species of stoneflies, Perla bipunctata and Dinocras cephalotes, which cohabit in the same stream. The study of digestive enzyme activity together with the analysis of gut contents can contribute to a better understanding of the ecology of these aquatic insects and their role in freshwater food webs. Thus, our results show that the two studied predator species inhabiting in the same stream present specializations on their feeding behaviors, facilitating their coexistence, and also differences in their capacity of use the resources. One of the main findings of this study is that D. cephalotes is able to assimilate a wider trophic resource spectrum and this could be one of the reasons why this species has a wider global distribution in all its geographical range. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. 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...

  6. Activity of pyrimidine degradation enzymes in normal tissues

    NARCIS (Netherlands)

    van Kuilenburg, A. B. P.; van Lenthe, H.; van Gennip, A. H.

    2006-01-01

    In this study, we measured the activity of dihydropyrimidine dehydrogenase (DPD), dihydropyrimidinase (DHP) and beta-ureidopropionase (beta-UP), using radiolabeled substrates, in 16 different tissues obtained at autopsy from a single patient. The activity of DPD could be detected in all tissues

  7. 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

  8. 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.

  9. A highly sensitive electrochemical glucose sensor structuring with nickel hydroxide and enzyme glucose oxidase

    International Nuclear Information System (INIS)

    Mathew, Manjusha; Sandhyarani, N.

    2013-01-01

    Graphical abstract: A combination of Ni 2+ /Ni 3+ redox couple and glucose oxidase has successfully been exploited for the realization of a highly sensitive glucose sensor for the first time. -- Highlights: • A multilayered glucose biosensor with enhanced sensitivity was fabricated. • Combination of Ni 2+ /Ni 3+ redox couple and glucose oxidase has been exploited for the first time. • Exhibits a lower detection limit of 100 nM with a high sensitivity of 16,840 μA mM −1 cm −2 . • The surface shows a low Michaelis–Menten constant value of 2.4 μM. • Detailed mechanism of sensing was proposed and justified. -- Abstract: A multilayered glucose biosensor with enhanced electron transport was fabricated via the sequential electrodeposition of chitosan gold nanocomposite (CGNC) and nickel hydroxide (Ni(OH) 2 ) on a bare gold electrode and subsequent immobilization of glucose oxidase. A thin film of Ni(OH) 2 deposited on CGNC modified gold electrode serves as an electrochemical redox probe as well as a matrix for the immobilization of glucose oxidase retaining its activity. Electron transport property of CGNC has been exploited to enhance the electron transport between the analyte and electrode. Electrochemical characteristics of the biosensor were studied by cyclic voltammetry and chronoamperometry. Under optimal conditions the biosensor exhibits a linear range from 1 μM to 100 μM with a limit of detection (lod) down to 100 nM. The sensor shows a low Michaelis-Menten constant value of 2.4 μM indicates the high affinity of enzyme to the analyte points to the retained activity of enzyme after immobilization. The present glucose sensor with the high selectivity, sensitivity and stability is promising for practical clinical applications

  10. 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.

  11. DNA topoisomerase II enzyme activity appears in mouse sperm ...

    African Journals Online (AJOL)

    Sperm suspensions of 4 male mice (A, B, C and D), having an initial motility grade of 3.5 were used to examine the presence of DNA topoisomerase II (top 2) activity in sperm heads. The initial percentage motile of male A was 75%, male B was 80%, male C was 70% and male D was 60%. Top 2 activity was examined by ...

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Blood selenium concentrations and enzyme activities related to glutathione metabolism in wild emperor geese

    Science.gov (United States)

    Franson, J. Christian; Hoffman, David J.; Schmutz, Joel A.

    2002-01-01

    In 1998, we collected blood samples from 63 emperor geese (Chen canagica) on their breeding grounds on the Yukon-Kuskokwim Delta (YKD) in western Alaska, USA. We studied the relationship between selenium concentrations in whole blood and the activities of glutathione peroxidase and glutathione reductase in plasma. Experimental studies have shown that plasma activities of these enzymes are useful biomarkers of selenium-induced oxidative stress, but little information is available on their relationship to selenium in the blood of wild birds. Adult female emperor geese incubating their eggs in mid-June had a higher mean concentration of selenium in their blood and a greater activity of glutathione peroxidase in their plasma than adult geese or goslings that were sampled during the adult flight feathermolting period in late July and early August. Glutathione peroxidase activity was positively correlated with the concentration of selenium in the blood of emperor geese, and the rate of increase relative to selenium was greater in goslings than in adults. The activity of glutathione reductase was greatest in the plasma of goslings and was greater in molting adults than incubating females but was not significantly correlated with selenium in the blood of adults or goslings. Incubating female emperor geese had high selenium concentrations in their blood, accompanied by increased glutathione peroxidase activity consistent with early oxidative stress. These findings indicate that further study of the effects of selenium exposure, particularly on reproductive success, is warranted in this species.

  14. Correlation between Pesticide Resistance and Enzyme Activity in the Diamondback Moth, Plutella xylostella

    Science.gov (United States)

    Gong, Ya-Jun; Wang, Ze-Hua; Shi, Bao-Cai; Kang, Zong-Jiang; Zhu, Liang; Jin, Gui-Hua; Weig, Shu-Jun

    2013-01-01

    The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most important pests that has developed high pesticide resistance. The resistances of five Chinese populations of this moth, four resistant strains (from Beijing, Henan, Fujian, and Guangdong) and one susceptible strain, to five pesticides were determined, and the activities of carboxylesterase, glutathione S-transferase, and acetylcholine esterase were tested in all five populations. The correlations between pesticide resistance and enzyme activity were analyzed. The results showed that the resistance status to the five pesticides was different among the five populations. The resistance ratios of the Beijing and Henan populations to spinosad were 5.84 and 8.22, respectively, and those to beta-cypermethrin were 4.91 and 4.98, respectively. These ratios were higher than those for the Fujian and Guangdong populations. The Fujian population was more sensitive to abamectin and chlorpyrifos than the susceptible population (the resistance ratios were 0.14 and 0.91, respectively); in fact, the median lethal concentration for P. xylostella was significantly higher for chlorpyrifos than that for any of the other four pesticides. The carboxylesterase activity in P. xylostella showed positive correlations with the resistance to spinosad, beta-cypermethrin, chlorpyrifos, and abamectin, but no correlation was observed between the carboxylesterase activity and resistance to emamectin benzoate, between glutathione S-transferase activity and resistance to any of the five pesticides tested, or between acetylcholine esterase activity and any of the pesticides except for emamectin benzoate. PMID:24766444

  15. [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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. 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

  1. Angiotensin-converting enzyme in Spodoptera littoralis: molecular characterization, expression and activity profile during development.

    Science.gov (United States)

    Lemeire, Els; Vanholme, Bartel; Van Leeuwen, Thomas; Van Camp, John; Smagghe, Guy

    2008-02-01

    The characterization of the full-length angiotensin-converting enzyme (ACE) cDNA sequence of the lepidopteran Spodoptera littoralis is reported in this study. The predicted open reading frame encodes a 647 amino acids long protein (SlACE) and shows 63.6% identity with the Bombyx mori ACE sequence. A 3D-model, consisting of 26 alpha-helices and three beta-sheets, was predicted for the sequence. SlACE expression was studied in the embryonic, larval and pupal stages of S. littoralis and in different tissues of the last larval stage by reverse-transcribed PCR. This revealed that the gene is expressed throughout the life cycle and especially in brain, gut and fat body tissue of the last stage. These results are in agreement with a role of ACE in the metabolism of neuropeptides and gut hormones. In addition, ACE activity has been studied in more detail during development, making use of a fluorescent assay. High ACE peptidase activity coincides with every transition state, from embryo to larva, from larva to larva and from larva to pupa. A peak value in activity occurs during the early pupal stage. These results indicate the importance of SlACE during metamorphosis and reveal the high correlation of ACE activity with the insect's development, which is regulated by growth and developmental hormones.

  2. 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)

  3. [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.

  4. 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.

  5. Enzyme Activity and Biomolecule Templating at Liquid and Solid Interfaces

    Energy Technology Data Exchange (ETDEWEB)

    Harvey W. Blanch

    2004-12-01

    There are two main components of this research program. The first involves studies of the adsorption and catalytic activity of proteins at fluid-fluid and fluid-solid interfaces; the second employs biological macromolecules as templates at the solid-liquid interface for controlled crystallization of inorganic materials, to provide materials with specific functionality.

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

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-03

    thylbenzothiazoline-6-sulphonic acid (ABTS, Sigma) and. Kalmış et al. 4315 guaiacol (Sigma) oxidizing activity was performed in Petri dishes. (90 mm diameter) with 15 ml of G.A.E. (Glucose Asparagine) agar medium (Dhoulib et al.

  7. Soil Rhizosphere Microbial Communities and Enzyme Activities under Organic Farming

    Science.gov (United States)

    This study investigated the activities of ß-glucosidase (C cycling, ß-glucosaminidase (C and N cycling), acid phosphatase (P cycling) and arylsulfatase (S cycling) under lettuce (Lactuca sativa), potato (Solanum Tuberosum), onion (Allium cepa L), broccoli (Brassica oleracea var. botrytis) and Tall f...

  8. Activities of some enzymes associated with oxygen metablolism ...

    African Journals Online (AJOL)

    STORAGESEVER

    2010-04-26

    Apr 26, 2010 ... SOD and CAT activities in 20 days seedlings were higher than those in ... content and cell permeability in leaves were lower than those in root which in turn .... The water soluble protein content of all crude SOD, CAT and MDA.

  9. Interference of aldehyde metabolizing enzyme with diamine oxidase/histaminase/activity as determined by /sup 14/C putrescine method

    Energy Technology Data Exchange (ETDEWEB)

    Fogel, W A [Polish Academy of Sciences, Cracow (Poland). Inst. of Pharmacology; Bieganski, T; Wozniak, J; Maslinski, C

    1978-01-01

    The ..delta../sup 1/ pyrroline formation, as an indicator of diamine oxidase activity according to Okuyama and Kobayashi /sup 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 ..delta../sup 1/ pyrroline was strongly increased in a dose-dependent manner. Also inhibition of aldehyde reductase by phenobarbital enhanced ..delta../sup 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 ..delta../sup 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.

  10. 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)

  11. 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.

  12. 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.

  13. 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.

  14. High activity waste disposal

    International Nuclear Information System (INIS)

    Gaul, W.C.

    1990-01-01

    Chem-Nuclear Environmental Services (CNES) has developed a container that is capable of containing high activity waste and can be shipped as a regular DOT Type A shipment. By making the container special form the amount of activity that can be transported in a Type A shipment is greatly enhanced. Special form material presents an extra degree of protection to the environment by requiring the package to be destroyed to get access to the radioactive material and must undergo specific testing requirements, whereas normal form material can allow access to the radioactive material. With the special form container up to 10 caries of radium can be transported in a single package. This paper will describe the considerations that were taken to develop these products

  15. 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

  16. 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

  17. Specificity of a prodrug-activating enzyme hVACVase: the leaving group effect.

    Science.gov (United States)

    Sun, Jing; Dahan, Arik; Walls, Zachary F; Lai, Longsheng; Lee, Kyung-Dall; Amidon, Gordon L

    2010-12-06

    Human valacyclovirase (hVACVase) is a prodrug-activating enzyme for amino acid prodrugs including the antiviral drugs valacyclovir and valganciclovir. In hVACVase-catalyzed reactions, the leaving group of the substrate corresponds to the drug moiety of the prodrug, making the leaving group effect essential for the rational design of new prodrugs targeting hVACVase activation. In this study, a series of valine esters, phenylalanine esters, and a valine amide were characterized for the effect of the leaving group on the efficiency of hVACVase-mediated prodrug activation. Except for phenylalanine methyl and ethyl esters, all of the ester substrates exhibited a relatively high specificity constant (k(cat)/K(m)), ranging from 850 to 9490 mM(-1)·s(-1). The valine amide Val-3-APG exhibited significantly higher K(m) and lower k(cat) values compared to the corresponding ester Val-3-HPG, indicating poor specificity for hVACVase. In conclusion, the substrate leaving group has been shown to affect both binding and specific activity of hVACVase-catalyzed activation. It is proposed that hVACVase is an ideal target for α-amino acid ester prodrugs with relatively labile leaving groups while it is relatively inactivate toward amide prodrugs.

  18. 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.

  19. 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.

  20. Effects of pesticide chemicals on the activity of metabolic enzymes: focus on thiocarbamates.

    Science.gov (United States)

    Mathieu, Cécile; Duval, Romain; Xu, Ximing; Rodrigues-Lima, Fernando; Dupret, Jean-Marie

    2015-01-01

    Thiocarbamates are chemicals widely used as pesticides. Occupational exposure is associated with acute intoxication. Populations can be exposed through food and water. Moreover, certain thiocarbamates are used clinically. The widespread use of thiocarbamates raises many issues regarding their toxicological and pharmacological impact. Thiocarbamates and their metabolites can modify biological macromolecules functions, in particular enzymes, through modification of cysteine residues, chelation of metal ions or modulation of the oxidative stress. Loss of enzyme activity can lead to the disruption of metabolic pathways, and explain, at least in part, the effects of these pesticides. Additionally, their reactivity and ability to easily cross biological barrier confer them a great interest for development of clinical applications. Many advances in the study of thiocarbamates metabolism and reactivity have led to a better knowledge of biological effects of these compounds. However, more data are needed on the determination of targets and specificity. Only few data concerning the exposure to a cocktail of pesticides/chemicals are available, raising the need to evaluate the toxic side effects of representative pesticides mixtures. Moreover, the dithiocarbamate Disulfiram has shown great potential in therapeutic applications and leads to the development of pharmacological thiocarbamates derivatives, highly specific to their target and easily distributed.

  1. Comparative analysis of carbohydrate active enzymes in Clostridium termitidis CT1112 reveals complex carbohydrate degradation ability.

    Directory of Open Access Journals (Sweden)

    Riffat I Munir

    Full Text Available Clostridium termitidis strain CT1112 is an anaerobic, gram positive, mesophilic, cellulolytic bacillus isolated from the gut of the wood-feeding termite, Nasutitermes lujae. It produces biofuels such as hydrogen and ethanol from cellulose, cellobiose, xylan, xylose, glucose, and other sugars, and therefore could be used for biofuel production from biomass through consolidated bioprocessing. The first step in the production of biofuel from biomass by microorganisms is the hydrolysis of complex carbohydrates present in biomass. This is achieved through the presence of a repertoire of secreted or complexed carbohydrate active enzymes (CAZymes, sometimes organized in an extracellular organelle called cellulosome. To assess the ability and understand the mechanism of polysaccharide hydrolysis in C. termitidis, the recently sequenced strain CT1112 of C. termitidis was analyzed for both CAZymes and cellulosomal components, and compared to other cellulolytic bacteria. A total of 355 CAZyme sequences were identified in C. termitidis, significantly higher than other Clostridial species. Of these, high numbers of glycoside hydrolases (199 and carbohydrate binding modules (95 were identified. The presence of a variety of CAZymes involved with polysaccharide utilization/degradation ability suggests hydrolysis potential for a wide range of polysaccharides. In addition, dockerin-bearing enzymes, cohesion domains and a cellulosomal gene cluster were identified, indicating the presence of potential cellulosome assembly.

  2. Exponential isothermal amplification of nucleic acids and amplified assays for proteins, cells, and enzyme activities.

    Science.gov (United States)

    Reid, Michael S; Le, X Chris; Zhang, Hongquan

    2018-04-27

    Isothermal exponential amplification techniques, such as strand-displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated isothermal amplification (LAMP), nucleic acid sequence-based amplification (NASBA), helicase-dependent amplification (HDA), and recombinase polymerase amplification (RPA), have great potential for on-site, point-of-care, and in-situ assay applications. These amplification techniques eliminate the need for temperature cycling required for polymerase chain reaction (PCR) while achieving comparable amplification yield. We highlight here recent advances in exponential amplification reaction (EXPAR) for the detection of nucleic acids, proteins, enzyme activities, cells, and metal ions. We discuss design strategies, enzyme reactions, detection techniques, and key features. Incorporation of fluorescence, colorimetric, chemiluminescence, Raman, and electrochemical approaches enables highly sensitive detection of a variety of targets. Remaining issues, such as undesirable background amplification resulting from non-specific template interactions, must be addressed to further improve isothermal and exponential amplification techniques. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. 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.

  4. A High Sensitivity Micro Format Chemiluminescence Enzyme Inhibition Assay for Determination of Hg(II

    Directory of Open Access Journals (Sweden)

    Kanchanmala Deshpande

    2010-06-01

    Full Text Available A highly sensitive and specific enzyme inhibition assay based on alcohol oxidase (AlOx and horseradish peroxidase (HRP for determination of mercury Hg(II in water samples has been presented. This article describes the optimization and miniaturization of an enzymatic assay using a chemiluminescence reaction. The analytical performance and detection limit for determination of Hg(II was optimized in 96 well plates and further extended to 384 well plates with a 10-fold reduction in assay volume. Inhibition of the enzyme activity by dissolved Hg(II was found to be linear in the range 5–500 pg.mL−1 with 3% CVin inter-batch assay. Due to miniaturization of assay in 384 well plates, Hg(II was measurable as low as 1 pg.mL−1 within15 min. About 10-fold more specificity of the developed assay for Hg(II analysis was confirmed by challenging with interfering divalent metal ions such as cadmium Cd(II and lead Pb(II. Using the proposed assay we could successfully demonstrate that in a composite mixture of Hg(II, Cd(II and Pb(II, inhibition by each metal ion is significantly enhanced in the presence of the others. Applicability of the proposed assay for the determination of the Hg(II in spiked drinking and sea water resulted in recoveries ranging from 100–110.52%.

  5. Fabrication of CuO nanoplatelets for highly sensitive enzyme-free determination of glucose

    Energy Technology Data Exchange (ETDEWEB)

    Wang Juan [School of Chemistry and Chemical Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510640 (China); Zhang Weide, E-mail: zhangwd@scut.edu.cn [School of Chemistry and Chemical Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510640 (China)

    2011-09-01

    Highlights: > Adhered growth of CuO nanoplatelets on Cu foils. > Enzyme-free glucose sensor with very high sensitivity. > Excellent stability and good anti-interference ability. - Abstract: CuO nanoplatelets were grown on Cu foils by a one step, template free process. The structure and morphology of the CuO nanoplatelets were characterized by X-ray diffraction, scanning and transmission electron microscopy. The CuO nanoplatelets grown on Cu foil were integrated to be an electrode for glucose sensing. The electrocatalytic activity of the CuO nanoplatelets electrode for glucose in alkaline media was investigated by cyclic voltammetry and chronoamperometry. The electrode exhibits a sensitivity of 3490.7 {mu}A mM{sup -1} cm{sup -2} to glucose which is much higher than that of most reported enzyme-free glucose sensors and the linear range was obtained over a concentration up to 0.80 mM with a detection limit of 0.50 {mu}M (signal/noise = 3). Exhilaratingly, the electrode based on the CuO nanoplatelets is resistant against poisoning by chloride ion, and the interference from the oxidation of common interfering species, such as uric acid, ascorbic acid, dopamine and carbonhydrate compounds, can also be effectively avoided. Finally, the electrode was applied to analyze glucose concentration in human serum samples.

  6. 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)

  7. Non-invasive imaging of tumors by monitoring autotaxin activity using an enzyme-activated near-infrared fluorogenic substrate.

    Directory of Open Access Journals (Sweden)

    Damian Madan

    Full Text Available Autotaxin (ATX, an autocrine motility factor that is highly upregulated in metastatic cancer, is a lysophospholipase D enzyme that produces the lipid second messenger lysophosphatidic acid (LPA from lysophosphatidylcholine (LPC. Dysregulation of the lysolipid signaling pathway is central to the pathophysiology of numerous cancers, idiopathic pulmonary fibrosis, rheumatoid arthritis, and other inflammatory diseases. Consequently, the ATX/LPA pathway has emerged as an important source of biomarkers and therapeutic targets. Herein we describe development and validation of a fluorogenic analog of LPC (AR-2 that enables visualization of ATX activity in vivo. AR-2 exhibits minimal fluorescence until it is activated by ATX, which substantially increases fluorescence in the near-infrared (NIR region, the optimal spectral window for in vivo imaging. In mice with orthotopic ATX-expressing breast cancer tumors, ATX activated AR-2 fluorescence. Administration of AR-2 to tumor-bearing mice showed high fluorescence in the tumor and low fluorescence in most healthy tissues with tumor fluorescence correlated with ATX levels. Pretreatment of mice with an ATX inhibitor selectively decreased fluorescence in the tumor. Together these data suggest that fluorescence directly correlates with ATX activity and its tissue expression. The data show that AR-2 is a non-invasive and selective tool that enables visualization and quantitation of ATX-expressing tumors and monitoring ATX activity in vivo.

  8. Activities of indigenous proteolytic enzymes in caprine milk of different somatic cell counts.

    Science.gov (United States)

    Albenzio, M; Santillo, A; Kelly, A L; Caroprese, M; Marino, R; Sevi, A

    2015-11-01

    Individual caprine milk with different somatic cell counts (SCC) were studied with the aim of investigating the percentage distribution of leukocyte cell types and the activities of indigenous proteolytic enzymes; proteolysis of casein was also studied in relation to cell type following recovery from milk. The experiment was conducted on 5 intensively managed dairy flocks of Garganica goats; on the basis of SCC, the experimental groups were denoted low (L-SCC; 1,501,000 cells/mL) SCC. Leukocyte distribution differed between groups; polymorphonuclear neutrophilic leukocytes were higher in M-SCC and H-SCC milk samples, the percentage macrophages was the highest in H-SCC, and levels of nonviable cells significantly decreased with increasing SCC. Activities of all the main proteolytic enzymes were affected by SCC; plasmin activity was the highest in H-SCC milk and the lowest in L-SCC, and elastase and cathepsin D activities were the highest in M-SCC. Somatic cell count influenced casein hydrolysis patterns, with less intact α- and β-casein in H-SCC milk. Higher levels of low electrophoretic mobility peptides were detected in sodium caseinate incubated with leukocytes isolated from L-SCC milk, independent of cell type, whereas among cells recovered from M-SCC milk, macrophages yielded the highest levels of low electrophoretic mobility peptides from sodium caseinate. The level of high electrophoretic mobility peptides was higher in sodium caseinate incubated with polymorphonuclear neutrophilic leukocytes and macrophages isolated from M-SCC, whereas the same fraction of peptides was always the highest, independent of leukocyte type, for cells recovered from H-SCC milk. In caprine milk, a level of 700,000 cells/mL represented the threshold for changes in leukocyte distribution, which is presumably related to the immune status of the mammary gland. Differences in the profile of indigenous lysosomal proteolytic enzymes in caprine milk may influence the integrity of casein

  9. 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...

  10. Bacterial Glycosyltransferases: Challenges and opportunities of a highly diverse enzyme class toward tailoring natural products

    Directory of Open Access Journals (Sweden)

    Jochen eSchmid

    2016-02-01

    Full Text Available The enzyme subclass of glycosyltransferases (EC 2.4 currently comprises 97 families as specified by CAZy classification. One of their important roles is in the biosynthesis of disaccharides, oligosaccharides and polysaccharides by catalyzing the transfer of sugar moieties from activated donor molecules to other sugar molecules. In addition glycosyltransferases also catalyze the transfer of sugar moieties onto aglycons, which is of great relevance for the synthesis of many high value natural products. Bacterial glycosyltransferases show a higher sequence similarity in comparison to mammalian ones. Even when most glycosyltransferases are poorly explored, state of the art technologies, such as protein engineering, domain swapping or computational analysis strongly enhance our understanding and utilization of these very promising classes of proteins. This perspective article will focus on bacterial glycosyltransferases, especially on classification, screening and engineering strategies to alter substrate specificity. The future development in these fields as well as obstacles and challenges will be highlighted and discussed.

  11. 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...

  12. [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.

  13. Polychlorinated biphenyl (PCB) induction of CYP3A4 enzyme activity in healthy Faroese adults

    International Nuclear Information System (INIS)

    Petersen, Maria Skaalum; Halling, Jonrit; Damkier, Per; Nielsen, Flemming; Grandjean, Philippe; Weihe, Pal; Brosen, Kim

    2007-01-01

    The CYP3A4 enzyme is, along with other cytochrome P450 enzymes, involved in the metabolism of environmental pollutants and is highly inducible by these substances. A commercial polychlorinated biphenyl (PCB) mixture, 1,1,1,-trichloro-2-(o-chlorophenyl), 2-(p'-chlorophenyl)ethane (o,p'-DDT) and 1,1,-dichloro-2,2-bis (p-chlorophenyl)ethene (p,p'-DDE) are known to induce CYP3A4 activity through activation of nuclear receptors, such as the pregnane X receptor. However, this induction of CYP3A4 has not yet been investigated in humans. Thus, the aim of the study was to determine the variability of the CYP3A4 phenotype in regard to increased concentrations of PCBs and other persistent organohalogen pollutants (POPs) in healthy Faroese adults. In 310 randomly selected Faroese residents aged 18-60 years, the CYP3A4 activity was determined based on the urinary 6β-hydroxycortisol/cortisol (6β-OHC/FC) ratio. POP exposures were assessed by measuring their concentrations in serum lipid. The results showed a unimodal distribution of the 6β-OHC/FC ratio with values ranging from 0.58 to 27.38. Women had a slightly higher 6β-OHC/FC ratio than men (p = 0.07). Confounder-adjusted multiple regression analysis showed significant associations between 6β-OHC/FC ratios and ΣPCB, PCB-TEQ and p,p'-DDE, o,p'-DDT and HCB, respectively, but the associations were statistically significant for men only

  14. Contrasting effects of biochar versus manure on soil microbial communities and enzyme activities in an Aridisol.

    Science.gov (United States)

    Elzobair, Khalid A; Stromberger, Mary E; Ippolito, James A; Lentz, Rodrick D

    2016-01-01

    Biochar can increase microbial activity, alter microbial community structure, and increase soil fertility in arid and semi-arid soils, but at relatively high rates that may be impractical for large-scale field studies. This contrasts with organic amendments such as manure, which can be abundant and inexpensive if locally available, and thus can be applied to fields at greater rates than biochar. In a field study comparing biochar and manure, a fast pyrolysis hardwood biochar (22.4 Mg ha(-1)), dairy manure (42 Mg ha(-1) dry wt), a combination of biochar and manure at the aforementioned rates, or no amendment (control) was applied to an Aridisol (n=3) in fall 2008. Plots were annually cropped to corn (Zea maize L.). Surface soils (0-30 cm) were sampled directly under corn plants in late June 2009 and early August 2012, and assayed for microbial community fatty acid methyl ester (FAME) profiles and six extracellular enzyme activities involved in soil C, N, and P cycling. Arbuscular mycorrhizal (AM) fungal colonization was assayed in corn roots in 2012. Biochar had no effect on microbial biomass, community structure, extracellular enzyme activities, or AM fungi root colonization of corn. In the short-term, manure amendment increased microbial biomass, altered microbial community structure, and significantly reduced the relative concentration of the AM fungal biomass in soil. Manure also reduced the percent root colonization of corn by AM fungi in the longer-term. Thus, biochar and manure had contrasting short-term effects on soil microbial communities, perhaps because of the relatively low application rate of biochar. Copyright © 2015 Elsevier Ltd. All rights reserved.

  15. Process for preparing multilayer enzyme coating on a fiber

    Science.gov (United States)

    Kim, Jungbae [Richland, WA; Kwak, Ja Hun [Richland, WA; Grate, Jay W [West Richland, WA

    2009-11-03

    A process for preparing high stability, high activity biocatalytic materials is disclosed and processes for using the same. The process involves coating of a material or fiber with enzymes and enzyme aggregate providing a material or fiber with high biocatalytic activity and stability useful in heterogeneous environments. In one illustrative approach, enzyme "seeds" are covalently attached to polymer nanofibers followed by treatment with a reagent that crosslinks additional enzyme molecules to the seed enzymes forming enzyme aggregates thereby improving biocatalytic activity due to increased enzyme loading and enzyme stability. This approach creates a useful new biocatalytic immobilized enzyme system with potential applications in bioconversion, bioremediation, biosensors, and biofuel cells.

  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. 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)

  18. Flow-Based Systems for Rapid and High-Precision Enzyme Kinetics Studies

    Directory of Open Access Journals (Sweden)

    Supaporn Kradtap Hartwell

    2012-01-01

    Full Text Available Enzyme kinetics studies normally focus on the initial rate of enzymatic reaction. However, the manual operation of steps of the conventional enzyme kinetics method has some drawbacks. Errors can result from the imprecise time control and time necessary for manual changing the reaction cuvettes into and out of the detector. By using the automatic flow-based analytical systems, enzyme kinetics studies can be carried out at real-time initial rate avoiding the potential errors inherent in manual operation. Flow-based systems have been developed to provide rapid, low-volume, and high-precision analyses that effectively replace the many tedious and high volume requirements of conventional wet chemistry analyses. This article presents various arrangements of flow-based techniques and their potential use in future enzyme kinetics applications.

  19. Alkylation of amide linkages and cleavage of the C chain in the enzyme-activated-substrate inhibition of alpha-chymotrypsin with N-nitrosamides

    International Nuclear Information System (INIS)

    Donadio, S.; Perks, H.M.; Tsuchiya, K.; White, E.H.

    1985-01-01

    Active-site-directed N-nitrosamides inhibit alpha-chymotrypsin through an enzyme-activated-substrate mechanism. In this work, the activation results in the release--in the active site--of benzyl carbonium ions, which alkylate and inhibit the enzyme. The final ratio of benzyl groups to enzyme molecules is 1.0, but the alkyl groups are scattered over a number of sites. Reduction and alkylation of the inhibited enzyme generate peptides insoluble in most media. Guanidine hydrochloride at 6 M proved a good solvent, and its use as an eluant on G-75 Sephadex permitted separation of the peptides. In the case of 14 C-labeled enzyme, such an approach has shown that all of the alkylation occurs on the C chain of the enzyme, the chain of which the active site is constructed. Chemical modification of the peptides with ethylenediamine and N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide rendered them soluble in dilute acid, permitting high-performance liquid chromatographic separation. Model studies have shown that the benzyl carbonium ions are highly reactive, alkylating amide linkages at both oxygen and nitrogen. Chromatography of this mixture and also 13 C NMR spectroscopy of the intact inhibited enzyme have shown that three major N-alkylations have occurred. Tryptic digestion of the C chain of chymotrypsin, which contains all of the alkylation sites, provides evidence that the stable N sites are principally located between residue 216 and residue 230

  20. Effect of long-term industrial waste effluent pollution on soil enzyme activities and bacterial community composition.

    Science.gov (United States)

    Subrahmanyam, Gangavarapu; Shen, Ju-Pei; Liu, Yu-Rong; Archana, Gattupalli; Zhang, Li-Mei

    2016-02-01

    Although numerous studies have addressed the influence of exogenous pollutants on microorganisms, the effect of long-term industrial waste effluent (IWE) pollution on the activity and diversity of soil bacteria was still unclear. Three soil samples characterized as uncontaminated (R1), moderately contaminated (R2), and highly contaminated (R3) receiving mixed organic and heavy metal pollutants for more than 20 years through IWE were collected along the Mahi River basin, Gujarat, western India. Basal soil respiration and in situ enzyme activities indicated an apparent deleterious effect of IWE on microbial activity and soil function. Community composition profiling of soil bacteria using 16S rRNA gene amplification and denaturing gradient gel electrophoresis (DGGE) method indicated an apparent bacterial community shift in the IWE-affected soils. Cloning and sequencing of DGGE bands revealed that the dominated bacterial phyla in polluted soil were affiliated with Firmicutes, Acidobacteria, and Actinobacteria, indicating that these bacterial phyla may have a high tolerance to pollutants. We suggested that specific bacterial phyla along with soil enzyme activities could be used as relevant biological indicators for long-term pollution assessment on soil quality. Graphical Abstract Bacterial community profiling and soil enzyme activities in long-term industrial waste effluent polluted soils.

  1. An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

    International Nuclear Information System (INIS)

    Ruan, Guihua; Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui; Du, Fuyou

    2016-01-01

    A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N_α-benzoyl-L-arginine ethyl ester to N_α-benzoyl-L-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. - Graphical abstract: Schematic illustration of preparation of hypercrosslinking polyHIPE immobilized enzyme reactor for on-column protein digestion. - Highlights: • A reactor was prepared and used for enzyme immobilization and continuous on-column protein digestion. • The new polyHIPE IMER was quite suit for protein digestion with good properties. • On-column digestion revealed that the IMER was easy regenerated by HCl without any structure destruction.

  2. An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

    Energy Technology Data Exchange (ETDEWEB)

    Ruan, Guihua, E-mail: guihuaruan@hotmail.com [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004 (China); Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Du, Fuyou, E-mail: dufu2005@126.com [Guangxi Key Laboratory of Electrochemical and Magnetochemical Functional Materials, College of Chemistry and Bioengineering, Guilin University of Technology, Guangxi 541004 (China); Guangxi Collaborative Innovation Center for Water Pollution Control and Water Safety in Karst Area, Guilin University of Technology, Guilin 541004 (China)

    2016-04-22

    A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of N{sub α}-benzoyl-L-arginine ethyl ester to N{sub α}-benzoyl-L-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. - Graphical abstract: Schematic illustration of preparation of hypercrosslinking polyHIPE immobilized enzyme reactor for on-column protein digestion. - Highlights: • A reactor was prepared and used for enzyme immobilization and continuous on-column protein digestion. • The new polyHIPE IMER was quite suit for protein digestion with good properties. • On-column digestion revealed that the IMER was easy regenerated by HCl without any structure destruction.

  3. 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.

  4. 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.

  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. Several genes encoding enzymes with the same activity are necessary for aerobic fungal degradation of cellulose in nature.

    Directory of Open Access Journals (Sweden)

    Peter K Busk

    Full Text Available The cellulose-degrading fungal enzymes are glycoside hydrolases of the GH families and lytic polysaccharide monooxygenases. The entanglement of glycoside hydrolase families and functions makes it difficult to predict the enzymatic activity of glycoside hydrolases based on their sequence. In the present study we further developed the method Peptide Pattern Recognition to an automatic approach not only to find all genes encoding glycoside hydrolases and lytic polysaccharide monooxygenases in fungal genomes but also to predict the function of the genes. The functional annotation is an important feature as it provides a direct route to predict function from primary sequence. Furthermore, we used Peptide Pattern Recognition to compare the cellulose-degrading enzyme activities encoded by 39 fungal genomes. The results indicated that cellobiohydrolases and AA9 lytic polysaccharide monooxygenases are hallmarks of cellulose-degrading fungi except brown rot fungi. Furthermore, a high number of AA9, endocellulase and β-glucosidase genes were identified, not in what are known to be the strongest, specialized lignocellulose degraders but in saprophytic fungi that can use a wide variety of substrates whereas only few of these genes were found in fungi that have a limited number of natural, lignocellulotic substrates. This correlation suggests that enzymes with different properties are necessary for degradation of cellulose in different complex substrates. Interestingly, clustering of the fungi based on their predicted enzymes indicated that Ascomycota and Basidiomycota use the same enzymatic activities to degrade plant cell walls.

  7. Co-ordinate regulation of sterol biosynthesis enzyme activity during accumulation of sterols in developing rape and tobacco seed.

    Science.gov (United States)

    Harker, Mark; Hellyer, Amanda; Clayton, John C; Duvoix, Annelyse; Lanot, Alexandra; Safford, Richard

    2003-02-01

    The activities of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, sterol methyl transferase 1 and sterol acyltransferase, key enzymes involved in phytosterol biosynthesis were shown to be co-ordinately regulated during oilseed rape ( Brassica napus L.) and tobacco ( Nicotiana tabacum L.) seed development. In both plants, enzyme activities were low during the initial stages of seed development, increasing towards mid-maturation where they remained stable for a time, before declining rapidly as the oilseeds reached maturity. During seed development, the level of total sterols increased 12-fold in tobacco and 9-fold in rape, primarily due to an increase in steryl ester production. In both seed tissues, stages of maximum enzyme activity coincided with periods of high rates of sterol production, indicating developmental regulation of the enzymes to be responsible for the increases in the sterol content observed during seed development. Consistent with previous studies the data presented suggest that sterol biosynthesis is regulated by two key steps, although there may be others. The first is the regulation of carbon flux into the isoprenoid pathway to cycloartenol. The second is the flux from cycloartenol to Delta(5)-end-product sterols. The implications of the results in terms of enhancing seed sterol levels by genetic modification are also discussed.

  8. Screening of SDS-degrading bacteria from car wash wastewater and study of the alkylsulfatase enzyme activity.

    Science.gov (United States)

    Shahbazi, Razieh; Kasra-Kermanshahi, Roha; Gharavi, Sara; Moosavi-Nejad, Zahra; Borzooee, Faezeh

    2013-06-01

    Sodium dodecyl sulfate (SDS) is one of the main surfactant components in detergents and cosmetics, used in high amounts as a detergent in products such as shampoos, car wash soap and toothpaste. Therefore, its bioremediation by suitable microorganisms is important. Alkylsulfatase is an enzyme that hydrolyses sulfate -ester bonds to give inorganic sulfate and alcohol. The purpose of this study was to isolate SDS-degrading bacteria from Tehran city car wash wastewater, study bacterial alkylsulfatase enzyme activity and identify the alkylsulfatase enzyme coding gene. Screening of SDS-degrading bacteria was carried out on basal salt medium containing SDS as the sole source of carbon. Amount of SDS degraded was assayed by methylene blue active substance (MBAS). Identification of the sdsA gene was carried by PCR and subsequent sequencing of the 16S rDNA gene and biochemical tests identified Pseudomonas aeruginosa. This bacterium is able to degrade 84% of SDS after four days incubation. Bacteria isolated from car wash wastewater were shown to carry the sdsA gene (670bp) and the alkylsulfatase enzyme specific activity expressed from this gene was determined to be 24.3 unit/mg. The results presented in this research indicate that Pseudomonas aeruginosa is a suitable candidate for SDS biodegradation.

  9. Impact of azadirachtin, an insecticidal allelochemical from neem on soil microflora, enzyme and respiratory activities.

    Science.gov (United States)

    Gopal, Murali; Gupta, Alka; Arunachalam, V; Magu, S P

    2007-11-01

    The effect of 10% azadirachtin granules (alcoholic extract of neem seed kernel mixed with China clay) was studied on the population of bacteria, actinomycetes, fungi, Azotobacter and nitrifying bacteria; soil dehydrogenase, phosphatase and respiratory activities on 0, 15th, 30th, 60th and 90th days after application in sandy loam soil collected from the fields. It was observed that baring the Azotobacter sp., azadirachtin at all the doses exerted a suppressive effect on the rest of the microbial communities and enzyme activities in the initial 15 day period. The population of bacteria, actinomycetes besides phosphatase and respiratory activities recovered after 60th day and subsequently increased significantly. The fungi and nitrifiers were most sensitive groups as their numbers were reduced significantly throughout the studies. The two times and five times recommended dose of azadirachtin had very high biocidal effects on the soil microorganisms and its activities. However, analysis of the data by the Shannon Weaver index showed that azadirachtin reduces both the form and functional microbial diversity at all doses.

  10. Discovery of Microorganisms and Enzymes Involved in High-Solids Decomposition of Rice Straw Using Metagenomic Analyses

    Science.gov (United States)

    D’haeseleer, Patrik; Khudyakov, Jane; Burd, Helcio; Hadi, Masood; Simmons, Blake A.; Singer, Steven W.; Thelen, Michael P.; VanderGheynst, Jean S.

    2013-01-01

    High-solids incubations were performed to enrich for microbial communities and enzymes that decompose rice straw under mesophilic (35°C) and thermophilic (55°C) conditions. Thermophilic enrichments yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome sequencing was performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Proteobacteria were found to dominate the mesophilic community while Actinobacteria were most abundant in the thermophilic community. Analysis of protein family representation in each metagenome indicated that cellobiohydrolases containing carbohydrate binding module 2 (CBM2) were significantly overrepresented in the thermophilic community. Micromonospora, a member of Actinobacteria, primarily housed these genes in the thermophilic community. In light of these findings, Micromonospora and other closely related Actinobacteria genera appear to be promising sources of thermophilic lignocellulolytic enzymes for rice straw deconstruction under high-solids conditions. Furthermore, these discoveries warrant future research to determine if exoglucanases with CBM2 represent thermostable enzymes tolerant to the process conditions expected to be encountered during industrial biofuel production. PMID:24205054

  11. Discovery of microorganisms and enzymes involved in high-solids decomposition of rice straw using metagenomic analyses.

    Directory of Open Access Journals (Sweden)

    Amitha P Reddy

    Full Text Available High-solids incubations were performed to enrich for microbial communities and enzymes that decompose rice straw under mesophilic (35°C and thermophilic (55°C conditions. Thermophilic enrichments yielded a community that was 7.5 times more metabolically active on rice straw than mesophilic enrichments. Extracted xylanase and endoglucanse activities were also 2.6 and 13.4 times greater, respectively, for thermophilic enrichments. Metagenome sequencing was performed on enriched communities to determine community composition and mine for genes encoding lignocellulolytic enzymes. Proteobacteria were found to dominate the mesophilic community while Actinobacteria were most abundant in the thermophilic community. Analysis of protein family representation in each metagenome indicated that cellobiohydrolases containing carbohydrate binding module 2 (CBM2 were significantly overrepresented in the thermophilic community. Micromonospora, a member of Actinobacteria, primarily housed these genes in the thermophilic community. In light of these findings, Micromonospora and other closely related Actinobacteria genera appear to be promising sources of thermophilic lignocellulolytic enzymes for rice straw deconstruction under high-solids conditions. Furthermore, these discoveries warrant future research to determine if exoglucanases with CBM2 represent thermostable enzymes tolerant to the process conditions expected to be encountered during industrial biofuel production.

  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. Characterization of angiotensin-converting enzyme inhibitory activity of fermented milk produced by Lactobacillus helveticus.

    Science.gov (United States)

    Chen, Yongfu; Li, Changkun; Xue, Jiangang; Kwok, Lai-yu; Yang, Jie; Zhang, Heping; Menghe, Bilige

    2015-08-01

    Hypertension affects up to 30% of the adult population in most countries. It is a known risk factor for cardiovascular diseases, including coronary heart disease, peripheral artery disease, and stroke. Owing to the increased health awareness of consumers, the application of angiotensin-converting enzyme (ACE)-inhibitory peptides produced by Lactobacillushelveticus to prevent or control high blood pressure has drawn wide attention. A total of 59 L. helveticus strains were isolated from traditional fermented dairy products and the ACE-inhibitory activity of the fermented milks produced with the isolated microorganisms was assayed. The ACE-inhibitory activity of 38 L. helveticus strains was more than 50%, and 3 strains (IMAU80872, IMAU80852, and IMAU80851) expressing the highest ACE-inhibitory activity were selected for further studies. Particularly, the gastrointestinal protease tolerance and thermostability of the ACE-inhibitory activity in the fermented milks were assessed. Based on these 2 criteria, IMAU80872 was found to be superior over the other 2 strains. Furthermore, IMAU80872 exhibited a high in vitro ACE-inhibitory activity at the following fermentation conditions: fermentation temperature at 40°C, inoculation concentration of 1×10(6) cfu/mL, and fermentation for 18h. Finally, by using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry analysis, we observed changes of the metabolome along the milk fermentation process of IMAU80872. Furthermore, 6 peptides were identified, which might have ACE-inhibitory activity. In conclusion, we identified a novel ACE-inhibitory L. helveticus strain suitable for the production of fermented milk or other functional dairy products. Copyright © 2015 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  14. 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.

  15. An investigation of the mimetic enzyme activity of two-dimensional Pd-based nanostructures

    Science.gov (United States)

    Wei, Jingping; Chen, Xiaolan; Shi, Saige; Mo, Shiguang; Zheng, Nanfeng

    2015-11-01

    In this work, we investigated the mimetic enzyme activity of two-dimensional (2D) Pd-based nanostructures (e.g. Pd nanosheets, Pd@Au and Pd@Pt nanoplates) and found that they possess intrinsic peroxidase-, oxidase- and catalase-like activities. These nanostructures were able to activate hydrogen peroxide or dissolved oxygen for catalyzing the oxidation of organic substrates, and decompose hydrogen peroxide to generate oxygen. More systematic investigations revealed that the peroxidase-like activities of these Pd-based nanomaterials were highly structure- and composition-dependent. Among them, Pd@Pt nanoplates displayed the highest peroxidase-like activity. Based on these findings, Pd-based nanostructures were applied for the colorimetric detection of H2O2 and glucose, and also the electro-catalytic reduction of H2O2. This work offers a promising prospect for the application of 2D noble metal nanostructures in biocatalysis.In this work, we investigated the mimetic enzyme activity of two-dimensional (2D) Pd-based nanostructures (e.g. Pd nanosheets, Pd@Au and Pd@Pt nanoplates) and found that they possess intrinsic peroxidase-, oxidase- and catalase-like activities. These nanostructures were able to activate hydrogen peroxide or dissolved oxygen for catalyzing the oxidation of organic substrates, and decompose hydrogen peroxide to generate oxygen. More systematic investigations revealed that the peroxidase-like activities of these Pd-based nanomaterials were highly structure- and composition-dependent. Among them, Pd@Pt nanoplates displayed the highest peroxidase-like activity. Based on these findings, Pd-based nanostructures were applied for the colorimetric detection of H2O2 and glucose, and also the electro-catalytic reduction of H2O2. This work offers a promising prospect for the application of 2D noble metal nanostructures in biocatalysis. Electronic supplementary information (ESI) available: TEM images, EDX and dispersion stability of Pd-based nanomaterials

  16. 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.

  17. 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.

  18. Modelling the Effects of Ageing Time of Starch on the Enzymatic Activity of Three Amylolytic Enzymes

    Science.gov (United States)

    Guerra, Nelson P.; Pastrana Castro, Lorenzo

    2012-01-01

    The effect of increasing ageing time (t) of starch on the activity of three amylolytic enzymes (Termamyl, San Super, and BAN) was investigated. Although all the enzymatic reactions follow michaelian kinetics, v max decreased significantly (P enzymes and the release of the reaction products to the medium. A similar effect was observed when the enzymatic reactions were carried out with unaged starches supplemented with different concentrations of gelatine [G]. The inhibition in the amylolytic activities was best mathematically described by using three modified forms of the Michaelis-Menten model, which included a term to consider, respectively, the linear, exponential, and hyperbolic inhibitory effects of t and [G]. PMID:22666116

  19. Antioxidation, angiotensin converting enzyme inhibition activity, nattokinase, and antihypertension of Bacillus subtilis (natto)-fermented pigeon pea

    OpenAIRE

    Bao-Hong Lee; Yi-Syuan Lai; She-Ching Wu

    2015-01-01

    Because of the high incidence of cardiovascular diseases in Asian countries, traditional fermented foods from Asia have been increasingly investigated for antiatherosclerotic effects. This study investigated the production of nattokinase, a serine fibrinolytic enzyme, in pigeon pea by Bacillus subtilis fermentation. B. subtilis 14714, B. subtilis 14715, B. subtilis 14716, and B. subtilis 14718 were employed to produce nattokinase. The highest nattokinase activity in pigeon pea was obtained us...

  20. ENZYME MARKERS ACTIVITY AND BILE FORMATION FUNCTION OF LIVER IN CASES OF TUBERCULOSTATICS AND HEXAVALENT CHROMIUM COMPOUNDS AFFECTION IN RATS

    OpenAIRE

    N. I. Burmas; L. S. Fira; P. H. Lyhackyy

    2016-01-01

    Background. Currently, the growing incidence of toxic lesions of the liver is associated with industrial chemicalization and uncontrolled use of hepatotoxic drugs in everyday life. There are about one thousand drugs with high or low hepatotoxicity, such as anti-TB drugs. Objective. In this research we studied the intracellular enzymes activity and bile formation function of the liver in rats of different ages in cases of tuberculostatic (isoniazid and rifampicin) affection and chromium (p...

  1. [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.

  2. 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)

  3. Comparative study of enzyme activity and heme reactivity in Drosophila melanogaster and Homo sapiens cystathionine β-synthases.

    Science.gov (United States)

    Su, Yang; Majtan, Tomas; Freeman, Katherine M; Linck, Rachel; Ponter, Sarah; Kraus, Jan P; Burstyn, Judith N

    2013-01-29

    Cystathionine β-synthase (CBS) is the first and rate-limiting enzyme in the transsulfuration pathway, which is critical for the synthesis of cysteine from methionine in eukaryotes. CBS uses coenzyme pyridoxal 5'-phosphate (PLP) for catalysis, and S-adenosylmethionine regulates the activity of human CBS, but not yeast CBS. Human and fruit fly CBS contain heme; however, the role for heme is not clear. This paper reports biochemical and spectroscopic characterization of CBS from fruit fly Drosophila melanogaster (DmCBS) and the CO/NO gas binding reactions of DmCBS and human CBS. Like CBS enzymes from lower organisms (e.g., yeast), DmCBS is intrinsically highly active and is not regulated by AdoMet. The DmCBS heme coordination environment, the reactivity, and the accompanying effects on enzyme activity are similar to those of human CBS. The DmCBS heme bears histidine and cysteine axial ligands, and the enzyme becomes inactive when the cysteine ligand is replaced. The Fe(II) heme in DmCBS is less stable than that in human CBS, undergoing more facile reoxidation and ligand exchange. In both CBS proteins, the overall stability of the protein is correlated with the heme oxidation state. Human and DmCBS Fe(II) hemes react relatively slowly with CO and NO, and the rate of the CO binding reaction is faster at low pH than at high pH. Together, the results suggest that heme incorporation and AdoMet regulation in CBS are not correlated, possibly providing two independent means for regulating the enzyme.

  4. 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

  5. Serum activities of liver enzymes in workers exposed to sub-TLV levels of dimethylformamide

    Directory of Open Access Journals (Sweden)

    Jinjiang He

    2015-04-01

    Full Text Available Objectives: The aim of this study has been to investigate serum activities of liver enzymes in workers exposed to sub-TLV levels of dimethylformamide (DMF. Material and Methods: Seventy-two workers and 72 healthy controls participated in the study. All subjects underwent complete physical examinations and abdominal ultrasound examination. Serum aspartate aminotransferase (AST, alanine aminotransferase (ALT, and c-glutamyl transpeptidase (c-GT were determined by an auto-chemistry analyzer. The data of airborne concentrations of DMF was obtained from the local Center of Disease Control and Prevention. The level of urine N-acetyl-S-(N-methylcarbamoylcysteine (AMCC was measured by means of high-performance liquid chromatography. Results: Time weighted average (TWA concentration of the DMF in workplace was 18.6 (range: 9.8–36.2 mg/m3. The concentration of the AMCC in workers’ urine was 28.32 (range: 1.8–58.6 mg/l and 9 workers’ AMCC exceeded the biological exposure index (40 mg/l. Thirty-one workers reported gastrointestinal symptoms (abdominal pain, nausea, anorexia and 10 workers reported headache, dizziness and/or palpitation in the exposed group. Serum analysis revealed that both the mean of serum activities of liver enzymes (ALT, AST and c-GT and the percentage of workers with abnormal liver function were significantly higher in the exposed group as compared to the controls. Conclusions: Dimethylformamide can cause liver damage even if air concentration is in the sub-threshold limit value (sub-TLV level. The protection of skin contact against the exposure to the DMF might be a critical issue as far as the occupational health is concerned.

  6. [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.

  7. 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.

  8. 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.

  9. 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

  10. 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

  11. 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...

  12. 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.

  13. 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

  14. 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.

  15. Inhibition of NEDD8-activating enzyme: a novel approach for the treatment of acute myeloid leukemia.

    Science.gov (United States)

    Swords, Ronan T; Kelly, Kevin R; Smith, Peter G; Garnsey, James J; Mahalingam, Devalingam; Medina, Ernest; Oberheu, Kelli; Padmanabhan, Swaminathan; O'Dwyer, Michael; Nawrocki, Steffan T; Giles, Francis J; Carew, Jennifer S

    2010-05-06

    NEDD8 activating enzyme (NAE) has been identified as an essential regulator of the NEDD8 conjugation pathway, which controls the degradation of many proteins with important roles in cell-cycle progression, DNA damage, and stress responses. Here we report that MLN4924, a novel inhibitor of NAE, has potent activity in acute myeloid leukemia (AML) models. MLN4924 induced cell death in AML cell lines and primary patient specimens independent of Fms-like tyrosine kinase 3 expression and stromal-mediated survival signaling and led to the stabilization of key NAE targets, inhibition of nuclear factor-kappaB activity, DNA damage, and reactive oxygen species generation. Disruption of cellular redox status was shown to be a key event in MLN4924-induced apoptosis. Administration of MLN4924 to mice bearing AML xenografts led to stable disease regression and inhibition of NEDDylated cullins. Our findings indicate that MLN4924 is a highly promising novel agent that has advanced into clinical trials for the treatment of AML.

  16. Effects of sodium pentaborate pentahydrate exposure on Chlorella vulgaris growth, chlorophyll content, and enzyme activities.

    Science.gov (United States)

    Chen, Xueqing; Pei, Yuansheng

    2016-10-01

    Sodium pentaborate pentahydrate (SPP) is a rare mineral. In this study, SPP was synthesized from boric acid and borax through low-temperature crystallization, and its effects on the growth of the alga, Chlorella vulgaris (C. vulgaris) were assessed. The newly synthesized SPP was characterized by chemical analysis, X-ray diffraction, Fourier-transform infrared spectroscopy, Raman spectroscopy, thermogravimetric analysis, and differential thermal analysis. The changes in C. vulgaris growth, chlorophyll content, and enzyme activities upon exposure to SPP for 168h were evaluated. Results showed that SPP treatment was detrimental to C. vulgaris growth during the first 24-120h of exposure. The harmful effects, however, diminished over time (168h), even at an effective medium concentration of 226.37mg BL(-1) (the concentration of boron applied per liter of culture medium). A similar trend was observed for chlorophyll content (chlorophyll a and b) and indicated that the photosynthesis of C. vulgaris was not affected and that high levels of SPP may even promote chlorophyll synthesis. Superoxide dismutase and catalase activities of C. vulgaris increased during 24-120h exposure to SPP, but these activities gradually decreased as culture time progressed. In other words, the initial detrimental effects of synthetic SPP on C. vulgaris were temporary and reversible. This research provides a scientific basis for applications of SPP in the environment. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. 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

  18. Early pharmacological inhibition of angiotensin-I converting enzyme activity induces obesity in adulthood

    Directory of Open Access Journals (Sweden)

    Kely ede Picoli Souza

    2015-04-01

    Full Text Available We have investigated early programming of body mass in order to understand the multifactorial etiology of obesity. Considering that the renin-angiotensin system is expressed and functional in the white adipose tissue (WAT and modulates its development, we reasoned whether early transitory inhibition of angiotensin-I converting enzyme activity after birth could modify late body mass development. Therefore, newborn Wistar rats were treated with enalapril (10 mg/kg of body mass or saline, starting at the first day of life until the age of 16 days. Between days 90th and 180th, a group of these animals received high fat diet (HFD. Molecular, biochemical, histological and physiological data were collected. Enalapril treated animals presented hyperphagia, overweight and increased serum level of triglycerides, total cholesterol and leptin, in adult life. Body composition analyses revealed higher fat mass with increased adipocyte size in these animals. Molecular analyses revealed that enalapril treatment increases neuropeptide Y (NPY and cocaine- and amphetamine-regulated transcript (CART gene expression in hypothalamus, fatty acid synthase (FAS and hormone-sensitive lipase (HSL gene expression in retroperitoneal WAT and decreases peroxixome proliferators-activated receptor (PPAR γ, PPARα, uncoupling protein (UCP 2 and UCP3 gene expression in WAT. The results of the current study indicate that enalapril administration during early postnatal development increases body mass, adiposity and serum lipids in adulthood associated with enhanced food intake and decreased metabolic activity in WAT, predisposing to obesity in adulthood.

  19. Improving a natural enzyme activity through incorporation of unnatural amino acids.

    Science.gov (United States)

    Ugwumba, Isaac N; Ozawa, Kiyoshi; Xu, Zhi-Qiang; Ely, Fernanda; Foo, Jee-Loon; Herlt, Anthony J; Coppin, Chris; Brown, Sue; Taylor, Matthew C; Ollis, David L; Mander, Lewis N; Schenk, Gerhard; Dixon, Nicholas E; Otting, Gottfried; Oakeshott, John G; Jackson, Colin J

    2011-01-19

    The bacterial phosphotriesterases catalyze hydrolysis of the pesticide paraoxon with very fast turnover rates and are thought to be near to their evolutionary limit for this activity. To test whether the naturally evolved turnover rate could be improved through the incorporation of unnatural amino acids and to probe the role of peripheral active site residues in nonchemical steps of the catalytic cycle (substrate binding and product release), we replaced the naturally occurring tyrosine amino acid at position 309 with unnatural L-(7-hydroxycoumarin-4-yl)ethylglycine (Hco) and L-(7-methylcoumarin-4-yl)ethylglycine amino acids, as well as leucine, phenylalanine, and tryptophan. Kinetic analysis suggests that the 7-hydroxyl group of Hco, particularly in its deprotonated state, contributes to an increase in the rate-limiting product release step of substrate turnover as a result of its electrostatic repulsion of the negatively charged 4-nitrophenolate product of paraoxon hydrolysis. The 8-11-fold improvement of this already highly efficient catalyst through a single rationally designed mutation using an unnatural amino acid stands in contrast to the difficulty in improving this native activity through screening hundreds of thousands of mutants with natural amino acids. These results demonstrate that designer amino acids provide easy access to new and valuable sequence and functional space for the engineering and evolution of existing enzyme functions.

  20. A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes

    DEFF Research Database (Denmark)

    Kracun, Stjepan Kresimir; Schückel, Julia; Westereng, Bjørge

    2015-01-01

    of carbohydrate-acting enzymes to be putatively identified. However, there is a paucity of methods for rapidly screening the biochemical activities of these enzymes, and this is a serious bottleneck in the development of enzyme-reliant bio-refining processes. Results: We have developed a new generation of multi...

  1. 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...

  2. 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.

  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. 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

  5. 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

  6. [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.

  7. 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.

  8. Variation of Enzyme Activities and Metabolite Levels in 24 Arabidopsis Accessions Growing in Carbon-Limited Conditions1[W

    Science.gov (United States)

    Cross, Joanna M.; von Korff, Maria; Altmann, Thomas; Bartzetko, Linda; Sulpice, Ronan; Gibon, Yves; Palacios, Natalia; Stitt, Mark

    2006-01-01

    Our understanding of the interaction of carbon (C) metabolism with nitrogen (N) metabolism and growth is based mainly on studies of responses to environmental treatments, and studies of mutants and transformants. Here, we investigate which metabolic parameters vary and which parameters change in a coordinated manner in 24 genetically diverse Arabidopsis (Arabidopsis thaliana) accessions, grown in C-limited conditions. The accessions were grown in short days, moderate light, and high nitrate, and analyzed for rosette biomass, levels of structural components (protein, chlorophyll), total phenols and major metabolic intermediates (sugars, starch, nitrate, amino acids), and the activities of seven representative enzymes from central C and N metabolism. The largest variation was found for plant weight, reducing sugars, starch at the end of the night, and several enzyme activities. High levels of one sugar correlated with high levels of other sugars and starch, and a trend to increased amino acids, slightly lower nitrate, and higher protein. The activities of enzymes at the interface of C and N metabolism correlated with each other, but were unrelated to carbohydrates, amino acid levels, and total protein. Rosette weight was unrelated or showed a weak negative trend to sugar and amino acid contents at the end of the day in most of the accessions, and was negatively correlated with starch at the end of the night. Rosette weight was positively correlated with several enzyme activities. We propose that growth is not related to the absolute levels of starch, sugars, and amino acids; instead, it is related to flux, which is indicated by the enzymatic capacity to use these central resources. PMID:17085515

  9. 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.

  10. 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.

  11. Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays (SOT)

    Science.gov (United States)

    Alginate Immobilization of Metabolic Enzymes (AIME) for High-Throughput Screening Assays DE DeGroot, RS Thomas, and SO SimmonsNational Center for Computational Toxicology, US EPA, Research Triangle Park, NC USAThe EPA’s ToxCast program utilizes a wide variety of high-throughput s...

  12. Effects of boarding return satellite on antioxidant enzyme activities during germination of hot pepper seed

    International Nuclear Information System (INIS)

    Li Shuifeng; Wang Bingliang; Guan Xueyu; Zhang Yan

    2006-01-01

    The effect of boarding return satellite on antioxidant enzyme activities during germination of hot pepper seed was studied. The results showed that the germination potentiality and germination rate of hot pepper seed after boarding return satellite were increased by 3.5% and 5.3%, respectively. During seed germination, soluble protein and MDA contents decreased, however, the SOD activities increased. SOD activity of treated seeds was higher than that of the control especially during the initial period of germination, while the content of soluble and MDA contents were much lower than those of control. The activities of SOD, G-POD, APX and CAT in 13d seedlings of treated seeds were increased by 14.29%, 25.23%, 1.84% and 21.52%, respectively. It was concluded that space flight enhanced antioxidant enzyme activities of seeds and seedlings, which were very important to prevent membrane lipid superoxide. (authors)

  13. Identification of novel esterase-active enzymes from hot environments by use of the host bacterium Thermus thermophilus

    Directory of Open Access Journals (Sweden)

    Benedikt eLeis

    2015-04-01

    Full Text Available Functional metagenomic screening strategies, which are independent of known sequence information, can lead to the identification of truly novel genes and enzymes. Since E. coli has been used exhaustively for this purpose as a host, it is important to establish alternative expression hosts and to use them for functional metagenomic screening for new enzymes. In this study we show that Thermus thermophilus HB27 is an excellent screening host and can be used as an alternative provider of truly novel biocatalysts. In a previous study we constructed the mutant strain BL03 that was no longer able to grow on defined minimal medium supplemented with tributyrin as the sole carbon source and could be used as a host to screen for metagenomic DNA fragments that could complement growth on tributyrin. Several thousand single fosmid clones from thermophilic metagenomic libraries from heated compost and hot spring water samples were subjected to a comparative screening for esterase activity in both T. thermophilus strain BL03 and E. coli EPI300. We scored a greater number of active clones in the thermophilic bacterium than in the mesophilic E. coli. From all clones functionally screened in E. coli, only two thermostable α/β-fold hydrolase enzymes with high amino acid sequence similarity to already characterized enzymes were identifiable. In contrast, five further fosmids were found that conferred lipolytic activities in T. thermophilus. Four open reading frames (ORFs were found which did not share significant similarity to known esterase enzymes. Two of the genes were expressed in both hosts and the novel thermophilic esterases, which based on their primary structures could not be assigned to known esterase or lipase families, were purified and preliminarily characterized. Our work underscores the benefit of using additional screening hosts other than E. coli for the identification of novel biocatalysts with industrial relevance.

  14. 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.

  15. 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

  16. 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

  17. Microbial respiration and extracellular enzyme activity in sediments from the Gulf of Mexico hypoxic zone

    Science.gov (United States)

    This study explores the relationship between sediment chemistry (TC, TN, TP) and microbial respiration (DHA) and extracellular enzyme activity (EEA) across the Gulf of Mexico (GOM) hypoxic zone. TC, TN, and TP were all positively correlated with each other (r=0.19-0.68). DHA was ...

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

    Czech Academy of Sciences Publication Activity Database

    Convigton, E. D.; Roitsch, Thomas; Dernastia, M.

    2016-01-01

    Roč. 63, č. 4 (2016), s. 757-762 ISSN 1318-0207 R&D Projects: GA MŠk(CZ) LO1415 Institutional support: RVO:67179843 Keywords : AGPase * carbohydrates * invertases * sucrose synthase * panel of enzyme activity assays * phytoplasma Subject RIV: EH - Ecology, Behaviour Impact factor: 0.983, year: 2016

  19. A study of the fine structure, enzyme activities and pattern of 14CO2 ...

    African Journals Online (AJOL)

    A detailed study of selected grasses has been made with respect to fine structures characteristics, enzyme activities associated with C-4 and C-3 pathway photosynthesis, and short term carbon dioxide-14 incorporation experiments. A good correlation was obtained between the fine structure, the carbon pathway and the ...

  20. Polymorphic drug metabolising enzymes : Assessment of activities by phenotyping and genotyping in clinical pharmacology

    NARCIS (Netherlands)

    Tamminga, Willem Jan

    2001-01-01

    Drug effects (pharmacodynamics) are determined by drug concentration at target site and the affinity of the drug for a target. Pharmacogenetics describes inherited differences in drug metabolising enzyme activities and differences in drug transporters and receptors.Answers were sought on the

  1. N-Glycosylation of Carnosinase Influences Protein Secretion and Enzyme Activity Implications for Hyperglycemia

    NARCIS (Netherlands)

    Riedl, Eva; Koeppel, Hannes; Pfister, Frederick; Peters, Verena; Sauerhoefer, Sibylle; Sternik, Paula; Brinkkoetter, Paul; Zentgraf, Hanswalter; Navis, Gerjan; Henning, Robert H.; Van Den Born, Jacob; Bakker, Stephan J. L.; Janssen, Bart; van der Woude, Fokko J.; Yard, Benito A.

    OBJECTIVE-The (CTG)(n) polymorphism in the serum carnosinase (CN-1) gene affects CN-1 secretion Since CN-1 is heavily glycosylated and glycosylation might influence protein secretion as well, we tested the role of N-glycosylation for CN-1 secretion and enzyme activity. We also tested whether CN-1

  2. 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)...

  3. 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.

  4. 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.

  5. 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.

  6. THE PRIMARY STRUCTURE OF HEVAMINE, AN ENZYME WITH LYSOZYME CHITINASE ACTIVITY FROM HEVEA-BRASILIENSIS LATEX

    NARCIS (Netherlands)

    JEKEL, PA; HARTMANN, JBH; BEINTEMA, JJ

    1991-01-01

    The primary structure of hevamine, an enzyme with lysozyme/chitinase activity from Hevea brasiliensis latex, has been determined predominantly with conventional non-automatic methods. The positions of three disulfide bridges have been determined. The sequence has about 60% identity with that of a

  7. 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

  8. 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)

  9. 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).

  10. Biomass and enzyme activity of two soil transects at King George Island, Maritime Antarctica

    Czech Academy of Sciences Publication Activity Database

    Tscherko, D.; Bölter, M.; Beyer, L.; Chen, J.; Elster, Josef; Kandeler, E.; Kuhn, D.; Blume, H. P.

    2003-01-01

    Roč. 35, č. 1 (2003), s. 34-47 ISSN 1523-0430 R&D Projects: GA ČR GA205/94/0156; GA AV ČR KSK6005114 Institutional research plan: CEZ:AV0Z6005908 Keywords : Maritime Antarctica * microbial soil biomass * enzyme activity Subject RIV: EF - Botanics Impact factor: 0.954, year: 2003

  11. 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.

  12. 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

  13. Detection of metals and polychlorobiphenyls and their correlation with detoxificant enzymes activity in Dicentrarchus labrax

    Directory of Open Access Journals (Sweden)

    I. Traversi

    2011-01-01

    Full Text Available Several pollutants released to the environment, are biotransformed into more soluble molecules, in liver, by several enzymes, as catalase (CAT and glutathione-S-tranferase (GST, which are fundamental for detoxification and excretion. The aim of this study was to investigate the relationships among xenobiotic levels and CAT and GST enzymatic activities, in reared European sea bass.

  14. Histochemical demonstration of creatine kinase activity using polyvinyl alcohol and auxiliary enzymes

    NARCIS (Netherlands)

    Frederiks, W. M.; Marx, F.; van Noorden, C. J.

    1987-01-01

    Creatine kinase activity (EC 2.7.3.2.) has been demonstrated in myocardium and skeletal muscle from rats by a method based on the incubation of cryostat sections with a polyvinyl alcohol-containing medium and the use of auxiliary enzymes. Hexokinase and glucose-6-phosphate dehydrogenase were spread

  15. 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)

  16. 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...

  17. 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.

  18. Phosphoglycerate Mutase Is a Highly Efficient Enzyme without Flux Control in Lactococcus lactis

    DEFF Research Database (Denmark)

    Solem, Christian; Petranovic, D.; Købmann, Brian

    2010-01-01

    The glycolytic enzyme phosphoglycerate mutase (PGM), which catalyzes the conversion of 3-phosphoglycerate to 2-phosphoglycerate, was examined in Lactococcus lactis with respect to its function, kinetics and glycolytic flux control. A library of strains with PGM activities ranging between 15-465% ...

  19. A High-Throughput (HTS) Assay for Enzyme Reaction Phenotyping in Human Recombinant P450 Enzymes Using LC-MS/MS.

    Science.gov (United States)

    Li, Xiaofeng; Suhar, Tom; Glass, Lateca; Rajaraman, Ganesh

    2014-03-03

    Enzyme reaction phenotyping is employed extensively during the early stages of drug discovery to identify the enzymes responsible for the metabolism of new chemical entities (NCEs). Early identification of metabolic pathways facilitates prediction of potential drug-drug interactions associated with enzyme polymorphism, induction, or inhibition, and aids in the design of clinical trials. Incubation of NCEs with human recombinant enzymes is a popular method for such work because of the specificity, simplicity, and high-throughput nature of this approach for phenotyping studies. The availability of a relative abundance factor and calculated intersystem extrapolation factor for the expressed recombinant enzymes facilitates easy scaling of in vitro data, enabling in vitro-in vivo extrapolation. Described in this unit is a high-throughput screen for identifying enzymes involved in the metabolism of NCEs. Emphasis is placed on the analysis of the human recombinant enzymes CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2B6, and CYP3A4, including the calculation of the intrinsic clearance for each. Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.

  20. 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)

  1. Mapping the active site helix-to-strand conversion of CxxxxC peroxiredoxin Q enzymes.

    Science.gov (United States)

    Perkins, Arden; Gretes, Michael C; Nelson, Kimberly J; Poole, Leslie B; Karplus, P Andrew

    2012-09-25

    Peroxiredoxins (Prx) make up a family of enzymes that reduce peroxides using a peroxidatic cysteine residue; among these, members of the PrxQ subfamily are proposed to be the most ancestral-like yet are among the least characterized. In many PrxQ enzymes, a second "resolving" cysteine is located five residues downstream from the peroxidatic Cys, and these residues form a disulfide during the catalytic cycle. Here, we describe three hyperthermophilic PrxQ crystal structures originally determined by the RIKEN structural genomics group. We reprocessed the diffraction data and conducted further refinement to yield models with R(free) values lowered by 2.3-7.2% and resolution extended by 0.2-0.3 Å, making one, at 1.4 Å, one of the best resolved peroxiredoxins to date. Comparisons of two matched thiol and disulfide forms reveal that the active site conformational change required for disulfide formation involves a transition of ~20 residues from a pair of α-helices to a β-hairpin and 3(10)-helix. Each conformation has ~10 residues with a high level of disorder providing slack that allows the dramatic shift, and the two conformations are anchored to the protein core by distinct nonpolar side chains that fill three hydrophobic pockets. Sequence conservation patterns confirm the importance of these and a few additional residues for function. From a broader perspective, this study raises the provocative question of how to make use of the valuable information in the Protein Data Bank generated by structural genomics projects but not described in the literature, perhaps remaining unrecognized and certainly underutilized.

  2. Dietary compounds that induce cancer preventive phase 2 enzymes activate apoptosis at comparable doses in HT29 colon carcinoma cells.

    Science.gov (United States)

    Kirlin, W G; Cai, J; DeLong, M J; Patten, E J; Jones, D P

    1999-10-01

    Dietary agents that induce glutathione S-transferases and related detoxification systems (Phase 2 enzyme inducers) are thought to prevent cancer by enhancing elimination of chemical carcinogens. The present study shows that compounds of this group (benzyl isothiocyanate, allyl sulfide, dimethyl fumarate, butylated hydroxyanisole) activated apoptosis in human colon carcinoma (HT29) cells in culture over the same concentration ranges that elicited increases in enzyme activity (5-25, 25-100, 10-100, 15-60 micromol/L, respectively). Pretreatment of cells with sodium butyrate, an agent that induces HT29 cell differentiation, resulted in parallel increases in Phase 2 enzyme activities and induction of apoptosis in response to the inducers. Cell death characteristics included apoptotic morphological changes, appearance of cells at sub-G1 phase on flow cytometry, caspase activation, DNA fragmentation and TUNEL-positive staining. The results suggest that dietary Phase 2 inducers may protect against cancer by a mechanism distinct from and in addition to that associated with enhanced elimination of carcinogens. If this occurs in vivo, diets high in such compounds could eliminate precancerous cells by apoptosis at time points well after initial exposure to chemical mutagens and carcinogens.

  3. Acute cadmium intoxication induces alpha-class glutathione S-transferase protein synthesis and enzyme activity in rat liver

    International Nuclear Information System (INIS)

    Casalino, Elisabetta; Sblano, Cesare; Calzaretti, Giovanna; Landriscina, Clemente

    2006-01-01

    Acute cadmium intoxication affects glutathione S-transferase (GST) in rat liver. It has been found that 24 h after i.p. cadmium administration to rats, at a dose of 2.5 mg CdCl 2 kg -1 body weight, the activity of this enzyme in liver cytosol increased by 40%. A less stimulatory effect persisted till 48 h and thereafter the enzyme activity normalized. Since, GST isoenzymes belong to different classes in mammalian tissues, we used quantitative immunoassays to verify which family of GST isoenzymes is influenced by this intoxication. Only alpha-class glutathione S-transferase (α-GST) proteins were detected in rat liver cytosol and their level increased by about 25%, 24 h after cadmium treatment. No pi-GST isoforms were found in liver cytosol from either normal or cadmium-treated rats. Co-administration of actinomycin D with cadmium normalized both the protein level and the activity of α-GST, suggesting that some effect occurs on enzyme transcription of these isoenzymes by this metal. On the other hand, it seems unlikely that the stimulatory effect is due to the high level of peroxides caused by lipid peroxidation, since Vitamin E administration strongly reduced the TBARS level, but did not cause any GST activity decrease

  4. Activities of some enzymes of lignin formation in reaction wood of Thuja orientalis, Metasequoia glyptostroboides and Robinia pseudoacacia.

    Science.gov (United States)

    Kutsuki, H; Higuchi, T

    1981-07-01

    The activities of the following five enzymes which are involved in the formation of lignin have been compared in reaction wood and in opposite wood: phenylalanine ammonia lyase (EC 4.3.1.5), caffeate 3-O-methyltransferase (EC 2.1.1.-), p-hydroxycinnamate: CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-) and peroxidase (EC 1.11.1.7). The activities of the four first-named enzymes in the compression wood of Thuja orientalis L. and Metasequoia glyptostroboides Hu et Cheng were 2.8±1.4-fold and 2.6±1.5-fold higher than those in opposite wood, respectively, whereas peroxidase had the same level of activity in either type of wood. On the other hand, no differences were observed in the activities of the five enzymes between tension and opposite woods of Robinia pseudoacacia L. These findings are well in accord with the chemical structure of lignin in the compression and tension woods of the three species studied: high content of lignin rich in condensed units in compression wood, and little difference in lignin between tension and opposite woods.

  5. The dogfish shark (Squalus acanthias) increases both hepatic and extrahepatic ornithine urea cycle enzyme activities for nitrogen conservation after feeding.

    Science.gov (United States)

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

    2006-01-01

    Urea not only is utilized as a major osmolyte in marine elasmobranchs but also constitutes their main nitrogenous waste. This study investigated the effect of feeding, and thus elevated nitrogen intake, on nitrogen metabolism in the Pacific spiny dogfish Squalus acanthias. We determined the activities of ornithine urea cycle (O-UC) and related enzymes in liver and nonhepatic tissues. Carbamoyl phosphate synthetase III (the rate-limiting enzyme of the O-UC) activity in muscle is high compared with liver, and the activities in both tissues increased after feeding. The contribution of muscle to urea synthesis in the dogfish body appears to be much larger than that of liver when body mass is considered. Furthermore, enhanced activities of the O-UC and related enzymes (glutamine synthetase, ornithine transcarbamoylase, arginase) were seen after feeding in both liver and muscle and were accompanied by delayed increases in plasma urea, trimethylamine oxide, total free amino acids, alanine, and chloride concentrations, as well as in total osmolality. The O-UC and related enzymes also occurred in the intestine but showed little change after feeding. Feeding did not change the rate of urea excretion, indicating strong N retention after feeding. Ammonia excretion, which constituted only a small percentage of total N excretion, was raised in fed fish, while plasma ammonia did not change, suggesting that excess ammonia in plasma is quickly ushered into synthesis of urea or protein. In conclusion, we suggest that N conservation is a high priority in this elasmobranch and that feeding promotes ureogenesis and growth. Furthermore, exogenous nitrogen from food is converted into urea not only by the liver but also by the muscle and to a small extent by the intestine.

  6. 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...

  7. Does cypermethrin affect enzyme activity, respiration rate and walking behavior of the maize weevil (Sitophilus zeamais)?

    Institute of Scientific and Technical Information of China (English)

    Ronnie Von Santos Veloso; Eliseu José G.Pereira; Raul Narciso C.Guedes; Maria Goreti A.Oliveira

    2013-01-01

    Insecticides cause a range of sub-lethal effects on targeted insects,which are frequently detrimental to them.However,targeted insects are able to cope with insecticides within sub-lethal ranges,which vary with their susceptibility.Here we assessed the response of three strains of the maize weevil Sitophilus zeamais Motschulsky (Coleoptera:Curculionidae) to sub-lethal exposure to the pyrethoid insecticide cypermethrin.We expected enzyme induction associated with cypermethrin resistance since it would aid the resistant insects in surviving such exposure.Lower respiration rate and lower activity were also expected in insecticide-resistant insects since these traits are also likely to favor survivorship under insecticide exposure.Curiously though,cypermethrin did not affect activity of digestive and energy metabolism enzymes,and even reduced the activity of some enzymes (particularly for cellulase and cysteine-proteinase activity in this case).There was strain variation in response,which may be (partially) related to insecticide resistance in some strains.Sub-lethal exposure to cypermethrin depressed proteolytic and mainly cellulolytic activity in the exposed insects,which is likely to impair their fitness.However,such exposure did not affect respiration rate and walking behavior of the insects (except for the susceptible strain where walking activity was reduced).Walking activity varies with strain and may minimize insecticide exposure,which should be a concern,particularly if associated with (physiological) insecticide resistance.

  8. 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.

  9. Climate and root proximity as dominant drivers of enzyme activity and C and N isotopic signature in soil

    Science.gov (United States)

    Stock, Svenja; Köster, Moritz; Dippold, Michaela; Boy, Jens; Matus, Francisco; Merino, Carolina; Nájera, Francisco; Spielvogel, Sandra; Gorbushina, Anna; Kuzyakov, Yakov

    2017-04-01

    The Chilean ecosystems provide a unique study area to investigate biotic controls on soil organic matter (SOM) decomposition and mineral weathering depending on climate (from hyper arid to temperate humid). Microorganisms play a crucial role in the SOM decomposition, nutrient release and cycling. By means of extracellular enzymes microorganisms break down organic compounds and provide nutrients for plants. Soil moisture (abiotic factor) and root carbon (biotic factor providing easily available energy source for microorganisms), are important factors for microbial decomposition of SOM and show strong gradients along the investigated climatic gradient. A high input of root carbon increases microbial activity and enzyme production, and facilitates SOM breakdown and nutrient release The aim of this study was to determine the potential enzymatic SOM decomposition and nutrient release depending on root proximity and precipitation. C and N contents, δ13C and δ15N values, and kinetics (Vmax, Km) of six extracellular enzymes, responsible for C, N, and P cycles, were quantified in vertical (soil depth) and horizontal (from roots to bulk soil) gradients in two climatic regions: within a humid temperate forest and a semiarid open forest. The greater productivity of the temperate forest was reflected by higher C and N contents compared to the semiarid forest. Regression lines between δ13C and -[ln(%C)] showed a stronger isotopic fractionation from top- to subsoil at the semiarid open forest, indicating a faster SOM turnover compared to the humid temperate forest. This is the result of more favorable soil conditions (esp. temperature and smaller C/N ratios) in the semiarid forest. Depth trends of δ15N values indicated N limitation in both soils, though the limitation at the temperate site was stronger. The activity of enzymes degrading cellulose and hemicellulose increased with C content. Activity of enzymes involved in C, N and P cycles decreased from top- to subsoil and

  10. Highly specific detection of genetic modification events using an enzyme-linked probe hybridization chip.

    Science.gov (United States)

    Zhang, M Z; Zhang, X F; Chen, X M; Chen, X; Wu, S; Xu, L L

    2015-08-10

    The enzyme-linked probe hybridization chip utilizes a method based on ligase-hybridizing probe chip technology, with the principle of using thio-primers for protection against enzyme digestion, and using lambda DNA exonuclease to cut multiple PCR products obtained from the sample being tested into single-strand chains for hybridization. The 5'-end amino-labeled probe was fixed onto the aldehyde chip, and hybridized with the single-stranded PCR product, followed by addition of a fluorescent-modified probe that was then enzymatically linked with the adjacent, substrate-bound probe in order to achieve highly specific, parallel, and high-throughput detection. Specificity and sensitivity testing demonstrated that enzyme-linked probe hybridization technology could be applied to the specific detection of eight genetic modification events at the same time, with a sensitivity reaching 0.1% and the achievement of accurate, efficient, and stable results.

  11. A high-throughput screening strategy for nitrile-hydrolyzing enzymes based on ferric hydroxamate spectrophotometry.

    Science.gov (United States)

    He, Yu-Cai; Ma, Cui-Luan; Xu, Jian-He; Zhou, Li

    2011-02-01

    Nitrile-hydrolyzing enzymes (nitrilase or nitrile hydratase/amidase) have been widely used in the pharmaceutical industry for the production of carboxylic acids and their derivatives, and it is important to build a method for screening for nitrile-hydrolyzing enzymes. In this paper, a simple, rapid, and high-throughput screening method based on the ferric hydroxamate spectrophotometry has been proposed. To validate the accuracy of this screening strategy, the nitrilases from Rhodococcus erythropolis CGMCC 1.2362 and Alcaligenes sp. ECU0401 were used for evaluating the method. As a result, the accuracy for assaying aliphatic and aromatic carboxylic acids was as high as the HPLC-based method. Therefore, the method may be potentially used in the selection of microorganisms or engineered proteins with nitrile-hydrolyzing enzymes.

  12. 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.

  13. 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.

  14. 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.