Sample records for b6-dependent enzymatic activities

  1. The B6 database: a tool for the description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families

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    Peracchi Alessio


    Full Text Available Abstract Background - Enzymes that depend on vitamin B6 (and in particular on its metabolically active form, pyridoxal 5'-phosphate, PLP are of great relevance to biology and medicine, as they catalyze a wide variety of biochemical reactions mainly involving amino acid substrates. Although PLP-dependent enzymes belong to a small number of independent evolutionary lineages, they encompass more than 160 distinct catalytic functions, thus representing a striking example of divergent evolution. The importance and remarkable versatility of these enzymes, as well as the difficulties in their functional classification, create a need for an integrated source of information about them. Description - The B6 database contains documented B6-dependent activities and the relevant protein families, defined as monophyletic groups of sequences possessing the same enzymatic function. One or more families were associated to each of 121 PLP-dependent activities with known sequences. Hidden Markov models (HMMs were built from family alignments and incorporated in the database. These HMMs can be used for the functional classification of PLP-dependent enzymes in genomic sets of predicted protein sequences. An example of such analyses (a census of human genes coding for PLP-dependent enzymes is provided here, whereas many more are accessible through the database itself. Conclusion - The B6 database is a curated repository of biochemical and molecular information about an important group of enzymes. This information is logically organized and available for computational analyses, providing a key resource for the identification, classification and comparative analysis of B6-dependent enzymes.

  2. Enzymatic activity of myccorrhizal fungi

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    Roman Pachlewski


    Full Text Available The investigations included assays of enzymatic activity of ectomycorrhizal fungi from the genera: Amanita, Cenococcum, Coltricia, Hebeloma, Lactarius, Rhizopogon, Russula, Suillus, Tricholoma and the pine ectendomycorrhizal strain MrgX. Among the 22 investigated strains of fungi 18 could decompose starch, 14 urea, 11 asparagine, 7 protein, 6 pectin and 3 ce1lulose. The most varied enzyme activities were found in Amanita muscaria, A. verna, Hebeloma, mesophaeum, ectendomycorrhizal isolate MrgX, Rhizopogon luteolus and Suillus bovinus, the highest cellolotytic activity was shown by the ectendomycorrhizal strain.

  3. Extracellular enzymatic activities of Bipolaris sorokiniana isolates. (United States)

    Geimba, Mercedes P; Brandelli, Adriano


    Several enzymatic activities were investigated in six isolates of the fungus Bipolaris sorokiniana, originating from different areas of Brazil. Among the glycosidases studied, beta-glucosidase, beta-N-acetylglucosaminidase, beta-xylosidase, cellobiohydrolase, and chitobiohydrolase were the major activities. In some isolates, beta-glucuronidase, beta-galactosidase, and alpha-mannosidase activities were also present. Polysaccharide-hydrolyzing enzymes, such as pectin lyase and carboxymethyl cellulase were detected in significant amounts, and their activities were variable among the different isolates. Other enzymes, namely phosphatases, proteinases and phenol oxidase, were also examined, showing variable amounts depending on the isolate. The pH dependence of all enzymes tested was investigated. Endoproteinase, carboxymethyl cellulase, and phenoloxidase had maximum activity in the pH range of 6-8, whilst all other enzymes showed maximum activity at pH 4-6.

  4. Effects of organic carbon sequestration strategies on soil enzymatic activities (United States)

    Puglisi, E.; Suciu, N.; Botteri, L.; Ferrari, T.; Coppolecchia, D.; Trevisan, M.; Piccolo, A.


    Greenhouse gases emissions can be counterbalanced with proper agronomical strategies aimed at sequestering carbon in soils. These strategies must be tested not only for their ability in reducing carbon dioxide emissions, but also for their impact on soil quality: enzymatic activities are related to main soil ecological quality, and can be used as early and sensitive indicators of alteration events. Three different strategies for soil carbon sequestration were studied: minimum tillage, protection of biodegradable organic fraction by compost amendment and oxidative polimerization of soil organic matter catalyzed by biometic porfirins. All strategies were compared with a traditional agricultural management based on tillage and mineral fertilization. Experiments were carried out in three Italian soils from different pedo-climatic regions located respectively in Piacenza, Turin and Naples and cultivated with maize or wheat. Soil samples were taken for three consecutive years after harvest and analyzed for their content in phosphates, ß-glucosidase, urease and invertase. An alteration index based on these enzymatic activities levels was applied as well. The biomimetic porfirin application didn't cause changes in enzymatic activities compared to the control at any treatment or location. Enzymatic activities were generally higher in the minimum tillage and compost treatment, while differences between location and date of samplings were limited. Application of the soil alteration index based on enzymatic activities showed that soils treated with compost or subjected to minimum tillage generally have a higher biological quality. The work confirms the environmental sustainability of the carbon sequestering agronomical practices studied.

  5. Enzymatic activity of rodents acclimated to cold and long scotophase (United States)

    Fourie, F. Le R.; Haim, A.


    Rodents representative of a diurnal species ( Rhabdomys pumilio) as well as a nocturnal species ( Praomys natalensis) were acclimated to cold (Ta = 8°C) at a photoperiod of LD 12:12 and a long scotophase (LD 8; 16) at a temperature of 25° C(Ta). Control groups were kept for both species at Ta = 25° C and LD 12:12 and winter acclimated individuals were obtained during July and August to serve as further reference. Blood samples obtained from the tail were analysed for enzymes representative of three major biochemical pathways. The enzymatic activity of LDH (glycolytic pathway), MDH (Krebs cycle) and G6PDH (hexose monophosphate shunt, as an indicator of gonadal activity) were monitored to represent metabolic activity of the respective cycles. Cold acclimated as well as winter acclimatized mice revealed similar enzymatic patterns for both species and significant increases in LDH and MDH were recorded with a concurrent decrease in G6PDH activity. Specimens exposed to long scotophase exhibited similar enzymatic patterns for both species studied, but enzymatic activity was higher than those of cold acclimated individuals. From these results it is concluded that cold as well as long scotophase induce metabolic adaptations through biochemical activity in the experimental animals. The effect of long scotophase is assumed to be an important factor in the induction of winter acclimatization.

  6. Enzymatic activity in extracts of allergy-causing astigmatid mites. (United States)

    Morgan, Marjorie S; Arlian, Larry G


    Many of the previously characterized allergens of house dust mites are known to be proteases, and this enzymatic activity is thought to contribute to their allergenicity. Other astigmatid mites, including stored-product mites and the ectoparasitic itch mite, Sarcoptes scabiei De Geer, are also known to be allergenic, but little or nothing is known about their enzymatic activities. The purpose of this study was to characterize the enzymatic activities present in extracts of the parasitic itch mite and from eight other species of free-living astigmatid mites. Extracts were prepared from one parasitic mite (S. scabiei), five stored-product mites (Chortoglyphus arcuatus (Troupeau), Lepidoglyphus destructor (Schrank), Blomia tropicalis Bronswijk, Cock, Oshima, Tyrophagus putrescentiae (Schrank), and Acarus siro L.), and three house dust mites [Dermatophagoidesfarinae Hughes, Dermatophagoides pteronyssinus (Troussart), and Euroglyphus maynei (Cooreman) ]. ApiZym strips were used to screen for the presence of 19 individual enzyme activities. Digestion of nine other substrates was evaluated by spectrophotometric or electrophoretic methods. All mite extracts exhibited some form of phosphatase, esterase, aminopeptidase, and glycosidase activity, although their substrate specificities varied considerably. Itch mite extract did not possess detectable serine peptidase activity nor was it able to hydrolyze gelatin or casein, whereas all other mite extracts exhibited these activities. Storage mite extracts possessed enzymes capable of degrading the widest range of substrates, whereas itch mite extract had the most limited proteolytic capacity. Extracts of nine species of allergy-causing astigmatid mites contain wide and diverse repertoires of enzymatic activities. These catalytic activities may be important contributors to the induction and manifestation of inflammatory and immune responses to mites in patients.

  7. Lipid protrusions membrane softness, and enzymatic activity

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    Jensen, Morten Østergaard; Høyrup, P.; Callisen, T.H.;


    The activity of phospholipase A(2) on lipid bilayers displays a characteristic lag burst behavior that has previously been shown to reflect the physical properties of the substrate. It has remained unclear which underlying molecular mechanism is responsible for this phenomenon. We propose here...... that protrusions of single lipid molecules out of the bilayer plane could provide such a mechanism. The proposal is supported by a combination of atomic-scale molecular dynamics simulations, theory, and experiments that have been performed in order to investigate the relationship between on the one side lipid...... protrusion modes and mechanical softness of phospholipid bilayers and on the other side the activity of enzymes acting on lipid bilayers composed of different unsaturated lipids. Specifically, our experiments show a correlation between the bilayer bending rigidity and the apparent Arrhenius activation energy...

  8. Spectrin’s chimeric E2/E3 enzymatic activity (United States)

    Goodman, Steven R; Petrofes Chapa, Rachel


    In this minireview, we cover the discovery of the human erythrocyte α spectrin E2/E3 ubiquitin conjugating/ligating enzymatic activity and the specific cysteines involved. We then discuss the consequences when this activity is partially inhibited in sickle cell disease and the possibility that the same attenuation is occurring in multiple organ dysfunction syndrome. We finish by discussing the reasons for believing that nonerythroid α spectrin isoforms (I and II) also have this activity and the importance of testing this hypothesis. If correct, this would suggest that the nonerythroid spectrin isoforms play a major role in protein ubiquitination in all cell types. This would open new fields in experimental biology focused on uncovering the impact that this enzymatic activity has upon protein–protein interactions, protein turnover, cellular signaling, and many other functions impacted by spectrin, including DNA repair. PMID:26283706


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    Simona Dunca


    Full Text Available In the thermoactinomycete strains subjected to examination the values of their recorded enzymatic activities (i.e. α-amy lase, protease, exo-β-1,4 – glucanase, endo -β-1,4 – glucanase and β-glucosidase were lower in the stationary cultures as compared to the stirred ones. The strain Thermomonospora fusca BB255 was found to be highly cellulase- producing and at the same time able to synthesize α-amy lases and proteases.

  10. Enzymatic activity of three sugarcane varieties under salt stress

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    Mercia F. Carvalho

    Full Text Available ABSTRACT Sugarcane is a species of great economic importance to Brazil. The study of enzymatic activity has helped to identify varieties of sugarcane tolerant to salt stress, for planting in Brazilian semiarid regions, where there is the occurrence of saline soils. The objective of this study was to evaluate the enzymatic activity of three varieties of sugarcane subjected to stress caused by salinity. The experiment was conducted in a greenhouse at the Federal Rural University of Pernambuco, in a completely randomized design with four replicates. The leaf blade samples were collected for analysis of soluble proteins and determination of the activity of catalase and ascorbate peroxidase. The protein content was higher for RB962962 and lower for the varieties RB92579 and RB867515. However, in RB867515, there was an increase in the activity of ascorbate peroxidase and catalase, while the varieties RB92579 and RB962962 showed a reduction in the activity of these enzymes. These results indicate that the variety RB867515 has a more efficient mechanism in plant protection against the action of reactive oxygen species.

  11. Nitric oxide synthase expression and enzymatic activity in multiple sclerosis

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    Broholm, H; Andersen, B; Wanscher, B


    We used post-mortem magnetic resonance imaging (MRI) guidance to obtain paired biopsies from the brains of four patients with clinical definite multiple sclerosis (MS). Samples were analyzed for the immunoreactivity (IR) of the three nitric oxide (NO) synthase isoforms [inducible, neuronal...... and endothelial nitric oxide synthase (NOS)], and enzymatic NO synthase activity. MRI guided biopsies documented more active plaques than macroscopic examination, and histological examination revealed further lesions. Inducible NOS (iNOS) was the dominant IR isoform, while reactive astrocytes were the dominant i......NOS expressing cells in active lesions. NOS IR expressing cells were widely distributed in plaques, in white and gray matter that appeared normal macroscopically, and on MR. Endothelial NOS (eNOS) was highly expressed in intraparenchymal vascular endothelial cells of MS patients. A control group matched for age...

  12. Controlling the enzymatic activity of a restriction enzyme by light. (United States)

    Schierling, Benno; Noël, Ann-Josée; Wende, Wolfgang; Hien, Le Thi; Volkov, Eugeny; Kubareva, Elena; Oretskaya, Tatiana; Kokkinidis, Michael; Römpp, Andreas; Spengler, Bernhard; Pingoud, Alfred


    For many applications it would be desirable to be able to control the activity of proteins by using an external signal. In the present study, we have explored the possibility of modulating the activity of a restriction enzyme with light. By cross-linking two suitably located cysteine residues with a bifunctional azobenzene derivative, which can adopt a cis- or trans-configuration when illuminated by UV or blue light, respectively, enzymatic activity can be controlled in a reversible manner. To determine which residues when cross-linked show the largest "photoswitch effect," i.e., difference in activity when illuminated with UV vs. blue light, > 30 variants of a single-chain version of the restriction endonuclease PvuII were produced, modified with azobenzene, and tested for DNA cleavage activity. In general, introducing single cross-links in the enzyme leads to only small effects, whereas with multiple cross-links and additional mutations larger effects are observed. Some of the modified variants, which carry the cross-links close to the catalytic center, can be modulated in their DNA cleavage activity by a factor of up to 16 by illumination with UV (azobenzene in cis) and blue light (azobenzene in trans), respectively. The change in activity is achieved in seconds, is fully reversible, and, in the case analyzed, is due to a change in V(max) rather than K(m).


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    Hong-wei Wang; Lin Yuan; Tie-liang Zhao; He Huang; Hong Chen; Di Wu


    The purpose of this research is to investigate the effects of the variously sulfated chitosans on lysozyme activity and structure.It was shown that the specific enzymatic activity of lysozyme remained almost similar to the native protein after being bound to 6-O-sulfated chitosan (6S-chitosan) and 3,6-O-sulfated chitosan (3,6S-chitosan),but decreased greatly after being bound to 2-N-6-O-sulfated chitosan (2,6S-chitosan).Meanwhile,among these sulfated chitosans,2,6S-chitosan induced the greatest conformational change in lysozyme as indicated by the fluorescence spectra.These findings demonstrated that when sulfated chitosans of different structures bind to lysozyme,lysozyme undergoes conformational change of different magnitudes,which results in corresponding levels of lysozyme activity.Further study on the interaction of sulfated chitosans with lysozyme by surface plasmon resonance (SPR) suggested that their affinities might be determined by their molecular structures.

  14. Enzymatic activities and protein profile of latex from Calotropis procera. (United States)

    Freitas, Cleverson Diniz T; Oliveira, Jefferson Soares; Miranda, Maria Raquel A; Macedo, Nívea Maria R; Sales, Maurício Pereira; Villas-Boas, Laurival A; Ramos, Márcio Viana


    The laticifer fluid of Calotropis procera is rich in proteins and there is evidence that they are involved in the pharmacological properties of the latex. However, not much is known about how the latex-containing proteins are produced or their functions. In this study, laticifer proteins of C. procera were pooled and examined by 1D and 2D electrophoresis, masses spectrometry (MALDI-TOF) and characterized in respect of proteolytic activity and oxidative enzymes. Soluble laticifer proteins were predominantly composed of basic proteins (PI>6.0) with molecular masses varying between 5 and 95 kDa. Proteins with a molecular mass of approximately 26,000 Da were more evident. Strong anti-oxidative activity of superoxide dismutase (EC (1007.74+/-91.89 Ug(-1)DM) and, to a lesser extent ascorbate peroxidase (EC (0.117(d)+/-0.013 microMol H(2)O(2)g(-1)min(-1)), were detected. However, catalase (EC was absent. The strong proteolytic activities of laticifer proteins from C. procera were shown to be shared by at least four distinct cysteine proteinases (EC that were isolated by gel filtration chromatography. Serine and metaloproteinases were not detected and aspartic proteinase activities were barely visible. Chitinases (EC were also isolated in a chitin column and their activities quantified. The presence of these enzymatic activities in latex from C. procera may confirm their involvement in resistance to phytopathogens and insects, mainly in its leaves where the latex circulates abundantly.


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    Lychman V. A.


    Full Text Available The results of a long-term research of the influence of various biologically active agents (a humic preparation Lignogumat and microbiological Baikal EM fertilizer on a structural state and the enzymatic activity of ordinary carbonated black soil are presented. It has been established that biologically active substances contribute to increased enzymatic activity, humus and improve the soil structure

  16. First results on enzymatic activities in two salt marsh soils under different hydromorphic level and vegetation

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    Carmen Trasar-Cepeda


    Full Text Available Salt-marsh soils are soils characterized by non-permanent hydric saturation that, depending on factors like duration of submersion periods, are dominated by different salt-tolerant plant species. The composition of microbial communities is an essential component in trophic dynamics and biogeochemical processes in salt marshes, and determines the level of enzymatic activities, which catalyze the conversion of complex molecules into simpler ones. Despite of this, the enzymatic activities in marsh-soils has not yet been investigated. The aim of this study was to analyze the enzymatic activities in two soil profiles of marsh-soils under different water saturation level and dominated by different plant species [Juncus maritimus Lam and Spartina maritima (Curtis Fernald (Sp]. In both soils, the enzymatic activities were much lower than the levels typically found in terrestrial ecosystems. The enzymatic activities were measured both in air-dried and in re-moistened and incubated soil samples. In air-dried samples, the enzymatic activities were higher in Juncus than in Spartina soil and tended to decrease with depth, being sharper the decrease in Juncus than in Spartina soil. Re-moistened and pre-incubated soils showed a general increase in all the enzymatic activities and throughout the whole soil profile, especially in Spartina soils. Hydrolase activities showed a strong and positive relationship with organic matter content both in air-dried and in re-moistened soil samples, higher in these latter. In general, oxidoreductase activities only showed this relationship in re-moistened soil samples. More studies, preferably using freshly collected soil samples, are needed to understand the relationship between enzymatic activities and these environmental conditions.

  17. Enzymatic activity of the cellulolytic complex produced by Trichoderma reesei. Enzymatic hydrolysis of cellulose; Actividad enzimatica del complejo celulolitico producido por Trichoderma reesei. Hidrolisis enzimatica de la celulosa

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    Alfonsel, M.; Negro, M. J.; Saez, R.; Martin, C.


    The enzymatic activity characterization of the cellulolytic complex obtained from Trichoderma reesei QM 9414 and the influence of the enzymatic hydrolysis conditions on the hydrolysis yield are studied. Pure cellulose and native or alkali pretreated biomass Onopordum nervosum have been used as substrates. The values of pH, temperature, substrate concentration and enzyme-substrate ratio for the optimum activity of that complex, evaluated as glucose and reducing sugars production, have been selected. Previous studies on enzymatic hydrolysis of 0. nervosum have shown a remarkable effect of the alkaline pretreatments on the final hydrolysis yield. (Author) 10 refs.

  18. Inhibitory effect of nicotinamide on enzymatic activity of selected fungal strains causing skin infection. (United States)

    Ciebiada-Adamiec, Anna; Małafiej, Eugeniusz; Ciebiada, Ireneusz


    Pathogenicity of fungi is connected with their ability to easily penetrate the host tissues, survive in the infected host organism and use the elements of the host tissues as nutrients. Hence, the co-occurrence of pathogenic properties with the high enzymatic activity, which is manifested through the production of various enzymes including extracellular enzymes, was observed. It can be expected that it is possible to decrease fungal pathogenicity by lowering their enzymatic activity. The aim of the study was to determine the effect of nicotinamide on enzymatic activity of the fungi, which are most frequently isolated in cases of skin infection. Enzymatic activity was analysed using 15 Candida albicans, 15 Trichophyton rubrum and 15 Trichophyton mentagrophytes strains. The strains used for the study were collected from the current diagnostic material. API ZYM tests were used in diagnostic analysis. MICs of nicotinamide were determined by the macrodilution method in liquid medium. In the case of Candida strains, the presence of nicotinamide in the broth had a significant effect on the decrease of enzymatic activity (P nicotinamide was observed in the case of dermatophytes (P nicotinamide exhibited biological activity towards C. albicans, T. rubrum and Trichophyton mentagrophytes, which resulted in a decrease in the activity of enzymes produced by the fungi.

  19. Controlling enzymatic activity and kinetics in swollen mesophases by physical nano-confinement (United States)

    Sun, Wenjie; Vallooran, Jijo J.; Zabara, Alexandru; Mezzenga, Raffaele


    Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them into a highly confined environment. We show that the enzymatic activity of a model enzyme, horseradish peroxidase (HRP), can be accurately controlled by relaxing its confinement within the cubic phases' water channels, when the aqueous channel diameters are systematically swollen with varying amount of hydration-enhancing sugar ester. The in-meso activity and kinetics of HRP are then systematically investigated by UV-vis spectroscopy, as a function of the size of the aqueous mesophase channels. The enzymatic activity of HRP increases with the swelling of the water channels. In swollen mesophases with water channel diameter larger than the HRP size, the enzymatic activity is more than double that measured in standard mesophases, approaching again the enzymatic activity of free HRP in bulk water. We also show that the physically-entrapped enzymes in the mesophases exhibit a restricted-diffusion-induced initial lag period and report the first observation of in-meso enzymatic kinetics significantly deviating from the normal Michaelis-Menten behaviour observed in free solutions, with deviations vanishing when enzyme confinement is released by swelling the mesophase.Bicontinuous lipid cubic mesophases are widely investigated as hosting matrices for functional enzymes to build biosensors and bio-devices due to their unique structural characteristics. However, the enzymatic activity within standard mesophases (in-meso) is severely hindered by the relatively small diameter of the mesophase aqueous channels, which provide only limited space for enzymes, and restrict them

  20. Enzymatic Activity in the Gastrointestinal Tract of Pimelodus maculatus (Teleostei, Siluriformes) in Two Neotropical Reservoirs with Different Trophic Conditions


    Silvana Duarte; Marcelo Bemquerer; Francisco Gerson de Araújo


    Enzymatic activities for digestion of proteins and carbohydrates were compared among three organs of the digestive system of Pimelodus maculatus in two reservoirs with different trophic conditions during the winter of 2006. The aim was to test the hypothesis that enzymatic activity for the digestion of proteins and carbohydrates differed among organs and that such activities differ between the trophic state of the environment. Enzymatic activities were determined through the assays of specifi...

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

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    Karina Cenciani


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

  2. Thrombolytic efficacy and enzymatic activity of rt-PA-loaded echogenic liposomes. (United States)

    Bader, Kenneth B; Bouchoux, Guillaume; Peng, Tao; Klegerman, Melvin E; McPherson, David D; Holland, Christy K


    Echogenic liposomes (ELIP), that can encapsulate both recombinant tissue-type plasminogen activator (rt-PA) and microbubbles, are under development to improve the treatment of thrombo-occlusive disease. However, the enzymatic activity, thrombolytic efficacy, and stable cavitation activity generated by this agent has yet to be evaluated and compared to another established ultrasound-enhanced thrombolytic scheme. A spectrophotometric method was used to compare the enzymatic activity of the rt-PA incorporated into ELIP (t-ELIP) to that of rt-PA. An in vitro flow model was employed to measure the thrombolytic efficacy and dose of ultraharmonic emissions from stable cavitation for 120-kHz ultrasound exposure of three treatment schemes: rt-PA, rt-PA and the perfluorocarbon-filled microbubble Definity(®), and t-ELIP. The enzymatic activity of rt-PA incorporated into t-ELIP was 28 % that of rt-PA. Thrombolytic efficacy of t-ELIP or rt-PA and Definity(®) was equivalent when the dose of t-ELIP was adjusted to produce comparable enzymatic activity. Sustained bubble activity was nucleated from Definity but not from t-ELIP exposed to 120-kHz ultrasound. These results emphasize the advantages of encapsulating a thrombolytic and the importance of incorporating an insoluble gas required to promote sustained, stable cavitation activity.

  3. Enzymatic activity of the intestine in effect of pesticides of pyrethroid group

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    Mukaddas Khamrakulova


    Full Text Available The purpose of investigation was to study the effect of pesticides from group of pyrethroids (e.g. decis on the enzymatic function in the homogenate of the mucosal membrane of the proximal and distal segments of the small intestine.Determination of the degree of the activity of hydrolytic enzymes in the homogenates of some parts of the intestine allowed to show effect of pesticide decis on the gradient of distribution of enzymatic activity along the intestine. For characteristic of the enzymatic activity of the small intestine there was performed study of the activity of dipeptidase, amylase, invertase and alkaline phosphatase in the homogenate of the mucosal membrane from proximal and distal parts in multiple effects of pesticide decis in toxic dose (1/20 LD50 during 4 months.Changes of the enzymatic activity in acute poisoning were depended on the time of pesticide exposure and site of the bowel. The different digestive enzymes have different response to effect ofpesticidesof pyrethroid group (decis, there are differences in the reactions of proximal and distal part of the small intestine and there is correlation between changes of activity of the majority hydrolases and administered dose of pesticides.

  4. Beta-glucosidase enzymatic activity of crystal polypeptide of the Bacillus thuringiensis strain 1.1. (United States)

    Papalazaridou, A; Charitidou, L; Sivropoulou, A


    The crystals of Bacillus thuringiensis strain 1.1 consist of the 140 kDa delta-endotoxin, which exhibits beta-glucosidase enzymatic activity, based on the following data. (i) Purified crystals exhibit beta-glucosidase enzymatic activity. When the crystals are reacted with specific antibodies directed either against the commercial (almond purified) beta-glucosidase or against the 140 kDa polypeptide, then considerable reduction of enzymatic activity is observed almost at the same level with both antibodies. (ii) Commercial beta-glucosidase and the 140 kDa crystal polypeptide share antigenic similarities; in Western immunoblots, the 140 kDa crystal polypeptide is recognized by anti-beta-glucosidase antibodies, and commercial beta-glucosidase is recognized by anti-140-kDa antibodies. (iii) The enzymatic properties of commercial beta-glucosidase and that resident in the crystals of B. thuringiensis strain 1.1 are very similar. Thus, both enzymes hydrolyze a wide range of substrates (aryl-beta-glucosides, disaccharides with alpha- or beta-linkage polysaccharides) and have an optimum activity at 40 degrees C and pH 5. Both enzymes are relatively thermostable and are resistant to end-product inhibition by glucose. Additionally, they show the same pattern of inhibition or activation by several chemical compounds. (iv) The crystals and commercial beta-glucosidase show almost equivalent levels of insecticidal activity against Drosophila melanogaster larvae and, furthermore, cause reduction in adult flies that emerge from larvae surviving treatment.

  5. Amphiphilic conetworks as activating carriers for the enhancement of enzymatic activity in supercritical CO2. (United States)

    Bruns, Nico; Bannwarth, Willi; Tiller, Joerg C


    Enzymatic reactions in supercritical carbon dioxide (scCO2) represent a way of combining the advantages of biocatalysis with the environmental benign nature of scCO2 as a solvent. Here we demonstrate that activities of enzymes in scCO2 can be greatly enhanced by incorporating them into amphiphilic conetworks (APCNs), a novel type of enzyme support. Two sets of hydrophilic/scCO2-philic APCNs, poly(2-hydroxyethyl acrylate)-linked by-poly(dimethylsiloxane) (PHEA-l-PDMS) and poly(2-hydroxyethyl acrylate)-linked by-perfluoropolyether (PHEA-l-PFPE), were prepared and loaded with the synthetically relevant lipase from Rhizomucor miehei. The effect of the APCNs' composition on the amount of the absorbed lipase was studied. It is observed that both sets of lipase-loaded APCNs enhance the catalytic activity of the enzyme in scCO2. The chemical nature of the scCO2-philic phase as well as the conetworkscomposition greatly influences the activity of the lipase in the conetworks. Activities obtained with PFPE-basedAPCNS were up to 10-fold higher than those obtained with PDMS-based conetworks. The highest specific activity measured corresponds to a 2,000-fold activation compared to the lyophilized enzyme powder. This activity is 10 times higher than the specific activity of the lipase immobilized on an optimized commercial carrier.

  6. Enzymatic hydrolysis of rice protein with papain and antioxidation activity of hydrolysate (United States)

    The enzymatic hydrolysis technology of rice protein and the antioxidant activity of the hydrolysate were studied. Substrate concentration,enzyme dose,pH value and temperature were selected as factors to optimize the hydrolysis parameters with single—factor and orthogonal tests. Results show the opti...

  7. Effects of Polyelectrolyte Complex Micelles and Their Components on the Enzymatic Activity of Lipase

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    Lindhoud, Saskia; Norde, Willem; Stuart, Martien Cohen


    The enzymatic activity of Hi-lipase embedded in complexes of poly-2-methylvinylpyridinium-co-poly(ethylene oxide) (P2MVP(41)-PEG(205)) and poly(acrylic acid)(PAA(139)) is studied as a function of the PAA(139) + P2MVP(41) - PEO(205) complex composition. The measurements revealed that there are severa

  8. Soil enzymatic activities and microbial community structure with different application rates of Cd and Pb

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    KHAN Sardar; CAO Qing; HESHAM Abd El-Latif; XIA Yue; HE Ji-zheng


    This study focused on the changes of soil microbial diversity and potential inhibitory effects of heavy metals on soil enzymatic activities at different application rates of Cd and Pb. The soil used for experiments was collected from Beijing and classified as endoaquepts. Pots containing 500 g of the soil with different Cd or/and Pb application rates were incubated for a period of 0, 2, 9, 12 weeks in a glasshouse and the soil samples were analyzed for individual enzymes, including catalase, alkaline phosphatase and dehydrogenase, and the changes of microbial community structure. Results showed that heavy metals slightly inhibited the enzymatic activities in all the samples spiked with heavy metals. The extent of inhibition increased significantly with increasing level of heavy metals, and varied with the incubation periods. The soil bacterial community structure, as determined by polymerase chain reaction-denaturing gradient gel electrophoresis techniques, was different in the contaminated samples as compared to the control. The highest community change was observed in the samples amended with high level of Cd. Positive correlations were observed among the three enzymatic activities, but negative correlations were found between the amounts of the heavy metals and the enzymatic activities.

  9. Enzymatic Hydrolysis of Oleuropein from Olea europea (Olive Leaf Extract and Antioxidant Activities

    Directory of Open Access Journals (Sweden)

    Jiao-Jiao Yuan


    Full Text Available Oleuropein (OE, the main polyphenol in olive leaf extract, is likely to decompose into hydroxytyrosol (HT and elenolic acid under the action of light, acid, base, high temperature. In the enzymatic process, the content of OE in olive leaf extract and enzyme are key factors that affect the yield of HT. A selective enzyme was screened from among 10 enzymes with a high OE degradation rate. A single factor (pH, temperature, time, enzyme quantity optimization process and a Box-Behnken design were studied for the enzymatic hydrolysis of 81.04% OE olive leaf extract. Additionally, enzymatic hydrolysis results with different substrates (38.6% and 81.04% OE were compared and the DPPH antioxidant properties were also evaluated. The result showed that the performance of hydrolysis treatments was best using hemicellulase as a bio-catalyst, and the high purity of OE in olive extract was beneficial to biotransform OE into HT. The optimal enzymatic conditions for achieving a maximal yield of HT content obtained by the regression were as follows: pH 5, temperature 55 °C and enzyme quantity 55 mg. The experimental result was 11.31% ± 0.15%, and the degradation rate of OE was 98.54%. From the present investigation of the antioxidant activity determined by the DPPH method, the phenol content and radical scavenging effect were both decreased after enzymatic hydrolysis by hemicellulase. However, a high antioxidant activity of the ethyl acetate extract enzymatic hydrolysate (IC50 = 41.82 μg/mL was demonstated. The results presented in this work suggested that hemicellulase has promising and attractive properties for industrial production of HT, and indicated that HT might be a valuable biological component for use in pharmaceutical products and functional foods.

  10. Effect of the addition of sand on the enzymatic activity of carbetidine soils contaminated with metals

    Energy Technology Data Exchange (ETDEWEB)

    Ramirez Vera, R.; Martin Calvarro, L.; Rodriguez Fernandez, T.; Ortiz Rosales, M. A.; Lopez Lafuente, A.


    Heavy metals can alter the composition, structure and metabolic activity of soil microbian communities. These alterations can be used as quick and sensible bio indicators of changes in the soil quality; therefore the study of enzymatic activity in the soil represents one of the prior objectives to determine its status. The activity of deshidrogenase, {beta}-galactosidase, alkaline phosphatase and urease, enzymes which are involved in the biogeochemical cycles of the main macro nutrients, has been analyzed. (Author)

  11. Surfactant-activated lipase hybrid nanoflowers with enhanced enzymatic performance (United States)

    Cui, Jiandong; Zhao, Yamin; Liu, Ronglin; Zhong, Cheng; Jia, Shiru


    Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to improve catalytic performance. However, activity of the most of the enzymes was declined after immobilization. Here, we develop a surfactant-activated lipase-inorganic flowerlike hybrid nanomaterials with rational design based on interfacial activation and self-assembly. The resulting surfactant-activated lipase-inorganic hybird nanoflower (activated hNF-lipase) exhibited 460% and 200% higher activity than native lipase and conventional lipase-inorganic hybird nanoflower (hNF-lipase). Furthermore, the activated hNF-lipase displayed good reusability due to its monodispersity and mechanical properties, and had excellent long-time stability. The superior catalytic performances were attributed to both the conformational modulation of surfactants and hierarchical structure of nanoflowers, which not only anchored lipases in an active form, but also decreased the enzyme-support negative interaction and mass-transfer limitations. This new biocatalytic system is promising to find widespread use in applications related to biomedicine, biosensor, and biodiesel. PMID:27297609


    Directory of Open Access Journals (Sweden)

    Figueiredo Márcia do Vale B.


    Full Text Available A greenhouse experiment was carried out aiming to study the effect of water stress on metabolic activity of cowpea nodules at different plant development stages. Cowpea plants were grown in pots with yellow latosol soil under three different matric potentials treatments: -7.0 (control-S1, -70.0 (S2 and <-85.0 KPa (S3. The experimental design was randomized blocks with sub-divided plots, each plot containing a different degree of water stress, divided in sub-plots for the four different developmental stages: E1 (0-15, E2 (15-30, E3 (20-35 and E4 (30-45 days after emmergence. Water stress treatments were applied by monitoring soil water potential using a set of porous cups. The effect of water stress was most harmful to cowpea when it was applied at E2 than at other symbiotic process stages. Shoot/root ratio decreased from 2.61 to 2.14 when matric potential treatment was <-85.0 and -70.0 KPa respectively. There was a reduction in the glutamine synthetase activity and phosphoenolpyruvate carboxilase activity with increased stress, while glutamine synthase activity was the enzyme most sensitive to water stress. Glutamate dehydrogenase activity increased in more negative matric potential, indicating that this enzyme is sufficiently activitye under water stress.

  13. Enzymatic assay for calmodulins based on plant NAD kinase activity

    Energy Technology Data Exchange (ETDEWEB)

    Harmon, A.C.; Jarrett, H.W.; Cormier, M.J.


    NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca/sup 2 -/-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K/sub 0.5/) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The A/sub 0.5/ s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K/sub 0.5/'s for the activation of Ca/sup 2 +/-ATPase ranged from 36.3 ng/mol for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca/sup 2 +/. Palmitic acid had a slightly stimulatory effect in the presence of Ca/sup 2 +/ (10% of maximum), but no effect in the absence of Ca/sup 2 +/. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures. 30 references, 1 figure, 4 tables.

  14. Extracellular enzymatic activities and physiological profiles of yeasts colonizing fruit trees. (United States)

    Molnárová, Jana; Vadkertiová, Renáta; Stratilová, Eva


    Yeasts form a significant and diverse part of the phyllosphere microbiota. Some yeasts that inhabit plants have been found to exhibit extracellular enzymatic activities. The aim of the present study was to investigate the ability of yeasts isolated from leaves, fruits, and blossoms of fruit trees cultivated in Southwest Slovakia to produce extracellular enzymes, and to discover whether the yeasts originating from these plant organs differ from each other in their physiological properties. In total, 92 strains belonging to 29 different species were tested for: extracellular protease, β-glucosidase, lipase, and polygalacturonase activities; fermentation abilities; the assimilation of xylose, saccharose and alcohols (methanol, ethanol, glycerol); and for growth in a medium with 33% glucose. The black yeast Aureobasidium pullulans showed the largest spectrum of activities of all the species tested. Almost 70% of the strains tested demonstrated some enzymatic activity, and more than 90% utilized one of the carbon compounds tested. Intraspecies variations were found for the species of the genera Cryptococcus and Pseudozyma. Interspecies differences of strains exhibiting some enzymatic activities and utilizing alcohols were also noted. The largest proportion of the yeasts exhibited β-glucosidase activity and assimilated alcohols independently of their origin. The highest number of strains positive for all activities tested was found among the yeasts associated with leaves. Yeasts isolated from blossoms assimilated saccharose and D-xylose the most frequently of all the yeasts tested. The majority of the fruit-inhabiting yeasts grew in the medium with higher osmotic pressure.

  15. Enzymatic activity toward poly(L-lactic acid) implants

    NARCIS (Netherlands)

    Schakenraad, J.M.; Hardonk, M.J.; Feijen, J.; Molenaar, I.; Nieuwenhuis, P.


    Tissue reactions toward biodegradable poly(L-lactic acid) implants were monitored by studying the activity pattern of seven enzymes as a function of time: alkaline phosphatase, acid phosphatase, -naphthyl acetyl esterase, -glucuronidase, ATP-ase, NADH-reductase, and lactate dehydrogenase. Cell types

  16. Soil disturbance increases soil microbial enzymatic activity in arid ecoregion (United States)

    Functional diversity of the soil microbial community is commonly used in the assessment of soil health as it relates to the activity of soil microflora involved in carbon cycling. Soil microbes in different microenvironments will have varying responses to different substrates, thus catabolic fingerp...

  17. Unbinding of Hyaluronan Accelerates the Enzymatic Activity of Bee Hyaluronidase


    Iliás, Attila; Liliom, Károly; Greiderer-Kleinlercher, Brigitte; Reitinger, Stephan; Lepperdinger, Günter


    Hyaluronan (HA), a polymeric glycosaminoglycan ubiquitously present in higher animals, is hydrolyzed by hyaluronidases (HAases). Here, we used bee HAase as a model enzyme to study the HA-HAase interaction. Located in close proximity to the active center, a bulky surface loop, which appears to obstruct one end of the substrate binding groove, was found to be functionally involved in HA turnover. To better understand kinetic changes in substrate interaction, binding of high molecular weight HA ...

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

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    Nelson P. Guerra


    Full Text Available 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, vmax decreased significantly (P<0.05 and KM increased (although not always significantly with the increase in t. The conformational changes produced in the starch chains as a consequence of the ageing seemed to affect negatively the diffusivity of the starch to the active site of the 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].

  19. An enzymatic atavist revealed in dual pathways for water activation.

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    Donghong Min


    Full Text Available Inosine monophosphate dehydrogenase (IMPDH catalyzes an essential step in the biosynthesis of guanine nucleotides. This reaction involves two different chemical transformations, an NAD-linked redox reaction and a hydrolase reaction, that utilize mutually exclusive protein conformations with distinct catalytic residues. How did Nature construct such a complicated catalyst? Here we employ a "Wang-Landau" metadynamics algorithm in hybrid quantum mechanical/molecular mechanical (QM/MM simulations to investigate the mechanism of the hydrolase reaction. These simulations show that the lowest energy pathway utilizes Arg418 as the base that activates water, in remarkable agreement with previous experiments. Surprisingly, the simulations also reveal a second pathway for water activation involving a proton relay from Thr321 to Glu431. The energy barrier for the Thr321 pathway is similar to the barrier observed experimentally when Arg418 is removed by mutation. The Thr321 pathway dominates at low pH when Arg418 is protonated, which predicts that the substitution of Glu431 with Gln will shift the pH-rate profile to the right. This prediction is confirmed in subsequent experiments. Phylogenetic analysis suggests that the Thr321 pathway was present in the ancestral enzyme, but was lost when the eukaryotic lineage diverged. We propose that the primordial IMPDH utilized the Thr321 pathway exclusively, and that this mechanism became obsolete when the more sophisticated catalytic machinery of the Arg418 pathway was installed. Thus, our simulations provide an unanticipated window into the evolution of a complex enzyme.

  20. Modulation of enzymatic activity of human mast cell tryptase and chymase by protease inhibitors

    Institute of Scientific and Technical Information of China (English)

    HEShao-Heng; CHENPu; CHENHan-Qiu


    AIM: To investigate the actions of protease inhibitors on the enzymatic activities of tryptase and chymase in similarexperimental systems. METHODS: Human lung tryptase and human skin chymase were purified by a similarprocedure involving high salt extraction of tryptase, heparin agarose affinity chromatography, and S-200 Sephacrylgel filtration chromatography. Actions of protease inhibitors on tryptase and chymase activities were examined byenzyme assays. RESULTS: The specific activities of tryptase and chymase were 2.1 kU/g protein and 4.9 kU/g protein, respectively. Both preparations showed a single diffuse band on SDS-PAGE. Among non-native proteaseinhibitors, N-(1-hydroxy-2-naphthoyl)-L- arginyl-L-prolinamide hydrochloride (HNAP), leupeptin, antipain,benzamidine, and protamine inhibited more than 90 % enzymatic activity of tryptase, whereas soy bean trypsininhibitor (SBTI), Z-Ile-Glu-Pro-Phe-CO2Me (ZIGPPM) and chymostatin inhibited more than 95 % enzymaticactivity of chymase. Native protease inhibitors α-antitrypsin and secretory leukocyte protease inhibitor (SLPI)inhibited more than 90 % enzymatic activity of chymase, but lactoferrin appeared to enhance chymase enzymaticactivity. All the 3 inhibitors had weak inhibitory actions on tryptase. CONCLUSION: The protease inhibitorstested had relatively good selectivity to either tryptase or chymase.

  1. Proconvertase proteolytic processing of an enzymatically active myeloperoxidase precursor. (United States)

    McCormick, Sally; Nelson, Angela; Nauseef, William M


    activity was retained in the secreted promyeloperoxidase.

  2. Impact of Heavy Metals in Enzymatic Activity of Soils from Hidalgo, Mexico

    Energy Technology Data Exchange (ETDEWEB)

    Reyes-Ortigoza, A. L.; Reyes-Solis, I. E.; Galicia-Palacios, M. S.; Montiel-Arteaga, S.


    The soils from Valle of Mezquital, Hidalgo, Mexico have been irrigated with waste waters from Mexico City for more than 88 years. the present investigation was made in order to know the relationship between heavy metal contents and time of irrigation with waste waters and production of CO{sub 2} and enzymatic activity in soils from Valle Mezquital for knowing the disponibility of nutrients and degradation of soils. (Author)

  3. Acetylation of malate dehydrogenase 1 promotes adipogenic differentiation via activating its enzymatic activity. (United States)

    Kim, Eun Young; Kim, Won Kon; Kang, Hyo Jin; Kim, Jeong-Hoon; Chung, Sang J; Seo, Yeon Soo; Park, Sung Goo; Lee, Sang Chul; Bae, Kwang-Hee


    Acetylation is one of the most crucial post-translational modifications that affect protein function. Protein lysine acetylation is catalyzed by acetyltransferases, and acetyl-CoA functions as the source of the acetyl group. Additionally, acetyl-CoA plays critical roles in maintaining the balance between carbohydrate metabolism and fatty acid synthesis. Here, we sought to determine whether lysine acetylation is an important process for adipocyte differentiation. Based on an analysis of the acetylome during adipogenesis, various proteins displaying significant quantitative changes were identified by LC-MS/MS. Of these identified proteins, we focused on malate dehydrogenase 1 (MDH1). The acetylation level of MDH1 was increased up to 6-fold at the late stage of adipogenesis. Moreover, overexpression of MDH1 in 3T3-L1 preadipocytes induced a significant increase in the number of cells undergoing adipogenesis. The introduction of mutations to putative lysine acetylation sites showed a significant loss of the ability of cells to undergo adipogenic differentiation. Furthermore, the acetylation of MDH1 dramatically enhanced its enzymatic activity and subsequently increased the intracellular levels of NADPH. These results clearly suggest that adipogenic differentiation may be regulated by the acetylation of MDH1 and that the acetylation of MDH1 is one of the cross-talk mechanisms between adipogenesis and the intracellular energy level.

  4. Development of Microreactor Array Chip-Based Measurement System for Massively Parallel Analysis of Enzymatic Activity (United States)

    Hosoi, Yosuke; Akagi, Takanori; Ichiki, Takanori

    Microarray chip technology such as DNA chips, peptide chips and protein chips is one of the promising approaches for achieving high-throughput screening (HTS) of biomolecule function since it has great advantages in feasibility of automated information processing due to one-to-one indexing between array position and molecular function as well as massively parallel sample analysis as a benefit of down-sizing and large-scale integration. Mostly, however, the function that can be evaluated by such microarray chips is limited to affinity of target molecules. In this paper, we propose a new HTS system of enzymatic activity based on microreactor array chip technology. A prototype of the automated and massively parallel measurement system for fluorometric assay of enzymatic reactions was developed by the combination of microreactor array chips and a highly-sensitive fluorescence microscope. Design strategy of microreactor array chips and an optical measurement platform for the high-throughput enzyme assay are discussed.

  5. The dual effects of Maillard reaction and enzymatic hydrolysis on the antioxidant activity of milk proteins. (United States)

    Oh, N S; Lee, H A; Lee, J Y; Joung, J Y; Lee, K B; Kim, Y; Lee, K W; Kim, S H


    The objective of this study was to determine the enhanced effects on the biological characteristics and antioxidant activity of milk proteins by the combination of the Maillard reaction and enzymatic hydrolysis. Maillard reaction products were obtained from milk protein preparations, such as whey protein concentrates and sodium caseinate with lactose, by heating at 55°C for 7 d in sodium phosphate buffer (pH 7.4). The Maillard reaction products, along with untreated milk proteins as controls, were hydrolyzed for 0 to 3h with commercial proteases Alcalase, Neutrase, Protamex, and Flavorzyme (Novozymes, Bagsværd, Denmark). The antioxidant activity of hydrolyzed Maillard reaction products was determined by reaction with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt, their 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity, and the ability to reduce ferric ions. Further characteristics were evaluated by the o-phthaldialdehyde method and sodium dodecyl sulfate-PAGE. The degree of hydrolysis gradually increased in a time-dependent manner, with the Alcalase-treated Maillard reaction products being the most highly hydrolyzed. Radical scavenging activities and reducing ability of hydrolyzed Maillard reaction products increased with increasing hydrolysis time. The combined products of enzymatic hydrolysis and Maillard reaction showed significantly greater antioxidant activity than did hydrolysates or Maillard reaction products alone. The hydrolyzed Maillard reaction products generated by Alcalase showed significantly higher antioxidant activity when compared with the other protease products and the antioxidant activity was higher for the whey protein concentrate groups than for the sodium caseinate groups. These findings indicate that Maillard reaction products, coupled with enzymatic hydrolysis, could act as potential antioxidants in the pharmaceutical, food, and dairy industries.

  6. Methods for determining enzymatic activity comprising heating and agitation of closed volumes

    Energy Technology Data Exchange (ETDEWEB)

    Thompson, David Neil; Henriksen, Emily DeCrescenzo; Reed, David William; Jensen, Jill Renee


    Methods for determining thermophilic enzymatic activity include heating a substrate solution in a plurality of closed volumes to a predetermined reaction temperature. Without opening the closed volumes, at least one enzyme is added, substantially simultaneously, to the closed volumes. At the predetermined reaction temperature, the closed volumes are agitated and then the activity of the at least one enzyme is determined. The methods are conducive for characterizing enzymes of high-temperature reactions, with insoluble substrates, with substrates and enzymes that do not readily intermix, and with low volumes of substrate and enzyme. Systems for characterizing the enzymes are also disclosed.

  7. Identification of an enzymatic activity that hydrolyzes protein-bound ADP-ribose in skeletal muscle. (United States)

    Chang, Y C; Soman, G; Graves, D J


    An enzymatic activity present in high-speed supernatant fluids of rat skeletal muscle was found that catalyzes the release of ADP-ribose from ADP-ribosylated-modified lysozyme. The nature of the product was proved by chromatographic studies and proton nuclear magnetic resonance spectroscopy. The enzyme activity is stimulated by Mg2+, dithioerythritol, and flouride. These results and those published earlier (Soman, G., Mickelson, J.R., Louis, C.F., and Graves, D.J. (1984) Biochem. Biophys. Res. Commun. 120, 973-980) show that ADP-ribosylation is a reversible process in skeletal muscle.

  8. Decipher the dynamic coordination between enzymatic activity and structural modulation at focal adhesions in living cells (United States)

    Lu, Shaoying; Seong, Jihye; Wang, Yi; Chang, Shiou-Chi; Eichorst, John Paul; Ouyang, Mingxing; Li, Julie Y.-S.; Chien, Shu; Wang, Yingxiao


    Focal adhesions (FAs) are dynamic subcellular structures crucial for cell adhesion, migration and differentiation. It remains an enigma how enzymatic activities in these local complexes regulate their structural remodeling in live cells. Utilizing biosensors based on fluorescence resonance energy transfer (FRET), we developed a correlative FRET imaging microscopy (CFIM) approach to quantitatively analyze the subcellular coordination between the enzymatic Src activation and the structural FA disassembly. CFIM reveals that the Src kinase activity only within the microdomain of lipid rafts at the plasma membrane is coupled with FA dynamics. FA disassembly at cell periphery was linearly dependent on this raft-localized Src activity, although cells displayed heterogeneous levels of response to stimulation. Within lipid rafts, the time delay between Src activation and FA disassembly was 1.2 min in cells seeded on low fibronectin concentration ([FN]) and 4.3 min in cells on high [FN]. CFIM further showed that the level of Src-FA coupling, as well as the time delay, was regulated by cell-matrix interactions, as a tight enzyme-structure coupling occurred in FA populations mediated by integrin αvβ3, but not in those by integrin α5β1. Therefore, different FA subpopulations have distinctive regulation mechanisms between their local kinase activity and structural FA dynamics.

  9. Low enzymatic activity haplotypes of the human catechol-O-methyltransferase gene: enrichment for marker SNPs.

    Directory of Open Access Journals (Sweden)

    Andrea G Nackley

    Full Text Available Catechol-O-methyltransferase (COMT is an enzyme that plays a key role in the modulation of catechol-dependent functions such as cognition, cardiovascular function, and pain processing. Three common haplotypes of the human COMT gene, divergent in two synonymous and one nonsynonymous (val(158met position, designated as low (LPS, average (APS, and high pain sensitive (HPS, are associated with experimental pain sensitivity and risk of developing chronic musculoskeletal pain conditions. APS and HPS haplotypes produce significant functional effects, coding for 3- and 20-fold reductions in COMT enzymatic activity, respectively. In the present study, we investigated whether additional minor single nucleotide polymorphisms (SNPs, accruing in 1 to 5% of the population, situated in the COMT transcript region contribute to haplotype-dependent enzymatic activity. Computer analysis of COMT ESTs showed that one synonymous minor SNP (rs769224 is linked to the APS haplotype and three minor SNPs (two synonymous: rs6267, rs740602 and one nonsynonymous: rs8192488 are linked to the HPS haplotype. Results from in silico and in vitro experiments revealed that inclusion of allelic variants of these minor SNPs in APS or HPS haplotypes did not modify COMT function at the level of mRNA folding, RNA transcription, protein translation, or enzymatic activity. These data suggest that neutral variants are carried with APS and HPS haplotypes, while the high activity LPS haplotype displays less linked variation. Thus, both minor synonymous and nonsynonymous SNPs in the coding region are markers of functional APS and HPS haplotypes rather than independent contributors to COMT activity.

  10. Evolution of enzymatic activities and carbon fractions throughout composting of plant waste. (United States)

    Jurado, M M; Suárez-Estrella, F; Vargas-García, M C; López, M J; López-González, J A; Moreno, J


    Many alternatives for the proper disposal of horticultural plant wastes have been studied, and composting is one of the most attractive due to its insignificant environmental impact and low cost. The quality of compost for agronomical use is related to the degree of organic matter maturation and stabilization. Traditional parameters as well as temperature, ratio C/N, cationic exchange capacity, extractable carbon, or evolution of humificated substances have been successfully used to assess compost maturity and stability. However, microorganisms frequently isolated during composting release a wide range of hydrolytic enzymes, whose activity could apparently give interesting information on the rate of decomposition of organic matter and, therefore, on the product stability. The aim of this work was to study the evolution of some important enzymatic activities during composting of agricultural wastes and their comparison with other chemical parameters commonly employed as quality and maturity indexes, to establish a relationship between the degradation intensity of specific organic carbon fractions throughout the process. In this work, the chemical and biochemical parameters of plant wastes were studied along a composting process of 189 days to evaluate their importance as tools for compost characterization. Results showed an intense enzymatic activity during the first 2-3 weeks of composting (bio-oxidative phase), because of the availability of easily decomposable organic compounds. From a biological point of view, a less intense phase was observed between second and third month of composting (mesophilic or cooling phase). Finally, chemical humification parameters were more closely associated with the period between 119 and 189 days (maturation phase). Significant correlations between the enzymatic activities as well as between enzyme activities and other more traditional parameters were also highlighted, indicating that both kind of indexes can be a reliable tool to

  11. Mass Spectrometric Detection of Bacterial Protein Toxins and Their Enzymatic Activity. (United States)

    Kalb, Suzanne R; Boyer, Anne E; Barr, John R


    Mass spectrometry has recently become a powerful technique for bacterial identification. Mass spectrometry approaches generally rely upon introduction of the bacteria into a matrix-assisted laser-desorption time-of-flight (MALDI-TOF) mass spectrometer with mass spectrometric recognition of proteins specific to that organism that form a reliable fingerprint. With some bacteria, such as Bacillus anthracis and Clostridium botulinum, the health threat posed by these organisms is not the organism itself, but rather the protein toxins produced by the organisms. One such example is botulinum neurotoxin (BoNT), a potent neurotoxin produced by C. botulinum. There are seven known serotypes of BoNT, A-G, and many of the serotypes can be further differentiated into toxin variants, which are up to 99.9% identical in some cases. Mass spectrometric proteomic techniques have been established to differentiate the serotype or toxin variant of BoNT produced by varied strains of C. botulinum. Detection of potent biological toxins requires high analytical sensitivity and mass spectrometry based methods have been developed to determine the enzymatic activity of BoNT and the anthrax lethal toxins produced by B. anthracis. This enzymatic activity, unique for each toxin, is assessed with detection of the toxin-induced cleavage of strategically designed peptide substrates by MALDI-TOF mass spectrometry offering unparalleled specificity. Furthermore, activity assays allow for the assessment of the biological activity of a toxin and its potential health risk. Such methods have become important diagnostics for botulism and anthrax. Here, we review mass spectrometry based methods for the enzymatic activity of BoNT and the anthrax lethal factor toxin.

  12. Relationship among antimutagenic, antioxidant and enzymatic activities of methanolic extract from common beans (Phaseolus vulgaris L). (United States)

    Cardador-Martínez, Anaberta; Albores, Arnulfo; Bah, Moustapha; Calderón-Salinas, Victor; Castaño-Tostado, Eduardo; Guevara-González, Ramón; Shimada-Miyasaka, Armando; Loarca-Piña, Guadalupe


    Common beans are rich in phenolic compounds, which can provide health benefits to the consumer. The objective of this work was to study the relationship among antimutagenicity, antioxidant and enzymatic activities of methanolic extract and trolox by principal components multivariate analysis. Antimutagenicity of phenolic compounds present in methanolic extract from the seed coat of common beans (P. vulgaris, Flor de Mayo Bajío cultivar) and trolox against AFB1 mutagenicity were evaluated in the Salmonella typhimurium microsuspension assay. Antioxidant capacity of methanolic extract and trolox were evaluated using beta-carotene and 1,1-diphenyl-2-picryhydrazyl (DPPH) in vitro model assays. Cythrome P450 activity was measured by fluorometric assay. For phenolic extract, trolox and phenolic extract+trolox, the inhibition on AFB1 mutagenicity in tester strain TA100 was 47, 59 and 69%, respectively. While in TA98 was 39, 48 and 68%. The inhibition of phenolic compounds, trolox and phenolic compounds+trolox on cytochrome P450 (CYP450) activity was 48, 59 and 88%, respectively. Correlation analysis showed that phenolic extract and trolox have high antimutagenic and antioxidant activity and also inhibited enzymatic activity. The results suggest that the primary mechanism of action of phenolic compounds in beans against AFB1 mutagenicity may be extra-cellular in the microsuspension assay.

  13. Enzymatic Activity Analysis and Catalytic Essential Residues Identification of Brucella abortus Malate Dehydrogenase

    Directory of Open Access Journals (Sweden)

    Xiangan Han


    Full Text Available Malate dehydrogenase (MDH plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA to L-malate (hereon referred to as MDH activity was analyzed. Michaelis Constant (Km and Maximum Reaction Velocity (Vmax of the reaction were determined to be 6.45×10−3 M and 0.87 mM L−1 min−1, respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu2+, Zn2+, and Pb2+, respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH’s roles in B. abortus metabolism.

  14. Enzymatic Synthesis of Agmatine by Immobilized Escherichia coli Cells with Arginine Decarboxylase Activity

    Institute of Scientific and Technical Information of China (English)

    ZHANG Wei-guo; ZHAO Gen-hai; LIU Jun-zhong; LIU Qian; JIAO Qing-cai


    A new method for the enzymatic synthesis of agmatine by immobilized Escherichia coli cells with arginine decarboxylase(ADC)activity was established and a series of optimal reaction conditions was set down.The arginine decarboxylase showed the maximum activity when the pyridoxal phosphate(PLP)concentration was 50 mmol/L,pH=7 and 45 ℃.The arginine decarboxylase exhibited the maximum production efficiency when the substrate concentration was 100 mmol/L and the reaction time was 15 h.It was also observed that the appropriate concentration of Mg2+,especially at 0.5 mmol/L promoted the arginine decarboxylase activity; Mn2+ had little effect on the arginine decarboxylase activity.The inhibition of Cu2+ and Zn2+ to the arginine decarboxylase activity was significant.The immobilized cells were continuously used 6 times and the average conversion rate during the six-time usage was 55.6%.The immobilized cells exhibited favourable operational stability.After optimization,the maximally cumulative amount of agmatine could be up to 20 g/L.In addition,this method can also catalyze D,L-arginine to agmatine,leaving the pure optically D-arginine simultaneously.The method has a very important guiding significance to the enzymatic preparation of agmatine.

  15. Enzymatic activity analysis and catalytic essential residues identification of Brucella abortus malate dehydrogenase. (United States)

    Han, Xiangan; Tong, Yongliang; Tian, Mingxing; Zhang, Yuxi; Sun, Xiaoqing; Wang, Shaohui; Qiu, Xusheng; Ding, Chan; Yu, Shengqing


    Malate dehydrogenase (MDH) plays important metabolic roles in bacteria. In this study, the recombinant MDH protein (His-MDH) of Brucella abortus was purified and its ability to catalyze the conversion of oxaloacetate (OAA) to L-malate (hereon referred to as MDH activity) was analyzed. Michaelis Constant (Km) and Maximum Reaction Velocity (Vmax) of the reaction were determined to be 6.45 × 10(-3) M and 0.87 mM L(-1)min(-1), respectively. In vitro studies showed that His-MDH exhibited maximal MDH activity in pH 6.0 reaction buffer at 40°C. The enzymatic activity was 100%, 60%, and 40% inhibited by Cu(2+), Zn(2+), and Pb(2+), respectively. In addition, six amino acids in the MDH were mutated to investigate their roles in the enzymatic activity. The results showed that the substitutions of amino acids Arg 89, Asp 149, Arg 152, His 176, or Thr 231 almost abolished the activity of His-MDH. The present study will help to understand MDH's roles in B. abortus metabolism.

  16. Enzymatic activities and effects of mycovirus infection on the virulence of Metarhizium anisopliae in Rhipicephalus microplus. (United States)

    Perinotto, Wendell M S; Golo, Patricia S; Coutinho Rodrigues, Caio J B; Sá, Fillipe A; Santi, Lucélia; Beys da Silva, Walter O; Junges, Angela; Vainstein, Marilene H; Schrank, Augusto; Salles, Cristiane M C; Bittencourt, Vânia R E P


    The present study aimed to evaluate the pathogenic potential of different Metarhizium anisopliae s.l. isolates and to determine whether differences in enzymatic activities of proteases, lipases and chitinases and infection with mycoviruses affect the control of Rhipicephalus microplus achieved by these fungal isolates. Engorged female ticks were exposed to fungal suspensions. The lipolytic and proteolytic activities in the isolates were evaluated using chromogenic substrates and the chitinolytic activity was determined using fluorescent substrates. A gel zymography was performed to determine the approximate size of serine proteases released by M. anisopliae isolates. To detect mycoviral infections, dsRNA was digested using both RNAse A and S1 endonuclease; samples were analyzed on an agarose gel. Four of the five isolates tested were infected with mycovirus; however, the level of control of R. microplus ticks achieved with the only isolate free of infection (isolate CG 347) was low. This finding suggests that mycoviral infection does not affect the virulence of fungi against ticks. Although all five isolates were considered pathogenic to R. microplus, the best tick control and the highest levels of enzymatic activity were achieved with the isolates CG 629 and CG 148. The in vitro activities of lipases, proteases and chitinases produced by M. anisopliae s.l. differed among isolates and may be related to their virulence.

  17. Effects of Cd and Pb pollution on soil enzymatic activities and soil microbiota

    Institute of Scientific and Technical Information of China (English)

    LIU Shuqing; YANG Zhixin; WANG Xiaomin; ZHANG Xiaogui; GAO Rutai; LIU Xia


    Based on a representative sampling method and pot experiment with different concentrations of Cd and Pd,the enzymatic activities(urease,phosphatase,catalase,invertase),population of bacteria,fungus and actinomycete in the soil,the Cd and Pd pollution status of soil samples(from the wastewater-irrigated area of Baoding suburb)were appraised.Unitary linear and nonlinear curve-fitting optimization models were applied in the research,and the relationship between Pb and Cd causing pollution and enzymatic activities of the tested soils were discussed.The research may provide a theoretical basis for protecting the environment in the region of Baiyangdian Lake,Hebei province,prevent soil pollution,and ascertain biochemical indexes,which reflect soil heavy metal pollution levels.The research results indicated that:(1)there was obvious accumulation of Pb and Cd in the wastewater-irrigated area,also the accumulation in wastewater-irrigated soil is more than that in fresh water-irrigated soil,and accumulation on surface layer was more than that in the lower layer.Pb and Cd contents in the tested soils exceeded the standards of soil background values for some major cities at home and abroad and the world soil Cd and Pb contents range.This means that the tested soil had reached a lightly polluted level;(2)there existed an obvious negative correlation between soil enzymatic activities and Pb and Cd contents in wastewaterirrigated soil,where the soil urease and catalase activities decreased obviously with the increase of Pb and Cd contents in soil.Therefore,the urease and catalase can be considered as biochemical indexes that reflect the degree of soil Pb and Cd pollution;(3)the pot experiments indicated that the influence of Cd on soil enzymatic activities was greater than that of Pb.Generally,the effect of Cd on soil phosphatase,urease,catalase is more obvious than that on invertase,while Pb has a more obvious effect on invertase than Cd;(4)pot experiments of triple cropping

  18. Enzymatic activity analysis of MMP-2 and 9 collected by swab from lower limb venous ulcers

    Directory of Open Access Journals (Sweden)

    Flávio Santos da Silva


    Full Text Available Metalloproteinases play a role in repair of venous ulcers of the lower limbs. The great majority of studies of metalloproteinase enzyme activity conducted to date have employed material from biopsies of ulcers. We evaluated the viability of using zymography to measure the enzyme activity of metalloproteinases 2 and 9 in samples of venous ulcer exudate collected on swabs. The method chosen for processing the samples proved viable in terms of its ability to provide adequate protein concentrations for analysis. Using zymography, we observed that the parameters that provided the best results for analysis of gelatinolytic activity were 0.125 to 0.5 μg of total protein content in the gels and enzymatic activation time of 19 hours (at 37 °C. Collection of venous ulcer fluid using swabs proved to be a simple, rapid and effective method for obtaining samples for measurement of gelatinolytic activity with a minimum degree of invasivity.

  19. Magnetically treated water irrigation effect on turnip seed germination, seedling growth and enzymatic activities

    Directory of Open Access Journals (Sweden)

    Zia ul Haq


    Full Text Available Pre-sowing magnetic field seed treatment effects on biological characteristics of vegetables and crops have been studied well. However, studies reporting irrigation with magnetically treated water are scanty. Therefore, the effect of irrigation with magnetically treated water on turnip seed germination, seedling growth and enzymatic activities was evaluated. The tap water was treated at 211 mT for 30, 45 and 60 min and used for irrigation of turnip seed and seedlings. Uniform and healthy turnip seed was sown under randomized complete block design (RCBD. The germination, emergence rate index, vigor index I and vigor index II increased up to 28.33%, 11.54%, 57.59% and 32.26%, respectively. The growth parameters such as seedling lengths, fresh & dry weights, chlorophyll content were also enhanced in response of irrigation with magnetically treated water. The seedlings irrigated with magnetically treated water showed 28.92%, 11.36% and 14.76% higher protein content, alpha amylase and protease activities, respectively vs control. Results revealed that irrigation with magnetically treated water has potential to improve turnip germination, seedling growth and enzymatic activities and this study is also extendable to other vegetables and crops for the improvement of germination and growth.

  20. EPSPS variability, gene expression, and enzymatic activity in glyphosate-resistant biotypes of Digitaria insularis. (United States)

    Galeano, E; Barroso, A A M; Vasconcelos, T S; López-Rubio, A; Albrecht, A J P; Victoria Filho, R; Carrer, H


    Weed resistance to herbicides is a natural phenomenon that exerts selection on individuals in a population. In Brazil, glyphosate resistance was recently detected in Digitaria insularis. The objective of this study was to elucidate mechanisms of weed resistance in this plant, including genetic variability, allelism, amino acid substitutions, gene expression, and enzymatic activity levels. Most of these have not previously been studied in this species. D. insularis DNA sequences were used to analyze genetic variability. cDNA from resistant and susceptible plants was used to identify mutations, alleles, and 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) expression, using real-time quantitative reverse transcription-polymerase chain reaction. In addition, EPSPS activity was measured. We found a decrease in genetic variability between populations related to glyphosate application. Substitutions from proline to threonine and tyrosine to cysteine led to a decrease in EPSPS affinity for the glyphosate. In addition, the EPSPS enzymatic activity was slightly higher in resistant plants, whereas EPSPS gene expression was almost identical in both biotypes, suggesting feedback regulation at different levels. To conclude, our results suggest new molecular mechanisms used by D. insularis to increase glyphosate resistance.

  1. Effect of untreated sewage effluent irrigation on heavy metal content, microbial population and enzymatic activities of soils in Aligarh. (United States)

    Bansal, O P; Singh, Gajraj; Katiyar, Pragati


    The study pertains to the impact of domestic and industrial sewage water irrigation on the chemical, biological and enzymatic activities in alluvial soils of Aligarh District. Results showed that soil enzymatic [dehydogenase (DHA), acid and alkaline phosphatase, urease and catalase] activities in the soils increased up to 14 days of incubation and thereafter inhibited significantly. The enzymatic activity were in the order sewage effluent > partial sewage effluent > ground water irrigated soils. Increase in soil enzymatic activities up to 2nd week of incubation was due to decomposition of organic matter. Maximum inhibition of enzymatic activities, after 14 days of incubation were found in sewage effluent irrigated soils and minimum in ground water irrigated soils. Similar trend was also seen for microbial population. Soil enzymatic activities and microbial population were significantly and positively correlated with soil organic matter. Results also indicated that the microbial population and enzymatic activities in sewage irrigated soils decreased continually with irrigation period. The average concentration of total heavy metals in sewage irrigated soils and partial sewage irrigated soils increased and was 3 and 2 times higher for Zn; 4.5 and 1.7 times higher for Cu; 3.8 and 2.4 times higher for Cr; 5.7 and 3.5 times higher for Pb; 3.5 and 2.2 times higher for Cd and 2.7 and 2.0 times higher for Ni respectively than that of ground water irrigated soils. Results also showed that though total heavy metals concentration increased with period of sewage irrigation but the concentration of diethylene triamine pentaacetic acid (DTPA) extractable heavy metals in partial sewage irrigated and sewage irrigated soils remained almost same, which might be due to deposition of heavy metals in crops grown on the soils.

  2. Non-enzymatic Glycation of Almond Cystatin Leads to Conformational Changes and Altered Activity. (United States)

    Siddiqui, Azad A; Sohail, Aamir; Bhat, Sheraz A; Rehman, Md T; Bano, Bilqees


    The non-enzymatic reaction between proteins and reducing sugars, known as glycation, leads to the formation of inter and intramolecular cross-links of proteins. Stable end products called as advanced Maillard products or advanced glycation end products (AGEs) have received tremendous attention since last decades. It was suggested that the formation of AGEs not only modify the conformation of proteins but also induces altered biological activity. In this study, cystatin purified from almond was incubated with three different sugars namely D-ribose, fructose and lactose to monitor the glycation process. Structural changes induced in cystatin on glycation were studied using UV-visible spectroscopy, fluorescence spectroscopy, CD and FTIR techniques. Glycated cystatin was found to migrate slower on electrophoresis as compared to control cystatin. Biological activity data of glycated cystatin showed that D-ribose was most effective in inducing conformational changes with maximum altered activity.

  3. Antibacterial and enzymatic activity of microbial community during wastewater treatment by pilot scale vermifiltration system. (United States)

    Arora, Sudipti; Rajpal, Ankur; Bhargava, Renu; Pruthi, Vikas; Bhatia, Akansha; Kazmi, A A


    The present study investigated microbial community diversity and antibacterial and enzymatic properties of microorganisms in a pilot-scale vermifiltration system during domestic wastewater treatment. The study included isolation and identification of diverse microbial community by culture-dependent method from a vermifilter (VF) with earthworms and a conventional geofilter (GF) without earthworms. The results of the four months study revealed that presence of earthworms in VF could efficiently remove biochemical oxygen demand (BOD), chemical oxygen demand (COD), total and fecal coliforms, fecal streptococci and other pathogens. Furthermore, the burrowing activity of earthworms promoted the aeration conditions in VF which led to the predominance of the aerobic microorganisms, accounting for complex microbial community diversity. Antibacterial activity of the isolated microorganisms revealed the mechanism behind the removal of pathogens, which is reported for the first time. Specifically, cellulase, amylase and protease activity is responsible for biodegradation and stabilization of organic matter.

  4. Presence of membranous vesicles in cat seminal plasma: ultrastructural characteristics, protein profile and enzymatic activity. (United States)

    Polisca, A; Troisi, A; Minelli, A; Bellezza, I; Fontbonne, A; Zelli, R


    This study sought to verify the presence of membranous vesicles in cat seminal plasma by means of transmission electron microscopy and to identify protein profile and some of the enzymatic activities associated with these particles. The transmission electron microscopy observations showed the existence of different sized vesicular membranous structures of more or less spherical shape. These vesicles were surrounded by single-, double- or multiple-layered laminar membranes. The vesicle diameters ranged from 16.3 to 387.4 nm, with a mean of 116.5 ± 70.7 nm. Enzyme activity determinations showed the presence of dipeptilpeptidase IV, aminopeptidase, alkaline and acid phosphatase. To our knowledge, this is the first report that identifies and characterizes the membranous vesicles in cat seminal plasma. However, further studies are necessary to identify the exact site of production of these membranous vesicles in the cat male genital tract and to determine their specific roles in the reproductive events of this species.

  5. Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases (United States)

    Tarhonskaya, Hanna; Rydzik, Anna M.; Leung, Ivanhoe K. H.; Loik, Nikita D.; Chan, Mun Chiang; Kawamura, Akane; McCullagh, James S. O.; Claridge, Timothy D. W.; Flashman, Emily; Schofield, Christopher J.


    Accumulation of (R)-2-hydroxyglutarate in cells results from mutations to isocitrate dehydrogenase that correlate with cancer. A recent study reports that (R)-, but not (S)-2-hydroxyglutarate, acts as a co-substrate for the hypoxia-inducible factor prolyl hydroxylases via enzyme-catalysed oxidation to 2-oxoglutarate. Here we investigate the mechanism of 2-hydroxyglutarate-enabled activation of 2-oxoglutarate oxygenases, including prolyl hydroxylase domain 2, the most important human prolyl hydroxylase isoform. We observe that 2-hydroxyglutarate-enabled catalysis by prolyl hydroxylase domain 2 is not enantiomer-specific and is stimulated by ferrous/ferric ion and reducing agents including L-ascorbate. The results reveal that 2-hydroxyglutarate is oxidized to 2-oxoglutarate non-enzymatically, likely via iron-mediated Fenton-chemistry, at levels supporting in vitro catalysis by 2-oxoglutarate oxygenases. Succinic semialdehyde and succinate are also identified as products of 2-hydroxyglutarate oxidation. Overall, the results rationalize the reported effects of 2-hydroxyglutarate on catalysis by prolyl hydroxylases in vitro and suggest that non-enzymatic 2-hydroxyglutarate oxidation may be of biological interest. PMID:24594748

  6. Enzymatic and metabolic activities of four anaerobic sludges and their impact on methane production from ensiled sorghum forage. (United States)

    Sambusiti, C; Rollini, M; Ficara, E; Musatti, A; Manzoni, M; Malpei, F


    Biochemical methane potential (BMP) tests were run on ensiled sorghum forage using four inocula (urban, agricultural, mixture of agricultural and urban, granular) and differences on their metabolic and enzymatic activities were also discussed. Results indicate that no significant differences were observed in terms of BMP values (258±14NmLCH4g(-1)VS) with a slightly higher value when agricultural sludge was used as inoculum. Significant differences can be observed among different inocula, in terms of methane production rate. In particular the fastest biomethanization occurred when using the urban sludge (hydrolytic kinetic constant kh=0.146d(-1)) while the slowest one was obtained from the agricultural sludge (kh=0.049d(-1)). Interestingly, positive correlations between the overall enzymatic activities and methane production rates were observed for all sludges, showing that a high enzymatic activity may favour the hydrolysis of complex substrate and accelerate the methanization process of sorghum.

  7. The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC) is enhanced by NPM-ALK

    DEFF Research Database (Denmark)

    Boccalatte, Francesco E; Voena, Claudia; Riganti, Chiara;


    documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampering the methotrexate-mediated transformylase activity inhibition...

  8. Mitochondrial Respiration Chain Enzymatic Activities in the Human Brain: Methodological Implications for Tissue Sampling and Storage. (United States)

    Ronsoni, Marcelo Fernando; Remor, Aline Pertile; Lopes, Mark William; Hohl, Alexandre; Troncoso, Iris H Z; Leal, Rodrigo Bainy; Boos, Gustavo Luchi; Kondageski, Charles; Nunes, Jean Costa; Linhares, Marcelo Neves; Lin, Kátia; Latini, Alexandra Susana; Walz, Roger


    Mitochondrial respiratory chain complexes enzymatic (MRCCE) activities were successfully evaluated in frozen brain samples. Epilepsy surgery offers an ethical opportunity to study human brain tissue surgically removed to treat drug resistant epilepsies. Epilepsy surgeries are done with hemodynamic and laboratory parameters to maintain physiology, but there are no studies analyzing the association among these parameters and MRCCE activities in the human brain tissue. We determined the intra-operative parameters independently associated with MRCCE activities in middle temporal neocortex (Cx), amygdala (AMY) and head of hippocampus (HIP) samples of patients (n = 23) who underwent temporal lobectomy using multiple linear regressions. MRCCE activities in Cx, AMY and HIP are differentially associated to trans-operative mean arterial blood pressure, O2 saturation, hemoglobin, and anesthesia duration to time of tissue sampling. The time-course between the last seizure occurrence and tissue sampling as well as the sample storage to biochemical assessments were also associated with enzyme activities. Linear regression models including these variables explain 13-17 % of MRCCE activities and show a moderate to strong effect (r = 0.37-0.82). Intraoperative hemodynamic and laboratory parameters as well as the time from last seizure to tissue sampling and storage time are associated with MRCCE activities in human samples from the Cx, AMYG and HIP. Careful control of these parameters is required to minimize confounding biases in studies using human brain samples collected from elective neurosurgery.

  9. 2-Bromopalmitate reduces protein deacylation by inhibition of acyl-protein thioesterase enzymatic activities.

    Directory of Open Access Journals (Sweden)

    Maria P Pedro

    Full Text Available S-acylation, the covalent attachment of palmitate and other fatty acids on cysteine residues, is a reversible post-translational modification that exerts diverse effects on protein functions. S-acylation is catalyzed by protein acyltransferases (PAT, while deacylation requires acyl-protein thioesterases (APT, with numerous inhibitors for these enzymes having already been developed and characterized. Among these inhibitors, the palmitate analog 2-brompalmitate (2-BP is the most commonly used to inhibit palmitoylation in cells. Nevertheless, previous results from our laboratory have suggested that 2-BP could affect protein deacylation. Here, we further investigated in vivo and in vitro the effect of 2-BP on the acylation/deacylation protein machinery, with it being observed that 2-BP, in addition to inhibiting PAT activity in vivo, also perturbed the acylation cycle of GAP-43 at the level of depalmitoylation and consequently affected its kinetics of membrane association. Furthermore, 2-BP was able to inhibit in vitro the enzymatic activities of human APT1 and APT2, the only two thioesterases shown to mediate protein deacylation, through an uncompetitive mechanism of action. In fact, APT1 and APT2 hydrolyzed both the monomeric form as well as the micellar state of the substrate palmitoyl-CoA. On the basis of the obtained results, as APTs can mediate deacylation on membrane bound and unbound substrates, this suggests that the access of APTs to the membrane interface is not a necessary requisite for deacylation. Moreover, as the enzymatic activity of APTs was inhibited by 2-BP treatment, then the kinetics analysis of protein acylation using 2-BP should be carefully interpreted, as this drug also inhibits protein deacylation.

  10. The diversity, extracellular enzymatic activities and photoprotective compounds of yeasts isolated in Antarctica

    Directory of Open Access Journals (Sweden)

    Aline B. M Vaz


    Full Text Available The diversity of yeasts collected from different sites in Antarctica (Admiralty Bay, King George Island and Port Foster Bay and Deception Island and their ability to produce extracellular enzymes and mycosporines were studied. Samples were collected during the austral summer season, between November 2006 and January 2007, from the rhizosphere of Deschampsia antarctica, ornithogenic (penguin guano soil, soil, marine and lake sediments, marine water and freshwater from lakes. A total of 89 isolates belonging to the following genera were recovered: Bensingtonia, Candida, Cryptococcus, Debaryomyces, Dioszegia, Exophiala, Filobasidium, Issatchenkia (Pichia, Kodamaea, Leucosporidium, Leucosporidiella, Metschnikowia, Nadsonia, Pichia, Rhodotorula, and Sporidiobolus, and the yeast-like fungi Aureobasidium, Leuconeurospora and Microglossum. Cryptococcus victoriae was the most frequently identified species. Several species isolated in our study have been previously reported to be Antarctic psychophilic yeasts, including Cr. antarcticus, Cr. victoriae, Dioszegia hungarica and Leucosporidium scottii. The cosmopolitan yeast species A. pullulans, C. zeylanoides, D. hansenii, I. orientalis, K. ohmeri, P. guilliermondii, Rh. mucilaginosa, and S. salmonicolor were also isolated. Five possible new species were identified. Sixty percent of the yeasts had at least one detectable extracellular enzymatic activity. Cryptococcus antarcticus, D. aurantiaca, D. crocea, D. hungarica, Dioszegia sp., E. xenobiotica, Rh. glaciales, Rh. laryngis, Microglossum sp. 1 and Microglossum sp. 2 produced mycosporines. Of the yeast isolates, 41.7% produced pigments and/or mycosporines and could be considered adapted to survive in Antarctica. Most of the yeasts had extracellular enzymatic activities at 4ºC and 20ºC, indicating that they could be metabolically active in the sampled substrates.

  11. Evaluation of the enzymatic activity of Trichoderma inhamatum (BOL-12QD as possible biocontroller

    Directory of Open Access Journals (Sweden)

    García-Espejo Cielo Noemí


    Full Text Available It is known that Trichoderma spp. acts as a natural biocontroller of pathogen fungi, is for this reason, that this research studies the potential of its hydrolytic enzyme activity. In this article, first we determined that the speed of growth of Trichoderma inhamatum cepa BOL-12QD is 9 hours. Later, we proposed a simple and sensitive method based in the use of basal media (BM with coloidal chitin as the only carbon resource and supplemented with bromocresol purple for the qualitative determination of chitinase activi-ty. On the other hand, it was determined the celullolytic and proteolytic activities of Trichoderma in-hamatum cepa BOL-12QD and it was observed that agitation, type and concentration of sustrate are determinant factors in enzymatic production. Then, we evaluated the cellulolytic activity of Trichoderma inhamatum cepa BOL-12QD in agitation and stationary using carboxymethylcellulose (CMC as sustrate, finding that using a 2% of sustrate the highest activity is registered at 8 days of incubation in agitation with a value of 99.23 IU/L. In relation to the results at stationary the optimal value is at the fourth day with a value of 92.76 IU/L. The protease activity it was determined taking in consideration variables of agitation and stationary, using different types and concentration of sustrate at 2%, 4% y 6% (w/v of meat extract, 1%, 3%, 5% (w/v of jelly and 1%, 2%, 4% (w/v of casein. The highest protease activity was obtained at the end of the sev-enth day with an enzymatic activity of 3075.45 IU/L at stationary using a concentration of 6% (w/v of meat extract, and using jelly at 3% (w/v at stationary it was found an activity of 568.36 IU/L on the tenth day and in agitation a value of 547.27 IU/L was reached on the twelveth day, while using casein at 1% (w/v at stationary an activity of 407.06 IU/L is reached in the fifth day, and in agitation at 4% (w/v of casein a value of 547.27 IU/L is obtained on the twelfth day, while using

  12. Enzymatic modification enhances the protective activity of citrus flavonoids against alcohol-induced liver disease. (United States)

    Park, Ho-Young; Choi, Hee-Don; Eom, Hyojin; Choi, Inwook


    Alcoholic liver disease (ALD) can be developed by a prolonged or large intake of alcohol in a short period of time. ALD is considered as a leading cause for a liver injury in modern dietary life. This study was aimed to investigate the effects of orally administrated citrus flavonoids (CFs) and their enzymatically modified ones (EM-CFs) to prevent ALD. Hesperidin and narirutin were extracted from peels of Citrus unshiu by ultra-sonication and purified further. These CFs were modified enzymatically through glycosylation and de-rhamnosylation by the actions of cyclodextrin glucanotransferase (CGTase) and hesperidinase, respectively. CFs and EM-CFs were fed to ICR mouse along with ethanol for 8 weeks, and changes in lipid contents, lipid peroxidation, GSH, antioxidant enzymes activity and proinflammatory cytokines in hepatic tissues were observed. Administration of CFs and EM-CFs along with alcohol significantly suppressed increases in prognostic parameters of a hepatocellular injury. Especially, EM-CFs fed groups maintained malondialdehyde, GSH levels and catalase activity in hepatic tissues close to those of the normal diet fed group. Abrupt increases in proinflammatory cytokines such as IκB-α, TNF-α, IL-1β and IL-6 in hepatocytes due to a chronic alcohol uptake were significantly suppressed by co-administration of EM-CFs. These results indicate that although the administration of CFs can alleviate ALD through preventing excessive lipid formation, protecting the antioxidant system and suppressing induction of inflammation in hepatocytes, their effectiveness can be further improved by glycosylation and de-rhamnosylation.

  13. Linking Microbial Enzymatic Activities and Functional Diversity of Soil around Earthworm Burrows and Casts. (United States)

    Lipiec, Jerzy; Frąc, Magdalena; Brzezińska, Małgorzata; Turski, Marcin; Oszust, Karolina


    The aim of this work was to evaluate the effect of earthworms (Lumbricidae) on the enzymatic activity and microbial functional diversity in the burrow system [burrow wall (BW) 0-3 mm, transitional zone (TZ) 3-7 mm, bulk soil (BS) > 20 mm from the BW] and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential) was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the BW or casts than in BS and TZ. Conversely, acid phosphomonoesterase had the largest value in the BS. Average Well Color Development in both the TZ and the BS (0.98-0.94 A590 nm) were more than eight times higher than in the BWs and casts. The lowest richness index in the BS (15 utilized substrates) increased by 86-113% in all the other compartments. The PC1 in principal component analysis mainly differentiated the BWs and the TZ. Utilization of all substrate categories was the lowest in the BS. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem.

  14. Linking microbial enzymatic activities and functional diversity of soil around earthworm burrows and casts

    Directory of Open Access Journals (Sweden)

    Jerzy Lipiec


    Full Text Available The aim of this work was to evaluate the effect of earthworms (Lumbricidae on the enzymatic activity and microbial functional diversity in the burrow system (burrow wall 0–3 mm, transitional zone 3–7 mm, bulk soil >20 mm from the burrow wall and cast aggregates of a loess soil under a pear orchard. The dehydrogenase, β-glucosidase, protease, alkaline phosphomonoesterase, and acid phosphomonoesterase enzymes were assessed using standard methods. The functional diversity (catabolic potential was assessed using the Average Well Color Development and Richness Index following the community level physiological profiling from Biolog Eco Plates. All measurements were done using soil from each compartment immediately after in situ sampling in spring. The enzymatic activites including dehydrogenase, protease, β-glucosidase and alkaline phosphomonoesterase were appreciably greater in the burrow wall or casts than in bulk soil and transitional zone. Conversely, acid phosphomonoesterase had the largest value in the bulk soil. Average Well Color Development in both the transitional zone and the bulk soil (0.98-0.94 A590nm were more than eight times higher than in the burrow walls and casts. The lowest richness index in the bulk soil (15 utilized substrates increased by 86-113% in all the other compartments. The PC1 in principal component analysis (PCA mainly differentiated the burrow walls and the transitional zone. Utilization of all substrate categories was the lowest in the bulk soil. The PC2 differentiated the casts from the other compartments. The enhanced activity of a majority of the enzymes and increased microbial functional diversity in most earthworm-influenced compartments make the soils less vulnerable to degradation and thus increases the stability of ecologically relevant processes in the orchard ecosystem.

  15. Conditional iron and pH-dependent activity of a non-enzymatic glycolysis and pentose phosphate pathway. (United States)

    Keller, Markus A; Zylstra, Andre; Castro, Cecilia; Turchyn, Alexandra V; Griffin, Julian L; Ralser, Markus


    Little is known about the evolutionary origins of metabolism. However, key biochemical reactions of glycolysis and the pentose phosphate pathway (PPP), ancient metabolic pathways central to the metabolic network, have non-enzymatic pendants that occur in a prebiotically plausible reaction milieu reconstituted to contain Archean sediment metal components. These non-enzymatic reactions could have given rise to the origin of glycolysis and the PPP during early evolution. Using nuclear magnetic resonance spectroscopy and high-content metabolomics that allowed us to measure several thousand reaction mixtures, we experimentally address the chemical logic of a metabolism-like network constituted from these non-enzymatic reactions. Fe(II), the dominant transition metal component of Archean oceanic sediments, has binding affinity toward metabolic sugar phosphates and drives metabolism-like reactivity acting as both catalyst and cosubstrate. Iron and pH dependencies determine a metabolism-like network topology and comediate reaction rates over several orders of magnitude so that the network adopts conditional activity. Alkaline pH triggered the activity of the non-enzymatic PPP pendant, whereas gentle acidic or neutral conditions favored non-enzymatic glycolytic reactions. Fe(II)-sensitive glycolytic and PPP-like reactions thus form a chemical network mimicking structural features of extant carbon metabolism, including topology, pH dependency, and conditional reactivity. Chemical networks that obtain structure and catalysis on the basis of transition metals found in Archean sediments are hence plausible direct precursors of cellular metabolic networks.

  16. A novel technical approach for the measurement of individual ACAT-1 and ACAT-2 enzymatic activity in the testis. (United States)

    Chen, Li; Lafond, Julie; Pelletier, R-Marc


    Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is implicated in the esterification of cholesterol when the latter is present at concentrations exceeding metabolic demands. Thus, ACAT contributes to the maintenance of cholesterol homeostasis which in testis is essential for the production of fertile gametes. However, the role of individual isoform of the enzyme in the maintenance of cholesterol homeostasis in the gonads has not been addressed yet because approaches to measure the enzymatic activity of each isoform were lacking. Here, we used the selective ACAT-1 inhibitor, K-604, to measure the individual enzymatic activity of ACAT-1 and ACAT-2 in enriched fractions of mouse seminiferous tubules. K-604 inhibited adult mouse ACAT-1 much more than ACAT-2 with IC(50) values of 100 and 1,000 microM, respectively, in the tubules. Next, the inhibitor concentration (100 microM) that inhibits the activity of ACAT-1 but not the activity of ACAT-2 was determined and applied to measure ACAT-1 and ACAT-2 enzymatic activities in mouse seminiferous tubule-enriched fractions. ACAT-2 activity reached 2173 CPMB/200 microg protein, while ACAT-1 enzymatic activity was 713 CPMB/200 microg proteins in the tubules. We also compared the effect of another inhibitor Manassantin B with K-604. Increasing the concentration (0-1,000 microM) of Manassantin B resulted in the inhibition of the activity of both ACAT-1 and ACAT-2. The results show that only K-604 is a useful tool to determine the individual ACAT-1 and ACAT-2 enzymatic activities in the seminiferous tubules.

  17. Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

    Directory of Open Access Journals (Sweden)

    R Adam Smith


    Full Text Available R Adam Smith, Sarah L Sewell, Todd D GiorgioDepartment of Biomedical Engineering, Vanderbilt University, Nashville, TN, USAAbstract: The development and in vitro performance of a modular nanoscale system capable of specific structural modification by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fluorescent species including a quantum dot base particle and fluorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fluorometric analysis. The model proximity-activated nanoparticles studied here exhibit significant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7 at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specific to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specific modification is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure.Keywords: quantum dot, MMP-7, protease, proximity activated nanoparticle

  18. Glutamic acid-149 is important for enzymatic activity of yeast inorganic pyrophosphatase. (United States)

    Gonzalez, M A; Cooperman, B S


    Modification of Saccharomyces cerevisiae inorganic pyrophosphatase (PPase) with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide is known to lead to a loss of enzymatic activity, the rate of which is decreased in the presence of ligands binding to the active site [Cooperman, B. S., & Chiu, N. Y. (1973) Biochemistry 12, 1676-1682; Heitman, P., & Uhlig, H. J. (1974) Acta Biol. Med. Ger. 32, 565-594]. In this work we show that, when such inactivation is carried out in the presence of [14C]glycine ethyl ester (GEE), GEE is covalently incorporated into PPase, incorporation into the most highly labeled tryptic peptide is site-specific, as evidenced by the reduction of such incorporation in the presence of the active site ligands Zn2+ and Pi, the extent of formation of this specifically labeled peptide correlates with the fractional loss of PPase activity, and the specifically labeled peptide corresponds to residues 145-153 and the position of incorporation within this peptide is Glu-149. The significance of our findings for the location of the active site and for the catalytic mechanism of PPase is briefly considered in the light of the 3-A X-ray crystallographic structure of Arutyunyun and his colleagues [Arutyunyun, E. G., et al. (1981) Dokl. Akad. Nauk SSSR 258, 1481-1485; Kuranova, I. P., et al. (1983) Bioorg. Khim. 9, 1611-1919; Terzyan, S. S., et al. (1984) Bioorg. Khim. 10, 1469-1482].

  19. The effect of chlorsulfurone and MCPB-Na on the enzymatic activity of microorganisms

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    Filimon Marioara Nicoleta


    Full Text Available herbicides, have a broad spectrum effect on weeds, in relatively low doses and with a much reduced toxicity on livestock. In this study were used two herbicides: dacsulfuron with the active substance chlorsulfuron (0.005 - 0.035 μg/g soil and butoxone with the active substance MCPB-Na (0.005 - 0.035 mg/L/g soil. The samples were collected from a depth of 0-20 cm from chernozem soil. The effect of herbicide was estimated by measuring the activity of catalase, actual and potential dehydrogenase, urease and cellulase activities. All samples being incubated for 10 days at 27°C using Sapp medium for isolation and study of cellulosolytic bacteria. The inhibitory effect of the tested herbicides was the most intense for the urease and dehydrogenase enzymatic activities. The most resistant cellulosolytic bacteria to the effects of dacsulfuron were Cellfalcicula fusca, Cellfalcicula viridis, Cellvibrio fulvus and Fuseaux veris and for butoxone Cellfalcicula mucosa, C. viridis and C. fulvus.

  20. Urease immobilized polymer hydrogel: Long-term stability and enhancement of enzymatic activity. (United States)

    Kutcherlapati, S N Raju; Yeole, Niranjan; Jana, Tushar


    A method has been developed in which an enzyme namely urease was immobilized inside hydrogel matrix to study the stability and enzymatic activity in room temperature (∼27-30°C). This urease coupled hydrogel (UCG) was obtained by amine-acid coupling reaction and this procedure is such that it ensured the wider opening of mobile flap of enzyme active site. A systematic comparison of urea-urease assay and the detailed kinetic data clearly revealed that the urease shows activity for more than a month when stored at ∼27-30°C in case of UCG whereas it becomes inactive in case of free urease (enzyme in buffer solution). The aqueous microenvironment inside the hydrogel, unusual morphological features and thermal behaviour were believed to be the reasons for unexpected behaviour. UCG displayed enzyme activity at basic pH and up to 60°C. UCG showed significant enhancement in activity against thermal degradation compared to free urease. In summary, this method is a suitable process to stabilize the biomacromolecules in standard room temperature for many practical uses.

  1. Enzymatic Activity of the Mycelium Compared with Oospore Development During Infection of Pea Roots by Aphanomyces euteiches

    DEFF Research Database (Denmark)

    Kjøller, Rasmus; Rosendahl, Søren


    To describe the disease cycle of the root pathogen Aphanomyces euteiches, enzymatic activity in the mycelium was compared with the development of oospores in pea roots. Plants were inoculated with two zoospore concentrations to achieve different disease levels. Hyphae were stained for fungal alka....... In these plants, oospores were found in 90% of the root length, while the enzymatic activity of the mycelium was low. This suggests that the pathogen mycelium is only active on living plants and does not grow saprophytically on dead plant material.......To describe the disease cycle of the root pathogen Aphanomyces euteiches, enzymatic activity in the mycelium was compared with the development of oospores in pea roots. Plants were inoculated with two zoospore concentrations to achieve different disease levels. Hyphae were stained for fungal...... concentration, the enzymatic activity of the pathogen mycelium peaked 10 to 14 days after inoculation, when oospore formation was initiated. Oospore formation was associated with a gradual increase in disease symptoms. At the last harvest, plants inoculated with the higher zoospore concentration had died...

  2. Effect of enzymatic mash treatment and storage on phenolic composition, antioxidant activity, and turbidity of cloudy apple juice. (United States)

    Oszmiański, Jan; Wojdylo, Aneta; Kolniak, Joanna


    The effects of different commercial enzymatic mash treatments on yield, turbidity, color, and polyphenolic and sediment of procyanidins content of cloudy apple juice were studied. Addition of pectolytic enzymes to mash treatment had positive effect on the production of cloud apple juices by improving polyphenolic contents, especially procyanidins and juice yields (68.3% in control samples to 77% after Pectinex Yield Mash). As summary of the effect of enzymatic mash treatment, polyphenol contents in cloudy apple juices significantly increased after Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL maceration were applied but no effect was observed after Pectinex Ultra-SPL I Panzym XXL use, compared to the control samples. The content of polymeric procyanidins represented 50-70% of total polyphenols, but in the present study, polymeric procyanidins were significantly lower in juices than in fruits and also affected by enzymatic treatment (Pectinex AFP L-4 and Panzym Yield Mash) compared to the control samples. The enzymatic treatment decreased procyanidin content in most sediment with the exception of Pectinex Smash XXL and Pectinex AFP L-4. Generally in samples that were treated by pectinase, radical scavenging activity of cloudy apple juices was increased compared to the untreated reference samples. The highest radical scavenging activity was associated with Pectinex Yield Mash, Pectinex Smash XXL, and Pectinex XXL enzyme and the lowest activity with Pectinex Ultra SP-L and Pectinex APFL-4. However, in the case of enzymatic mash treatment cloudy apple juices showed instability of turbidity and low viscosity. These results must be ascribed to the much higher hydrolysis of pectin by enzymatic preparation which is responsible for viscosity. During 6 months of storage at 4 degrees C small changes in analyzed parameters of apple juices were observed.

  3. Membrane phospholipid augments cytochrome P4501a enzymatic activity by modulating structural conformation during detoxification of xenobiotics.

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    Manik C Ghosh

    Full Text Available Cytochrome P450 is a superfamily of membrane-bound hemoprotein that gets involved with the degradation of xenobiotics and internal metabolites. Accumulated body of evidence indicates that phospholipids play a crucial role in determining the enzymatic activity of cytochrome P450 in the microenvironment by modulating its structure during detoxification; however, the structure-function relationship of cytochrome P4501A, a family of enzymes responsible for degrading lipophilic aromatic hydrocarbons, is still not well defined. Inducibility of cytochrome P4501A in cultured catfish hepatocytes in response to carbofuran, a widely used pesticide around the world, was studied earlier in our laboratory. In this present investigation, we observed that treating catfish with carbofuran augmented total phospholipid in the liver. We examined the role of phospholipid on the of cytochrome P4501A-marker enzyme which is known as ethoxyresorufin-O-deethylase (EROD in the context of structure and function. We purified the carbofuran-induced cytochrome P4501A protein from catfish liver. Subsequently, we examined the enzymatic activity of purified P4501A protein in the presence of phospholipid, and studied how the structure of purified protein was influenced in the phospholipid environment. Membrane phospholipid appeared to accelerate the enzymatic activity of EROD by changing its structural conformation and thus controlling the detoxification of xenobiotics. Our study revealed the missing link of how the cytochrome P450 restores its enzymatic activity by changing its structural conformation in the phospholipid microenvironment.

  4. Interaction with Single-stranded DNA-binding Protein Stimulates Escherichia coli Ribonuclease HI Enzymatic Activity. (United States)

    Petzold, Christine; Marceau, Aimee H; Miller, Katherine H; Marqusee, Susan; Keck, James L


    Single-stranded (ss) DNA-binding proteins (SSBs) bind and protect ssDNA intermediates formed during replication, recombination, and repair reactions. SSBs also directly interact with many different genome maintenance proteins to stimulate their enzymatic activities and/or mediate their proper cellular localization. We have identified an interaction formed between Escherichia coli SSB and ribonuclease HI (RNase HI), an enzyme that hydrolyzes RNA in RNA/DNA hybrids. The RNase HI·SSB complex forms by RNase HI binding the intrinsically disordered C terminus of SSB (SSB-Ct), a mode of interaction that is shared among all SSB interaction partners examined to date. Residues that comprise the SSB-Ct binding site are conserved among bacterial RNase HI enzymes, suggesting that RNase HI·SSB complexes are present in many bacterial species and that retaining the interaction is important for its cellular function. A steady-state kinetic analysis shows that interaction with SSB stimulates RNase HI activity by lowering the reaction Km. SSB or RNase HI protein variants that disrupt complex formation nullify this effect. Collectively our findings identify a direct RNase HI/SSB interaction that could play a role in targeting RNase HI activity to RNA/DNA hybrid substrates within the genome.

  5. Ultrasound-assisted enzymatic extraction and antioxidant activity of polysaccharides from pumpkin (Cucurbita moschata). (United States)

    Wu, Hao; Zhu, Junxiang; Diao, Wenchao; Wang, Chengrong


    An efficient ultrasound-assisted enzymatic extraction (UAEE) of Cucurbita moschata polysaccharides (CMCP) was established and the CMCP antioxidant activities were studied. The UAEE operating parameters (extraction temperature, ultrasonic power, pH, and liquid-to-material ratio) were optimized using the central composite design (CCD) and the mass transfer kinetic study in UAEE procedure was used to select the optimal extraction time. Enzymolysis and ultrasonication that were simultaneously conducted was selected as the UAEE synergistic model and the optimum extraction conditions with a maximum polysaccharide yield of 4.33 ± 0.15% were as follows: extraction temperature, 51.5 °C; ultrasonic power, 440 W; pH, 5.0; liquid-to-material ratio, 5.70:1 mL/g; and extraction time, 20 min. Evaluation of the antioxidant activity in vitro suggested that CMCP has good potential as a natural antioxidant used in the food or medicine industry because of their high reducing power and positive radical scavenging activity for DPPH radical.


    Directory of Open Access Journals (Sweden)

    Glevitzky Mirel


    Full Text Available This paper aims at determining the main quality parameters of grain mill products in the Transylvania region, also studying and emphasizing the enzymatic activity of flour. Determination of quality characteristics of grain mill products entails establishing physical, chemical and sensory parameters and assessing them against the limits imposed by law. Analysis was performed on samples formed by mixing basic medium extracted from different batches. Incremental size, sampling tools, how to extract them, the training sample and laboratory environments, packaging and labeling of samples were performed according to STAS 1068 69. Determination of the fall (Falling Number, an empirical test that relies on the ability of endogenous ?-amylase to reduce viscosity of the treated warm flour suspension is used, large scale milling and bakery industry to predict and assess the Baking quality of flour. In sprouted wheat, characterised by a low Falling number, dextrin produced by the action of ?-amylase leads to a sticky bread core. Experiments suggest that the values fall turnover (FN does not shrink in direct proportion to the percentage of germinating seeds. Amylolytic activity depends on the stage of sprouting of grains. Lack of ?-amylase activity can be corrected by adding malt grain ?-amylase or fungal ?-amylase.

  7. Enzymatic activity of endophytic bacterial isolates of Jacaranda decurrens Cham. (Carobinha-do-campo

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    Aysha Jussara Ivonilde Carrim


    Full Text Available The objectives of this work was to isolate endophytic bacteria from Jacaranda decurrens Cham. and screening of some enzymes of biotechnological interest. Ten (10 bacterial species were isolated and identified from the leaves and steams. All the isolates presented enzymatic activity, which was ranked as follows: proteolytic (60% and amilolytic activity (60%, lipolytic (40%, esterasic (40%. However, cellulolytic and pectinolytic activities were not detected. This is the first report on the isolation and identification of endophytic bacteria from Jacaranda decurrens Cham.O objetivo deste trabalho foi isolar bactérias endofíticas de Jacaranda decurrens Cham. e selecionar algumas enzimas de interesse biotecnológico. Dez espécies de bactérias foram isoladas e identificadas de caule e folhas. Todos os isolados de apresentaram atividade enzimática, e a maior predominância foi de atividade proteolítica (60% e atividade amilolítica (60%, seguidas atividades lipolítica (40% e esterásica (40%. Contudo, as atividades celulolítica e pectinolítica não foram detectadas. Este é o primeiro relato de isolamento e identificação de bactérias presentes em Jacaranda decurrens Cham.

  8. New Biocatalyst with Multiple Enzymatic Activities for Treatment of Complex Food Wastewaters

    Directory of Open Access Journals (Sweden)

    Olga Senko


    Full Text Available The cells of filamentous fungus R. oryzae entrapped in the polyvinyl alcohol cryogelare capable of producing various extracellular hydrolytic enzymes (proteases, amylases, lipases and are used for the treatment of complex wastewaters of food industry. Five types of media simulating the wastewater of various food enterprises were treated under batch conditions for 600 h. Fats containing mostly residues of unsaturated fatty acids, as well as casein, glucose, sucrose, starch, soybean flour and various salts were the main components of the treated wastewaters. The immobilized cells concurrently possessed lipolytic, amylolytic and proteolytic activities. The level of each enzymatic activity depended on the wastewater content. The physiological state of immobilized cells was monitored by bioluminescent method. The intracellular adenosine triphosphate (ATP concentration determined in the granules with immobilized cells was high enough and almost constant for all the period of biocatalyst application confirming thereby the active metabolic state of the cells. The study of mechanical strength of biocatalyst granules allowed revealing the differences in the values of modulus of biocatalyst elasticity at the beginning and at the end of its use for the wastewater treatment. The decrease in chemical oxygen demand of the tested media after their processing by immobilized biocatalyst was 68–79 % for one working cycle.

  9. Metabolic activation of pyrrolizidine alkaloids: insights into the structural and enzymatic basis. (United States)

    Ruan, Jianqing; Yang, Mengbi; Fu, Peter; Ye, Yang; Lin, Ge


    Pyrrolizidine alkaloids (PAs) are natural toxins widely distributed in plants. The toxic potencies of different PAs vary significantly. PAs are mono- or diesters of necine acids with a necine base. On the basis of the necine bases, PAs are classified into three types: retronecine-type, otonecine-type, and platynecine-type. Hepatotoxic PAs contain an unsaturated necine base. PAs exert hepatotoxicity through metabolic activation by hepatic cytochromes P450s (CYPs) to generate reactive intermediates which form pyrrole-protein adducts. These adducts provide a mechanism-based biomarker to assess PA toxicity. In the present study, metabolic activation of 12 PAs from three structural types was investigated first in mice to demonstrate significant variations in hepatic metabolic activation of different PAs. Subsequently, the structural and enzymatic factors affecting metabolic activation of these PAs were further investigated by using human liver microsomes and recombinant human CYPs. Pyrrole-protein adducts were detected in the liver and blood of mice and the in vitro systems treated with toxic retronecine-type and otonecine-type PAs having unsaturated necine bases but not with a platynecine-type PA containing a saturated necine base. Retronecine-type PAs produced more pyrrole-protein adducts than otonecine-type PAs with similar necine acids, demonstrating that the structure of necine base affected PA toxic potency. Among retronecine-type PAs, open-ring diesters generated the highest amount of pyrrole-protein adducts, followed by macrocyclic diesters, while monoesters produced the least. Only CYP3A4 and CYP3A5 activated otonecine-type PAs, while all 10 CYPs studied showed the ability to activate retronecine-type PAs. Moreover, the contribution of major CYPs involved also varied significantly among retronecine-type PAs. In conclusion, our findings provide a scientific basis for predicting the toxicities of individual PAs in biological systems based on PA structural

  10. Enzymatically synthesized inorganic polymers as morphogenetically active bone scaffolds: application in regenerative medicine. (United States)

    Wang, Xiaohong; Schröder, Heinz C; Müller, Werner E G


    In recent years a paradigm shift in understanding of human bone formation has occurred that starts to change current concepts in tissue engineering of bone and cartilage. New discoveries revealed that fundamental steps in biomineralization are enzyme driven, not only during hydroxyapatite deposition, but also during initial bioseed formation, involving the transient deposition and subsequent transformation of calcium carbonate to calcium phosphate mineral. The principal enzymes mediating these reactions, carbonic anhydrase and alkaline phosphatase, open novel targets for pharmacological intervention of bone diseases like osteoporosis, by applying compounds acting as potential activators of these enzymes. It is expected that these new findings will give an innovation boost for the development of scaffolds for bone repair and reconstruction, which began with the use of bioinert materials, followed by bioactive materials and now leading to functional regenerative tissue units. These new developments have become possible with the discovery of the morphogenic activity of bioinorganic polymers, biocalcit, bio-polyphosphate and biosilica that are formed by a biogenic, enzymatic mechanism, a driving force along with the development of novel rapid-prototyping three-dimensional (3D) printing methods and bioprinting (3D cell printing) techniques that may allow a fabrication of customized implants for patients suffering in bone diseases in the future.

  11. Proteomic analysis of tylosin-resistant Mycoplasma gallisepticum reveals enzymatic activities associated with resistance. (United States)

    Xia, Xi; Wu, Congming; Cui, Yaowen; Kang, Mengjiao; Li, Xiaowei; Ding, Shuangyang; Shen, Jianzhong


    Mycoplasma gallisepticum is a significant pathogenic bacterium that infects poultry, causing chronic respiratory disease and sinusitis in chickens and turkeys, respectively. M. gallisepticum infection poses a substantial economic threat to the poultry industry, and this threat is made worse by the emergence of antibiotic-resistant strains. The mechanisms of resistance are often difficult to determine; for example, little is known about antibiotic resistance of M. gallisepticum at the proteome level. In this study, we performed comparative proteomic analyses of an antibiotic (tylosin)-resistant M. gallisepticum mutant and a susceptible parent strain using a combination of two-dimensional differential gel electrophoresis and nano-liquid chromatography-quadrupole-time of flight mass spectrometry. Thirteen proteins were identified as differentially expressed in the resistant strain compared to the susceptible strain. Most of these proteins were related to catalytic activity, including catalysis that promotes the formylation of initiator tRNA and energy production. Elongation factors Tu and G were over-expressed in the resistant strains, and this could promote the binding of tRNA to ribosomes and catalyze ribosomal translocation, the coordinated movement of tRNA, and conformational changes in the ribosome. Taken together, our results indicate that M. gallisepticum develops resistance to tylosin by regulating associated enzymatic activities.

  12. Phenylpropanoid Glycoside Analogues: Enzymatic Synthesis, Antioxidant Activity and Theoretical Study of Their Free Radical Scavenger Mechanism (United States)

    López-Munguía, Agustín; Hernández-Romero, Yanet; Pedraza-Chaverri, José; Miranda-Molina, Alfonso; Regla, Ignacio; Martínez, Ana; Castillo, Edmundo


    Phenylpropanoid glycosides (PPGs) are natural compounds present in several medicinal plants that have high antioxidant power and diverse biological activities. Because of their low content in plants (less than 5% w/w), several chemical synthetic routes to produce PPGs have been developed, but their synthesis is a time consuming process and the achieved yields are often low. In this study, an alternative and efficient two-step biosynthetic route to obtain natural PPG analogues is reported for the first time. Two galactosides were initially synthesized from vanillyl alcohol and homovanillyl alcohol by a transgalactosylation reaction catalyzed by Kluyveromyces lactis β-galactosidase in saturated lactose solutions with a 30%–35% yield. To synthesize PPGs, the galactoconjugates were esterified with saturated and unsaturated hydroxycinnamic acid derivatives using Candida antarctica Lipase B (CaL-B) as a biocatalyst with 40%–60% yields. The scavenging ability of the phenolic raw materials, intermediates and PPGs was evaluated by the 2,2-diphenyl-1-picrylhydrazyl radical (DPPH•) method. It was found that the biosynthesized PPGs had higher scavenging abilities when compared to ascorbic acid, the reference compound, while their antioxidant activities were found similar to that of natural PPGs. Moreover, density functional theory (DFT) calculations were used to determine that the PPGs antioxidant mechanism proceeds through a sequential proton loss single electron transfer (SPLET). The enzymatic process reported in this study is an efficient and versatile route to obtain PPGs from different phenylpropanoid acids, sugars and phenolic alcohols. PMID:21674039

  13. Haematology, genotoxicity, enzymatic activity and histopathology as biomarkers of metal pollution in the shrew Crocidura russula

    Energy Technology Data Exchange (ETDEWEB)

    Sanchez-Chardi, A. [Departament Biologia Animal, Facultat de Biologia, Universitat Barcelona, Av. Diagonal 645, 08028 Barcelona (Spain); Servei de Microscopia, Facultat de Ciencies, Ed. C, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona (Spain)], E-mail:; Marques, C.C.; Gabriel, S.I. [Centro de Biologia Ambiental, Departamento de Biologia Animal, Faculdade de Ciencias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa (Portugal); Capela-Silva, F. [Centro de Investigacao em Ciencias e Tecnologias da Saude, Departamento de Biologia, Universidade de Evora, 7002-552 Evora (Portugal); Cabrita, A.S. [Centro de Histofisiologia, Instituto de Patologia Experimental, Faculdade de Medicina, Universidade de Coimbra, 3004-504 Coimbra (Portugal); Lopez-Fuster, M.J.; Nadal, J. [Departament Biologia Animal, Facultat de Biologia, Universitat Barcelona, Av. Diagonal 645, 08028 Barcelona (Spain); Mathias, M.L. [Centro de Biologia Ambiental, Departamento de Biologia Animal, Faculdade de Ciencias, Universidade de Lisboa, Campo Grande, 1749-016 Lisboa (Portugal)


    Haematological (WBC, RBC, Hgb and Hct) and genotoxicity (MNT) parameters, hepatic enzymatic activities (GST, GPx and GR), and a histopathological evaluation of liver, kidneys and gonads were assessed as general biomarkers of metal pollution in the shrew Crocidura russula inhabiting a pyrite mining area. Specimens exposed to metals presented a few significant alterations when compared with reference animals: GST activity decreased; micronuclei increased; and evident liver alterations related to metal exposure were observed. On the basis of all the parameters studied, age was an important factor that partly explained the observed variation, whereas sex was the least important factor. Significant correlations were also found between heavy metal concentrations and biomarkers evaluated, demonstrating the great influence of these metals in the metabolic alterations. To the best of our knowledge, these data constitute the first measurements of a battery of biomarkers in shrews from a mine site and are among the few available for insectivorous mammals. - Metals from an abandoned pyrite mine produce alterations in haematological parameters, GST, MNT, and histopathology in shrews.

  14. Highly efficient recombinant production and purification of streptococcal cysteine protease streptopain with increased enzymatic activity. (United States)

    Lane, Michael D; Seelig, Burckhard


    Streptococcus pyogenes produces the cysteine protease streptopain (SpeB) as a critical virulence factor for pathogenesis. Despite having first been described seventy years ago, this protease still holds mysteries which are being investigated today. Streptopain can cleave a wide range of human proteins, including immunoglobulins, the complement activation system, chemokines, and structural proteins. Due to the broad activity of streptopain, it has been challenging to elucidate the functional results of its action and precise mechanisms for its contribution to S. pyogenes pathogenesis. To better study streptopain, several expression and purification schemes have been developed. These methods originally involved isolation from S. pyogenes culture but were more recently expanded to include recombinant Escherichia coli expression systems. While substantially easier to implement, the latter recombinant approach can prove challenging to reproduce, often resulting in mostly insoluble protein and poor purification yields. After extensive optimization of a wide range of expression and purification conditions, we applied the autoinduction method of protein expression and developed a two-step column purification scheme that reliably produces large amounts of purified soluble and highly active streptopain. This method reproducibly yielded 3 mg of streptopain from 50 mL of expression culture at >95% purity, with an activity of 5306 ± 315 U/mg, and no remaining affinity tags or artifacts from recombinant expression. This improved method therefore enables the facile production of the important virulence factor streptopain at higher yields, with no purification scars that might bias functional studies, and with an 8.1-fold increased enzymatic activity compared to previously described procedures.


    Directory of Open Access Journals (Sweden)

    K. S. Daljit Singh


    Full Text Available Substantial clearance of forests and conversion of forest into various land use types contribute to deterioration of soil fertility and associated nutrients loss. Soils from natural and rehabilitated forest in Chikus Forest Reserve and also enrichment planting forest and secondary forest of Tapah Hill Forest Reserve, Perak, Malaysia were selected in order to assess the influence of land use change on biological properties. This study was carried out to provide fundamental information on soil biological properties and also to compare the differences between natural forest, mono-rehabilitated forest, mixed planting forest and natural regenerated forest (secondary forest. Six subplots (20×20 m were established at each study plot and soil samples were collected at the depths of 0-15 cm (topsoil and 15-30 cm (subsoil. Soil microbial population was determined using spread-plate technique. Fluorescein Diacetate (FDA hydrolysis was used to assess the amount of microbial enzymatic activity for each forest plot. Soil Microbial Biomass C (MBC and N (MBN were extracted using chloroform fumigation extraction technique and the amount of MBC was determined by dichromate digestion, while MBN via Kjeldahl digestion technique. Soil acidity was determined by pH meter and moisture content was elucidated using gravimetric method. The levels of microbial population of bacterial and fungal at natural significantly exceeded the corresponding values of rehabilitated and secondary forest. However, microbial population is much higher in rehabilitated forest of Tapah Hill compared to that of secondary forest and also Chikus Forest Reserve planted forest which proves that rehabilitation activities do help increase the level of microbial community in the soils. Longer period of time after planting as in enrichment planting compared to mono planting of S. leprosula plantation showed that restoring and recovery of the planted forest needed time. Deforestation activities

  16. Molecular Docking Studies and Anti-enzymatic Activities of Thai Mango Seed Kernel Extract Against Snake Venoms

    Directory of Open Access Journals (Sweden)

    Patchreenart Saparpakorn


    Full Text Available The ethanolic extract from seed kernels of Thai mango (MSKE (Mangifera indica L. cv. ‘Fahlun’ (Anacardiaceae and its major phenolic principle (pentagalloyl glucopyranose exhibited dose-dependent inhibitory effects on enzymatic activities of phospholipase A2 (PLA2, hyaluronidase and L-amino acid oxidase (LAAO of Calloselasma rhodostoma (CR and Naja naja kaouthia (NKvenoms by in vitro tests. The anti-hemorrhagic and anti-dermonecrotic activities of MSKE against both venoms were clearly supported by in vivo tests. Molecular docking studies indicated that the phenolic molecules of the MSKE could selectively bind to the active sites or their proximity, or modify conserved residues that are critical for the catalysis of PLA2, and selectively bind to the LAAO binding pocket of both CR and NK venoms and thereby inhibit their enzymatic activities. The results imply a potential use of MSKE against snake venoms.

  17. Molecular docking studies and anti-enzymatic activities of Thai mango seed kernel extract against snake venoms. (United States)

    Leanpolchareanchai, Jiraporn; Pithayanukul, Pimolpan; Bavovada, Rapepol; Saparpakorn, Patchreenart


    The ethanolic extract from seed kernels of Thai mango (MSKE) (Mangifera indica L. cv. 'Fahlun') (Anacardiaceae) and its major phenolic principle (pentagalloyl glucopyranose) exhibited dose-dependent inhibitory effects on enzymatic activities of phospholipase A(2) (PLA(2)), hyaluronidase and L-amino acid oxidase (LAAO) of Calloselasma rhodostoma (CR) and Naja naja kaouthia (NK)venoms by in vitro tests. The anti-hemorrhagic and anti-dermonecrotic activities of MSKE against both venoms were clearly supported by in vivo tests. Molecular docking studies indicated that the phenolic molecules of the MSKE could selectively bind to the active sites or their proximity, or modify conserved residues that are critical for the catalysis of PLA(2), and selectively bind to the LAAO binding pocket of both CR and NK venoms and thereby inhibit their enzymatic activities. The results imply a potential use of MSKE against snake venoms.

  18. Purification, enzymatic properties, and active site environment of a novel manganese(III)-containing acid phosphatase. (United States)

    Sugiura, Y; Kawabe, H; Tanaka, H; Fujimoto, S; Ohara, A


    A new manganese-containing acid phosphatase has been isolated and crystallized from sweet potato tubers. The pure enzyme contains one atom of manganese per Mr = 110,000 polypeptide and shows phosphatase activity toward various phosphate substrates. The pH optimum of the enzyme was 5.8 and the enzyme activity was inhibited by Cu2+, Zn2+, Hg2+, AsO43-, and MoO42-. This stable metalloenzyme is red-violet in color with an intense absorption band at 515 nm (epsilon - 2460). Our electronic, circular dichroism, and electron spin resonance findings strongly indicate that the Mn-valence state of the native enzyme is trivalent. When the Mn-enzyme is excited by the 5145 A line of Ar+ laser, prominent Raman lines at 1230, 1298, 1508, and 1620 cm-1 were detected. This Raman spectrum can probably be interpreted in terms of internal vibration of a coordinated tyrosine phenolate anion. The tryptophan-modified enzyme showed a positive Raman band at 370 cm-1, which is preferentially assigned to a Mn(III)-S streching mode. The modification of the Mn-enzyme by N-bromosuccinimide led to a large decrease in the fluorescence intensity of 335 nm which was dominated by its tryptophan residues within a considerable hydrophobic environment. The acid phosphatase activity was significantly decreased by the tryptophan modification. With respect to the active site donor sets, the Mn(III)-containing acid phosphatase is distinctly different from the Zn(II)-containing alkaline phosphatase. Of interest is also the appreciable similarity of some enzymatic and spectroscopic properties between the present enzyme and uteroferrin.

  19. Direct calorimetric analysis of the enzymatic activity of yeast cytochrome c oxidase. (United States)

    Morin, P E; Freire, E


    The kinetic and thermodynamic parameters associated with the enzymatic reaction of yeast cytochrome c oxidase with its biological substrate, ferrocytochrome c, have been measured by using a titration microcalorimeter to monitor directly the rate of heat production or absorption as a function of time. This technique has allowed determination of both the energetics and the kinetics of the reaction under a variety of conditions within a single experiment. Experiments performed in buffer systems of varying ionization enthalpies allow determination of the net number of protons absorbed or released during the course of the reaction. For cytochrome c oxidase the intrinsic enthalpy of reaction was determined to be -16.5 kcal/mol with one (0.96) proton consumed for each ferrocytochrome c molecule oxidized. Activity measurements at salt concentrations ranging from 0 to 200 mM KCl in the presence of 10 mM potassium phosphate, pH 7.40, and 0.5 mM EDTA display a biphasic dependence of the electron transferase activity upon ionic strength with a peak activity observed near 50 mM KCl. The ionic strength dependence was similar for both detergent-solubilized and membrane-reconstituted cytochrome c oxidase. Despite the large ionic strength dependence of the kinetic parameters, the enthalpy measured for the reaction was found to be independent of ionic strength. Additional experiments involving direct transfer of the enzyme from low to high salt conditions produced negligible enthalpy changes that remained constant within experimental error throughout the salt concentrations studied (0-200 mM KCl). These results indicate that the salt effect on the enzyme activity is of entropic origin and further suggest the absence of a major conformational change in the enzyme due to changes in ionic strength.(ABSTRACT TRUNCATED AT 250 WORDS)

  20. Modulation of Enzymatic Activities of Dual Functional Peroxiredoxin by Gamma Irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Hong, Sung Hyun; Lee, Seung Sik; Park, Chul Hong; Chung, Byung Yeoup [Korea Atomic Energy Research Institute, Jeongeup (Korea, Republic of)


    Recently, enzymes have frequently been used as catalysts in various bio-industrial, commercial, and pharmaceutical applications, because they are more stable, more efficient, and less toxic than the synthetic catalysts. However, one of their major disadvantages is their low thermostability, which leads the researchers to develop new forms of industrially important enzymes with increased resistance to inactivation and aggregation. This study describes a strategy for modifying the molecular chaperone activity of peroxiredoxin (Prx) by using gamma irradiation. Prxs are a ubiquitous family of antioxidant enzymes. Upon oxidation of their peroxidatic Cys, the molecules undergo a structural conversion from a low-molecular-weight (LMW) species acting as a peroxidase to a high-molecular-weight (HMW) complex functioning as a chaperone. In the present study, we examined the effect of gamma irradiation on PP1084 with respect to its protein structure and enzymatic function. The use of gamma irradiation as a physical treatment can increase the cohesive strength of the protein by forming cross-links. The aims of the present work were (1) to improve the chaperone activity of PP1084 by gamma irradiation, (2) to identify the 'optimal' intensity of gamma irradiation, and (3) to investigate the influence of gamma irradiation on protein hydrophobicity as related to chaperone function. Following PP1084 treatment with 30 kGy gamma irradiation, the PP1084 chaperone activity enhanced by about 3-4-fold compared with nonirradiated PP1084, while the peroxidase activity decreased. Ongoing research efforts are addressing the physical modifications of PP1084 protein by gamma irradiation


    Directory of Open Access Journals (Sweden)

    L. M. Kurtasova


    Full Text Available We have observed forty-four patients with metastatic renal cancer before and after interferon therapy. Immune markers of of peripheral blood lymphocytes were determined by flow cytometry. Activity of NAD (P-dependent dehydrogenase in blood lymphocytes was studied by means of bioluminescence technique. Changes of immune marker profiles and enzymatic activities of peripheral blood lymphocytes were found in patients with renal cancer after a course of interferon therapy.

  2. Molecular Docking Studies and Anti-enzymatic Activities of Thai Mango Seed Kernel Extract Against Snake Venoms


    Patchreenart Saparpakorn; Pimolpan Pithayanukul; Jiraporn Leanpolchareanchai; Rapepol Bavovada


    The ethanolic extract from seed kernels of Thai mango (MSKE) (Mangifera indica L. cv. ‘Fahlun’) (Anacardiaceae) and its major phenolic principle (pentagalloyl glucopyranose) exhibited dose-dependent inhibitory effects on enzymatic activities of phospholipase A2 (PLA2), hyaluronidase and L-amino acid oxidase (LAAO) of Calloselasma rhodostoma (CR) and Naja naja kaouthia (NK)venoms by in vitro tests. The anti-hemorrhagic and anti-dermonecrotic activities of MSKE against both venoms were clearly ...

  3. Enzymatic Activity of the Mycelium Compared with Oospore Development During Infection of Pea Roots by Aphanomyces euteiches. (United States)

    Kjøller, R; Rosendahl, S


    ABSTRACT To describe the disease cycle of the root pathogen Aphanomyces euteiches, enzymatic activity in the mycelium was compared with the development of oospores in pea roots. Plants were inoculated with two zoospore concentrations to achieve different disease levels. Hyphae were stained for fungal alkaline phosphatase activity in the roots. Additionally, enzyme activity was measured after electrophoresis of an A. euteiches-specific glucose-6-phosphate isozyme. Development of oospores in the roots was measured after staining the oospores with trypan blue. In plants inoculated with the higher zoospore concentration, the enzymatic activity of the pathogen mycelium peaked 10 to 14 days after inoculation, when oospore formation was initiated. Oospore formation was associated with a gradual increase in disease symptoms. At the last harvest, plants inoculated with the higher zoospore concentration had died. In these plants, oospores were found in 90% of the root length, while the enzymatic activity of the mycelium was low. This suggests that the pathogen mycelium is only active on living plants and does not grow saprophytically on dead plant material.

  4. The Use of Adenovirus Dodecahedron in the Delivery of an Enzymatic Activity in the Cell

    Directory of Open Access Journals (Sweden)



    Full Text Available Penton-dodecahedron (Pt-Dd derived from adenovirus type 3 is a symmetric complex of pentameric penton base plus fiber which can be produced in the baculovirus system at a high concentration. The size of Pt-Dd is smaller than the virus, but this virus-like particle (VLP has the major proteins recognized by specific receptors on the surface of almost all types of cell. In this study, by direct observation with fluorescence microscopy on a fixed and living cell, the intracellular trafficking and localization of Pt-Dd labeled with fluorescence dyes in the cytoplasm of HeLa Tub-GFP showed a rapid internalization characteristic. Subsequently, the linkage of horseradish peroxidase (HRP with Pt-Dd as the vector demonstrated an efficient system to deliver this enzyme into the cell without interfering its enzymatic activity as shown by biochemical and cellular experiments. These results were supported by additional studies using Bs-Dd or free form of the HRP used as the control. Overall, this study strengthens the potential role of Pt-Dd as an alternative vector for delivering therapeutic agents.

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

    DEFF Research Database (Denmark)

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


    . The basic design of the sensor relies on two DNA strands that hybridize to form a hairpin structure with a fluorophore-quencher pair. The quencher moiety is released from the sensor upon reaction with human topoisomerase I thus enabling real-time optical measurement of enzymatic activity. The sensor......Sensors capable of quantitative real-time measurements may present the easiest and most accurate way to study enzyme activities. Here we present a novel DNA-based sensor for specific and quantitative real-time measurement of the enzymatic activity of the essential human enzyme, topoisomerase I...... is specific for topoisomerase I even in raw cell extracts and presents a simple mean of following enzyme kinetics using standard laboratory equipment such as a qPCR machine or fluorimeter. Human topoisomerase I is a well-known target for the clinically used anti-cancer drugs of the camptothecin family...

  6. How metal substitution affects the enzymatic activity of catechol-o-methyltransferase.

    Directory of Open Access Journals (Sweden)

    Manuel Sparta

    Full Text Available Catechol-O-methyltransferase (COMT degrades catecholamines, such as dopamine and epinephrine, by methylating them in the presence of a divalent metal cation (usually Mg(II, and S-adenosyl-L-methionine. The enzymatic activity of COMT is known to be vitally dependent on the nature of the bound metal: replacement of Mg(II with Ca(II leads to a complete deactivation of COMT; Fe(II is slightly less than potent Mg(II, and Fe(III is again an inhibitor. Considering the fairly modest role that the metal plays in the catalyzed reaction, this dependence is puzzling, and to date remains an enigma. Using a quantum mechanical / molecular mechanical dynamics method for extensive sampling of protein structure, and first principle quantum mechanical calculations for the subsequent mechanistic study, we explicate the effect of metal substitution on the rate determining step in the catalytic cycle of COMT, the methyl transfer. In full accord with experimental data, Mg(II bound to COMT is the most potent of the studied cations and it is closely followed by Fe(II, whereas Fe(III is unable to promote catalysis. In the case of Ca(II, a repacking of the protein binding site is observed, leading to a significant increase in the activation barrier and higher energy of reaction. Importantly, the origin of the effect of metal substitution is different for different metals: for Fe(III it is the electronic effect, whereas in the case of Ca(II it is instead the effect of suboptimal protein structure.

  7. Detection of anti-oxidant enzymatic activities and purification of glutathione transferases from Angiostrongylus cantonensis. (United States)

    Morassutti, Alessandra L; Pinto, Paulo M; Dutra, Bibiana K; Oliveira, Guendalina Turcato; Ferreira, Henrique B; Graeff-Teixeira, Carlos


    There are several anti-oxidant enzyme families that play pivotal roles in facilitating the survival of parasites. Glutathione transferases (GSTs) are members of the anti-oxidant family that can detoxify a broad range of exogenous or endogenous compounds including reactive oxidative species. GSTs have been studied as vaccine candidates, immunodiagnostic markers and as treatment targets. Helminths of the genus Angiostrongylus live inside arteries of vertebrates and two main species are associated with accidental human infections: Angiostrongylus costaricensis adult worms live inside the mesenteric arteries and larvae of Angiostrongylus cantonensis become trapped in the central nervous system vasculature. Since the interactions between angiostrongylid nematodes and their vertebrate hosts are poorly understood, this study characterized the anti-oxidant enzymatic activities of A. cantonensis from female worms by collecting excreted and secreted (ES) and total extract (TE) molecules. Catalase (CAT) and superoxide dismutase (SOD) activities were found both in the ES and TE while glutathione peroxidase (GPX) and GST were found only in the TE. GSTs were purified by glutathione agarose affinity column (AcGST) and the pool of eluted GSTs was analyzed by mass spectrometry (LC-MS/MS) and de novo sequencing (Masslynx software). Sequences from two peptides (AcGSTpep1 and AcGSTpep2) present high identity to the N-terminal and C-terminal from sigma class GSTs of nematodes. It is known that these GST enzymes are associated with host immune regulation. Furthermore, understanding the role of parasite-derived anti-oxidant molecules is important in understanding host-parasite interactions.

  8. Effect of combined pollution by heavy metals on soil enzymatic activities in areas polluted by tailings from Pb-Zn-Ag mine

    Institute of Scientific and Technical Information of China (English)

    CHEN Cheng-li; LIAO Min; HUANG Chang-yong


    Some enzymatic activities were determined in the areas polluted by tailings from Tiantai Pb-Zn-Ag Mine in Zhejiang Province of China. The results indicated the soil enzymatic activities decreased significantly with increase of concentrations of heavy metals or the distance away from mining tailing center, especially dehydrogenase and urease activities. Multivariate regression analysis between heavy metal contents and soil enzymatic activities indicated that single dehydrogenase activity was very significantly correlated to combined effect of soil heavy metals in mine area. Moreover, single urease, protease and acid phosphatase activities were significantly related to the combined effect of heavy metals. The results suggest it is feasible to use soil enzymatic activities to indicate the pollution situation by combined heavy metals in the soil of mine area.

  9. Amendment application in a multi-contaminated mine soil: Effects on soil enzymatic activities and ecotoxicological characteristics


    Manzano, Rebeca; Esteban, Elvira; Peñalosa, Jesús Manuel; Alvarenga, Paula M L F


    The final publication is available at Springer via Several amendments were tested on soils obtained from an arsenopyrite mine, further planted with Arrhenatherum elatius and Festuca curvifolia, in order to assess their ability to improve soil's ecotoxicological characteristics. The properties used to assess the effects were: soil enzymatic activities (dehydrogenase, β-glucosidase, acid phosphatase, urease, protease and cellulase), terrestrial bio...

  10. Positive regulation of the enzymatic activity of gastric H(+),K(+)-ATPase by sialylation of its β-subunit. (United States)

    Fujii, Takuto; Watanabe, Midori; Shimizu, Takahiro; Takeshima, Hiroshi; Kushiro, Keiichiro; Takai, Madoka; Sakai, Hideki


    The gastric proton pump (H(+),K(+)-ATPase) consists of a catalytic α-subunit (αHK) and a glycosylated β-subunit (βHK). βHK glycosylation is essential for the apical trafficking and stability of αHK in gastric parietal cells. Here, we report the properties of sialic acids at the termini of the oligosaccharide chains of βHK. Sialylation of βHK was found in LLC-PK1 cells stably expressing αHK and βHK by staining of the cells with lectin-tagged fluorescent polymeric nanoparticles. This sialylation was also confirmed by biochemical studies using sialic acid-binding lectin beads and an anti-βHK antibody. The sialic acids of βHK are cleaved enzymatically by neuraminidase (sialidase) and nonenzymatically by an acidic solution (pH5). Interestingly, the enzymatic activity of H(+),K(+)-ATPase was significantly decreased by cleavage of the sialic acids of βHK. In contrast, βHK was not sialylated in the gastric tubulovesicles prepared from the stomach of fed hogs. The H(+),K(+)-ATPase activity in these tubulovesicles was not significantly altered by neuraminidase. Importantly, the sialylation of βHK was observed in the gastric samples prepared from the stomach of famotidine (a histamine H2 receptor antagonist)-treated rats, but not histamine (an acid secretagogue)-treated rats. The enzymatic activity of H(+),K(+)-ATPase in the samples of the famotidine-treated rats was significantly higher than in the histamine-treated rats. The effects of famotidine were weakened by neuraminidase. These results indicate that βHK is sialylated at neutral or weakly acidic pH, but not at acidic pH, suggesting that the sialic acids of βHK positively regulate the enzymatic activity of αHK.

  11. The synthetic curcuminoid BHMC restores endotoxin-stimulated HUVEC dysfunction:Specific disruption on enzymatic activity of p38 MAPK. (United States)

    Tham, Chau Ling; Hazeera Harith, Hanis; Wai Lam, Kok; Joong Chong, Yi; Singh Cheema, Manraj; Roslan Sulaiman, Mohd; Hj Lajis, Nordin; Ahmad Israf, Daud


    2,6-bis-(4-hydroxyl-3-methoxybenzylidine)cyclohexanone (BHMC) has been proven to selectively inhibit the synthesis of proinflammatory mediators in lipopolysaccharide-induced U937 monocytes through specific interruption of p38 Mitogen-Activated Protein Kinase enzymatic activity and improves the survival rate in a murine lethal sepsis model. The present study addressed the effects of BHMC upon lipopolysaccharide-induced endothelial dysfunction in human umbilical vein endothelial cells to determine the underlying mechanisms. The cytotoxicity effect of BHMC on HUVEC were determined by MTT assay. The effects of BHMC on endothelial dysfunction induced by lipopolysaccharide such as endothelial hyperpermeability, monocyte-endothelial adhesion, transendothelial migration, up-regulation of adhesion molecules and chemokines were evaluated. The effects of BHMC at transcriptional and post-translational levels were determined by Reverse Transcriptase-Polymerase Chain Reaction and Western Blots. The mode of action of BHMC was dissected by looking into the activation of Nuclear Factor-kappa B and Mitogen-Activated Protein Kinases. BHMC concentration-dependently reduced endothelial hyperpermeability, leukocyte-endothelial cell adhesion and monocyte transendothelial migration through inhibition of the protein expression of adhesion molecules (Intercellular Adhesion Molecule-1 and Vascular Cell Adhesion Molecule-1) and secretion of chemokines (Monocyte Chemotactic Protein-1) at the transcriptional level. BHMC restored endothelial dysfunction via selective inhibition of p38 Mitogen-Activated Protein Kinase enzymatic activity which indirectly prevents the activation of Nuclear Factor-kappaB and Activator Protein-1 transcription factors. These findings further support earlier observations on the inhibition of BHMC on inflammatory events through specific disruption of p38 Mitogen-Activated Protein Kinase enzymatic activity and provide new insights into the inhibitory effects of BHMC on

  12. Effects of partial decerebration and hypophyseal allograft in the thymus of chicken embryos: thymostimulin localization and enzymatic activities

    Directory of Open Access Journals (Sweden)

    M Aita


    Full Text Available Changes in chicken embryo thymus after partial decerebration (including the hypophysis and hypophyseal allograft were investigated. Chicken embryos were partially decerebrated at 36-40 hr of incubation and on day 12 received a hypophyseal allograft from 18-day-old donor embryos. The embryonic thymuses were collected on day 18 and examined with histological methods, tested for the anti-thymostimulin- like immune-reaction, and for histoenzymatic activities and compared with normal and sham-operated embryos at the same age. After partial decerebration, the thymic cortical and medullary compartments diminished markedly in size. Anti-thymostimulin, succinic dehydrogenase and ATPase enzymatic activities tested, yielded negative reactions. In partially decerebrated hypophyseal allografted embryos, the same thymic compartments improved and anti-thymostimulin-like immune-reaction and enzymatic activities partially recovered. These findings confirmed the key role of hypophysis in thymic ontogenic development and provided new information in metabolic enzymatic pathways and synthesis of a thymostimulin-like substance in the thymus

  13. Enzymatic conversion of diacetylated sophoroselipid into acetylated glucoselipid: surface-active properties of novel bolaform biosurfactants. (United States)

    Imura, Tomohiro; Masuda, Yuma; Minamikawa, Hiroyuki; Fukuoka, Tokuma; Konishi, Masaaki; Morita, Tomotake; Sakai, Hideki; Abe, Masahiko; Kitamoto, Dai


    Sophoroselipids (SL) are bolaform biosurafactants which are abundantly produced by microorganisms from renewable resources. In this study, four kinds of bolaform biosurfactants were produced, and these derivatives were chemoenzymatically synthesized from "acid form" diacetylated SL (SLdiAc), which are preferentially produced by Candida floricola TM 1502 which was newly found in our group. The effects of the structure of sugar moiety on their surface-active properties were investigated by surface tension measurement. After microbial production of SLdiAc from oleic acid/glucose, SLdiAc was converted into acetylated glucoselipid (GLAc). Among twelve species of glucosidases, pectinase and pectolyase including polygalacturonase were found to cleave the beta-1,2-glycosidic linked disaccharide, especially pectolyase produced GLAc effectively at 40 degrees C and pH 4.0. The structure of the major component of purified GLAc was assigned as 17- [(beta-D-glucopyranosyl)oxy]-cis-9-octadecenoate 6'-acetate by using NMR analyses, MALDI-TOF/MS and GC-MS. Glucoselipid (GL) without acetyl group was also enzymatically converted from SL obtained from alkaline hydrolysis of SLdiAc. Interestingly, the estimated CMC values of SLdiAc, SL, GLAc, GL indicated almost the same values despite their difference in hydrophilic structure. Although the difference in monosaccaride and disaccharide also did not affect gammaCMC, the presence of acetyl group on sugar moiety was found to lower the gammaCMC value slightly, suggesting that the acetyl group on produced bolaform biosurfactant is likely to play more important role to reduce the free energy of air/water interface.

  14. Enhanced enzymatic activity of glycerol-3-phosphate dehydrogenase from the cryophilic Saccharomyces kudriavzevii. (United States)

    Oliveira, Bruno M; Barrio, Eladio; Querol, Amparo; Pérez-Torrado, Roberto


    During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes and we observed a higher expression of GPD1 gene in S. kudriavzevii compared to S. cerevisiae in micro-vinification conditions. We observed higher enzymatic activity of Gpd1p in S. kudriavzevii in response to osmotic and cold stress. Also, we determined that S. kudriavzevii Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. Finally, we evaluated the glycerol production with S. cerevisiae, S. kudriavzevii or a recombinant Gpd1p variant in the same background and observed that the S. kudriavzevii enzyme produced increased glycerol levels at 12 or 28°C. This suggests that glycerol is increased in S. kudriavzevii mainly due to increased V max of the Gpd1p enzyme. All these differences indicate that S. kudriavzevii has changed the metabolism to promote the branch of the glycolytic pathway involved in glycerol production to adapt to low temperature environments and maintain the NAD(+)/NADH ratio in alcoholic fermentations. This knowledge is industrially relevant due to the potential use, for example, of S. cerevisiae-S. kudriavzevii hybrids in the wine industry where glycerol content is an important quality parameter.

  15. New eutectic ionic liquids for lipase activation and enzymatic preparation of biodiesel. (United States)

    Zhao, Hua; Baker, Gary A; Holmes, Shaletha


    The enzymatic preparation of biodiesel has been hampered by the lack of suitable solvents with desirable properties such as high lipase compatibility, low cost, low viscosity, high biodegradability, and ease of product separation. Recent interest in using ionic liquids (ILs) as advanced reaction media has led to fast reaction rates and high yields in the enzymatic synthesis of biodiesel. However, conventional (i.e., cation-anion paired) ILs based on imidazolium and other quaternary ammonium salts remain too expensive for wide application at industrial scales. In this study, we report on newly-synthesized eutectic ILs derived from choline acetate or choline chloride coupled with biocompatible hydrogen-bond donors, such as glycerol. These eutectic solvents have favorable properties including low viscosity, high biodegradability, and excellent compatibility with Novozym(®) 435, a commercial immobilized Candida antarctica lipase B. Furthermore, in a model biodiesel synthesis system, we demonstrate high reaction rates for the enzymatic transesterification of Miglyol(®) oil 812 with methanol, catalyzed by Novozym(®) 435 in choline acetate/glycerol (1:1.5 molar ratio). The high conversion (97%) of the triglyceride obtained within 3 h, under optimal conditions, suggests that these novel eutectic solvents warrant further exploration as potential media in the enzymatic production of biodiesel.

  16. An antiapoptotic role for telomerase RNA in human immune cells independent of telomere integrity or telomerase enzymatic activity. (United States)

    Gazzaniga, Francesca S; Blackburn, Elizabeth H


    Telomerase is a ribonucleoprotein complex that adds telomeric DNA to the ends of linear chromosomes. It contains two core canonical components: the essential RNA component, hTR, which provides the template for DNA synthesis, and the reverse transcriptase protein component, hTERT. Low telomerase activity in circulating peripheral blood mononuclear cells has been associated with a variety of diseases. It is unknown, however, whether telomerase, in addition to its long-term requirement for telomere maintenance, is also necessary for short-term immune cell proliferation and survival. We report that overexpression of enzymatically inactive hTR mutants protected against dexamethasone-induced apoptosis in stimulated CD4 T cells. Furthermore, hTR knockdown reproducibly induced apoptosis in the absence of any detectable telomere shortening or DNA damage response. In contrast, hTERT knockdown did not induce apoptosis. Strikingly, overexpression of hTERT protein caused apoptosis that was rescued by overexpression of enzymatically inactive hTR mutants. Hence, we propose that hTR can function as a noncoding RNA that protects from apoptosis independent of its function in telomerase enzymatic activity and long-term telomere maintenance in normal human immune cells. These results imply that genetic or environmental factors that alter hTR levels can directly affect immune cell function to influence health and disease.

  17. 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


    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.

  18. Novel roles for well-known players: from tobacco mosaic virus pests to enzymatically active assemblies

    Directory of Open Access Journals (Sweden)

    Claudia Koch


    , e.g., for monitoring blood sugar concentrations, might profit particularly from the presence of TMV rods: Their surfaces were recently shown to stabilize enzymatic activities upon repeated consecutive uses and over several weeks. This review gives the reader a ride through strikingly diverse achievements obtained with TMV-based particles, compares them to the progress with related viruses, and focuses on latest results revealing special advantages for enzyme-based biosensing formats, which might be of high interest for diagnostics employing 'systems-on-a-chip'.

  19. Mixing active-site components: a recipe for the unique enzymatic activity of a telomere resolvase. (United States)

    Bankhead, Troy; Chaconas, George


    The ResT protein, a telomere resolvase from Borrelia burgdorferi, processes replication intermediates into linear replicons with hairpin ends by using a catalytic mechanism similar to that for tyrosine recombinases and type IB topoisomerases. We have identified in ResT a hairpin binding region typically found in cut-and-paste transposases. We show that substitution of residues within this region results in a decreased ability of these mutants to catalyze telomere resolution. However, the mutants are capable of resolving heteroduplex DNA substrates designed to allow spontaneous destabilization and prehairpin formation. These findings support the existence of a hairpin binding region in ResT, the only known occurrence outside a transposase. The combination of transposase-like and tyrosine-recombinase-like domains found in ResT indicates the use of a composite active site and helps explain the unique breakage-and-reunion reaction observed with this protein. Comparison of the ResT sequence with other known telomere resolvases suggests that a hairpin binding motif is a common feature in this class of enzyme; the sequence motif also appears in the RAG recombinases. Finally, our data support a mechanism of action whereby ResT induces prehairpin formation before the DNA cleavage step.

  20. Effects of different bulking agents on the maturity, enzymatic activity, and microbial community functional diversity of kitchen waste compost. (United States)

    Wang, Xiaojuan; Zhang, Wenwei; Gu, Jie; Gao, Hua; Qin, Qingjun


    Aerobic composting is an effective method for the disposal and utilization of kitchen waste. However, the addition of a bulking agent is necessary during kitchen waste composting because of its high moisture content and low C/N ratio. In order to select a suitable bulking agent, we investigated the influence of leaf litter (LL), sawdust (SD), and wheat straw (WS) on the enzymatic activity, microbial community functional diversity, and maturity indices during the kitchen waste composting process. The results showed that the addition of WS yielded the highest maturity (the C/N ratio decreased from 25 to 13, T value = 0.5, and germination index (GI) = 114.7%), whereas the compost containing SD as a bulking agent had the lowest maturity (GI = 32.4%). The maximum cellulase and urease activities were observed with the WS treatment on day 8, whereas the SD treatment had the lowest cellulase activity and the LL treatment had the lowest urease activity. The compost temperature and microbial activity (as the average well color development) showed that bulking the composts with SD prolonged the composting process. The diversity index based on the community-level physiological profile showed that the composts bulked with LL and WS had greater microbial community functional diversity compared with those bulked with SD. Thus, the maturity indexes and enzymatic activities suggest that WS is a suitable bulking agent for use in kitchen waste composting systems.

  1. Effect of R119G Mutation on Human P5CR1 Dynamic Property and Enzymatic Activity (United States)

    Li, Linhua; Ye, Yujia; Sang, Peng; Yin, Yirui; Hu, Wei; Wang, Jing; Zhang, Chao; Li, Deyun; Wan, Wen; Li, Rui; Li, Longjun; Ma, Linling; Xie, Yuehui


    Pyrroline-5-carboxylate reductase (P5CR1) is a universal housekeeping enzyme that catalyzes the reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)+. The enzymatic cycle between P5C and proline is important for function in amino acid metabolism, apoptosis, and intracellular redox potential balance in mitochondria. Autosomal recessive cutis laxa (ARCL) results from a mutation in P5CR1 encoded by PYCR1. Specifically, the R119G mutation is reported to be linked to ARCL although it has not yet been characterized. We synthesized R119G P5CR1 and compared it to WT P5CR1. Foldx prediction of WT and R119G mutant P5CR1 protein stability suggests that the R119G mutation could significantly reduce protein stability. We also performed enzymatic activity assays to determine how the mutation impacts P5CR1 enzymatic function. The results of these experiments show that mutagenesis of R119 to G decreases P5CR1 catalytic efficiency for 3,4-dehydro-L-proline relative to WT. Mutagenesis and kinetic studies reveal that the activity of the mutant decreases as temperature increases from 5°C to 37°C, with almost no activity at 37°C, indicating that this mutation impairs P5CR1 function in vivo. Conversely, WT P5CR1 retains its activity after incubation at 37°C and has essentially no remaining activity at 75°C. Taken together, our experimental results indicate the R119G mutation could be an involving pathomechanism for ARCL. PMID:28194412

  2. Design of a VLP-nanovehicle for CYP450 enzymatic activity delivery


    Sánchez-Sánchez, Lorena; Tapia-Moreno, Alejandro; Juarez-Moreno, Karla; Patterson, Dustin P; Cadena-Nava, Ruben D.; Douglas, Trevor; Vazquez-Duhalt, Rafael


    Background The intracellular delivery of enzymes for therapeutic use has a promising future for the treatment of several diseases such as genetic disorders and cancer. Virus-like particles offer an interesting platform for enzymatic delivery to targeted cells because of their great cargo capacity and the enhancement of the biocatalyst stability towards several factors important in the practical application of these nanoparticles. Results We have designed a nano-bioreactor based on the encapsu...

  3. Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique

    Institute of Scientific and Technical Information of China (English)

    Shuai CHEN; Hua-liang JIANG; Li-li CHEN; Hai-bin LUO; Tao SUN; Jing CHEN; Fei YE; Jian-hua CAI; Jing-kang SHEN; Xu SHEN


    Aim: To characterize enzymatic activity of severe acute respiratory syndrome(SARS) coronavirus (CoV) 3C-like protease (3CLpro) and its four site-directed mutants. Methods: Based on the fluorescence resonance energy transfer (FRET)principle using 5-[(2'-aminoethyl)-amino] naphthelenesulfonic acid (EDANS) and 4-[[4-(dimethylamino) phenyl] azo] benzoic acid (Dabcyl) as the energy transfer pair, one fluorogenic substrate was designed for the evaluation of SARS-CoV 3CLpro proteolytic activity. Results: The kinetic parameters of the fluorogenic substrate have been determined as Km=404 μmol.L-1, kcat=1.08 min-1, and kcat/Km=2.7 gered activity switches, and site-directed mutagenesis analysis of SARS-CoV 3CLpro revealed that substitutions of His41, Cys145, and His163 resulted in complete loss of enzymatic activity, while replacement of Met162 with Ala caused strongly increased activity. Conclusion: This present work has provided valuable information for understanding the catalytic mechanism of SARS-CoV 3CLpro. This FRET-based assay might supply an ideal approach for the exploration SARSCoV 3CLpro putative inhibitors.

  4. Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii

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    Z. C. Pedri

    Full Text Available Abstract Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L–1 at 20 days of cultivation.

  5. Influence of nitrogen sources on the enzymatic activity and grown by Lentinula edodes in biomass Eucalyptus benthamii. (United States)

    Pedri, Z C; Lozano, L M S; Hermann, K L; Helm, C V; Peralta, R M; Tavares, L B B


    Lignocellulose is the most abundant environmental component and a renewable organic resource in soil. There are some filamentous fungi which developed the ability to break down and use cellulose, hemicellulose and lignin as an energy source. The objective of this research was to analyze the effect of three nitrogen resources (ammonium sulfate, saltpetre, soybean) in the holocellulolitic activity of Lentinula edodes EF 50 using as substrate sawdust E. benthamii. An experimental design mixture was applied with repetition in the central point consisting of seven treatments (T) of equal concentrations of nitrogen in ammonium sulfate, potassium nitrate and soybean. The enzymatic activity of avicelase, carboxymetilcellulase, β-glucosidase, xylanases and manganese peroxidase was determined. The humidity, pH, water activity (aw) and qualitative analysis of mycelial growth in 8 times of cultivation were evaluated. The results showed negative effect on enzyme production in treatments with maximum concentration of ammonium sulfate and potassium nitrate. The treatments with cooked soybean flour expressed higher enzymatic activities in times of 3, 6 and 9 days of culture, except in the activity of manganese peroxidase. The highest production was observed in the treatment with ammonium sulfate, and soybean (83.86 UI.L-1) at 20 days of cultivation.

  6. Long-term effects of fertilizer on soil enzymatic activity of wheat field soil in Loess Plateau, China. (United States)

    Hu, Weigang; Jiao, Zhifang; Wu, Fasi; Liu, Yongjun; Dong, Maoxing; Ma, Xiaojun; Fan, Tinglu; An, Lizhe; Feng, Huyuan


    The effects of long-term (29 years) fertilization on local agro-ecosystems in the Loess Plateau of northwest China, containing a single or combinations of inorganic (Nitrogen, N; Phosphate, P) and organic (Mature, M Straw, S) fertilizer, including N, NP, SNP, M, MNP, and a control. The soil enzymes, including dehydrogenase, urease, alkaline phosphatase, invertase and glomalin, were investigated in three physiological stages (Jointing, Dough, and Maturity) of wheat growth at three depths of the soil profile (0-15, 16-30, 31-45 cm). We found that the application of farmyard manure and straw produced the highest values of soil enzymatic activity, especially a balanced applied treatment of MNP. Enzymatic activity was lowest in the control. Values were generally highest at dough, followed by the jointing and maturity stages, and declined with soil profile depth. The activities of the enzymes investigated here are significantly correlated with each other and are correlated with soil nutrients, in particular with soil organic carbon. Our results suggest that a balanced application of fertilizer nutrients and organic manure (especially those containing P) has positive effects on multiple soil chemical parameters, which in turn enhances enzyme activity. We emphasize the role of organic manure in maintaining soil organic matter and promoting biological activity, as its application can result in a substantial increase in agricultural production and can be sustainable for many years.

  7. Changes in Enzymatic Activity and Quality Attributes of Late-mature Peaches in Response to Controlled Atmosphere Conditions

    Institute of Scientific and Technical Information of China (English)

    TIAN Shi-ping; XU Yong; JIANG Ai-li; WANG Yi


    Late-mature peaches (Prunus persica L. Batsch, cv. Dongxuemi ) were stored in air,modification atmosphere (MA) and controlled atmospheres (CA) at 1℃ to investigate enzymatic activities,flesh browning, quality attributes and fruit storability during storage periods. Peaches stored in CA conditions showed significant lower activities of polyphenol oxidase (PPO) and peroxidase (POD) compared to that kept in MA and in air during the early period of storage. CA treatments indicated a better result in maintaining fruit firmness, color and titratable acidity than MA and control treatments. But there was no significant difference in vitamin C and total soluble solids of peaches stored in CA, MA and air. Increasing PPO activity obviously stimulated flesh browning. Peach fruits stored in CA conditions showed a lower activity of POD and higher firmness in the early period of storage. The peaches stored in 5%CO2 + 5%O2 and 10%CO2 + 5%O2 for 92 days had a good quality without any off-flavor. The results indicated CA conditions obviously inhibited the enzymatic browning, delayed senescence and maintained quality of late-mature peaches.

  8. Effects of phenanthrene on hepatic enzymatic activities in tilapia (Oreochromis niloticus ♀ × O.aureus ♂)

    Institute of Scientific and Technical Information of China (English)

    XU Wenju; LI Yuanyou; WU Qingyang; WANG Shuqi; ZHENG Huaiping; LIU Wenhua


    The effects of phenanthrene (Phe) on hepatosomatic index (HSI) and hepatic enzymatic activities in hybrid tilapia (Oreochromis niloticus ♀× O.aureus ♂) were investigated via the static freshwater exposure at dosage of 50,100 or 400 μg/L for 4-14 d.Compared with the control group,HSI was significantly decreased (P<0.05) at 400 μg/L at day 14.Increased enzymatic activities (P<0.05) were observed for catalase (CAT),glutathione peroxidase μgPx) and superoxide dismutase (SOD) at either 100 or 400 μg/L at day 8 and 14,as well as for CAT at 50 μg/L at day 14,except for GPx at 400 μg/L at day 8.Ethoxyresorufin O-deethylase (EROD) activity was significantly increased (P<0.05) at all dosage at day 4 as well as at 50 μg/L at day 8,but significantly decreased at either 100 or 400 μg/L at day 14 (P<0.05).Glutathione-S-transferase μgST) activity was not affected.The results suggest that CAT,GPx,SOD and EROD,as well as HSI in tilapia may be used as the biomarkers or indexes for evaluating or monitoring the pollution of polycyclic aromatic hydrocarbons (PAHs) such as Phe.

  9. Pressure Modulation of the Enzymatic Activity of Phospholipase A2, A Putative Membrane-Associated Pressure Sensor. (United States)

    Suladze, Saba; Cinar, Suleyman; Sperlich, Benjamin; Winter, Roland


    Phospholipases A2 (PLA2) catalyze the hydrolysis reaction of sn-2 fatty acids of membrane phospholipids and are also involved in receptor signaling and transcriptional pathways. Here, we used pressure modulation of the PLA2 activity and of the membrane's physical-chemical properties to reveal new mechanistic information about the membrane association and subsequent enzymatic reaction of PLA2. Although the effect of high hydrostatic pressure (HHP) on aqueous soluble and integral membrane proteins has been investigated to some extent, its effect on enzymatic reactions operating at the water/lipid interface has not been explored, yet. This study focuses on the effect of HHP on the structure, membrane binding and enzymatic activity of membrane-associated bee venom PLA2, covering a pressure range up to 2 kbar. To this end, high-pressure Fourier-transform infrared and high-pressure stopped-flow fluorescence spectroscopies were applied. The results show that PLA2 binding to model biomembranes is not significantly affected by pressure and occurs in at least two kinetically distinct steps. Followed by fast initial membrane association, structural reorganization of α-helical segments of PLA2 takes place at the lipid water interface. FRET-based activity measurements reveal that pressure has a marked inhibitory effect on the lipid hydrolysis rate, which decreases by 75% upon compression up to 2 kbar. Lipid hydrolysis under extreme environmental conditions, such as those encountered in the deep sea where pressures up to the kbar-level are encountered, is hence markedly affected by HHP, rendering PLA2, next to being a primary osmosensor, a good candidate for a sensitive pressure sensor in vivo.

  10. Functionalized Graphene Oxide with Chitosan for Protein Nanocarriers to Protect against Enzymatic Cleavage and Retain Collagenase Activity (United States)

    Emadi, Fatemeh; Amini, Abbas; Gholami, Ahmad; Ghasemi, Younes


    Proteins have short half-life because of enzymatic cleavage. Here, a new protein nanocarrier made of graphene oxide (GO) + Chitosan (CS) is proposed to successfully prevent proteolysis in protein and simultaneously retain its activity. Bovine serum albumin (BSA) and collagenase were loaded on GO and GO-CS to explore the stability and activity of proteins. SEM, AFM, TEM, DSC, UV-Vis, FT-IR, RBS, Raman, SDS-PAGE and zymography were utilized as characterization techniques. The protecting role of GO and GO-CS against enzymatic cleavage was probed by protease digestion analysis on BSA, where the protease solution was introduced to GO-BSA and GO-CS-BSA at 37 °C for 0.5-1-3-6 hours. Characterizations showed the successful synthesis of few layers of GO and the coverage by CS. According to gelatin zymographic analysis, the loaded collagenase on GO and GO-CS lysed the gelatin and created non-staining bands which confirmed the activity of loaded collagenase. SDS-PAGE analysis revealed no significant change in the intact protein in the GO-BSA and GO-CS-BSA solution after 30-minute and 1-hour exposure to protease; however, free BSA was completely digested after 1 hour. After 6 hours, intact proteins were detected in GO-BSA and GO-CS-BSA solutions, while no intact protein was detected in the free BSA solution. PMID:28186169

  11. Evaluation of Oil Removal Efficiency and Enzymatic Activity in Some fungal Strains for Bioremediation of Petroleum-Polluted Soils

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    Fariba Mohsenzadeh


    Full Text Available Background: Petroleum pollution is a global disaster and there are several soil cleaning methods including bioremediation.Methods: In a field study, fugal strains were isolated from oil-contaminated sites of Arak refinery (Iran and their growth ability was checked in potato dextrose agar (PDA media containing 0-10% v/v crude oil, the activity of three enzymes (Catalase, Peroxidase and Phenol Oxidase was evaluated in the fungal colonies and bioremediation ability of the fungi was checked in the experimental pots containing 3 kg sterilized soil and different concentrations of petroleum (0-10% w/w.Results: Four fungal strains, Acromonium sp., Alternaria sp., Aspergillus terreus and Penicillium sp., were selected asthe most resistant ones. They were able to growth in the subjected concentrations and Alternaria sp. showed thehighest growth ability in the petroleum containing media. The enzyme assay showed that the enzymatic activity was increased in the oil-contaminated media. Bioremediation results showed that the studied fungi were able to decrease petroleum pollution. The highest petroleum removing efficiency of Aspergillus terreus, Penicillium sp.,Alternaria sp. and Acromonium sp. was evaluated in the 10%, 8%, 8% and 2% petroleum pollution respectively.Conclusions: Fungi are important microorganisms in decreasing of petroleum pollution. They have bioremediation potency that is related to their enzymatic activities.

  12. Evaluation of oil removal efficiency and enzymatic activity in some fungal strains for bioremediation of petroleum-polluted soils

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    Mohsenzadeh Fariba


    Full Text Available Abstract Background Petroleum pollution is a global disaster and there are several soil cleaning methods including bioremediation. Methods In a field study, fugal strains were isolated from oil-contaminated sites of Arak refinery (Iran and their growth ability was checked in potato dextrose agar (PDA media containing 0-10% v/v crude oil, the activity of three enzymes (Catalase, Peroxidase and Phenol Oxidase was evaluated in the fungal colonies and bioremediation ability of the fungi was checked in the experimental pots containing 3 kg sterilized soil and different concentrations of petroleum (0-10% w/w. Results Four fungal strains, Acromonium sp., Alternaria sp., Aspergillus terreus and Penicillium sp., were selected as the most resistant ones. They were able to growth in the subjected concentrations and Alternaria sp. showed the highest growth ability in the petroleum containing media. The enzyme assay showed that the enzymatic activity was increased in the oil-contaminated media. Bioremediation results showed that the studied fungi were able to decrease petroleum pollution. The highest petroleum removing efficiency of Aspergillus terreus, Penicillium sp., Alternaria sp. and Acromonium sp. was evaluated in the 10%, 8%, 8% and 2% petroleum pollution respectively. Conclusions Fungi are important microorganisms in decreasing of petroleum pollution. They have bioremediation potency that is related to their enzymatic activities.

  13. Contrasting effects of untreated textile wastewater onto the soil available nitrogen-phosphorus and enzymatic activities in aridisol. (United States)

    Arif, Muhammad Saleem; Riaz, Muhammad; Shahzad, Sher Muhammad; Yasmeen, Tahira; Buttler, Alexandre; Garcıa-Gil, Juan Carlos; Roohi, Mahnaz; Rasool, Akhtar


    Water shortage and soil qualitative degradation are significant environmental problems in arid and semi-arid regions of the world. The increasing demand for water in agriculture and industry has resulted in the emergence of wastewater use as an alternative in these areas. Textile wastewater is produced in surplus amounts which poses threat to the environment as well as associated flora and fauna. A 60-day incubation study was performed to assess the effects of untreated textile wastewater at 0, 25, 50, 75, and 100% dilution levels on the physico-chemical and some microbial and enzymatic properties of an aridisol soil. The addition of textile wastewater provoked a significant change in soil pH and electrical conductivity and soil dehydrogenase and urease activities compared to the distilled-water treated control soil. Moreover, compared to the control treatment, soil phosphomonoesterase activity was significantly increased from 25 to 75% application rates, but decreased at 100% textile wastewater application rate. Total and available soil N contents increased significantly in response to application of textile wastewater. Despite significant increases in the soil total P contents after the addition of textile wastewater, soil available P content decreased with increasing concentration of wastewater. Changes in soil nutrient contents and related enzymatic activities suggested a dynamic match between substrate availability and soil N and P contents. Aridisols have high fixation and low P availability, application of textile wastewater to such soils should be considered only after careful assessment.

  14. Aerobic and anaerobic enzymatic activity of orange roughy (Hoplostethus atlanticus) and alfonsino (Beryx splendens) from the Juan Fernandez seamounts area. (United States)

    Saavedra, L M; Quiñones, R A; Gonzalez-Saldía, R R; Niklitschek, E J


    The aerobic and anaerobic enzymatic activity of two important commercial bathypelagic species living in the Juan Fernández seamounts was analyzed: alfonsino (Beryx splendens) and orange roughy (Hoplostethus atlanticus). These seamounts are influenced by the presence of an oxygen minimum zone (OMZ) located between 160 and 250 m depth. Both species have vertical segregation; alfonsino is able to stay in the OMZ, while orange roughy remains at greater depths. In this study, we compare the aerobic and anaerobic capacity of these species, measuring the activity of key metabolic enzymes in different body tissues (muscle, heart, brain and liver). Alfonsino has higher anaerobic potential in its white muscle due to greater lactate dehydrogenase (LDH) activity (190.2 μmol NADH min(-1) g ww(-1)), which is related to its smaller body size, but it is also a feature shared with species that migrate through OMZs. This potential and the higher muscle citrate synthase and electron transport system activities indicate that alfonsino has greater swimming activity level than orange roughy. This species has also a high MDH/LDH ratio in its heart, brain and liver, revealing a potential capacity to conduct aerobic metabolism in these organs under prolonged periods of environmental low oxygen conditions, preventing lactic acid accumulation. With these metabolic characteristics, alfonsino may have increased swimming activity to migrate and also could stay for a period of time in the OMZ. The observed differences between alfonsino and orange roughy with respect to their aerobic and anaerobic enzymatic activity are consistent with their characteristic vertical distributions and feeding behaviors.

  15. Study on the enzymatic activity of Caspase-3 in response to alginic acid decomposing bacteria in Laminaria japonica Aresch.(Phaeophyta)

    Institute of Scientific and Technical Information of China (English)

    Wang Gaoge; Lin Wei; Yan Xiaojun; Duan Delin


    Caspase-3 is the major factor in apoptosis triggered by various stimuli, and plays a critical role during the apoptosis process. By using CaspGLOWTM fluorescein active caspase-3 staining method, caspase-3 enzymatic activities were detected in response to alginic acid bacteria in Laminaria japonica sporophytic tissues. Results showed that caspase-3 enzymatic activities were observed at 5 min after the infection. Caspase-3 enzymatic activity increased with the infection time, and had a tendency of moving from the infection site to outside. By applying caspase-specific peptide inhibitor Z-VAD-FMK, caspase-3 activation could be effectively abolished in the infected tissues. Our results indicate that programmed cell death (PCD) may be involved in the infected Laminaria japonica sporophytic tissues, and provide the evidence that defense mechanisms in algae may have similar caspase cascade events in animals.

  16. Effect of salinity tolerant PDH45 transgenic rice on physicochemical properties, enzymatic activities and microbial communities of rhizosphere soils. (United States)

    Sahoo, Ranjan Kumar; Tuteja, Narendra


    The effect of genetically modified (GM) plants on environment is now major concern worldwide. The plant roots of rhizosphere soil interact with variety of bacteria which could be influenced by the transgene in GM plants. The antibiotic resistance genes in GM plants may be transferred to soil microbes. In this study we have examined the effect of overexpression of salinity tolerant pea DNA helicase 45 (PDH45) gene on microbes and enzymatic activities in the rhizosphere soil of transgenic rice IR64 in presence and absence of salt stress in two different rhizospheric soils (New Delhi and Odisha, India). The diversity of the microbial community and soil enzymes viz., dehydrogenase, alkaline phosphatase, urease and nitrate reductase was assessed. The results revealed that there was no significant effect of transgene expression on rhizosphere soil of the rice plants. The isolated bacteria were phenotyped both in absence and presence of salt and no significant changes were found in their phenotypic characters as well as in their population. Overall, the overexpression of PDH45 in rice did not cause detectable changes in the microbial population, soil enzymatic activities and functional diversity of the rhizosphere soil microbial community.

  17. Glutathione peroxidase 3 of Saccharomyces cerevisiae suppresses non-enzymatic proteolysis of glutamine synthetase in an activity-independent manner. (United States)

    Lee, Phil Young; Kho, Chang Won; Lee, Do Hee; Kang, Sunghyun; Kang, Seongman; Lee, Sang Chul; Park, Byoung Chul; Cho, Sayeon; Bae, Kwang-Hee; Park, Sung Goo


    Glutathione peroxidase 3 (Gpx3) is ubiquitously expressed and is important antioxidant enzyme in yeast. It modulates the activities of redox-sensitive thiol proteins, particularly those involved in signal transduction pathway and protein translocation. Through immunoprecipitation/two-dimensional gel electrophoresis (IP-2DE), MALDI-TOF mass spectrometry, and a pull down assay, we found glutamine synthetase (GS; EC as a candidate interacting protein with Gpx3. GS is a key enzyme in nitrogen metabolism and ammonium assimilation. It has been known that GS is non-enzymatically cleaved by ROS generated by MFO (thiol/ Fe(3+)/O(2) mixed-function oxidase) system. In this study, it is demonstrated that GS interacts with Gpx3 through its catalytic domain both in vivo and in vitro regardless of redox state. In addition, Gpx3 helps to protect GS from inactivation and degradation via oxidative stress in an activity-independent manner. Based on the results, it is suggested that Gpx3 protects GS from non-enzymatic proteolysis, thereby contributing to cell homeostasis when cell is exposed to oxidative stress.

  18. Oxidant and enzymatic antioxidant status (gene expression and activity) in the brain of chickens with cold-induced pulmonary hypertension (United States)

    Hassanpour, Hossein; Khalaji-Pirbalouty, Valiallah; Nasiri, Leila; Mohebbi, Abdonnaser; Bahadoran, Shahab


    To evaluate oxidant and antioxidant status of the brain (hindbrain, midbrain, and forebrain) in chickens with cold-induced pulmonary hypertension, the measurements of lipid peroxidation, protein oxidation, antioxidant capacity, enzymatic activity, and gene expression (for catalase, glutathione peroxidase, and superoxide dismutases) were done. There were high lipid peroxidation/protein oxidation and low antioxidant capacity in the hindbrain of cold-induced pulmonary hypertensive chickens compared to control ( P hypertensive chickens, superoxide dismutase activity was decreased (forebrain, midbrain, and hindbrain), while catalase activity was increased (forebrain and midbrain) ( P hypertensive chickens. Probably, these situations in the oxidant and antioxidant status of the brain especially hindbrain may change its function at cardiovascular center and sympathetic nervous system to exacerbate pulmonary hypertension.

  19. Alternations in the liver enzymatic activity of Common carp, Cyprinus carpio in response to parasites, Dactylogyrus spp. and Gyrodactylus spp. (United States)

    Rastiannasab, Abulhasan; Afsharmanesh, Shiva; Rahimi, Ruhollah; Sharifian, Iman


    The present study was carried out to investigate the effects of parasites, monogenea, Dactylogyrus spp. and Gyrodactylus spp. on some enzymatic and biochemical components of liver in healthy and infected common carp, Cyprinus carpio. For this purpose, 10 healthy and 10 infected fish were collected from farm. The blood samples were taken and after separation of serum, the values of Aspartate aminotransferase (AST), Alanine aminotransferase (ALT) enzymes activities as well as Creatinine and Urea were measured. Based on obtained results, the values of AST, ALT enzymes activities as well as Creatinine and Urea were higher in the infected fish compared to non-infected fish. In conclusion; our results reveals that infection with external parasites, Dactylogyrus spp. and Gyrodactylus spp. can causes some dysfunctions in liver and kidney of common carp.

  20. Lactones 42. Stereoselective enzymatic/microbial synthesis of optically active isomers of whisky lactone. (United States)

    Boratyński, Filip; Smuga, Małgorzata; Wawrzeńczyk, Czesław


    Two different methods, enzyme-mediated reactions and biotrasformations with microorganisms, were applied to obtain optically pure cis- and trans-isomers of whisky lactone 4a and 4b. In the first method, eight alcohol dehydrogenases were investigated as biocatalysts to enantioselective oxidation of racemic erythro- and threo-3-methyloctane-1,4-diols (1a and 1b). Oxidation processes with three of them, alcohol dehydrogenases isolated from horse liver (HLADH) as well as recombinant from Escherichia coli and primary alcohol dehydrogenase (PADH I), were characterized by the highest degree of conversion with moderate enantioselectivity (ee=27-82%) of the reaction. In all enzymatic reactions enantiomerically enriched not naturally occurring isomers of trans-(-)-(4R,5S)-4b or cis-(+)-(4R,5R)-4a were formed preferentially. In the second strategy, based on microbial lactonization of γ-oxoacids, naturally occurring opposite isomers of whisky lactones were obtained. Trans-(+)-(4S,5R)-isomer (ee=99%) of whisky lactone 4b was stereoselectively formed as the only product of biotransformations of 3-methyl-4-oxooctanoic acid (5) catalyzed by Didimospheria igniaria KCH6651, Laetiporus sulphurens AM525, Chaetomium sp.1 KCH6670 and Saccharomyces cerevisiae AM464. Biotransformation of γ-oxoacid 5, in the culture of Beauveria bassiana AM278 and Pycnidiella resinae KCH50 afforded a mixtures of trans-(+)-(4S,5R)-4b with enantiomeric excess ee=99% and cis-(-)-(4S,5S)-4a with enantiomeric excesses ee=77% and ee=45% respectively.

  1. The influence of a microbial inoculum on the enzymatic activity of peat and morphological features of the French marigold

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    Agnieszka Wolna-Maruwka


    Full Text Available The objective of the study was to determine the dynamics of the enzymatic activity in a substrate containing a microbiological inoculum (Effective Microorganisms – EM intended to improve French marigold growth and flowering. The material used in the study was peat substrate of 5.5–6.0 pH into which plants were planted and then inoculated with different doses of the EM biofertilizer (1 : 10, 1 : 50, 1 : 100. Samples of the substrate on which experimental plants were cultivated were collected during the following three phases: the phase of seedling planting, the phase of vegetative growth and the phase of flowering. The scope of investigations comprised determination of the activity of dehydrogenases, urease and acid phosphatase activity by the spectrophotometric method. In addition, the following plant morphological analyses were performed: plant height, shoot number and length, number of buds and inflorescences, and leaf greenness index (SPAD. It was found that the phase of plant development was proved to be the main determinant of the activity of the enzymes under study. The EM biofertilizer had positive effect on the acid phosphatase activity. However, the preparation did not have stimulating effect on the activity of urease or dehydrogenases. The EM biofertilizer concentrated at 1 : 100 and applied into the soil and foliage resulted in a larger number of darker leaves and greater number of inflorescences on the plants.

  2. Behavior of some enzymatic systems to the action of the cytostatic active EGlCP glucanic biopreparation upon HeLa neoplastic cells

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    Daniela Gherghel


    Full Text Available Interference of an autochthonous cytostatic active EGlCP glucanic biopreparation (in dose of 1.5 mg/mL with the activity of some key enzymes, involved in the development of active transmembranary transport, of the intermediary and energetic metabolism, as well as in cellular answer to the oxidative stress, of HeLa neoplastic cells has been investigated. The study revealed: the intensification of the membranary Na+-K+-ATP-ase, of the cellular Mg2+-ATP-ase, of the superoxide dismutase activities; the operating level attenuation of the of catalase, peroxidase, glutathion peroxidase, lactate dehydrogenase, alkaline phosphatase, acid phosphatase; the diminution of the malondialdehyde content. This functional interference with some cell enzymatic biomolecules has also induced the perturbation of the diverse membrane and metabolic processes, which was incompatible with the survival of HeLa tumoral cells The modulations of the cellular enzymatic equipment activity can be the consequences of the glucanic components direct (with the molecules of the miscellaneous enzymes or indirect interactions ( with membrane or genetic apparatus with some cell, subcell and molecular structures, implicated in the control and regulation of the biosynthesis and activity of the enzymatic biomolecules. The central element, which induces this enzymatic imbalance, appears to be the excess generation of the free radicals in the tumoral cells’ metabolism aggressed by glucanic constituents.

  3. Quantification of ricin, RCA and comparison of enzymatic activity in 18 Ricinus communis cultivars by isotope dilution mass spectrometry. (United States)

    Schieltz, David M; McWilliams, Lisa G; Kuklenyik, Zsuzsanna; Prezioso, Samantha M; Carter, Andrew J; Williamson, Yulanda M; McGrath, Sara C; Morse, Stephen A; Barr, John R


    The seeds of the Ricinus communis (Castor bean) plant are the source of the economically important commodity castor oil. Castor seeds also contain the proteins ricin and R. communis agglutinin (RCA), two toxic lectins that are hazardous to human health. Radial immunodiffusion (RID) and the enzyme linked immunosorbent assay (ELISA) are two antibody-based methods commonly used to quantify ricin and RCA; however, antibodies currently used in these methods cannot distinguish between ricin and RCA due to the high sequence homology of the respective proteins. In this study, a technique combining antibody-based affinity capture with liquid chromatography and multiple reaction monitoring (MRM) mass spectrometry (MS) was used to quantify the amounts of ricin and RCA independently in extracts prepared from the seeds of eighteen representative cultivars of R. communis which were propagated under identical conditions. Additionally, liquid chromatography and MRM-MS was used to determine rRNA N-glycosidase activity for each cultivar and the overall activity in these cultivars was compared to a purified ricin standard. Of the cultivars studied, the average ricin content was 9.3 mg/g seed, the average RCA content was 9.9 mg/g seed, and the enzymatic activity agreed with the activity of a purified ricin reference within 35% relative activity.

  4. Lactogenic Activity of an Enzymatic Hydrolysate from Octopus vulgaris and Carica papaya in SD Rats. (United States)

    Cai, Bingna; Chen, Hua; Sun, Han; Sun, Huili; Wan, Peng; Chen, Deke; Pan, Jianyu


    The traditional Chinese medicine theory believes that octopus papaya soup can stimulate milk production in lactating women. The objective of this study was to determine whether dietary supplementation with an enzymatic hydrolysate of Octopus vulgaris and Carica papaya (EHOC) could increase milk production and nutritional indexes in Sprague Dawley (SD) rats. Female SD rats (n = 24) were fed a control diet (n = 8), EHOC-supplemented diet, or a positive control diet (Shengruzhi) from day 10 of pregnancy to day 10 of lactation. Maternal serum, mammary gland (day 10 of lactation), milk, and pup weight (daily) were collected for analysis. Results showed that the EHOC diet obviously elevated daily milk yield and pup weight compared to the control group (P < .05). The EHOC diet was found to increase the concentration of prolactin (PRL), progesterone (P), estradiol (E2), and growth hormone (GH) significantly in the circulation and mammary gland. Mammary glands of EHOC-treated dams showed clear lobuloalveolar development and proliferation of myoepithelial cells, but no striking variations were observed among the groups. Furthermore, the nutrition content and immune globulin concentration in the milk of EHOC-supplemented dams were higher than those of the control group, especially the cholesterol, glucose, and IgG were higher by 44.98% (P < .001), 42.76% (P < .01), and 42.23% (P < .01), respectively. In conclusion, this article demonstrates that EHOC administration has beneficial effects on milk production in the dams and on performance of the dam and pup. These results indicate that EHOC could be explored as a potentially lactogenic nutriment for lactating women.

  5. Protein-Rich Fraction of Cnidoscolus urens (L. Arthur Leaves: Enzymatic Characterization and Procoagulant and Fibrinogenolytic Activities

    Directory of Open Access Journals (Sweden)

    Yamara A. S. de Menezes


    Full Text Available Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L. Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogenolytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.

  6. Influence of Different Lignocellulose Sources on Endo-1,4-β-Glucanase Gene Expression and Enzymatic Activity of Bacillus amyloliquefaciens B31C

    Directory of Open Access Journals (Sweden)

    Rosangela Di Pasqua


    Full Text Available Conversion of cellulose into fermentable sugars for ethanol production is currently performed by enzymatic hydrolysis catalyzed by cellulases. The cellulases are produced by a wide variety of microorganisms, playing a major role in the recycling of biomass. The endo-1,4-β-glucanase (CelB31C from Bacillus amyloliquefaciens B31C, isolated from compost and previously selected on the basis of highest cellulase activity levels among Bacillus isolated, was characterized as being a potential candidate for a biocatalyst in lignocellulose conversion for second-generation bioethanol production. The aim of this work was to evaluate the changes in production of enzymatic activity of the endo-1,4-β-glucanase (CelB31C and the expression of its gene (bglC using a carboxymethylcellulase activity assay and qRT-PCR analysis, respectively, during growth of B. amyloliquefaciens B31C on different cellulose sources: carboxymethylcellulose (CMC, pure cellulose from Arundo donax, pretreated Arundo donax biomass (Chemtex, and microcrystalline cellulose (Avicel. The results showed that both the expression of bglC gene and the enzymatic activity production are related to the type of cellulose source. The strain showed a high enzymatic activity on lignocellulosic biomass and on microcrystalline cellulose. Furthermore, the highest gene expression occurred during the exponential phase of growth, except in the presence of Avicel.

  7. Antifungal hydroxy fatty acids produced during sourdough fermentation: microbial and enzymatic pathways, and antifungal activity in bread. (United States)

    Black, Brenna A; Zannini, Emanuele; Curtis, Jonathan M; Gänzle, Michael G


    Lactobacilli convert linoleic acid to hydroxy fatty acids; however, this conversion has not been demonstrated in food fermentations and it remains unknown whether hydroxy fatty acids produced by lactobacilli have antifungal activity. This study aimed to determine whether lactobacilli convert linoleic acid to metabolites with antifungal activity and to assess whether this conversion can be employed to delay fungal growth on bread. Aqueous and organic extracts from seven strains of lactobacilli grown in modified De Man Rogosa Sharpe medium or sourdough were assayed for antifungal activity. Lactobacillus hammesii exhibited increased antifungal activity upon the addition of linoleic acid as a substrate. Bioassay-guided fractionation attributed the antifungal activity of L. hammesii to a monohydroxy C(18:1) fatty acid. Comparison of its antifungal activity to those of other hydroxy fatty acids revealed that the monohydroxy fraction from L. hammesii and coriolic (13-hydroxy-9,11-octadecadienoic) acid were the most active, with MICs of 0.1 to 0.7 g liter(-1). Ricinoleic (12-hydroxy-9-octadecenoic) acid was active at a MIC of 2.4 g liter(-1). L. hammesii accumulated the monohydroxy C(18:1) fatty acid in sourdough to a concentration of 0.73 ± 0.03 g liter(-1) (mean ± standard deviation). Generation of hydroxy fatty acids in sourdough also occurred through enzymatic oxidation of linoleic acid to coriolic acid. The use of 20% sourdough fermented with L. hammesii or the use of 0.15% coriolic acid in bread making increased the mold-free shelf life by 2 to 3 days or from 2 to more than 6 days, respectively. In conclusion, L. hammesii converts linoleic acid in sourdough and the resulting monohydroxy octadecenoic acid exerts antifungal activity in bread.

  8. Dynamics of microbiological parameters, enzymatic activities and worm biomass production during vermicomposting of effluent treatment plant sludge of bakery industry. (United States)

    Yadav, Anoop; Suthar, S; Garg, V K


    This paper reports the changes in microbial parameters and enzymatic activities during vermicomposting of effluent treatment plant sludge (ETPS) of bakery industry spiked with cow dung (CD) by Eisenia fetida. Six vermibins containing different ratios of ETPS and CD were maintained under controlled laboratory conditions for 15 weeks. Total bacterial and total fungal count increased upto 7th week and declined afterward in all the bins. Maximum bacterial and fungal count was 31.6 CFU × 10(6) g(-1) and 31 CFU × 10(4) g(-1) in 7th week. Maximum dehydrogenase activity was 1921 μg TPF g(-1) h(-1) in 9th week in 100 % CD containing vermibin, whereas maximum urease activity was 1208 μg NH4 (-)N g(-1) h(-1) in 3rd week in 100 % CD containing vermibin. The enzyme activity and microbial counts were lesser in ETPS containing vermibins than control (100 % CD). The growth and fecundity of the worms in different vermibins were also investigated. The results showed that initially biomass and fecundity of the worms increased but decreased at the later stages due to non-availability of the palatable feed. This showed that quality and palatability of food directly affect biological parameters of the system.

  9. Enzymatic activity and immunoreactivity of Aca s 4, an alpha-amylase allergen from the storage mite Acarus siro

    Directory of Open Access Journals (Sweden)

    Pytelková Jana


    Full Text Available Abstract Background Enzymatic allergens of storage mites that contaminate stored food products are poorly characterized. We describe biochemical and immunological properties of the native alpha-amylase allergen Aca s 4 from Acarus siro, a medically important storage mite. Results A. siro produced a high level of alpha-amylase activity attributed to Aca s 4. This enzyme was purified and identified by protein sequencing and LC-MS/MS analysis. Aca s 4 showed a distinct inhibition pattern and an unusual alpha-amylolytic activity with low sensitivity to activation by chloride ions. Homology modeling of Aca s 4 revealed a structural change in the chloride-binding site that may account for this activation pattern. Aca s 4 was recognized by IgE from house dust mite-sensitive patients, and potential epitopes for cross-reactivity with house dust mite group 4 allergens were found. Conclusions We present the first protein-level characterization of a group 4 allergen from storage mites. Due to its high production and IgE reactivity, Aca s 4 is potentially relevant to allergic hypersensitivity.

  10. The Acid Sphingomyelinase Sequence Variant p.A487V Is Not Associated With Decreased Levels of Enzymatic Activity. (United States)

    Rhein, Cosima; Naumann, Julia; Mühle, Christiane; Zill, Peter; Adli, Mazda; Hegerl, Ulrich; Hiemke, Christoph; Mergl, Roland; Möller, Hans-Jürgen; Reichel, Martin; Kornhuber, Johannes


    Rare loss-of-function mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene are known to dramatically decrease the catalytic activity of acid sphingomyelinase (ASM), resulting in an autosomal recessive lysosomal storage disorder known as Niemann-Pick disease (NPD) type A and B. In contrast to the general low frequency of those deleterious mutations, we found a relatively high frequency for the proposed type B NPD variant c.1460C>T (p.A487V) in our sample of 58 patients suffering from Major Depressive Disorder. We therefore investigated the biochemical consequences of this variant more closely. Our in vivo data derived from blood cell analyses indicated cellular ASM activity levels in the normal range. The secreted ASM activity levels in blood plasma were slightly lower, but still above those levels reported for type B NPD patients. In vitro expression studies of this ASM variant in different cell lines confirmed these results, showing cellular and secreted enzymatic activities equivalent to those of wild-type ASM and similar expression levels. Thus, we conclude that the ASM variant c.1460C>T (p.A487V) is not a rare missense mutation but an SMPD1 sequence variant that yields a protein with functional catalytic characteristics.

  11. Effects of Multiglycoside of Tripterygium Wilfordii (GTW) on Spermatogenic Ceils and Enzymatic Activities in Male Rats

    Institute of Scientific and Technical Information of China (English)

    田健; 周孝瑚


    This study was undertaken to observe consecutively the morphology of testis and epididymis and the changes of enzymatic activities of AKP, ACP, 3β-HSD and LDH-C4 in male rats treated with GTW 30 to 80 days. In addition, male antifertility effect and its possible reversibility were also observed. The results showed that GTW is a potential testicular toxicant in the animals. It can cause damage of the sperm cells with close relation to the treated time and dosage, Sloughing of sperm cells and many multinucleated giant cells in the seminiferous tubules were found on day 40 and 50 after the treatment. The tubules were hyperatrophic and the sperm cells were almost absent at the end of the study(80 days), No obvious morphological alteration was observed in the epididymal epithelial tissue, But changes of quality and number df the spermatozoa in epididymides were found prior to those of testes. Reduced number and abnormal cauda sperm were observed 30 days after the treatment, The ACP activity of Sertoli cells increased slightly, whereas the activities of ACP and LDH-C4 decreased gradually as the treated time prolonged. The 3β-HSD of Leydig cells was changed or subtle by m-phase or treatment and dramatically decreased at the end of treatment, The infertility caused by GTW was reversible 8 weeks after cessation of the treatment.

  12. Extracellular enzymatic activities of cold-adapted bacteria from polar oceans and effect of temperature and salinity on cell growth

    Institute of Scientific and Technical Information of China (English)

    Zeng Yinxin; Yu Yong; Chen Bo; Li Huirong


    The potential of 324 bacteria isolated from different habitats in polar oceans to produce a variety of extracellular enzymatic activities at low temperature was investigated. By plate assay, lipase, protease, amylase, gelatinase, agarase, chitinase or cellulase were detected. Lipases were generally present by bacteria living in polar oceans. Protease-producing bacteria held the second highest proportion in culturable isolates. Strains producing amylase kept a relative stable proportion of around 30% in different polar marine habitats. All 50 Arctic sea-ice bacteria producing proteases were cold-adapted strains, however, only 20% were psychrophilic. 98% of them could grow at 3% NaCl, and 56% could grow without NaCl. On the other hand, 98% of these sea-ice bacteria produced extracellular proteases with optimum temperature at or higher than 35℃, well above the upper temperature limit of cell growth. Extracellular enzymes including amylase, agarase, cellulase and lipase released by bacteria from seawater or sediment in polar oceans, most expressed maximum activities between 25 and 35℃. Among extracellular enzymes released by bacterial strain BSw20308, protease expressed maximum activity at 40℃, higher than 35℃ of polysaccharide hydrolases and 25℃ of lipase.

  13. beta-carotene does not change markers of enzymatic and nonenzymatic antioxidant activity in human blood

    DEFF Research Database (Denmark)

    Castenmiller, J.J.M.; Lauridsen, Søren T.; Dragsted, Lars O.;


    and erythrocyte enzyme activities were assessed, and differences among experimental groups were tested. Consumption of spinach resulted in greater (P catalase activity and serum alpha-tocopherol concentration compared...... to an increased carotenoid (lutein and zeaxanthin) intake, but beta-carotene is unlikely to be a causative factor. Lower erythrocyte catalase activity after intervention with spinach products may be related to other constituents in spinach such as flavonoids....

  14. Seasonal Dynamics of Enzymatic Activities and Functional Diversity in Soils under Different Organic Management (United States)

    Soil microbial activity and diversity fluctuate seasonally under annual organic amendment for improving soil quality. We investigated the effects of municipal compost (MC), poultry litter (PL), and cover crops of spring oats and red clover (RC) on soil enzyme activities, and soil bacterial community...

  15. Aldehyde PEGylation of laccase from Trametes versicolor in route to increase its stability: effect on enzymatic activity. (United States)

    Mayolo-Deloisa, Karla; González-González, Mirna; Simental-Martínez, Jesús; Rito-Palomares, Marco


    Laccase is a multicopper oxidase that catalyzes the oxidation of phenolic compounds. Laccase can be used in bioremediation, beverage (wine, fruit juice, and beer) processing, ascorbic acid determination, sugar beet pectin gelation baking, and as a biosensor. Recently, the antiproliferative activity of laccase toward tumor cells has been reported. Because of the potential applications of this enzyme, the efforts for enhancing and stabilizing its activity have increased. Thus, the PEGylation of laccase can be an alternative. PEGylation is the covalent attachment of one or more molecules of methoxy poly(ethylene glycol) (mPEG) to a protein. Normally, during the PEGylation reaction, the activity is reduced but the stability increases; thus, it is important to minimize the loss of activity. In this work, the effects of molar ratio (1:4, 1:8, and 1:12), concentration of laccase (6 and 12 mg/ml), reaction time (4 and 17 h), molecular weight, and type of mPEG (20, 30, 40 kDa and 40 kDa-branched) were analyzed. The activity was measured using three substrates: ABTS, 2,6-dimethoxyphenol, and syringaldazine. The best conditions for laccase PEGylation were 12 mg/ml of laccase, molar ratio 1:4, and 4 h reaction time. Under these conditions, the enzyme was able to maintain nearly 100% of its enzymatic activity with ABTS. The PEGylation of laccase has not been extensively explored, so it is important to analyze the effects of this bioconjugation in route to produce a robust modified enzyme.

  16. Effects of Straw Processing Methods and Irrigation Sources on Enzymatic Activity of Soils under Winter Wheat

    Institute of Scientific and Technical Information of China (English)

    Zhiwei LU; Guofeng WAN; Zijun YANG; Lei HOU; Wenhui ZHANG


    [Objective] The aim was to study on effects of "straw returning and ir-re- turning" and "irrigation with ground water and water in the Yellow River" on changes of enzyme activity in soils under wheat at different developmental stages. [Method] Jimai 22, a kind of winter wheat, was made use of in fields to study on effects of " straw returning and Jr-returning" and "irrigation with ground water and water in the Yellow River" on changes of enzyme activity in soils under wheat in different devel- opmental stages. [Result] With advancement of developmental stage, urease activity of wheat in the four groups all showed the trend of "increasing-decreasing-increas- ing" and activities of invertase and phosphatase both changed from increasing to de- creasing. In addition, urease activities of soils in wheat roots were improved by straw returning in four developmental stages. Meanwhile, activity of soil enzyme was better promoted by irrigation with ground water than with water in the Yellow River, except in grain-filling stage. Before developmental stage, different processing meth- ods had a significant effect on phosphatase activity, for example, straw returning and ground water significantly enhanced activities of two kinds of phosphatase and pro- moted P absorption and transferring by plants and microorganisms in jointing stage; activity of acid phosphatase was higher in the group where irrigation with ground water and straw returning were adopted than those in the rest three groups in boot- ing stage. [Conclusion] The research laid a foundation for dynamic relationship among activity of soil enzyme, crop growth and microorganisms.

  17. Effect of non-enzymatic glycation on esterase activities of hemoglobin and myoglobin. (United States)

    Sen, Subhrojit; Bose, Tania; Roy, Anjana; Chakraborti, Abhay Sankar


    Heme proteins--hemoglobin and myoglobin possess esterase activities. Studies with purified hemoglobin from normal individuals and diabetic patients revealed that the esterase activity as measured from hydrolysis of p-nitrophenyl acetate (p-NPA) was higher in diabetic condition and increased progressively with extent of the disease. HbA(1c), the major glycated hemoglobin, which increases proportionately with blood glucose level in diabetes mellitus, exhibited more esterase activity than the non-glycated hemoglobin fraction, HbA(0), as demonstrated spectrophotometrically as well as by activity staining. Glycation influenced esterase activity of hemoglobin by increasing the affinity for the substrate and the rate of the reaction. Both HbA(0) and HbA(1c)-mediated catalysis of p-NPA hydrolysis was pH-dependent. Esterase activity of in vitro-glycated myoglobin (GMb) was also higher than that of its non-glycated analog (Mb). The amplified esterase activities of hemoglobin and myoglobin might be associated with glycation-induced structural modifications of the proteins.

  18. Influence of the isolation procedure on coriander leaf volatiles with some correlation to the enzymatic activity. (United States)

    To Quynh, Cung Thi; Iijima, Yoko; Kubota, Kikue


    Coriander leaves (Coriandrum sativum L.) have become popular worldwide because of their pleasant and delicate aroma. By a hot water extraction method, in which coriander leaves were cut before suspending in boiling water for 2 min, the contents of the main volatile compounds such as alkanals and 2-alkenals from C10 to C14 decreased, while the levels of corresponding alcohols increased in comparison to those obtained by solvent extraction. To investigate the reasons for this variation, an enzyme activity was assayed. By using aliphatic aldehyde as a substrate and NADPH as a coenzyme, strong activity of an aliphatic aldehyde reductase was found for the first time in this herb in the relatively wide pH range of 5.0-9.0, with the maximum activity at pH 8.5. Additionally, the aliphatic aldehyde dehydrogenase, responsible for acid formation, was also found to have a relatively weak activity compared to that of reductase.

  19. Pyridoxine Supplementation Improves the Activity of Recombinant Glutamate Decarboxylase and the Enzymatic Production of Gama-Aminobutyric Acid.

    Directory of Open Access Journals (Sweden)

    Yan Huang

    Full Text Available Glutamate decarboxylase (GAD catalyzes the irreversible decarboxylation of L-glutamate to the valuable food supplement γ-aminobutyric acid (GABA. In this study, GAD from Escherichia coli K12, a pyridoxal phosphate (PLP-dependent enzyme, was overexpressed in E. coli. The GAD produced in media supplemented with 0.05 mM soluble vitamin B6 analog pyridoxine hydrochloride (GAD-V activity was 154.8 U mL-1, 1.8-fold higher than that of GAD obtained without supplementation (GAD-C. Purified GAD-V exhibited increased activity (193.4 U mg-1, 1.5-fold higher than that of GAD-C, superior thermostability (2.8-fold greater than that of GAD-C, and higher kcat/Km (1.6-fold higher than that of GAD-C. Under optimal conditions in reactions mixtures lacking added PLP, crude GAD-V converted 500 g L-1 monosodium glutamate (MSG to GABA with a yield of 100%, and 750 g L-1 MSG with a yield of 88.7%. These results establish the utility of pyridoxine supplementation and lay the foundation for large-scale enzymatic production of GABA.

  20. Identification of a novel enzymatic activity from lactic acid bacteria able to degrade biogenic amines in wine. (United States)

    Callejón, S; Sendra, R; Ferrer, S; Pardo, I


    The main objectives of this study were the search for enzymatic activities responsible for biogenic amine (BA) degradation in lactic acid bacteria (LAB) strains isolated from wine, their identification, and the evaluation of their applicability for reducing BAs in wine. Fifty-three percent of the 76 LAB cell extracts showed activity against a mixture of histamine, tyramine, and putrescine when analyzed in-gel. The quantification of the degrading ability for each individual amine was tested in a synthetic medium and wine. Most of the bacteria analyzed were able to degrade the three amines in both conditions. The highest percentages of degradation in wine were those of putrescine: up to 41% diminution in 1 week. Enzymes responsible for amine degradation were isolated and purified from Lactobacillus plantarum J16 and Pediococcus acidilactici CECT 5930 strains and were identified as multicopper oxidases. This is the first report of an efficient BA reduction in wine by LAB. Furthermore, the identity of the enzymes involved has been revealed.

  1. Quantifying protein adsorption and function at nanostructured materials: enzymatic activity of glucose oxidase at GLAD structured electrodes. (United States)

    Jensen, Uffe B; Ferapontova, Elena E; Sutherland, Duncan S


    Nanostructured materials strongly modulate the behavior of adsorbed proteins; however, the characterization of such interactions is challenging. Here we present a novel method combining protein adsorption studies at nanostructured quartz crystal microbalance sensor surfaces (QCM-D) with optical (surface plasmon resonance SPR) and electrochemical methods (cyclic voltammetry CV) allowing quantification of both bound protein amount and activity. The redox enzyme glucose oxidase is studied as a model system to explore alterations in protein functional behavior caused by adsorption onto flat and nanostructured surfaces. This enzyme and such materials interactions are relevant for biosensor applications. Novel nanostructured gold electrode surfaces with controlled curvature were fabricated using colloidal lithography and glancing angle deposition (GLAD). The adsorption of enzyme to nanostructured interfaces was found to be significantly larger compared to flat interfaces even after normalization for the increased surface area, and no substantial desorption was observed within 24 h. A decreased enzymatic activity was observed over the same period of time, which indicates a slow conformational change of the adsorbed enzyme induced by the materials interface. Additionally, we make use of inherent localized surface plasmon resonances in these nanostructured materials to directly quantify the protein binding. We hereby demonstrate a QCM-D-based methodology to quantify protein binding at complex nanostructured materials. Our approach allows label free quantification of protein binding at nanostructured interfaces.

  2. Nanonets Derived from Turnip Mosaic Virus as Scaffolds for Increased Enzymatic Activity of Immobilized Candida antarctica Lipase B. (United States)

    Cuenca, Sol; Mansilla, Carmen; Aguado, Marta; Yuste-Calvo, Carmen; Sánchez, Flora; Sánchez-Montero, Jose M; Ponz, Fernando


    Elongated flexuous plant viral nanoparticles (VNPs) represent an interesting platform for developing different applications in nanobiotechnology. In the case of potyviruses, the virion external surface is made up of helically arrayed domains of the viral structural coat protein (CP), repeated over 2000 times, in which the N- and C-terminal domains of each CP are projected toward the exterior of the external virion surface. These characteristics provide a chemical environment rich in functional groups susceptible to chemical conjugations. We have conjugated Candida antarctica lipase B (CALB) onto amino groups of the external surface of the potyvirus turnip mosaic virus (TuMV) using glutaraldehyde as a conjugating agent. Using this approach, TuMV virions were transformed into scaffolds for CALB nanoimmobilization. Analysis of the resulting structures revealed the formation of TuMV nanonets onto which large CALB aggregates were deposited. The functional enzymatic characterization of the CALB-bearing TuMV nanonets showed that CALB continued to be active in the nanoimmobilized form, even gaining an increased relative specific activity, as compared to the non-immobilized form. These novel virus-based nanostructures may provide a useful new approach to enzyme nanoimmobilization susceptible to be industrially exploited.

  3. Extracellular endoglucanase activity from Paenibacillus polymyxa BEb-40: production, optimization and enzymatic characterization. (United States)

    Gastelum-Arellanez, Argel; Paredes-López, Octavio; Olalde-Portugal, Víctor


    Endoglucanase activity produced by Paenibacillus polymyxa BEb-40 was studied. In submerged culture with minimal medium supplemented with carboxymethylcellulose (CMC), this microorganism produced up to 0.37 U/mL endoglucanase activity with high specific activity (14.3 U/mgtotal protein). Detection of endoglucanase activity through zymography revealed at least 14 isoenzymes with molecular weights between 38 and 220 kDa. This high variety of secreted endoglucanases has not been described previously in Paenibacillus genus. The optimum conditions, determined by response surface methodology, were 48 °C and pH 3.4, which allowed an increase of 33.7 % in the relative endoglucanase activity obtained with respect to the standard conditions. Nevertheless, high levels of hydrolysis of at least 70 % of the maximum activity could be obtained at wide ranges of pH (2-9) and temperature (40-60 °C). Under optimal conditions, high levels of CMC hydrolysis were reached, of about 40 %, after only 12 h of reaction with substrate/total protein ratios between 19 and 76. Kinetic analysis revealed that endoglucanase activity followed a mixed inhibition model (K m = 8.4 mM, K ic = 0.03 mM, K iu = 0.35 mM, V max = 33.3 U/mgtotal protein). These results allow to consider P. polymyxa BEb-40 as a promising microorganism for the production of endoglucanases, with possibilities of application in the breakdown of lignocellulosic biomass. The high specific activity at wide ranges of pH and temperature can allow its use in a wide variety of processes, under both acidic and alkaline conditions, as well as in mesophilic and thermophilic temperatures, further reducing the amount of enzymes used.

  4. Enzymatic activity of a novel halotolerant lipase from Haloarcula hispanica 2TK2

    Directory of Open Access Journals (Sweden)

    Ozgen Melis


    Full Text Available A strain of Haloarcula hispanica isolated from Tuzkoy salt mine, Turkey exhibited extracellular lipolytic activity. Important parameters such as carbon sources and salt concentration for lipase production were investigated. Optimal conditions for the enzyme production from Haloarcula hispanica 2TK2 were determined. It was observed that the lipolytic activity of Haloarcula hispanica was stimulated by some of the carbon sources. The high lipase acitivity values were obtained in the presence of 2% (v/v walnut oil (6.16 U/ml, 1% (v/v fish oil (5.07 U/ml, 1% (v/v olive oil (4.52 U/ml and 1% (w/v stearic acid (4.88 U/ml at 4M NaCl concentration. Lipase was partially purified by ammonium sulfate precipitation and ultrafiltration. Optimal temperature and pH values were determined as 45°C and 8.0, respectively. Lipase activity decreased with the increasing salt concentration, but 85% activity of the enzyme was maintained at 5M NaCl concentration. The enzyme preserved 41% of its relative activity at 90°C. The partially purified lipase maintained its activity in the presence of surfactants such as Triton X-100 and SDS. Therefore, the lipase which is an extremozyme may have potential applications especially in detergent industry.

  5. Irradiation effect on enzymatic activity of papain with {sup 60}Co-{gamma} rays

    Energy Technology Data Exchange (ETDEWEB)

    Furuta, Masakazu; Ohashi, Isao; Oka, Masahito; Hayashi, Toshio [Osaka Prefecture Univ., Sakai (Japan). Research Inst. for Advanced Science and Technology


    An investigation was made on the durability of enzyme activity against {sup 60}Co-{gamma} irradiation at a dose up to 55 kGy/h using dry powder and aqueous solution of papain preparations on the market. Hybrid materials including bioactive molecules combined with biocompatible synthetic polymers are expected to have biocompatible properties and also biomimetic functions as a component of artificial organs for human body. The activity of papain in an aqueous solution was rapidly decreased at the early stage of irradiation through oxidation of SH group at its active site with active oxygen produced by the irradiation and then, partially recovered since SH group was reproduced in an anoxic state after O{sub 2} consumption in the solution irradiated at a high dose. A usual radiation method for sterilization was found applicable to decontamination of dry and frozen preparations of papain. When suitable conditions for radiation were chosen and N{sub 2} gas was purged to suppress the formation of free radicals, it was possible to keep the enzyme activity at more than 50% of the initial activity after radiation at 30 kGy. (M.N.)

  6. Substrate binding of gelatinase B induces its enzymatic activity in the presence of intact propeptide. (United States)

    Bannikov, Gregory A; Karelina, Tatiana V; Collier, Ivan E; Marmer, Barry L; Goldberg, Gregory I


    Expression of gelatinase B (matrix metalloprotease 9) in human placenta is developmentally regulated, presumably to fulfill a proteolytic function. Here we demonstrate that gelatinolytic activity in situ, in tissue sections of term placenta, is co-localized with gelatinase B. Judging by molecular mass, however, all the enzyme extracted from this tissue was found in a proform. To address this apparent incongruity, we examined the activity of gelatinase B bound to either gelatin- or type IV collagen-coated surfaces. Surprisingly, we found that upon binding, the purified proenzyme acquired activity against both the fluorogenic peptide (7-methoxycoumarin-4-yl)-acetic acid (MCA)-Pro-Leu-Gly-Leu-3-(2,4-dinitrophenyl)-l-2,3-diaminopropionyl-Ala-Arg-NH(2) and gelatin substrates, whereas its propeptide remained intact. These results suggest that although activation of all known matrix metalloproteases in vitro is accomplished by proteolytic processing of the propeptide, other mechanisms, such as binding to a ligand or to a substrate, may lead to a disengagement of the propeptide from the active center of the enzyme, causing its activation.

  7. Influence of linker length and composition on enzymatic activity and ribosomal binding of neomycin dimers. (United States)

    Watkins, Derrick; Kumar, Sunil; Green, Keith D; Arya, Dev P; Garneau-Tsodikova, Sylvie


    The human and bacterial A site rRNA binding as well as the aminoglycoside-modifying enzyme (AME) activity against a series of neomycin B (NEO) dimers is presented. The data indicate that by simple modifications of linker length and composition, substantial differences in rRNA selectivity and AME activity can be obtained. We tested five different AMEs with dimeric NEO dimers that were tethered via triazole, urea, and thiourea linkages. We show that triazole-linked dimers were the worst substrates for most AMEs, with those containing the longer linkers showing the largest decrease in activity. Thiourea-linked dimers that showed a decrease in activity by AMEs also showed increased bacterial A site binding, with one compound (compound 14) even showing substantially reduced human A site binding. The urea-linked dimers showed a substantial decrease in activity by AMEs when a conformationally restrictive phenyl linker was introduced. The information learned herein advances our understanding of the importance of the linker length and composition for the generation of dimeric aminoglycoside antibiotics capable of avoiding the action of AMEs and selective binding to the bacterial rRNA over binding to the human rRNA.

  8. ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes. (United States)

    Boschin, Giovanna; Scigliuolo, Graziana Maria; Resta, Donatella; Arnoldi, Anna


    The objective of this investigation was to compare the angiotensin converting enzyme (ACE)-inhibitory activity of the hydrolysates obtained by pepsin digestion of proteins of some legumes, such as chickpea, common bean, lentil, lupin, pea, and soybean, by using the same experimental procedure. The ACE-inhibitory activity was measured by using the tripeptide hippuryl-histidyl-leucine (HHL), as model peptide, and HPLC-DAD, as analytical method. The peptide mixtures of all legumes were active, with soybean and lupin the most efficient, with IC50 values of 224 and 226 μg/ml, respectively. Considering the promising results obtained with lupin, and aiming to identify the protein(s) that release(s) the peptides responsible for the activity, the peptides obtained from the pepsin digestion of some industrial lupin protein isolates and purified protein fractions were tested. The most active mixture, showing an IC50 value of 138 μg/ml, was obtained hydrolysing a mixture of lupin α+β conglutin.

  9. Cellulase-producing bacteria from Thai higher termites, Microcerotermes sp.: enzymatic activities and ionic liquid tolerance. (United States)

    Taechapoempol, Kitipong; Sreethawong, Thammanoon; Rangsunvigit, Pramoch; Namprohm, Weerachart; Thamprajamchit, Bandhit; Rengpipat, Sirirat; Chavadej, Sumaeth


    The three highest hydrolysis-capacity-value isolates of Bacillus subtilis (A 002, M 015, and F 018) obtained from Thai higher termites, Microcerotermes sp., under different isolation conditions (aerobic, anaerobic, and anaerobic/aerobic) were tested for cellulase activities--FPase, endoglucanase, and β-glucosidase--at 37 °C and pH 7.2 for 24 h. Their tolerance to an ionic liquid, 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), was also investigated. The results showed that the isolate M 015 provided the highest endoglucanase activity whereas the highest FPase and β-glucosidase activities were observed for the isolate F 018. The isolate F 018 also showed the highest tolerance to [BMIM]Cl in the range of 0.1-1.0 vol.%. In contrast, the isolate A 002 exhibited growth retardation in the presence of 0.5-1.0 vol.% [BMIM]Cl.

  10. Effect of Static Magnetic Field on α-Amylase Activity and Enzymatic Reaction

    Institute of Scientific and Technical Information of China (English)

    JIA Shaoyi; LIU Yong; WU Songhai; WANG Zhibin


    The effect of magnetic field on α-amylase was studied. Under the experimental conditions, α-amylase solution was treated by 0.15 T, 0.30 T and 0.45 T static magnetic fields for a known period of time, then the activ-ity, kinetic parameters, and the secondary conformation were investigated. The results showed that there was a con-siderable effect of the magnetic exposure on the α-amylase. The activity was increased by 27%, 34.1%, 37.8% compared with the control. It was also found that both kinetic parameters Km and Vm could be decreased due to the increasing magnetic field, Km decreased from 2.20×102 to 0.87×102, whereas Vm decreased from 2.0×103 g/min to 1.1×103g/min. At the same time, there were some irregular changes in α-amylase secondary conformation.

  11. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Monika B Dolinska

    Full Text Available BACKGROUND: Tyrosinase (TYR catalyzes the rate-limiting, first step in melanin production and its gene (TYR is mutated in many cases of oculocutaneous albinism (OCA1, an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. METHODOLOGY/PRINCIPAL FINDINGS: The intra-melanosomal domain of human tyrosinase (residues 19-469 and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. CONCLUSIONS/SIGNIFICANCE: The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

  12. Enzymatic activity induced by interactions with a nanofabricated hydrophobic Si surface (United States)

    Nishiyama, Katsuhiko


    The binding of peptides of 2-10 glycine residues (2-10Gly) to papain on nanofabricated hydrophobic Si surfaces was investigated by molecular dynamics and docking simulations. 5Gly, 7Gly, 9Gly, and 10Gly were distributed on sites near the active center of papain on the Si surface, while 6-10Gly were distributed on sites near the active center of free papain. The Si surface changed the substrate specificity of papain, and modification of this surface should allow full control of substrate specificity. Molecular surgery of proteins in cells may be realized using papain on specially designed surfaces.

  13. Intestinal morphology and enzymatic activity in newly weaned piglets fed contrasting fiber levels and fiber properties

    DEFF Research Database (Denmark)

    Eskildsen, Maria


    ABSTRACT: The main objective of this study was to determinetheeffectoffibersourceandconcentrationon morphological characteristics, mucin staining pattern, and mucosal enzyme activities in the gastrointestinal tract of pigs. The experiment included 50 pigs from 10 litters weaned at 4 wk of age (BW ...... the small intestine, indicating that the pigs fed the pectinKey words: digestive enzyme, fiber, gut morphology, mucin, pig ......ABSTRACT: The main objective of this study was to determinetheeffectoffibersourceandconcentrationon morphological characteristics, mucin staining pattern, and mucosal enzyme activities in the gastrointestinal tract of pigs. The experiment included 50 pigs from 10 litters weaned at 4 wk of age (BW 8...

  14. Long-term effects of nickel oxide nanoparticles on performance, microbial enzymatic activity, and microbial community of a sequencing batch reactor. (United States)

    Wang, Sen; Li, Zhiwei; Gao, Mengchun; She, Zonglian; Guo, Liang; Zheng, Dong; Zhao, Yangguo; Ma, Bingrui; Gao, Feng; Wang, Xuejiao


    The nitrogen and phosphorus removal, microbial enzymatic activity, and microbial community of a sequencing batch reactor (SBR) were evaluated under long-term exposure to nickel oxide nanoparticles (NiO NPs). High NiO NP concentration (over 5 mg L(-1)) affected the removal of chemical oxygen demand, nitrogen, and phosphorus. The presence of NiO NP inhibited the microbial enzymatic activities and reduced the nitrogen and phosphorus removal rates of activated sludge. The microbial enzymatic activities of the activated sludge showed a similar variation trend to the nitrogen and phosphorus removal rates with the increase in NiO NP concentration from 0 to 60 mg L(-1). The Ni content in the effluent and activated sludge showed an increasing trend with the increase in NiO NP concentration. Some NiO NPs were absorbed on the sludge surface or penetrate the cell membrane into the interior of microbial cells in the activated sludge. NiO NP facilitated the increase in reactive oxygen species by disturbing the balance between the oxidation and anti-oxidation processes, and the variation in lactate dehydrogenase demonstrated that NiO NP could destroy the cytomembrane and cause variations in the microbial morphology and physiological function. High-throughput sequencing demonstrated that the microbial community of SBR had some obvious changes at 0-60 mg L(-1) NiO NPs at the phyla, class and genus levels.

  15. Chemical Characterization, Antioxidant and Enzymatic Activity of Brines from Scandinavian Marinated Herring Products

    DEFF Research Database (Denmark)

    Gringer, Nina; Osman, Ali; Nielsen, Henrik Hauch;


    Brines generated during the last marination step in the production of marinated herring (Clupea harengus) were chemically characterized and analyzed for antioxidant and enzyme activities. The end-products were vinegar cured, spice cured and traditional barrel-salted herring with either salt...

  16. Insecticidal effects of Moroccan plant extracts on development, energy reserves and enzymatic activities of Plodia interpunctella

    Energy Technology Data Exchange (ETDEWEB)

    Bouayard, N.; Rharrabe, K.; Ghailani, N. N.; Jbilou, R.; Castanera, P.; Ortego, F.


    The aim of this work was to study the effects of methanol extracts of ten plant species used in traditional medicine in Morocco (Peganum harmala, Ajuga iva, Rosmarinus officinalis, Lavandula stoechas, Lavandula dentata, Cistus ladanifer, Cistus salviaefolius, Cistus monspeliensis, Centaurium erythraea and Launaea arborescens) on Plodia interpunctella Hubner (Lepidoptera: Pyralidae) larvae. Firstly, we studied the effects of the ingestion of these extracts at 500 ppm on post-embryonic development parameters. Most plant extracts provoked a notable decrease of larval weight 8 days after treatment (up to 33% weight loss with C. erythraea) and caused significant alterations on pupation (ranging from 5% to 85%) and adult emergence (below 2.5% with R. officinalis, C. erythraea and A. iva). The plant extracts that showed strongest effects on post-embryonic development were selected to test their effects on the following physiological parameters: larval reserve substances (at 500 ppm); and midgut activities of hydrolytic and detoxification enzymes (at 500, 750 and 1000 ppm). All treatments provoked a significant reduction of protein and carbon hydrate larval contents, the inhibition of proteases and {alpha}-amylase activities in a dose depended manner, and the induction of glutathione S-transferase and esterase (using MtB as substrate) activities, whereas the activity of cytochrome P450 monooxygenases and esterases (using 1-NA as substrate) increase or decrease depending on the extract concentration and the plant analyzed. (Author) 65 refs.

  17. Insecticidal effects of Moroccan plant extracts on development, energy reserves and enzymatic activities of Plodia interpunctella

    Directory of Open Access Journals (Sweden)

    N. Bouayad


    Full Text Available The aim of this work was to study the effects of methanol extracts of ten plant species used in traditional medicine in Morocco (Peganum harmala, Ajuga iva, Rosmarinus officinalis, Lavandula stoechas, Lavandula dentata, Cistus ladanifer, Cistus salviaefolius, Cistus monspeliensis, Centaurium erythraea and Launaea arborescens on Plodia interpunctella Hübner (Lepidoptera: Pyralidae larvae. Firstly, we studied the effects of the ingestion of these extracts at 500 ppm on post-embryonic development parameters. Most plant extracts provoked a notable decrease of larval weight 8 days after treatment (up to 33% weight loss with C. erythraea and caused significant alterations on pupation (ranging from 5% to 85% and adult emergence (below 2.5% with R. officinalis, C. erythraea and A. iva. The plant extracts that showed strongest effects on post-embryonic development were selected to test their effects on the following physiological parameters: larval reserve substances (at 500 ppm; and midgut activities of hydrolytic and detoxification enzymes (at 500, 750 and 1000 ppm. All treatments provoked a significant reduction of protein and carbon hydrate larval contents, the inhibition of proteases and α-amylase activities in a dose depended manner, and the induction of glutathione S-transferase and esterase (using MtB as substrate activities, whereas the activity of cytochrome P450 monooxygenases and esterases (using 1-NA as substrate increase or decrease depending on the extract concentration and the plant analyzed.


    This study was undertaken to examine the effects of the triazole antifungal agent fluconazole on the expression of hepatic cytochrome P450 (Cyp) genes and the activities of Cyp enzymes in male Sprague-Dawley rats and male CD-1 mice. Alkoxyresorufin O-dealkylation (AROD) methods w...

  19. The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells. (United States)

    Mattox, Mildred L; D'Angelo, June A; Grimes, Zachary M; Fiebiger, Edda; Dickinson, Bonny L


    Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs.

  20. Glycolytic enzymatic activities in developing seeds involved in the differences between standard and low oil content sunflowers (Helianthus annuus L.). (United States)

    Troncoso-Ponce, M Adrián; Garcés, Rafael; Martínez-Force, Enrique


    As opposed to other oilseeds, developing sunflower seeds do not accumulate starch initially. They rely on the sucrose that comes from the mother plant to synthesise lipid precursors. Glycolysis is the principal source of carbon skeletons and reducing power for lipid biosynthesis. In this work, glycolytic initial metabolites and enzyme activities from developing seed of two different sunflower lines, of high and low oil content, were compared during storage lipid synthesis. These two lines showed different kinetic lipid accumulation in the developing embryos. Fatty acids levels during the initial and final stage of lipid synthesis were higher in CAS-6 than in ZEN-8. The analysis of the photosynthate and sugars content suggests that, although the hexoses levels were quite similar in both lines, the amount of sucrose produced by the mother plant and available for lipid synthesis was higher in CAS-6. Although, a smaller amount of sucrose is available in the ZEN-8 line, its seeds maintain the levels of intermediate sugars in the initial steps of glycolysis due to an increase in the levels of the invertase, hexokinase and phosphoglucose isomerase activities in ZEN-8, with respect to CAS-6. Also, a readjustment in the final part of this metabolic route took place, with the activities of phosphoglycerate kinase and enolase in CAS-6 being higher, allowing increased synthesis of phosphoenolpiruvate, the intermediate carbon donor for fatty acid synthesis. In addition, recently, it has been shown that Arabidopsis mutants with a lower fat content in their seeds have a higher amount of sucrose. These data together point to these last two enzymatic activities, phosphoglycerate kinase and enolase, as being responsible for the lower fat content in the ZEN-8 line.

  1. Intestinal morphology and enzymatic activity in newly weaned pigs fed contrasting fiber concentrations and fiber properties. (United States)

    Hedemann, M S; Eskildsen, M; Laerke, H N; Pedersen, C; Lindberg, J E; Laurinen, P; Knudsen, K E Bach


    The main objective of this study was to determine the effect of fiber source and concentration on morphological characteristics, mucin staining pattern, and mucosal enzyme activities in the gastrointestinal tract of pigs. The experiment included 50 pigs from 10 litters weaned at 4 wk of age (BW 8.6 +/- 1.4 kg) and divided into 5 treatment groups. Diets containing fiber of various physico-chemical properties and concentrations were formulated to contain 73, 104, or 145 g of dietary fiber/kg of DM. The diets were based on raw wheat and barley flours. Pectin and barley hulls, representing soluble and insoluble fiber sources, respectively, were used to increase the fiber concentration. The pigs were fed the experimental diets for 9 d, and then the pigs were euthanized and the entire gastrointestinal tract was removed. Tissue samples were taken from the mid and distal small intestine and from the mid colon. Inclusion of pectin in the diets significantly decreased (P fiber content, whereas sucrase activity was increased in pigs fed the pectin-containing diets. The activity of the peptidases, aminopeptidase N and dipeptidylpeptidase IV, was increased when feeding high fiber diets, whereas the activity of gamma-glutamyl transpeptidase remained unaffected by the experimental diets. In conclusion, the reduced feed intake observed with the pectin-containing diets could explain the lower villous height and crypt depth observed in this study. However, direct effects of pectin also are possible, and thus further study is warranted. Feeding pigs high insoluble fiber diets improved gut morphology by increasing villi length and increased mucosal enzyme activity when compared with pigs fed pectin-containing diets. The mucin content as determined by staining characteristics suggests that pigs fed high insoluble fiber diets might be better protected against pathogenic bacteria than pigs fed diets high in soluble fiber.

  2. Mitomycin C-DNA adducts generated by DT-diaphorase. Revised mechanism of the enzymatic reductive activation of mitomycin C. (United States)

    Suresh Kumar, G; Lipman, R; Cummings, J; Tomasz, M


    Mitomycin C (MC) was reductively activated by DT-diaphorase [DTD; NAD(P)H:quinone oxidoreductase] from rat liver carcinoma cells in the presence of Micrococcus lysodeicticus DNA at pH 5.8 and 7.4. The resulting alkylated MC-DNA complexes were digested to the nucleoside level and the covalent MC-nucleoside adducts were separated, identified, and quantitatively analyzed by HPLC. In analogous experiments, two other flavoreductases, NADH-cytochrome c reductase and NADPH-cytochrome c reductase, as well as two chemical reductive activating agents Na2S2O4 and H2/PtO2 were employed as activators for the alkylation of DNA by MC. DTD as well as all the other activators generated the four known major guanine-N2-MC adducts at both pHs. In addition, at the lower pH, the guanine-N7-linked adducts of 2,7-diaminomitosene were detectable in the adduct patterns. At a given pH all the enzymatic and chemical reducing agents generated very similar adduct patterns which, however, differed dramatically at the acidic as compared to the neutral pH. Overall yield of MC adducts was 3-4-fold greater at pH 7.4 than at 5. 8 except in the case of DTD when it was 4-fold lower. Without exception, however, cross-link adduct yields were greater at the acidic pH (2-10-fold within the series). The ratio of adducts of bifunctional activation to those of monofunctional activation was 6-20-fold higher at the acidic as compared to the neutral pH. A comprehensive mechanism of the alkylation of DNA by activated MC was derived from the DNA adduct analysis which complements earlier model studies of the activation of MC. The mechanism consists of three competing activation pathways yielding three different DNA-reactive electrophiles 11, 12, and 17 which generate three unique sets of DNA adducts as endproducts. The relative amounts of these adducts are diagnostic of the relative rates of the competing pathways in vitro, and most likely, in vivo. Factors that influence the relative rates of individual pathways

  3. Enzymatic activity of soluble and membrane tethered peptide pro-hormone convertase 1. (United States)

    Bruzzaniti, Angela; Mains, Richard E


    Pro-hormone convertases PC1 and PC2 perform endoproteolytic cleavages of precursors in peptide-containing secretory granules. PC1 and PC2 are soluble, secreted with bioactive peptides. Evolutionarily related PCs have membrane tethers, not secreted. We tethered PC1 to the transmembrane-cytoplasmic domains (CD) of a granule enzyme (peptidylglycine-alpha-amidating monooxygenase; PAM) and Golgi-localized PC8. The tethered PC1 is far more stable to elevated temperature and denaturants than soluble PC1, and more active. Both tethers allow PC1 to visit the cell surface transiently, cleaving soluble molecules outside the cell. Both membrane-bound PC1 chimeras cleave membrane PAM into soluble active fragments when PAM is expressed on adjacent cells.

  4. Antioxidant Defense by Thioredoxin Can Occur Independently of Canonical Thiol-Disulfide Oxidoreductase Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Miryoung Song


    Full Text Available The thiol-disulfide oxidoreductase CXXC catalytic domain of thioredoxin contributes to antioxidant defense in phylogenetically diverse organisms. We find that although the oxidoreductase activity of thioredoxin-1 protects Salmonella enterica serovar Typhimurium from hydrogen peroxide in vitro, it does not appear to contribute to Salmonella’s antioxidant defenses in vivo. Nonetheless, thioredoxin-1 defends Salmonella from oxidative stress resulting from NADPH phagocyte oxidase macrophage expression during the innate immune response in mice. Thioredoxin-1 binds to the flexible linker, which connects the receiver and effector domains of SsrB, thereby keeping this response regulator in the soluble fraction. Thioredoxin-1, independently of thiol-disulfide exchange, activates intracellular SPI2 gene transcription required for Salmonella resistance to both reactive species generated by NADPH phagocyte oxidase and oxygen-independent lysosomal host defenses. These findings suggest that the horizontally acquired virulence determinant SsrB is regulated post-translationally by ancestrally present thioredoxin.

  5. Lipase catalyzed esterification of glycidol in nonaqueous solvents: solvent effects on enzymatic activity. (United States)

    Martins, J F; de Sampaio, T C; de Carvalho, I B; Barreiros, S


    We studied the effect of organic solvents on the kinetics of porcine pancreatic lipase (pp) for the resolution of racemic glycidol through esterification with butyric acid. We quantified ppl hydration by measuring water sorption isotherms for the enzyme in the solvents/mixtures tested. The determination of initial rates as a function of enzyme hydration revealed that the enzyme exhibits maximum apparent activity in the solvents/mixtures at the same water content (9% to 11% w/w) within the associated experimental error. The maximum initial rates are different in all the media and correlate well with the logarithm of the molar solubility of water in the media, higher initial rates being observed in the solvents/mixtures with lower water solubilities. The data for the mixtures indicate that ppl apparent activity responds to bulk property of the solvent. Measurements of enzyme particle sizes in five of the solvents, as function of enzyme hydration, revealed that mean particle sizes increased with enzyme hydration in all the solvents, differences between solvents being more pronounced at enzyme hydration levels close to 10%. At this hydration level, solvents having a higher water content lead to lower reaction rates; these are the solvents where the mean enzyme particle sizes are greater. Calculation of the observable modulus indicates there are no internal diffusion limitations. The observed correlation between changes in initial rates and changes in external surface area of the enzyme particles suggests that interfacial activation of ppl is only effective at the external surface of the particles. Data obtained for the mixtures indicate that ppl enantioselectivity depends on specific solvent-enzyme interactions. We make reference to ppl hydration and activity in supercritical carbon dioxide.

  6. Variations of the enzymatic activity of the rat's gum glycolytic cycle following administration of sweeteners. (United States)

    Seri, S; D'Alessandro, A; Seri, M


    The effects of saccharose and isomalt on the rat's gum carbohydrate metabolism have been studied through evaluation of the activity of hexokinase (HK), phosphoglucoisomerase (PGI), lactatedehydrogenase (LDH), glucose-6-phosphate-dehydrogenase (G6PD), phosphofructokinase (PFK) and phosphoglucomutase (PGM). The values obtained by administering a drink sweetened with 10% isomalt are significantly lower (p isomalt on the rat's gum glycolytic cycle enzymes was found to be lower than that detected for other polyalcohols.

  7. Effect of high pressures on the enzymatic activity of commercial milk protein coagulants (United States)

    Wiśniewska, Krystyna; Reps, Arnold; Jankowska, Agnieszka


    This study was aimed at determining the effect of high pressures in the range of 100-1000 MPa/15 min, applied in 100 MPa increments, on the coagulating and proteolytic activity of commercial coagulants produced with genetic engineering methods: Maxiren, Chymogen, Chymax and of a natural rennin preparation, Hala. The coagulating activity of Hala preparation differed compared with the other preparations, due to greater resistance to high pressures, especially in the range of 500-600 MPa. The preparations produced with genetic engineering methods lost their capability for milk protein coagulation by 500 MPa. Pressurization at 200 MPa contributed to their reduced capability for casein macroproteolysis. In contrast, an increase in Chymax, Chymogen, Maxiren and Hala preparations' hydrolytic capability for the macroproteolysis of isoelectric casein was observed upon pressure treatment at 100 and 400 MPa and for microproteolysis after pressure treatment at 200 MPa. Storage (48 h/5°C) of the pressurized preparations had an insignificant effect on their coagulating and proteolytic activities.

  8. Effects of six selected antibiotics on plant growth and soil microbial and enzymatic activities

    Energy Technology Data Exchange (ETDEWEB)

    Liu Feng [State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, 511 Kehua Street, Tianhe District, Guangzhou 510640 (China); Ying Guangguo, E-mail: [State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, 511 Kehua Street, Tianhe District, Guangzhou 510640 (China); Tao Ran; Zhao Jianliang; Yang Jifeng [State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, 511 Kehua Street, Tianhe District, Guangzhou 510640 (China); Zhao Lanfeng [College of Resource and Environmental Science, South China Agricultural University, Guangzhou 510642 (China)


    The potential impact of six antibiotics (chlortetracycline, tetracycline and tylosin; sulfamethoxazole, sulfamethazine and trimethoprim) on plant growth and soil quality was studied by using seed germination test on filter paper and plant growth test in soil, soil respiration and phosphatase activity tests. The phytotoxic effects varied between the antibiotics and between plant species (sweet oat, rice and cucumber). Rice was most sensitive to sulfamethoxazole with the EC10 value of 0.1 mg/L. The antibiotics tested inhibited soil phosphatase activity during the 22 days' incubation. Significant effects on soil respiration were found for the two sulfonamides (sulfamethoxazole and sulfamethazine) and trimethoprim, whereas little effects were observed for the two tetracyclines and tylosin. The effective concentrations (EC10 values) for soil respiration in the first 2 days were 7 mg/kg for sulfamethoxazole, 13 mg/kg for sulfamethazine and 20 mg/kg for trimethoprim. Antibiotic residues in manure and soils may affect soil microbial and enzyme activities. - Terrestrial ecotoxicological effects of antibiotics are related to their sorption and degradation behavior in soil.

  9. Antimicrobial and antioxidant activities of clove essential oil and eugenyl acetate produced by enzymatic esterification. (United States)

    Vanin, Adriana B; Orlando, Tainara; Piazza, Suelen P; Puton, Bruna M S; Cansian, Rogério L; Oliveira, Debora; Paroul, Natalia


    This work reports the maximization of eugenyl acetate production by esterification of essential oil of clove in a solvent-free system using Novozym 435 as catalyst. The antimicrobial and antioxidant activities of clove essential oil and eugenyl acetate produced were determined. The conditions that maximized eugenyl acetate production were 60 °C, essential oil of clove to acetic anhydride ratio of 1:5, 150 rpm, and 10 wt% of enzyme, with a conversion of 99.87 %. A kinetic study was performed to assess the influence of substrates' molar ratio, enzyme concentration, and temperature on product yield. Results show that an excess of anhydride, enzyme concentration of 5.5 wt%, 50 °C, and essential oil of clove to acetic anhydride ratio of 1:5 afforded nearly a complete conversion after 2 h of reaction. Comparing the antibacterial activity of the essential oil of clove before and after esterification, we observed a decrease in the antimicrobial activity of eugenyl acetate, particularly with regard to minimum inhibitory concentration (MIC). Both eugenyl acetate and clove essential oil were most effective to the gram-negative than gram-positive bacteria group. The results showed a high antioxidant potential for essential oil before and particularly after the esterification reaction thus becoming an option for the formulation of new antioxidant products.

  10. Differential coral bleaching-Contrasting the activity and response of enzymatic antioxidants in symbiotic partners under thermal stress. (United States)

    Krueger, Thomas; Hawkins, Thomas D; Becker, Susanne; Pontasch, Stefanie; Dove, Sophie; Hoegh-Guldberg, Ove; Leggat, William; Fisher, Paul L; Davy, Simon K


    Mass coral bleaching due to thermal stress represents a major threat to the integrity and functioning of coral reefs. Thermal thresholds vary, however, between corals, partly as a result of the specific type of endosymbiotic dinoflagellate (Symbiodinium sp.) they harbour. The production of reactive oxygen species (ROS) in corals under thermal and light stress has been recognised as one mechanism that can lead to cellular damage and the loss of their symbiont population (Oxidative Theory of Coral Bleaching). Here, we compared the response of symbiont and host enzymatic antioxidants in the coral species Acropora millepora and Montipora digitata at 28°C and 33°C. A. millepora at 33°C showed a decrease in photochemical efficiency of photosystem II (PSII) and increase in maximum midday excitation pressure on PSII, with subsequent bleaching (declining photosynthetic pigment and symbiont density). M. digitata exhibited no bleaching response and photochemical changes in its symbionts were minor. The symbiont antioxidant enzymes superoxide dismutase, ascorbate peroxidase, and catalase peroxidase showed no significant upregulation to elevated temperatures in either coral, while only catalase was significantly elevated in both coral hosts at 33°C. Increased host catalase activity in the susceptible coral after 5days at 33°C was independent of antioxidant responses in the symbiont and preceded significant declines in PSII photochemical efficiencies. This finding suggests a potential decoupling of host redox mechanisms from symbiont photophysiology and raises questions about the importance of symbiont-derived ROS in initiating coral bleaching.

  11. Divergence in the enzymatic activities of a tomato and Solanum pennellii alcohol acyltransferase impacts fruit volatile ester composition. (United States)

    Goulet, Charles; Kamiyoshihara, Yusuke; Lam, Nghi B; Richard, Théo; Taylor, Mark G; Tieman, Denise M; Klee, Harry J


    Tomato fruits accumulate a diverse set of volatiles including multiple esters. The content of ester volatiles is relatively low in tomato fruits (Solanum lycopersicum) and far more abundant in the closely related species Solanum pennellii. There are also qualitative variations in ester content between the two species. We have previously shown that high expression of a non-specific esterase is critical for the low overall ester content of S. lycopersicum fruit relative to S. pennellii fruit. Here, we show that qualitative differences in ester composition are the consequence of divergence in enzymatic activity of a ripening-related alcohol acyltransferase (AAT1). The S. pennellii AAT1 is more efficient than the tomato AAT1 for all the alcohols tested. The two enzymes have differences in their substrate preferences that explain the variations observed in the volatiles. The results illustrate how two related species have evolved to precisely adjust their volatile content by modulating the balance of the synthesis and degradation of esters.

  12. Long-term effects of cupric oxide nanoparticles (CuO NPs) on the performance, microbial community and enzymatic activity of activated sludge in a sequencing batch reactor. (United States)

    Wang, Sen; Li, Zhiwei; Gao, Mengchun; She, Zonglian; Ma, Bingrui; Guo, Liang; Zheng, Dong; Zhao, Yangguo; Jin, Chunji; Wang, Xuejiao; Gao, Feng


    The long-term effects of cupric oxide nanoparticles (CuO NPs) on the performance, microbial activity and microbial community of activated sludge were investigated in a sequencing batch reactor (SBR). The SBR performance had no evident change at 0-10 mg/L CuO NPs, whereas the CuO NPs concentration at 30-60 mg/L affected the COD, NH4(+)-N and soluble orthophosphate (SOP) removal, nitrogen and phosphorus removal rate and microbial enzymatic activity of activated sludge. Some CuO NPs might be absorbed on the surface of activated sludge or penetrate the microbial cytomembrane into the microbial cell interior of activated sludge. Compared to 0 mg/L CuO NPs, the reactive oxygen species (ROS) production and lactate dehydrogenase (LDH) release increased by 43.6% and 56.4% at 60 mg/L CuO NPs, respectively. The variations of ROS production and LDH release demonstrated that CuO NPs could induce the toxicity towards the microorganisms and destroy the integrity of microbial cytomembrane in the activated sludge. High throughput sequencing of 16S rDNA indicated that CuO NPs could evidently impact on the microbial richness, diversity and composition of activated sludge in the SBR.

  13. Comparison of mitochondrial and nucleolar RNase MRP reveals identical RNA components with distinct enzymatic activities and protein components. (United States)

    Lu, Qiaosheng; Wierzbicki, Sara; Krasilnikov, Andrey S; Schmitt, Mark E


    RNase MRP is a ribonucleoprotein endoribonuclease found in three cellular locations where distinct substrates are processed: the mitochondria, the nucleolus, and the cytoplasm. Cytoplasmic RNase MRP is the nucleolar enzyme that is transiently relocalized during mitosis. Nucleolar RNase MRP (NuMRP) was purified to homogeneity, and we extensively purified the mitochondrial RNase MRP (MtMRP) to a single RNA component identical to the NuMRP RNA. Although the protein components of the NuMRP were identified by mass spectrometry successfully, none of the known NuMRP proteins were found in the MtMRP preparation. Only trace amounts of the core NuMRP protein, Pop4, were detected in MtMRP by Western blot. In vitro activity of the two enzymes was compared. MtMRP cleaved only mitochondrial ORI5 substrate, while NuMRP cleaved all three substrates. However, the NuMRP enzyme cleaved the ORI5 substrate at sites different than the MtMRP enzyme. In addition, enzymatic differences in preferred ionic strength confirm these enzymes as distinct entities. Magnesium was found to be essential to both enzymes. We tested a number of reported inhibitors including puromycin, pentamidine, lithium, and pAp. Puromycin inhibition suggested that it binds directly to the MRP RNA, reaffirming the role of the RNA component in catalysis. In conclusion, our study confirms that the NuMRP and MtMRP enzymes are distinct entities with differing activities and protein components but a common RNA subunit, suggesting that the RNA must be playing a crucial role in catalytic activity.

  14. Induction of ferroxidase enzymatic activity by copper reduces MPP+-evoked neurotoxicity in rats. (United States)

    Rubio-Osornio, Moisés; Montes, Sergio; Heras-Romero, Yessica; Guevara, Jorge; Rubio, Carmen; Aguilera, Penélope; Rivera-Mancia, Susana; Floriano-Sánchez, Esaú; Monroy-Noyola, Antonio; Ríos, Camilo


    Parkinson's disease (PD) is a neurodegenerative disorder characterized by decreased dopamine, intracellular inclusions (Lewy bodies) and brain iron deposits. PD has also been related with reduced ferroxidase activity, diminished antioxidant defenses and lipid peroxidation. Striatal injection of 1-methyl-4-phenylpyridinium (MPP(+)) into rodents reproduces the major biochemical characteristics of PD, including oxidative stress. Copper (Cu) plays an important role as prosthetic group of several proteins involved in iron metabolism and antioxidant responses, such as ceruloplasmin (Cp). In the present study, intraperitoneal CuSO4 injection (10μmol/kg) produced an insignificant increase of Cu content in striatum and midbrain (17.5% and 7%, respectively). After 10 and 11h, Cu induced 6- and 4-fold increase Cp mRNA in midbrain and striatum, respectively. Cu-supplement also produced a time-dependent increase ferroxidase activity in striatal tissue, reaching a maximum 16h after Cu treatment in midbrain; while, ferrous iron content diminished 18% in striatum and 8% in midbrain. In regard the PD model, we found that MPP(+) (10μg/8μL, intrastriatal), induced a significant (P<0.05) reduction of striatal ferroxidase activity; this effect was reverted by Cu pre-treatment 16h before MPP(+). Likewise, Cu-supplement prevented lipid fluorescent products formation in striatum, evaluated (P<0.01) 6h after MPP(+). In the long term, apomorphine-evoked circling behavior was evaluated 6 days after MPP(+) injury; Cu pre-treatment significantly reduced (P<0.05) the apomorphine-induced ipsilateral turns in MPP(+)-lesioned rats. These results suggest that Cu-induced expression of Cp could be an interesting scope against the deleterious effects of iron deposits in PD.

  15. Screening for Genes Coding for Putative Antitumor Compounds, Antimicrobial and Enzymatic Activities from Haloalkalitolerant and Haloalkaliphilic Bacteria Strains of Algerian Sahara Soils

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    Okba Selama


    Full Text Available Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications.

  16. Screening for genes coding for putative antitumor compounds, antimicrobial and enzymatic activities from haloalkalitolerant and haloalkaliphilic bacteria strains of Algerian Sahara Soils. (United States)

    Selama, Okba; Amos, Gregory C A; Djenane, Zahia; Borsetto, Chiara; Laidi, Rabah Forar; Porter, David; Nateche, Farida; Wellington, Elizabeth M H; Hacène, Hocine


    Extreme environments may often contain unusual bacterial groups whose physiology is distinct from those of normal environments. To satisfy the need for new bioactive pharmaceuticals compounds and enzymes, we report here the isolation of novel bacteria from an extreme environment. Thirteen selected haloalkalitolerant and haloalkaliphilic bacteria were isolated from Algerian Sahara Desert soils. These isolates were screened for the presence of genes coding for putative antitumor compounds using PCR based methods. Enzymatic, antibacterial, and antifungal activities were determined by using cultural dependant methods. Several of these isolates are typical of desert and alkaline saline soils, but, in addition, we report for the first time the presence of a potential new member of the genus Nocardia with particular activity against the yeast Saccharomyces cerevisiae. In addition to their haloalkali character, the presence of genes coding for putative antitumor compounds, combined with the antimicrobial activity against a broad range of indicator strains and their enzymatic potential, makes them suitable for biotechnology applications.

  17. Research on the qualitative enzymatic activities in sediments from Secu and Gozna-Văliug dam reservoirs, Caraş-Severin county (Romania

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    Mihail Drăgan-Bularda


    Full Text Available Six sediment samples (three from Secu Lake and three from Gozna-Văliug Lake werecollected and analyzed qualitatively, enzymologically. In the sediment samples, the followingenzymatic activities have been qualitatively determined: four oligase activities (maltase, saccharase,lactase and cellobiase and five polyase activities (amylase, cellulose, dextranase, glicogenase andinulinase. The studied activities were determined in each sample and they were found to displayvariations in the intensities of the processes depending on the sampling place. Generally, the highestintensity of qualitative enzymatic activities was registered in case of oligases.

  18. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Fuconate Dehydratase from Xanthomonas campestris

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    Yew,W.; Fedorov, A.; Fedorov, E.; Rakus, J.; Pierce, R.; Almo, S.; Gerlt, J.


    Many members of the mechanistically diverse enolase superfamily have unknown functions. In this report the authors use both genome (operon) context and screening of a library of acid sugars to assign the L-fuconate dehydratase (FucD) function to a member of the mandelate racemase (MR) subgroup of the superfamily encoded by the Xanthomonas campestris pv. campestris str. ATCC 33913 genome (GI: 21233491). Orthologues of FucD are found in both bacteria and eukaryotes, the latter including the rTS beta protein in Homo sapiens that has been implicated in regulating thymidylate synthase activity. As suggested by sequence alignments and confirmed by high-resolution structures in the presence of active site ligands, FucD and MR share the same active site motif of functional groups: three carboxylate ligands for the essential Mg2+ located at the ends of th third, fourth, and fifth-strands in the (/)7-barrel domain (Asp 248, Glu 274, and Glu 301, respectively), a Lys-x-Lys motif at the end of the second-strand (Lys 218 and Lys 220), a His-Asp dyad at the end of the seventh and sixth-strands (His 351 and Asp 324, respectively), and a Glue at the end of the eighth-strand (Glu 382). The mechanism of the FucD reaction involves initial abstraction of the 2-proton by Lys 220, acid catalysis of the vinylogous-elimination of the 3-OH group by His 351, and stereospecific ketonization of the resulting 2-keto-3-deoxy-L-fuconate product. Screening of the library of acid sugars revealed substrate and functional promiscuity: In addition to L-fuconate, FucD also catalyzes the dehydration of L-galactonate, D-arabinonate, D-altronate, L-talonate, and D-ribonate. The dehydrations of L-fuconate, L-galactonate, and D-arabinonate are initiated by abstraction of the 2-protons by Lys 220. The dehydrations of L-talonate and D-ribonate are initiated by abstraction of the 2-protons by His 351; however, protonation of the enediolate intermediates by the conjugate acid of Lys 220 yields L

  19. Evolution of Enzymatic Activities in the Enolase Superfamily: L-Rhamnonate Dehydratase

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    Rakus,J.; Fedorov, A.; Fedorov, E.; Glaner, M.; Hubbard, B.; Delli, J.; Babbitt, P.; Almo, S.; Gerlt, J.


    The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy-l-mannonate) has the 'best' kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg2+; the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy-l-rhamnonate (obtained by reduction of the product with NaBH4). Like other members of the enolase superfamily, RhamD contains an N-terminal a + {beta} capping domain and a C-terminal ({beta}/a)7{beta}-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining '20s loop' in the capping domain is extended in length and the '50s loop' is truncated. The ligands for the Mg2+ are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth {beta}-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth {beta}-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg2+-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and

  20. Phospholipase C Produced by Clostridium botulinum Types C and D:Comparison of Gene, Enzymatic, and Biological Activities with Those of Clostridium perfringens Alpha-toxin

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    Full Text Available Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs, the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

  1. E. coli-Derived L-Asparaginase Retains Enzymatic and Cytotoxic Activity In Vitro for Canine and Feline Lymphoma after Cold Storage

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    Jackie M. Wypij


    Full Text Available Background. L-asparaginase is effective in treating canine and feline lymphoma, however chemotherapy poses a significant financial cost to veterinary clients, limiting therapy for many pets. Single dose vials result in significant drug wastage, and drug shortages limit consistent availability for pets. Hypothesis. E. coli-derived asparaginase retains enzymatic and antineoplastic activity in canine and feline lymphoma cells after cold storage. Methods. E. coli-derived asparaginase was cold-stored: refrigeration (7–14 days and freezing (14 days–six months, one to three freeze/thaw cycles. Enzymatic activity of asparaginase was measured via a modified asparagine assay. Effects of cold-stored asparaginase on cell proliferation and cytotoxicity were measured in feline (MYA-1, F1B and canine (17–71, OSW lymphoma cells. Results. Cold-stored E. coli-derived asparaginase retains antineoplastic activity in all four cell lines tested. Cold-stored E. coli-derived L-asparaginase depletes asparagine and retains enzymatic activity. Duration of refrigeration, duration of freezing, and number of freeze-thaw cycles have minimal effect on asparaginase enzyme activity. Conclusions and Clinical Importance. This study establishes a scientific basis for long-term cold storage of reconstituted E. coli-derived asparaginase that may result in better utilization of limited drug resources and improve financial feasibility of E. coli-derived asparaginase as a therapeutic option for pets with lymphoma.

  2. Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin. (United States)

    Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji


    Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

  3. Calcium pentosan polysulfate directly inhibits enzymatic activity of ADAMTS4 (aggrecanase-1) in osteoarthritic chondrocytes. (United States)

    Takizawa, Masayuki; Yatabe, Taku; Okada, Aiko; Chijiiwa, Miyuki; Mochizuki, Satsuki; Ghosh, Peter; Okada, Yasunori


    Aggrecanases that include ADAMTS1, 4, 5, 8, 9 and 15 are considered to play key roles in aggrecan degradation in osteoarthritic cartilage. Here we demonstrate that calcium pentosan polysulfate (CaPPS) directly inhibits the aggrecanase activity of ADAMTS4 without affecting the mRNA expression of the ADAMTS species in interleukin-1alpha-stimulated osteoarthritic chondrocytes. Synthetic peptides corresponding to specific regions of the thrombospondin type 1 repeat, cysteine-rich or spacer domain of ADAMTS4 inhibit the binding to immobilized CaPPS. These data suggest that CaPPS could function as chondroprotective agent for the treatment of osteoarthritis by inhibition of ADAMTS4 through interaction with the C-terminal ancillary domain.

  4. Factors influencing the rate of non-enzymatic activation of carboxylic and amino acids by ATP (United States)

    Mullins, D. W., Jr.; Lacey, J. C., Jr.


    The nonenzymatic formation of adenylate anhydrides of carboxylic and amino acids is discussed as a necessary step in the origin of the genetic code and protein biosynthesis. Results of studies are presented which have shown the rate of activation to depend on the pKa of the carboxyl group, the pH of the medium, temperature, the divalent metal ion catalyst, salt concentration, and the nature of the amino acid. In particular, it was found that of the various amino acids investigated, phenylalanine had the greatest affinity for the adenine derivatives adenosine and ATP. Results thus indicate that selective affinities between amino acids and nucleotides were important during prebiotic chemical evolution, and may have played a major role in the origin of protein synthesis and genetic coding.

  5. Antioxidant activities of buttermilk proteins, whey proteins, and their enzymatic hydrolysates. (United States)

    Conway, Valérie; Gauthier, Sylvie F; Pouliot, Yves


    The oxygen radical absorbance capacities (ORAC) and metal chelating capacities (MCC) of protein concentrates prepared from buttermilk and cheese whey by ultrafiltration were compared with those of skim milk protein. Samples were also heat-denatured and hydrolyzed by pepsin for 2 h followed by trypsin for 3 h. The highest MCC was obtained for hydrolyzed skim milk protein. ORAC values ranged from 554.4 to 1319.6 μmol Trolox equivalents/g protein, with the highest value obtained for hydrolyzed buttermilk protein. Liquid-phase isoelectric focusing (IEF) of this hydrolysate yielded peptide fractions with lower ORAC values. LC-MS analysis of the hydrolyzed skim milk and buttermilk proteins and IEF fractions of the latter showed that peptides derived from milk fat globule membrane proteins, primarily butyrophilin, could be responsible for the superior antioxidant activity of buttermilk. These results suggest overall that hydrolyzed buttermilk protein could be used as a source of natural antioxidants.

  6. Detection of contaminating enzymatic activity in plant-derived recombinant biotechnology products. (United States)

    Brinson, Robert G; Giulian, Gary G; Kelman, Zvi; Marino, John P


    Residual impurities in recombinantly produced protein biologics, such as host cell proteins (HCP), can potentially cause unwanted toxic or immunogenic responses in patients. Additionally, undetected impurities found in recombinant proteins used in cell culture may adversely impact basic research and biotechnology applications. Currently, the enzyme-linked immunosorbent assay (ELISA) is the standard for detection of residual HCP contamination in recombinantly produced biologics. Alternatively, two-dimensional liquid chromatography coupled to mass spectrometry is being developed as a tool for assessing this critical quality attribute. Both of these methods rely on the direct detection of HCPs and some previous knowledge of the contaminant. For contaminating enzymes, the mass level of the impurity may fall below the threshold of detection of these methods and underestimate the true impact. To address this point, here we demonstrate facile detection and characterization of contaminating phytase activity in rice-derived recombinant human serum albumin (rHSA) using a sensitive, label-free nuclear magnetic resonance (NMR) spectroscopy assay. We observed varying degrees of phytase contamination in biotechnology-grade rHSA from various manufacturers by monitoring the degradation of adenosine-5'-triphosphate and myo-inositol-1,2,3,4,5,6-hexakisphosphate by (31)P NMR. The observed lot-to-lot variability may result in irreproducible cell culture results and should be evaluated as a possible critical quality attribute in plant-derived biotherapeutics.

  7. Modulation of enzymatic activities by n-3 polyunsaturated fatty acids to support cardiovascular health. (United States)

    Siddiqui, Rafat A; Harvey, Kevin A; Zaloga, Gary P


    Epidemiological evidence from Greenland Eskimos and Japanese fishing villages suggests that eating fish oil and marine animals can prevent coronary heart disease. Dietary studies from various laboratories have similarly indicated that regular fish oil intake affects several humoral and cellular factors involved in atherogenesis and may prevent atherosclerosis, arrhythmia, thrombosis, cardiac hypertrophy and sudden cardiac death. The beneficial effects of fish oil are attributed to their n-3 polyunsaturated fatty acid (PUFA; also known as omega-3 fatty acids) content, particularly eicosapentaenoic acid (EPA; 20:5, n-3) and docosahexaenoic acid (DHA; 22:6, n-3). Dietary supplementation of DHA and EPA influences the fatty acid composition of plasma phospholipids that, in turn, may affect cardiac cell functions in vivo. Recent studies have demonstrated that long-chain omega-3 fatty acids may exert beneficial effects by affecting a wide variety of cellular signaling mechanisms. Pathways involved in calcium homeostasis in the heart may be of particular importance. L-type calcium channels, the Na+-Ca2+ exchanger and mobilization of calcium from intracellular stores are the most obvious key signaling pathways affecting the cardiovascular system; however, recent studies now suggest that other signaling pathways involving activation of phospholipases, synthesis of eicosanoids, regulation of receptor-associated enzymes and protein kinases also play very important roles in mediating n-3 PUFA effects on cardiovascular health. This review is therefore focused on the molecular targets and signaling pathways that are regulated by n-3 PUFAs in relation to their cardioprotective effects.

  8. Toxicity of perfluorooctanoic acid towards earthworm and enzymatic activities in soil. (United States)

    He, Wenxiang; Megharaj, Mallavarapu; Naidu, Ravi


    Perfluorooctanoic acid (PFOA) is a widespread persistent organic contaminant in the environment that has recently raised much of regulatory and public concern. Therefore, assessment of its ecological risk is a top priority research. Hence, this study investigated the toxicity of PFOA to beneficial microbial processes in the soil such as activities of dehydrogenase, urease and potential nitrification in addition to earthworm survival, weight loss and PFOA bioaccumulation in two contrasting soils. In general, PFOA caused inhibition of all the measured microbial processes in a dose-dependent manner and the inhibition was higher in Williamtown (WT) soil than Edinburgh (EB) soil. Thus, WT soil being sandy in nature with low clay content showed higher PFOA bioavailability and hence showed higher toxicity. There was no mortality in earthworms exposed up to 100 mg PFOA/kilogram soil in both the soils; however, there was a significant weight loss from 25 mg/kg onwards. This study clearly demonstrates that soil contamination of PFOA can lead to adverse effects on soil health.

  9. Toward an Automatic Determination of Enzymatic Reaction Mechanisms and Their Activation Free Energies. (United States)

    Zinovjev, Kirill; Ruiz-Pernía, J Javier; Tuñón, Iñaki


    We present a combination of the string method and a path collective variable for the exploration of the free energy surface associated to a chemical reaction in condensed environments. The on-the-fly string method is employed to find the minimum free energy paths on a multidimensional free energy surface defined in terms of interatomic distances, which is a convenient selection to study bond forming/breaking processes. Once the paths have been determined, a reaction coordinate is defined as a measure of the advance of the system along these paths. This reaction coordinate can be then used to trace the reaction Potential of Mean Force from which the activation free energy can be obtained. This combination of methodologies has been here applied to the study, by means of Quantum Mechanics/Molecular Mechanics simulations, of the reaction catalyzed by guanidinoacetate methyltransferase. This enzyme catalyzes the methylation of guanidinoacetate by S-adenosyl-l-methionine, a reaction that involves a methyl transfer and a proton transfer and for which different reaction mechanisms have been proposed.

  10. Dimerization and enzymatic activity of fungal 17β-hydroxysteroid dehydrogenase from the short-chain dehydrogenase/reductase superfamily

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    Kristan Katja


    Full Text Available Abstract Background 17β-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17β-HSDcl is a member of the short-chain dehydrogenase/reductase (SDR superfamily. SDR proteins usually function as dimers or tetramers and 17β-HSDcl is also a homodimer under native conditions. Results We have investigated here which secondary structure elements are involved in the dimerization of 17β-HSDcl and examined the importance of dimerization for the enzyme activity. Sequence similarity with trihydroxynaphthalene reductase from Magnaporthe grisea indicated that Arg129 and His111 from the αE-helices interact with the Asp121, Glu117 and Asp187 residues from the αE and αF-helices of the neighbouring subunit. The Arg129Asp and His111Leu mutations both rendered 17β-HSDcl monomeric, while the mutant 17β-HSDcl-His111Ala was dimeric. Circular dichroism spectroscopy analysis confirmed the conservation of the secondary structure in both monomers. The three mutant proteins all bound coenzyme, as shown by fluorescence quenching in the presence of NADP+, but both monomers showed no enzymatic activity. Conclusion We have shown by site-directed mutagenesis and structure/function analysis that 17β-HSDcl dimerization involves the αE and αF helices of both subunits. Neighbouring subunits are connected through hydrophobic interactions, H-bonds and salt bridges involving amino acid residues His111 and Arg129. Since the substitutions of these two amino acid residues lead to inactive monomers with conserved secondary structure, we suggest dimerization is a prerequisite for catalysis. A detailed understanding of this dimerization could lead to the development of compounds that will specifically prevent dimerization, thereby serving as a new type of inhibitor.

  11. Common HEXB polymorphisms reduce serum HexA and HexB enzymatic activities, potentially masking Tay-Sachs disease carrier identification. (United States)

    Vallance, Hilary; Morris, Tara J; Coulter-Mackie, Marion; Lim-Steele, Joyce; Kaback, Michael


    A DNA-proven Tay-Sachs disease (TSD) carrier and his brother were found to have serum percent Hexosaminidase A (%HexA) enzymatic activities in the non-carrier range, while the leukocyte %HexA profiles clearly identified them as TSD heterozygotes. Both their serum HexA and HexB enzymatic activities were below reference range, suggesting inheritance of mutations in both the HEXA (alpha-subunit) and HEXB (beta-subunit) genes. DNA sequencing revealed that both individuals, carried the common HEXA 1277_1278insTATC mutation, and two common HEXB polymorphisms: [619A>G (+) delTG]. To determine if these HEXB polymorphisms reduce HexA and HexB enzymatic activities, 69 DNA samples from subjects previously screened enzymatically in both serum and leukocytes for TSD carrier status were selected for either high, mid-range or low serum Total Hex (defined as the sum of HexA and HexB) activities and were tested for the HEXB mutations. Further, three additional TSD carriers ascertained by the atypical pattern of normal serum %HexA but carrier leukocyte %HexA, were found to have the [delTG (+) 619A>G] genotype. In addition, the frequency of the [delTG (+) 619A>G] genotype was significantly higher (P G] haplotype in the Ashkenazi Jewish population (approximately 10%), up to 10% of TSD carriers may have normal serum %HexA values with low total Hex. Accordingly, serum %HexA should not be the sole criterion used for carrier status determination. Where total Hex activity is reduced, further testing with leukocyte Hex profiles is indicated.

  12. Sulfated polysaccharide extracted of the green algae Caulerpa racemosa increase the enzymatic activity and paw edema induced by sPLA2 from Crotalus durissus terrificus venom

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    Camila L. Pires


    Full Text Available Sulfated polysaccharides derived from seaweed have shown great potential for use in the development of new drugs. In this study, we observed that a low-molecular-weight sulfated polysaccharide from Caulerpa racemosa, termed CrSP, could interact with secretory phospholipase A2 (sPLA2 isolated from Crotalus durissus terrificus venom. When native sPLA2 (14 kDa was incubated with CrSP, they formed a molecular complex (sPLA2:CrSP with a molecular mass of 32 kDa, approximately. Size exclusion chromatography experiments suggested that CrSP formed a stable complex with sPLA2. We belived that sPLA2 and SPCr are involved an ionic interaction between negatively charged CrSP and the positively charged basic amino acid residues of sPLA2, because this interaction induced significant changes in sPLA2 enzymatic and pharmacological activities. CrSP caused a significant increase in sPLA2 enzymatic and bactericidal activity and increased its edematogenic effect. A pharmacological assay showed that the myotoxic activity of sPLA2:CrSP is unrelated to its enzymatic activity and that sPLA2:CrSP may have a practical application as a natural antibacterial agent for use in humans and commercially raised animals.

  13. Evolution of Enzymatic Activities in the Enolase Superfamily: D-Mannonate Dhydratase from Novosphingobium aromaticivorans

    Energy Technology Data Exchange (ETDEWEB)

    Rakus,J.; Fedorov, A.; Fedorov, E.; Glasner, M.; Vick, J.; Babbitt, P.; Almo, S.; Gerlt, J.


    The d-mannonate dehydratase (ManD) function was assigned to a group of orthologous proteins in the mechanistically diverse enolase superfamily by screening a library of acid sugars. Structures of the wild type ManD from Novosphingobium aromaticivorans were determined at pH 7.5 in the presence of Mg2+ and also in the presence of Mg2+ and the 2-keto-3-keto-d-gluconate dehydration product; the structure of the catalytically active K271E mutant was determined at pH 5.5 in the presence of the d-mannonate substrate. As previously observed in the structures of other members of the enolase superfamily, ManD contains two domains, an N-terminal a+{beta} capping domain and a ({beta}/a)7{beta}-barrel domain. The barrel domain contains the ligands for the essential Mg2+, Asp 210, Glu 236, and Glu 262, at the ends of the third, fourth, and fifth {beta}-strands of the barrel domain, respectively. However, the barrel domain lacks both the Lys acid/base catalyst at the end of the second {beta}-strand and the His-Asp dyad acid/base catalyst at the ends of the seventh and sixth {beta}-strands, respectively, that are found in many members of the superfamily. Instead, a hydrogen-bonded dyad of Tyr 159 in a loop following the second {beta}-strand and Arg 147 at the end of the second {beta}-strand are positioned to initiate the reaction by abstraction of the 2-proton. Both Tyr 159 and His 212, at the end of the third {beta}-strand, are positioned to facilitate both syn-dehydration and ketonization of the resulting enol intermediate to yield the 2-keto-3-keto-d-gluconate product with the observed retention of configuration. The identities and locations of these acid/base catalysts as well as of cationic amino acid residues that stabilize the enolate anion intermediate define a new structural strategy for catalysis (subgroup) in the mechanistically diverse enolase superfamily. With these differences, we provide additional evidence that the ligands for the essential Mg2+ are the only

  14. Extraction and quantitation of coumarin from cinnamon and its effect on enzymatic browning in fresh apple juice: a bioinformatics approach to illuminate its antibrowning activity. (United States)

    Thada, Rajarajeshwari; Chockalingam, Shivashri; Dhandapani, Ramesh Kumar; Panchamoorthy, Rajasekar


    Enzymatic browning by polyphenoloxidase (PPO) affects food quality and taste in fruits and vegetables. Thus, the study was designed to reduce browning in apple juice by coumarin. The ethanolic extract of cinnamon was prepared and its coumarin content was quantitated by HPLC, using authentic coumarin (AC) as standard. The effect of cinnamon extract (CE) and AC on enzymatic browning, its time dependent effects, and the specific activity of PPO and peroxidase (POD) were studied in apple juice. The docking of coumarin with PPO and POD was also performed to elucidate its antibrowning mechanism. The CE (73%) and AC (82%) showed better reduction in browning, maintained its antibrowning effect at all time points, and significantly (p < 0.05) reduced the specific activity of PPO and POD when compared with controls. Coumarin showed strong interaction with binding pockets of PPO and POD, suggesting its potential use as inhibitor to enzyme mediated browning in apple juice.

  15. Covalent immobilization of porcine pancreatic lipase on carboxyl-activated magnetic nanoparticles: Characterization and application for enzymatic inhibition assays

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, Yuan-Ting [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China); University of Chinese Academy of Sciences, Beijing 100049 (China); Ren, Xiao-Yun [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China); Liu, Yi-Ming [Department of Chemistry and Biochemistry, Jackson State University, 1400 Lynch St., Jackson, MS 39217 (United States); Wei, Ying [Changzhi Medical College, Changzhi 046000 (China); Qing, Lin-Sen [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China); Liao, Xun, E-mail: [Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041 (China)


    Using carboxyl functionalized silica-coated magnetic nanoparticles (MNPs) as carrier, a novel immobilized porcine pancreatic lipase (PPL) was prepared through the 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS) coupling reaction. Transmission electron microscopic images showed that the synthesized nanoparticles (Fe{sub 3}O{sub 4}–SiO{sub 2}) possessed three dimensional core–shell structures with an average diameter of ∼ 20 nm. The effective enzyme immobilization onto the nanocomposite was confirmed by atomic force microscopic (AFM) analysis. Results from Fourier-transform infrared spectroscopy (FT-IR), Bradford protein assay, and thermo-gravimetric analysis (TGA) indicated that PPL was covalently attached to the surface of magnetic nanoparticles with a PPL immobilization yield of 50 mg enzyme/g MNPs. Vibrating sample magnetometer (VSM) analysis revealed that the MNPs-PPL nanocomposite had a high saturation magnetization of 42.25 emu·g{sup −1}. The properties of the immobilized PPL were investigated in comparison with the free enzyme counterpart. Enzymatic activity, reusability, thermo-stability, and storage stability of the immobilized PPL were found significantly superior to those of the free one. The K{sub m} and the V{sub max} values (0.02 mM, 6.40 U·mg{sup −1} enzyme) indicated the enhanced activity of the immobilized PPL compared to those of the free enzyme (0.29 mM, 3.16 U·mg{sup −1} enzyme). Furthermore, at an elevated temperature of 70 °C, immobilized PPL retained 60% of its initial activity. The PPL-MNPs nanocomposite was applied in the enzyme inhibition assays using orlistat, and two natural products isolated from oolong tea (i.e., EGCG and EGC) as the test compounds. - Highlights: • Porcine pancreatic lipase was firstly covalently immobilized onto carboxylated MNPs. • Immobilized porcine pancreatic lipase (PPL) was characterized by various techniques. • MNPs-PPL showed higher activity

  16. The effect of Eulaliopsis binata on the physi-chemical properties, microbial biomass, and enzymatic activities in Cd-Pb polluted soil. (United States)

    Yu, Hui; Xiang, Yanci; Zou, Dongsheng


    Pot culture experiment using mining wasteland soil was carried out to study the effect of Eulaliopsis binata on the heavy-metal polluted soil with the growth of 90, 180, 270, and 360 days. Soil nutritional components, heavy metal, microbial biomass, and enzymatic activities were analyzed in this study, and the control group had no plants. The results showed that heavy metal contents decreased with E. binata growth, extractable Cd and Pb decreased 28 and 15 % after 1 year, but the difference was not significant compared with the control. While soil nutritional components, microbial biomass and enzymatic activities increased significantly as compared with the control. Comparing with pre-experiment, soil organic matter, N, P, K, microbial biomass C, N, P, invertase, urease, acid phosphatase, and catalase increased 0.9, 1.1, 3.0, 1.1, 0.4, 0.3, and 0.5 times, respectively. The indexes of soil nutritional components, microbial biomass, and enzymatic activities are positively correlated to each other, while they are negatively correlated to heavy metal content respectively. E. binata has positive influence on Cd-Pb pollution soil and broad application prospects in remediating heavy-metal polluted soil.

  17. Enzymatic activity profile of a Brazilian culture collection of Candida albicans isolated from diabetics and non-diabetics with oral candidiasis. (United States)

    Sanitá, Paula Volpato; Zago, Chaiene Evelin; Pavarina, Ana Cláudia; Jorge, Janaina Habib; Machado, Ana Lúcia; Vergani, Carlos Eduardo


    The secretion of hydrolytic enzymes is a fundamental virulence factor of Candida albicans to develop disease. The objective of this study was to characterise the virulence of 148 clinical isolates of C. albicans from oral candidiasis by assessing the expression of phospholipase (PL) and secreted aspartyl proteinase (SAP). Isolates were obtained from healthy subjects (HS) and diabetics (DOC) and non-diabetics with oral candidiasis (NDOC). An aliquot (5 μl) of each cell suspension was inoculated on PL and SAP agar plates and incubated. Enzymes secretion was detected by the formation of an opaque halo around the colonies and enzymatic activity (PZ) was determined by the ratio between colony diameter and colony diameter plus the halo zone. Statistical comparisons were made by a one-way anova followed by Tukey's post hoc test (α = 0.05). The clinical sources of C. albicans had significant effect (P < 0.001) on the PZ values of both enzymes. For PL, clinical isolates from NDOC and DOC had highest enzymatic activity than those from HS (P < 0.05), with no significant differences between them (P = 0.506). For SAP, C. albicans from NDOC showed the lower enzymatic activity (P < 0.001). There were no significant differences between isolates from HS and DOC (P = 0.7051). C. albicans isolates from NDOC and DOC patients showed an increased production of PL.

  18. Enzymatic activities and stable isotope patterns of ectomycorrhizal fungi in relation to phylogeny and exploration types in an afrotropical rain forest. (United States)

    Tedersoo, Leho; Naadel, Triin; Bahram, Mohammad; Pritsch, Karin; Buegger, Franz; Leal, Miguel; Kõljalg, Urmas; Põldmaa, Kadri


    Ectomycorrhizal (ECM) fungi obtain both mineral and simple organic nutrients from soil and transport these to plant roots. Natural abundance of stable isotopes (¹⁵N and ¹³C) in fruit bodies and potential enzymatic activities of ECM root tips provide insights into mineral nutrition of these mutualistic partners. By combining rDNA sequence analysis with enzymatic and stable isotope assays of root tips, we hypothesized that phylogenetic affinities of ECM fungi are more important than ECM exploration type, soil horizon and host plant in explaining the differences in mineral nutrition of trees in an African lowland rainforest. Ectomycorrhizal fungal species belonging to extraradical mycelium-rich morphotypes generally displayed the strongest potential activities of degradation enzymes, except for laccase. The signature of ¹⁵N was determined by the ECM fungal lineage, but not by the exploration type. Potential enzymatic activities of root tips were unrelated to ¹⁵N signature of ECM root tip. The lack of correlation suggests that these methods address different aspects in plant nutrient uptake. Stable isotope analysis of root tips could provide an additional indirect assessment of fungal and plant nutrition that enables enhancement of taxonomic coverage and control for soil depth and internal nitrogen cycling in fungal tissues.

  19. Studies on gene structure, enzymatic activity and regulatory mechanism of acetohydroxy acid isomeroreductase from G2 pea

    Institute of Scientific and Technical Information of China (English)

    XU Yunjian (徐云剑); GU Xuesong (顾雪松); LI Jun (李珺); LI Qing (李 晴); Peter J. Davies; ZHU Yuxian (朱玉贤)


    The AAIR genomic DNA of G2 pea (Pisum sativum L.) was amplified by PCR method. Sequence analysis showed that it was composed of 8 introns and 9 exons with three of the introns containing specific A/T-rich endogenous promoter regions. Molecular hybridization experiments revealed that the expression of AAIR remained at a high level before and after flowering if grown in short day growth chambers. However, when grown under long day conditions, the level of AAIR expression declined very rapidly after flowering. This variation of AAIR expression is consistent with the change of enzymatic activity of acetohydroxy acid isomeroreductase. Functional complementation experiments carried out using an acetohydroxy acid isomeroreductase deficient E. coli strain showed that these cells could not grow on M9 medium without addition of branched-chain amino acids unless they were transformed with the AAIR expression vector. Further study revealed that overexpression of the pea AAIR cDNA in acetohydroxy acid isomeroreductase deficient E. coli strain enhanced significantly its branched-chain amino acid biosynthetic capacity. Results from gel shift experiments showed that fractions of pea nuclear protein extracts could bind specifically to some A/T rich regions present in introns of the AAIR gene. The A/T-rich-region-binding proteins remained at a steady level in the non-senescing apical buds of short-day grown G2 pea. In the rapid-senescing apical buds of long-day grown G2 pea, the levels of these proteins declined rapidly after flower initiation. Therefore, the nuclear protein binding capacities to endogenous promoter regions may constitute an important mechanism to regulate AAIR gene expression.

  20. Enzymatic activity of cell-free extracts from Burkholderia oxyphila OX-01 bio-converts (+)-catechin and (-)-epicatechin to (+)-taxifolin. (United States)

    Otsuka, Yuichiro; Matsuda, Motoki; Sonoki, Tomonori; Sato-Izawa, Kanna; Goodell, Barry; Jelison, Jody; Navarro, Ronald R; Murata, Hitoshi; Nakamura, Masaya


    This study characterized the enzymatic ability of a cell-free extract from an acidophilic (+)-catechin degrader Burkholderia oxyphila (OX-01). The crude OX-01 extracts were able to transform (+)-catechin and (-)-epicatechin into (+)-taxifolin via a leucocyanidin intermediate in a two-step oxidation. Enzymatic oxidation at the C-4 position was carried out anaerobically using H2O as an oxygen donor. The C-4 oxidation occurred only in the presence of the 2R-catechin stereoisomer, with the C-3 stereoisomer not affecting the reaction. These results suggest that the OX-01 may have evolved to target both (+)-catechin and (-)-epicatechin, which are major structural units in plants.

  1. Studies on the Effects of Polyaspartate Protease Fertilizer Enhancer in the Absorptions of Soil Nutrition and the Enzymatic Activities of Crops

    Institute of Scientific and Technical Information of China (English)

    JIANG Guoliang; YANG Dong; LIU Yun; ZHANG Guanghua; LI Zhongjun; ZHANG Xinhua


    The effects of polyaspartate protease fertilizer enhancer, made from oyster shell proteins, on the absorption of soil nutrition and the enzymatic activities of crops were studied. It has been found that the enhancer contributes 30%, 50 % and 50% augmentation of nitrogen (N), phosphate (P) and potassium (K) absorption respectively and about 20% of nitrate reductase and peroxide enzyme activities of crops. These results show that polyaspartate protease fertilizer enhancer could improve significantly the absorption and utilization efficiencies of soil nutrition and the activities of nitrate reductase and peroxide enzyme of crops, thus elevating the utilization rates of chemical fertilizers to a certain extent.

  2. Responses of metabolic and antioxidant enzymatic activities in gill, liver and plasma of Catla catla during methyl parathion exposure

    Directory of Open Access Journals (Sweden)

    B.D. Abhijith


    Conclusion: The results of the present investigation suggest that gill is the most sensitive organ to MP toxicity. The alterations of the enzymatic parameters can be effectively used as potential biomarkers for monitoring of the organophosphorus pesticides in aquatic environment. Further, MP should be used with caution in order to protect natural waters and aquatic organisms.

  3. Antibacterial activity of hen egg white lysozyme modified by heat and enzymatic treatments against oenological lactic acid bacteria and acetic acid bacteria. (United States)

    Carrillo, W; García-Ruiz, A; Recio, I; Moreno-Arribas, M V


    The antimicrobial activity of heat-denatured and hydrolyzed hen egg white lysozyme against oenological lactic acid and acetic acid bacteria was investigated. The lysozyme was denatured by heating, and native and heat-denatured lysozymes were hydrolyzed by pepsin. The lytic activity against Micrococcus lysodeikticus of heat-denatured lysozyme decreased with the temperature of the heat treatment, whereas the hydrolyzed lysozyme had no enzymatic activity. Heat-denatured and hydrolyzed lysozyme preparations showed antimicrobial activity against acetic acid bacteria. Lysozyme heated at 90°C exerted potent activity against Acetobacter aceti CIAL-106 and Gluconobacter oxydans CIAL-107 with concentrations required to obtain 50% inhibition of growth (IC50) of 0.089 and 0.013 mg/ml, respectively. This preparation also demonstrated activity against Lactobacillus casei CIAL-52 and Oenococcus oeni CIAL-91 (IC50, 1.37 and 0.45 mg/ml, respectively). The two hydrolysates from native and heat-denatured lysozyme were active against O. oeni CIAL-96 (IC50, 2.77 and 0.3 mg/ml, respectively). The results obtained suggest that thermal and enzymatic treatments increase the antibacterial spectrum of hen egg white lysozyme in relation to oenological microorganisms.

  4. Optimizing the enzymatic synthesis of beta-D-galactopyranosyl-D-xyloses for their use in the evaluation of lactase activity in vivo. (United States)

    Hermida, Carmen; Corrales, Guillermo; Cañada, Francisco J; Aragón, Juan J; Fernández-Mayoralas, Alfonso


    Disaccharides 2-O-, 3-O-, and 4-O-beta-D-galactopyranosyl-D-xyloses (2, 3, and 1, respectively) were obtained by beta-galactosidase-catalyzed reactions for their use in the evaluation of intestinal lactase activity in vivo. Their administration to suckling rats followed by determination of the derived D-xylose in the urine and measurement of lactase activity in intestinal homogenates showed 1 to be the most suitable disaccharide for a potential test of the deficiency of intestinal lactase. The synthesis of 1 was further studied by evaluating the effect of different variables on the yield and regioselectivity of the enzymatic galactosylation, and the purification process was optimized.

  5. Nitric oxide-induced calcium release: activation of type 1 ryanodine receptor, a calcium release channel, through non-enzymatic posttranslational modification by nitric oxide

    Directory of Open Access Journals (Sweden)

    Sho eKakizawa


    Full Text Available Nitric oxide (NO is a typical gaseous messenger involved in a wide range of biological processes. In our classical knowledge, effects of NO are largely achieved by activation of soluble guanylyl cyclase to form cyclic guanosine-3’, 5’-monophosphate. However, emerging evidences have suggested another signaling mechanism mediated by NO: S-nitrosylation of target proteins.S-nitrosylation is a covalent addition of an NO group to a cysteine thiol/sulfhydryl (RSH, and categorized into non-enzymatic posttranslational modification of proteins, contrasted to enzymatic posttranslational modification of proteins, such as phosphorylation mediated by various protein kinases.Very recently, we found novel intracellular calcium (Ca2+ mobilizing mechanism, NO-induced Ca2+ release (NICR in cerebellar Purkinje cells. NICR is mediated by type 1 ryanodine receptor (RyR1, a Ca2+ release channel expressed in endoplasmic-reticular membrane. Furthermore, NICR is indicated to be dependent on S-nitrosylation of RyR1, and involved in synaptic plasticity in the cerebellum. In this review, molecular mechanisms and functional significance of NICR, as well as non-enzymatic posttranslational modification of proteins by gaseous signals, are described.

  6. Divergence in Enzymatic Activities in the Soybean GST Supergene Family Provides New Insight into the Evolutionary Dynamics of Whole-Genome Duplicates. (United States)

    Liu, Hai-Jing; Tang, Zhen-Xin; Han, Xue-Min; Yang, Zhi-Ling; Zhang, Fu-Min; Yang, Hai-Ling; Liu, Yan-Jing; Zeng, Qing-Yin


    Whole-genome duplication (WGD), or polyploidy, is a major force in plant genome evolution. A duplicate of all genes is present in the genome immediately following a WGD event. However, the evolutionary mechanisms responsible for the loss of, or retention and subsequent functional divergence of polyploidy-derived duplicates remain largely unknown. In this study we reconstructed the evolutionary history of the glutathione S-transferase (GST) gene family from the soybean genome, and identified 72 GST duplicated gene pairs formed by a recent Glycine-specific WGD event occurring approximately 13 Ma. We found that 72% of duplicated GST gene pairs experienced gene losses or pseudogenization, whereas 28% of GST gene pairs have been retained in the soybean genome. The GST pseudogenes were under relaxed selective constraints, whereas functional GSTs were subject to strong purifying selection. Plant GST genes play important roles in stress tolerance and detoxification metabolism. By examining the gene expression responses to abiotic stresses and enzymatic properties of the ancestral and current proteins, we found that polyploidy-derived GST duplicates show the divergence in enzymatic activities. Through site-directed mutagenesis of ancestral proteins, this study revealed that nonsynonymous substitutions of key amino acid sites play an important role in the divergence of enzymatic functions of polyploidy-derived GST duplicates. These findings provide new insights into the evolutionary and functional dynamics of polyploidy-derived duplicate genes.

  7. Effect of land-use types on soil enzymatic activities and chemical properties in semi-deciduous forest areas of Central-West Côte d'Ivoire

    Directory of Open Access Journals (Sweden)

    Gonnety, JT.


    Full Text Available Enzymatic activities play a key role in the biochemical functioning of soils. As a consequence, they have been proposed as indicators of soil quality. This study was conducted at the Oumé benchmark site (Central-West, Côte d'Ivoire, and aimed at measuring the enzymatic activities involved in the phosphorus (acid phosphatase and alkaline phosphatase, nitrogen (N-acetyl-β-D glucosaminidase and carbon (β-glucosidase and N-acetyl-β-D glucosaminidase cycles. Soil from four main agro-ecological units (a secondary forest, a 20 year-old cocoa plantation, a 2 year-old Chromolaena odorata-based fallow and a continuous maize crop, representative of land-use systems in the area, were sampled for the measurement of enzymatic activities and chemical characteristics. Results showed that the enzymatic activity values were the highest in the fallow soil, whereas the maize crop displayed the lowest levels of enzymatic activity in soil. Moreover, soil from C. odorata fallow displayed the highest values of C, N, exchangeable bases (Mg2+, K+ contents, and CEC, and the lowest C:N ratio, which are characteristics of good quality soil. A Principal Component Analysis revealed a marked relationship between C, N and enzymatic activity levels, showing that these enzymes are suitable for monitoring soil quality in semi-deciduous forest areas in Central-West Côte d'Ivoire.

  8. Modulation of the pharmacological effects of enzymatically-active PLA2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

    Directory of Open Access Journals (Sweden)

    Nagano Celso S


    Full Text Available Abstract Background An interaction between lectins from marine algae and PLA2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2, isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA2 isolated from rattlesnake venom (Crotalus durissus cascavella, to better understand the enzymatic and pharmacological mechanisms of the PLA2 and its complex. Results This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa, its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24–26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm, but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap. PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48

  9. Highly Sensitive Spectrofluorimetric Determination of Riboflavin Based on the Generation of Active Oxygen Coupled with Enzymatic Reaction

    Institute of Scientific and Technical Information of China (English)


    A spectrofluorimetric method for the determining riboflavin (VB2) based on its enhancement on the fluorescence of hemoglobin-catalyzed enzymatic reaction was proposed. The proposed method consisted of two reactions. One was the photochemical reaction of VB2, the other was a hemoglobin-catalyzed enzymatic reaction. The optimal experimental conditions for the determinations were established. The linear range of the method was 5.0×10-9-1.0×10-7mol/L of VB2. The detection limit was calculated to be 3.65×10-9 mol/L. The relative standard deviation of this method was 2.3 % at 7.0×10-8 mol/L for 11 determinations.

  10. 鳙鱼活性多肽酶法制备工艺研究%Enzymatic Extraction of Active Polypeptide from Hypophthalmichthys nobilis

    Institute of Scientific and Technical Information of China (English)

    张雪; 陈复生; 隋继学


      为优化鳙鱼活性多肽酶法制备工艺,分析了鳙鱼肉糜预处理温度和酶解温度对水解度的影响,确定了最佳的预处理条件为85℃水浴中加热预处理20 min,酶解温度设为55~75℃.经均匀设计实验优选和最优条件验证实验证实,以氮溶指数为指标的最优酶解条件为:酶解时间8.0 h,固液比1∶4.25,蛋白酶A用量3‰,酶解温度75℃,产物氮溶指数达80.54%;以多肽得率为指标的最优酶解条件为:酶解时间8.0 h,固液比1∶2,蛋白酶A用量3‰,酶解温度75℃,产物多肽得率达11.92%;以产物总抗氧化指数为指标的最优酶解条件为:酶解时间1.0 h,固液比1∶6,蛋白酶A用量3‰,酶解温度55℃,所得产物总抗氧化指数达87.42‰.%For optimizating the enzymatic extraction of active polypeptide from Hypophthalmichthys nobilis,we investigated the effects of minced meat pretreatment temperature and enzymatic temperature on the degree of hydrolysis,and determined that the optimal pretreatment conditions was in a water bath of 85℃to heat pretreatment 20 min. Through uniform design and verification experiment,we confirmed that the optimal enzymatic hydrolysis conditions by using optimal conditionsuse nitrogen solubility index as an indicator were enzymatic hydrolysis time 8.0 h,the solid-liquid ratio 1∶4.25,dosage of protease A 3‰,hydrolysis temperature 75℃,then nitrogen solubility index of the product reached 80.54%. The optimal hydrolysis conditions of using polypeptide yield as indicators were enzymatic hydrolysis time 8.0 h,solid-liquid ratio of 1∶2,dosage of protease A 3‰,the temperature of enzymatic hydrolysis 75℃,then the polypeptide yield of product reached 11.92%. The optimal enzymatic hydrolysis conditions of using the total antioxidant index of the product as indicators were the enzymatic time 1.0 h,solid-liquid ratio 1∶6,dosage of protease A 3‰,hydrolysis temperature 55 ℃,and then the total antioxidant index

  11. Impact of the redox-cycling herbicide diquat on transcript expression and antioxidant enzymatic activities of the freshwater snail Lymnaea stagnalis

    Energy Technology Data Exchange (ETDEWEB)

    Bouetard, Anthony, E-mail: [INRA, UMR INRA-Agrocampus Ouest ESE 0985, Equipe Ecotoxicologie et Qualite des Milieux Aquatiques, 65 rue de Saint-Brieuc, 35042 Rennes cedex (France); Besnard, Anne-Laure; Vassaux, Daniele; Lagadic, Laurent; Coutellec, Marie-Agnes [INRA, UMR INRA-Agrocampus Ouest ESE 0985, Equipe Ecotoxicologie et Qualite des Milieux Aquatiques, 65 rue de Saint-Brieuc, 35042 Rennes cedex (France)


    The presence of pesticides in the environment results in potential unwanted effects on non-target species. Freshwater organisms inhabiting water bodies adjacent to agricultural areas, such as ditches, ponds and marshes, are good models to test such effects as various pesticides may reach these habitats through several ways, including aerial drift, run-off, and drainage. Diquat is a non-selective herbicide used for crop protection or for weed control in such water bodies. In this study, we investigated the effects of diquat on a widely spread aquatic invertebrate, the holarctic freshwater snail Lymnaea stagnalis. Due to the known redox-cycling properties of diquat, we studied transcript expression and enzymatic activities relative to oxidative and general stress in the haemolymph and gonado-digestive complex (GDC). As diquat is not persistent, snails were exposed for short times (5, 24, and 48 h) to ecologically relevant concentrations (22.2, 44.4, and 222.2 {mu}g l{sup -1}) of diquat dibromide. RT-qPCR was used to quantify the transcription of genes encoding catalase (cat), a cytosolic superoxide dismutase (Cu/Zn-sod), a selenium-dependent glutathione peroxidase (gpx), a glutathione reductase (gred), the retinoid X receptor (rxr), two heat shock proteins (hsp40 and hsp70), cortactin (cor) and the two ribosomal genes r18S and r28s. Enzymatic activities of SOD, Gpx, Gred and glutathione S-transferase (GST) were investigated in the GDC using spectrophoto/fluorometric methods. Opposite trends were obtained in the haemolymph depending on the herbicide concentration. At the lowest concentration, effects were mainly observed after 24 h of exposure, with over-transcription of cor, hsp40, rxr, and sod, whereas higher concentrations down-regulated the expression of most of the studied transcripts, especially after 48 h of exposure. In the GDC, earlier responses were observed and the fold-change magnitude was generally much higher: transcription of all target genes increased

  12. The importance of the interaction of CheD with CheC and the chemoreceptors compared to its enzymatic activity during chemotaxis in Bacillus subtilis.

    Directory of Open Access Journals (Sweden)

    Wei Yuan

    Full Text Available Bacillus subtilis use three systems for adaptation during chemotaxis. One of these systems involves two interacting proteins, CheC and CheD. CheD binds to the receptors and increases their ability to activate the CheA kinase. CheD also binds CheC, and the strength of this interaction is increased by phosphorylated CheY. CheC is believed to control the binding of CheD to the receptors in response to the levels of phosphorylated CheY. In addition to their role in adaptation, CheC and CheD also have separate enzymatic functions. CheC is a CheY phosphatase and CheD is a receptor deamidase. Previously, we demonstrated that CheC's phosphatase activity plays a minor role in chemotaxis whereas its ability to bind CheD plays a major one. In the present study, we demonstrate that CheD's deamidase activity also plays a minor role in chemotaxis whereas its ability to bind CheC plays a major one. In addition, we quantified the interaction between CheC and CheD using surface plasmon resonance. These results suggest that the most important features of CheC and CheD are not their enzymatic activities but rather their roles in adaptation.

  13. The Membrane-anchored Serine Protease Prostasin (CAP1/PRSS8) Supports Epidermal Development and Postnatal Homeostasis Independent of Its Enzymatic Activity

    DEFF Research Database (Denmark)

    Peters, Diane E; Szabo, Roman; Friis, Stine


    prostasin catalytically inactive (Prss8(Cat-/Cat-) mice), develop barrier function and are healthy when followed for up to 20 weeks. This striking difference could not be explained by genetic modifiers or by maternal effects, as these divergent phenotypes were displayed by Prss8(-/-) and Prss8(Cat-/Cat......-) mice born within the same litter. Furthermore, Prss8(Cat-/Cat-) mice were able to regenerate epidermal covering following cutaneous wounding. This study provides the first demonstration that essential in vivo functions of prostasin are executed by a non-enzymatic activity of this unique membrane...

  14. Similar potential ATP-P production and enzymatic activities in the microplankton community off Concepción (Chile) under oxic and suboxic conditions (United States)

    González, Rodrigo R.; Gutiérrez, Marcelo H.; Quiñones, Renato A.


    The effects of the oxygen minimum zone on the metabolism of the heterotrophic microplankton community (0.22-100 μm) in the Humboldt Current System, as well as the factors controlling its biomass production, remain unknown. Here we compare the effect of four sources of dissolved organic carbon (glucose, oxaloacetate, glycine, leucine) on microbial biomass production (such as ATP-P) and the potential enzymatic activities involved in catabolic pathways under oxic and suboxic conditions. Our results show significant differences ( p malate dehydrogenase has the highest signal of NADH oxidization activity in the microbial assemblage. Furthermore, for all experiments, the malate dehydrogenase activity data set had a significant relationship with ATP-P production. These findings suggest that the microbial community inhabiting the oxygen minimum zone has the same or greater potential growth than the community inhabiting more oxygenated strata of the water column and that malate dehydrogenase is the activity that best represents the metabolic potential of the community.

  15. Monitoring enzymatic ATP hydrolysis by EPR spectroscopy. (United States)

    Hacker, Stephan M; Hintze, Christian; Marx, Andreas; Drescher, Malte


    An adenosine triphosphate (ATP) analogue modified with two nitroxide radicals is developed and employed to study its enzymatic hydrolysis by electron paramagnetic resonance spectroscopy. For this application, we demonstrate that EPR holds the potential to complement fluorogenic substrate analogues in monitoring enzymatic activity.

  16. The stay-green phenotype of TaNAM-RNAi wheat plants is associated with maintenance of chloroplast structure and high enzymatic antioxidant activity. (United States)

    Checovich, Mariana L; Galatro, Andrea; Moriconi, Jorge I; Simontacchi, Marcela; Dubcovsky, Jorge; Santa-María, Guillermo E


    TaNAM transcription factors play an important role in controlling senescence, which in turn, influences the delivery of nitrogen, iron and other elements to the grain of wheat (Triticum aestivum) plants, thus contributing to grain nutritional value. While lack or diminished expression of TaNAMs determines a stay-green phenotype, the precise effect of these factors on chloroplast structure has not been studied. In this work we focused on the events undergone by chloroplasts in two wheat lines having either control or diminished TaNAM expression due to RNA interference (RNAi). It was found that in RNAi plants maintenance of chlorophyll levels and maximal photochemical efficiency of photosystem II were associated with lack of chloroplast dismantling. Flow cytometer studies and electron microscope analysis showed that RNAi plants conserved organelle ultrastructure and complexity. It was also found that senescence in control plants was accompanied by a low leaf enzymatic antioxidant activity. Lack of chloroplast dismantling in RNAi plants was associated with maintenance of protein and iron concentration in the flag leaf, the opposite being observed in control plants. These data provide a structural basis for the observation that down regulation of TaNAMs confers a functional stay-green phenotype and indicate that the low export of iron and nitrogen from the flag leaf of these plants is concomitant, within the developmental window studied, with lack of chloroplast degradation and high enzymatic antioxidant activity.

  17. Impact of salinity-tolerant MCM6 transgenic tobacco on soil enzymatic activities and the functional diversity of rhizosphere microbial communities. (United States)

    Chaudhry, Vasvi; Dang, Hung Quang; Tran, Ngoc Quang; Mishra, Aradhana; Chauhan, Puneet Singh; Gill, Sarvajeet Singh; Nautiyal, Chandra Shekhar; Tuteja, Narendra


    The development of genetically modified plants for agriculture has provided numerous economic benefits, but has also raised concern over the potential impact of transgenic plants upon the environment. The rhizosphere is the soil compartment that is directly under the influence of living roots; it constitutes a complex niche likely to be exploited by a wide variety of bacteria potentially influenced by the introduction of transgenes in genetically modified plants. In the present study, the impact of overexpression of the salinity stress-tolerant minichromosome maintenance complex subunit 6 (MCM6) gene upon functional diversity and soil enzymatic activity in the rhizosphere of transgenic tobacco in the presence and absence of salt stress was examined. The diversity of culturable bacterial communities and soil enzymes, namely, dehydrogenases and acid phosphatases, was assessed and revealed no significant (or only minor) alterations due to transgenes in the rhizosphere soil of tobacco plants. Patterns in principal components analysis showed clustering of transgenic and non-transgenic tobacco plants according to the fingerprint of their associated bacterial communities. However, the presence of MCM6 tobacco did not cause changes in microbial populations, soil enzymatic activities or the functional diversity of the rhizosphere soil microbial community.

  18. Evaluation of Biological and Enzymatic Activity of Soil in a Tropical Dry Forest: Desierto de la Tatacoa (Colombia) with Potential in Mars Terraforming and Other Similar Planets (United States)

    Moreno Moreno, A. N.


    Desierto de la Tatacoa has been determined to be a tropical dry forest bioma, which is located at 3° 13" N 75° 13" W. It has a hot thermal floor with 440 msnm of altitude; it has a daily average of 28° C, and a maximum of 40° C, Its annual rainfall total can be upwards of 1250 mm. Its solar sheen has a daily average of 5.8 hours and its relative humidity is between 60% and 65%. Therefore, the life forms presents are very scant, and in certain places, almost void. It was realized a completely random sampling of soil from its surface down to 6 inches deep, of zones without vegetation and with soils highly loaded by oxides of iron in order to determine the number of microorganisms per gram and its subsequent identification. It was measured the soil basal respiration. Besides, it was determined enzymatic activity (catalase, dehydrogenase, phosphatase and urease). Starting with the obtained results, it is developes an alternative towards the study of soil genesis in Mars in particular, and recommendations for same process in other planets. Although the information found in the experiments already realized in Martian soil they demonstrate that doesnt exist any enzymatic activity, the knowledge of the same topic in the soil is proposed as an alternative to problems like carbonic fixing of the dense Martian atmosphere of CO2, the degradation of inorganic compounds amongst other in order to prepare the substratum for later colonization by some life form.

  19. Crystal structure of β1→6-galactosidase from Bifidobacterium bifidum S17: trimeric architecture, molecular determinants of the enzymatic activity and its inhibition by α-galactose. (United States)

    Godoy, Andre Schutzer; Camilo, Cesar Moises; Kadowaki, Marco Antonio; Muniz, Heloisa Dos S; Espirito Santo, Melissa; Murakami, Mario Tyago; Nascimento, Alessandro S; Polikarpov, Igor


    In a search for better comprehension of β-galactosidase function and specificity, we solved the crystal structures of the GH42 β-galactosidase BbgII from Bifidobacterium bifidum S17, a well-adapted probiotic microorganism from the human digestive tract, and its complex with d-α-galactose. BbgII is a three-domain molecule that forms barrel-shaped trimers in solution. BbgII interactions with d-α-galactose, a competitive inhibitor, showed a number of residues that are involved in the coordination of ligands. A combination of site-directed mutagenesis of these amino acid residues with enzymatic activity measurements confirmed that Glu161 and Glu320 are fundamental for catalysis and their substitution by alanines led to catalytically inactive mutants. Mutation Asn160Ala resulted in a two orders of magnitude decrease of the enzyme kcat without significant modification in its Km , whereas mutations Tyr289Phe and His371Phe simultaneously decreased kcat and increased Km values. Enzymatic activity of Glu368Ala mutant was too low to be detected. Our docking and molecular dynamics simulations showed that the enzyme recognizes and tightly binds substrates with β1→6 and β1→3 bonds, while binding of the substrates with β1→4 linkages is less favorable.

  20. Feeding indices and enzymatic activities of carob moth Ectomyelois ceratoniae (Zeller (Lepidoptera: pyrallidae on two commercial pistachio cultivars and an artificial diet

    Directory of Open Access Journals (Sweden)

    Naeimeh Teimouri


    Full Text Available Feeding indices and enzymatic activities of Ectomyelois ceratoniae (Zeller were studied in a growth chamber under controlled conditions (29 ± 2 °C, relative humidity of 70 ± 5% and a photoperiod of 16:8 (L:D hours on two commercial Pistachio cultivars (Akbari and Kalequchi and an artificial diet. Feeding indices of E. ceratoniae larvae differed significantly on three hosts (P < 0.05. The relative consumption rate was calculated to be 5.36 ± 0.009, 11.10 ± 1.49 and 10.631 ± 0.599 (mg/mg/day on artificial diet, Akbari and Kalequchi cultivars, respectively. Carob moth larvae reared on Akbari cultivar showed the highest efficiency of conversion of digested food (ECD (5.64 ± 0.43. The highest amount of efficiency of conversion of ingested food (ECI was obtained on artificial diet but approximate digestibility (AD was the lowest on this diet. The highest enzymatic activities of alpha-amylase, general proteases and lipase were observed in the midgut of larvae reared on artificial diet. Total protein and lipid value were highest in larvae that were reared on artificial diet.

  1. Mutations in Mtr4 Structural Domains Reveal Their Important Role in Regulating tRNAiMet Turnover in Saccharomyces cerevisiae and Mtr4p Enzymatic Activities In Vitro. (United States)

    Li, Yan; Burclaff, Joseph; Anderson, James T


    RNA processing and turnover play important roles in the maturation, metabolism and quality control of a large variety of RNAs thereby contributing to gene expression and cellular health. The TRAMP complex, composed of Air2p, Trf4p and Mtr4p, stimulates nuclear exosome-dependent RNA processing and degradation in Saccharomyces cerevisiae. The Mtr4 protein structure is composed of a helicase core and a novel so-called arch domain, which protrudes from the core. The helicase core contains highly conserved helicase domains RecA-1 and 2, and two structural domains of unclear functions, winged helix domain (WH) and ratchet domain. How the structural domains (arch, WH and ratchet domain) coordinate with the helicase domains and what roles they are playing in regulating Mtr4p helicase activity are unknown. We created a library of Mtr4p structural domain mutants for the first time and screened for those defective in the turnover of TRAMP and exosome substrate, hypomodified tRNAiMet. We found these domains regulate Mtr4p enzymatic activities differently through characterizing the arch domain mutants K700N and P731S, WH mutant K904N, and ratchet domain mutant R1030G. Arch domain mutants greatly reduced Mtr4p RNA binding, which surprisingly did not lead to significant defects on either in vivo tRNAiMet turnover, or in vitro unwinding activities. WH mutant K904N and Ratchet domain mutant R1030G showed decreased tRNAiMet turnover in vivo, as well as reduced RNA binding, ATPase and unwinding activities of Mtr4p in vitro. Particularly, K904 was found to be very important for steady protein levels in vivo. Overall, we conclude that arch domain plays a role in RNA binding but is largely dispensable for Mtr4p enzymatic activities, however the structural domains in the helicase core significantly contribute to Mtr4p ATPase and unwinding activities.

  2. Enzymatic Browning: a practical class

    Directory of Open Access Journals (Sweden)

    Maria Teresa Pedrosa Silva Clerici


    Full Text Available This paper presents a practical class about the enzymes polyphenol oxidases, which have been shown to be responsible for the enzymatic browning of fruits and vegetables. Vegetables samples were submitted to enzymatic inactivation process with chemical reagents, as well as by bleaching methods of applying heat by conventional oven and microwave oven. Process efficiency was assessed qualitatively by both observing the guaiacol peroxidase activity and after the storage period under refrigeration or freezing. The practical results obtained in this class allow exploring multidisciplinary knowledge in food science, with practical applications in everyday life.

  3. Enzymatic Treatment of Whey Proteins in Cow's Milk Results in Differential Inhibition of IgE-Mediated Mast Cell Activation Compared to T-Cell Activation

    NARCIS (Netherlands)

    Knipping, Karen; van Esch, Betty C. A. M.; van Ieperen-van Dijk, Adrie G.; van Hoffen, Els; van Baalen, Ton; Knippels, Leon M. J.; van der Heide, Sicco; Dubois, Anthony E. J.; Garssen, Johan; Knol, Edward F.


    Background: Cow's milk (CM) hydrolysates are frequently used as milk substitutes for children with CM allergy. In hydrolysates, allergenic epitopes within CM proteins are diminished by enzymatic treatment. The aim of this study was to examine the allergenic and immunogenic properties of whey protein

  4. Inhibition of ALK enzymatic activity in T-cell lymphoma cells induces apoptosis and suppresses proliferation and STAT3 phosphorylation independently of Jak3

    DEFF Research Database (Denmark)

    Marzec, Michal; Kasprzycka, Monika; Ptasznik, Andrzej;


    Aberrant expression of the ALK tyrosine kinase as a chimeric protein with nucleophosmin (NPM) and other partners plays a key role in malignant cell transformation of T-lymphocytes and other cells. Here we report that two small-molecule, structurally related, quinazoline-type compounds, WHI-131...... and WHI-154, directly inhibit enzymatic activity of NPM/ALK as demonstrated by in vitro kinase assays using a synthetic tyrosine-rich oligopeptide and the kinase itself as the substrates. The inhibition of NPM/ALK activity resulted in malignant T cells in suppression of their growth, induction...... of apoptosis and inhibition of tyrosine phosphorylation of STAT3, the key effector of the NPM/ALK-induced oncogenesis. We also show that the STAT3 tyrosine phosphorylation is mediated in the malignant T cells by NPM/ALK independently of Jak3 kinase as evidenced by the presence of STAT3 phosphorylation...

  5. Enzymatic nanoreactors for environmentally benign biotransformations. 1. Formation and catalytic activity of supramolecular complexes of laccase and linear-dendritic block copolymers. (United States)

    Gitsov, Ivan; Hamzik, James; Ryan, Joseph; Simonyan, Arsen; Nakas, James P; Omori, Shigetoshi; Krastanov, Albert; Cohen, Tomer; Tanenbaum, Stuart W


    We describe the construction of enzymatic nanoreactors through noncovalent envelopment of a glycoprotein by amphiphilic linear-dendritic AB or ABA copolymers. The synthetic procedure is based on the regioselective adsorption of dendritic poly(benzyl ether)-block-linear poly(ethylene glycol)-block-dendritic poly(benzyl ether) or linear poly(ethylene oxide)-block-dendritic poly(benzyl ether) copolymers onto the oxidative enzyme laccase from Trametes versicolor in aqueous medium. The complexes formed have improved catalytic activity compared with the native enzyme (77-85 nkat/mL vs 60 nkat/mL, respectively) and are more stable at elevated temperatures up to 70 degrees C. Experiments with deglycosylated laccase confirm that the glycoside fragments in the native enzyme serve as the anchor sites for the linear-dendritic copolymers. The enzymatic nanoreactors are able to effectively oxidize series of substrates: phenolic compounds (syringaldazine) and hydrophobic polyaromatic hydrocarbons (anthracene and benzo[a]pyrene) under "green" chemistry conditions.

  6. 煤矿周边农田土壤酶活性的研究%Study on Soil Enzymatic Activities in Areas around Coal Mine

    Institute of Scientific and Technical Information of China (English)

    刘文权; 袁敏; 温娟; 孙贻超; 邵晓龙; 宋文华; 王兴; 邢志杰


    采用奈氏试剂比色法、磷钼酸比色法等,对山东裕隆煤矿周边农田脲酶、蔗糖酶等土壤酶活性进行了测定,分析了土壤重金属污染状况与土壤酶活性的相关关系.结果表明,煤矿周边土壤环境受到不同程度的Cr、Hg、Pb、Zn污染,从矿区中心到外围重金属污染程度逐渐减轻,而土壤酶活性则不断提高,其中以脲酶活性增加最明显.多元回归模型显著性结果表明,重金属元素对土壤酶活性有不同的作用.可见,采用酶活性构筑的土壤信息系统的总体酶活性来表征矿区土壤的重金属污染状况是可行的.%Soil enzymatic activities such as urease and invertase were determined in areas by Nesslers reagent calorimetric method and Mo-Phosphate colorimetric method in Yulong coal mine,Shandong Province.The index of heavy metal pollution and the relationship between the activities of soil enzymes were analyzed.The results showed that the contents of heavy metals in contaminated soils were higher than those in non-polluted soils and the enzyme activities were decreased significantly with increasing contamination.The result of multivariate regression model test indicated that the elements of soil heavy metals have different influence on the soil enzymatic activities.It is feasible to use this index as primary biochemical parameter to evaluate heavy metals compound pollution.

  7. A δ38 deletion variant of human transketolase as a model of transketolase-like protein 1 exhibits no enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Stefan Schneider

    Full Text Available Besides transketolase (TKT, a thiamin-dependent enzyme of the pentose phosphate pathway, the human genome encodes for two closely related transketolase-like proteins, which share a high sequence identity with TKT. Transketolase-like protein 1 (TKTL1 has been implicated in cancerogenesis as its cellular expression levels were reported to directly correlate with invasion efficiency of cancer cells and patient mortality. It has been proposed that TKTL1 exerts its function by catalyzing an unusual enzymatic reaction, a hypothesis that has been the subject of recent controversy. The most striking difference between TKTL1 and TKT is a deletion of 38 consecutive amino acids in the N-terminal domain of the former, which constitute part of the active site in authentic TKT. Our structural and sequence analysis suggested that TKTL1 might not possess transketolase activity. In order to test this hypothesis in the absence of a recombinant expression system for TKTL1 and resilient data on its biochemical properties, we have engineered and biochemically characterized a "pseudo-TKTL1" Δ38 deletion variant of human TKT (TKTΔ38 as a viable model of TKTL1. Although the isolated protein is properly folded under in vitro conditions, both thermal stability as well as stability of the TKT-specific homodimeric assembly are markedly reduced. Circular dichroism and NMR spectroscopic analysis further indicates that TKTΔ38 is unable to bind the thiamin cofactor in a specific manner, even at superphysiological concentrations. No transketolase activity of TKTΔ38 can be detected for conversion of physiological sugar substrates thus arguing against an intrinsically encoded enzymatic function of TKTL1 in tumor cell metabolism.

  8. Enzymatic Modification of Sphingomyelin

    DEFF Research Database (Denmark)

    Due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. Currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost......-efficient, high yield production methods are of great interest. In the present study, the potential of producing ceramide through the enzymatic hydrolysis of sphingomyelin have been studied. sphingomyelin is a ubiquitous membrane-lipid and rich in dairy products or by-products. It has been verified...... that sphingomyelin modification gives a feasible approach to the potential production of ceramide. The reaction system has been improved through system evaluation and the optimization of several important factors, and phospholipase C from Clostridium perfringens shows higher activity towards the hydrolysis reaction...

  9. In vitro evaluation of the prebiotic activity of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel. (United States)

    Mandalari, G; Nueno Palop, C; Tuohy, K; Gibson, G R; Bennett, R N; Waldron, K W; Bisignano, G; Narbad, A; Faulds, C B


    The prebiotic effect of a pectic oligosaccharide-rich extract enzymatically derived from bergamot peel was studied using pure and mixed cultures of human faecal bacteria. This was compared to the prebiotic effect of fructo-oligosaccharides (FOS). Individual species of bifidobacteria and lactobacilli responded positively to the addition of the bergamot extract, which contained oligosaccharides in the range of three to seven. Fermentation studies were also carried out in controlled pH batch mixed human faecal cultures and changes in gut bacterial groups were monitored over 24 h by fluorescent in situ hybridisation, a culture-independent microbial assessment. Addition of the bergamot oligosaccharides (BOS) resulted in a high increase in the number of bifidobacteria and lactobacilli, whereas the clostridial population decreased. A prebiotic index (PI) was calculated for both FOS and BOS after 10 and 24 h incubation. Generally, higher PI scores were obtained after 10 h incubation, with BOS showing a greater value (6.90) than FOS (6.12).

  10. Enzymatic acylation of isoorientin and isovitexin from bamboo-leaf extracts with fatty acids and antiradical activity of the acylated derivatives. (United States)

    Ma, Xiang; Yan, Rian; Yu, Shuqi; Lu, Yuyun; Li, Zhuo; Lu, Haohao


    This study enzymatically acrylates two flavonoids from bamboo-leaf extracts, isoorientin and isovitexin, with different fatty acids as acyl donors using Candida antarctica lipase B (CALB). The conversion yield ranged from 35 to 80% for fatty acids with different chain lengths. Higher isoorientin and isovitexin conversion yields (>75%) were obtained using lauric acid in tert-amyl-alcohol as the reaction medium. (1)H and (13)C nuclear magnetic resonance spectroscopy analysis showed that, in the presence of CALB, acylation occurred at the isoorientin and isovitexin primary hydroxyl group of glucose moiety and only monoesters were detected. Introducing an acyl group into isoorientin and isovitexin significantly improved their lipophilicity but reduced their antiradical activity.

  11. Humanized-single domain antibodies (VH/VHH) that bound specifically to Naja kaouthia phospholipase A2 and neutralized the enzymatic activity. (United States)

    Chavanayarn, Charnwit; Thanongsaksrikul, Jeeraphong; Thueng-In, Kanyarat; Bangphoomi, Kunan; Sookrung, Nitat; Chaicumpa, Wanpen


    Naja kaouthia (monocled cobra) venom contains many isoforms of secreted phospholipase A2 (sPLA(2)). The PLA(2) exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/V(H)H) that bound specifically to the P3 and P5 were selected from a humanized-camel VH/V(H)H phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3) produced humanized-V(H)H, while another clone (P3-7) produced humanized-VH. At the optimal venom:antibody ratio, the VH/V(H)H purified from the E. coli homogenates neutralized PLA(2) enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/V(H)H covered the areas around the PLA(2) catalytic groove and inserted their Complementarity Determining Regions (CDRs) into the enzymatic cleft. It is envisaged that the VH/V(H)H would ameliorate/abrogate the principal toxicity of the venom PLA(2) (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis), if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

  12. Phytase production by Rhizopus microsporus var. microsporus biofilm: characterization of enzymatic activity after spray drying in presence of carbohydrates and nonconventional adjuvants. (United States)

    Sato, Vanessa Sayuri; Jorge, João Atílio; Oliveira, Wanderley Pereira; Souza, Claudia Regina Fernandez; Guimarães, Luis Henrique Souza


    Microbial phytases are enzymes with biotechnological interest for the feed industry. In this article, the effect of spray-drying conditions on the stability and activity of extracellular phytase produced by R. microsporus var. microsporus biofilm is described. The phytase was spray-dried in the presence of starch, corn meal (>150 μm), soy bean meal (SB), corn meal (drying adjuvants. The residual enzyme activity after drying ranged from 10.7% to 60.4%, with SB and CM standing out as stabilizing agents. Water concentration and residual enzyme activity were determined in obtained powders as a function of the drying condition. When exposed to different pH values, the SB and CM products were stable, with residual activity above 50% in the pH range from 4.5 to 8.5 for 60 min. The use of CM as drying adjuvant promoted the best retention of enzymatic activity compared with SB. Spray drying of the R. microsporus var. microsporus phytase using different drying adjuvants showed interesting results, being quite feasible with regards their biotechnological applications, especially for poultry diets.

  13. Enzymatic profile of Haemophilus ducreyi

    Energy Technology Data Exchange (ETDEWEB)

    Casin, I.M.; Sanson-Le Pors, M.J.; Gorce, M.F.; Ortenberg, M.; Perol, Y. (Universite Paris - 7, Hopital Saint-Louis, 75 - Paris (France))

    The enzymatic activities of two reference strains of Haemophilus ducreyi and thirty clinical isolates were investigated by conventional biochemical tests and the API-ZYM test kit system which included 97 synthetic substrates. No strains converted acid to porphyrins, but they all reduced nitrates to nitrites. All strains possessed aminopeptidase activity against ..beta..-naphthylamide derivatives of L-alanine, L-arginine, L-glutamine, glycine, L-leucine, L-lysine and L-serine. No trypsin or chymotrypsin-like activities were detected. All strains had phosphatase activity with broad pH range, and phosphoamidase activity. No glycosidase was detected by the substrates tested.

  14. Changes of apparent enzymatic activities and physical and chemical properties of steam-exploded corn stover during enzymatic hydrolysis%汽爆玉米秸秆酶解时表观酶活及物料理化性质的变化

    Institute of Scientific and Technical Information of China (English)

    张霞; 李红伟; 马晓建


    Based on engineering practice, the sugar concentration, power and apparent enzymatic activities change of enzymolysis suspension in enzymolysis process of steam-exploded corn stover (SECS) were studied to promote the industrialization process of corn stover producing butanol. At the same time, the changes of SECS physical and chemical properties during enzymatic hydrolysis were studied by biological microscope, laser scattering particle size distribution analysis (LSPSDA), X-ray diffraction (XRD), and scanning electron microscopy (SEM). Enzymatic hydrolysis was performed in a reactor with standard commercial cellulase as the enzyme. Enzymatic hydrolysis took place at 50℃, 100 r/min (four oblique leaves T agitator), enzyme loading of 60 IU/g corn stover, a solid:liquid ratio 3:10, and 48 h duration. Before the enzyme was added, the pH of the residues was adjusted to 4.8 with sodium hydroxide. After enzymatic hydrolysis the samples were taken from the reactor and centrifuged, and the supernatant phase was collected and analyzed for sugar concentrations, cellulase apparent enzymatic activities, and the physical and chemical properties of SECS. Sugar concentrations were tested by 3,5-Dinitrosalicylicacid(DNS). Cellulase apparent enzymatic activities were determined by the filter-paper method. Scanning electron microscopy and X-ray diffraction analysis were conducted. The results show: In the first reaction, the sugar concentration in the enzymatic hydrolysis liquid increased very quickly, especially in the first hour. The change in sugar concentration was not very clear later in the hydrolysis reaction due to such factors as substrate loss, changes in substrate properties, cellulose inactivation, decreasing cellulose synergy, and so on. The sugar concentration after enzymatic hydrolysis 48 h was 56.25 g/L. The stirring power decreased during enzymatic hydrolysis and declined most quickly after one hour of enzymatic hydrolysis, then began to decline more slowly. The

  15. Copy number variation in sulfotransferase isoform 1A1 (SULT1A1 is significantly associated with enzymatic activity in Japanese subjects

    Directory of Open Access Journals (Sweden)

    Yu X


    Full Text Available Xinfeng Yu,1 Takahiro Kubota,2 Ishwori Dhakal,1 Setsuo Hasegawa,3 Suzanne Williams,1 Shogo Ozawa,4 Susan Kadlubar11Division of Medical Genetics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas, USA; 2Chiba Institute of Science, Chiba, Japan; 3Sekino Clinical Pharmacology Clinic, Tokyo, Japan; 4Iwate Medical University, Iwate, JapanAbstract: Sulfotransferase isoform 1A1 (SULT1A1 plays a key role in the metabolism of a variety of endo- and xenobiotics and it's activity could influence response to drugs. Our previous studies have focused on the impact of genetic variants of SULT1A1 on enzymatic activity in Caucasians and African-Americans. However, the contribution of genetic variants to SULT1A1 activity in Asians has not been explored. In this study, we investigated the collective effects of both SULT1A1 copy number variants (CNVs and single nucleotide polymorphisms (SNPs in the promoter region, coding region, and 3´ untranslated region on SULT1A1 activity in Japanese subjects. SNPs in the SULT1A1 promoter and 3´ untranslated region were not associated with SULT1A1 activity (P > 0.05. SULT1A1*1/2 (Arg213His was marginally associated with SULT1A1 activity (P = 0.037. However, SULT1A1 CNVs were strongly associated with SULT1A1 activity (trend test P = 0.008 and accounted for 10% of the observed variability in activity for Japanese subjects. In conclusion, SULT1A1 CNVs play a pivotal role in determination of SULT1A1 activity in Japanese subjects, highlighting the influence of ethnic differences in SULT1A1 genetic variants on drug metabolism and therapeutic efficacy.Keywords: CNV, genotype, phenotype, SULT1A1, single nucleotide polymorphisms

  16. Kinetics of wheat straw solid-state fermentation with Trametes versicolor and Pleurotus ostreatus - lignin and polysaccharide alteration and production of related enzymatic activities

    Energy Technology Data Exchange (ETDEWEB)

    Valmaseda, M.; Martinez, M.J.; Martinez, A.T. (Consejo Superior de Investigaciones Cientificas, Madrid (Spain). Centro des Investigaciones Biologicas)


    The kinetics of straw solid-state fermentation (SSF) with Trametes versicolor and Pleurotus ostreatus was investigated to characterize the delignification processes by these white-rot fungi. Two sucessive phases could be defined during straw transformation, characterized by changes in respiratory activity, changes in lignin and polysaccharide content and composition, increase in in-vitro digestibility, and enzymatic activities produced by the fungi. Lignin composition was analysed after CuO alkaline degradation, and decreases in syringyl/guaiacyl and syringyl/p-hydroxyphenyl ratios and cinnamic acid content were observed during the fungal treatment. An increase in the phenolic acid yield, revealing fungal degradation of sidechains in lignin, was produced by P. ostreatus. The highest xylanase level was produced by P. ostreatus, and exocellulase activity was nearly absent from straw treated with this fungus. Laccase activity was found in straw treated with both fungi, but lignin peroxidase was only detected during the initial phase of straw transformation with T. versicolor. High levels of H{sub 2}O{sub 2}-producing acryl-alcohol oxidase occurred throughout the straw SSF with P. ostreatus. (orig.).

  17. Kresoxim methyl dissipation kinetics and its residue effect on soil extra-cellular and intra-cellular enzymatic activity in four different soils of India. (United States)

    Sabale, Rupali P; Shabeer T P, Ahammed; Utture, Sagar C; Banerjee, Kaushik; Oulkar, Dasharath P; Adsule, Pandurang G; Deshmukh, Madhukar B


    The rate of degradation of kresoxim methyl and its effect on soil extra-cellular (acid phosphatase, alkaline phosphatase and β-glucosidase) and intra-cellular (dehydrogenase) enzymes were explored in four different soils of India. In all the tested soils, the degradation rate was faster at the beginning, which slowed down with time indicating a non-linear pattern of degradation. Rate of degradation in black soil was fastest followed by saline, brown and red soils, respectively and followed 1st or 1st + 1st order kinetics with half-life ranging between 1-6 days for natural soil and 1-19 days for sterile soils. The rate of degradation in natural against sterilized soils suggests that microbial degradation might be the major pathway of residue dissipation. Although small changes in enzyme activities were observed, kresoxim methyl did not have any significant deleterious effect on the enzymatic activity of the various test soils in long run. Simple correlation studies between degradation percentage and individual enzyme activities did not establish any significant relationships. The pattern and change of enzyme activity was primarily due to the effect of the incubation period rather than the effect of kresoxim methyl itself.

  18. Characteristics of maize biochar with different pyrolysis temperatures and its effects on organic carbon, nitrogen and enzymatic activities after addition to fluvo-aquic soil. (United States)

    Wang, Xiubin; Zhou, Wei; Liang, Guoqing; Song, Dali; Zhang, Xiaoya


    In this study, the characteristics of maize biochar produced at different pyrolysis temperatures (300, 450 and 600°C) and its effects on organic carbon, nitrogen and enzymatic activities after addition to fluvo-aquic soil were investigated. As pyrolysis temperature increased, ash content, pH, electrical conductivity, surface area, pore volume and aromatic carbon content of biochar increased while yield, ratios of oxygen:carbon and hydrogen: carbon and alkyl carbon content decreased. During incubation, SOC, total N, and ammonium-N contents increased in all biochar-amended treatments compared with the urea treatment; however, soil nitrate-N content first increased and then decreased with increasing pyrolysis temperature of the applied biochar. Extracellular enzyme activities associated with carbon transformation first increased and then decreased with biochars pyrolyzed at 450 and 600°C. Protease activity markedly increased with increased pyrolysis temperatures, whereas pyrolysis temperature had limited effect on soil urease activity. The results indicated that the responses of extracellular enzymes to biochar were dependent on the pyrolysis temperature, the enzyme itself and incubation time as well.

  19. Photoelectrochemical enzymatic biosensors. (United States)

    Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan


    Enzymatic biosensors have been valuable bioanalytical devices for analysis of diverse targets in disease diagnosis, biological and biomedical research, etc. Photoelectrochemical (PEC) bioanalysis is a recently emerged method that promptly becoming a subject of new research interests due to its attractive potential for future bioanalysis with high sensitivity and specificity. PEC enzymatic biosensors integrate the inherent sensitivities of PEC bioanalysis and the selectivity of enzymes and thus share their both advantages. Currently, PEC enzymatic biosensors have become a hot topic of significant research and the recent impetus has grown rapidly as demonstrated by increased research papers. Given the pace of advances in this area, this review will make a thorough discussion and survey on the fundamentals, sensing strategies, applications and the state of the art in PEC enzymatic biosensors, followed by future prospects based on our own opinions. We hope this work could provide an accessible introduction to PEC enzymatic biosensors for any scientist.

  20. Propeptide of carboxypeptidase Y provides a chaperone-like function as well as inhibition of the enzymatic activity

    DEFF Research Database (Denmark)

    Winther, Jakob R.; Sørensen, P


    The zymogen of the vacuolar carboxypeptidase Y from Saccharomyces cerevisiae was purified and characterized with respect to activation as well as refolding in vitro. The purified procarboxypeptidase Y has no detectable activity but can be efficiently activated by proteinase K from Tritirachium al...

  1. Heterotrophic bacterial production and extracellular enzymatic activity in sinking particulate matter in the western North Pacific Ocean

    Directory of Open Access Journals (Sweden)

    Namiha eYamada


    Full Text Available Heterotrophic activities on sinking particulate matter (SPM play an important role in SPM fluxes in the ocean. To demonstrate regional differences in heterotrophic activities on SPM, we measured heterotrophic bacterial production (HBP in seawater and SPM as well as potential extracellular enzyme activity (EEA in SPM on a transect along 155°E in the western North Pacific Ocean in the subarctic (44°N, the Kuroshio Extension area (35°N, and the subtropical gyre (20°N. Depth-integrated HBP in seawater from the surface to 500 m was comparable between the locations, whereas HBP in SPM at 44°N was substantially lower than at the other sites. We found the highest particulate organic carbon (POC export flux and export efficiency to bathypelagic depths, and the lowest water temperatures, at 44°N. We found significant correlations between leucine aminopeptidase (LAPase activity, ß-glucosidase (BGase activity, POC flux and particulate organic nitrogen flux. LAPase activity was two orders of magnitude higher than BGase activity, with a BGase:LAPase activity ratio of 0.027. There were no significant correlations between HBP and EEA in SPM except for lipase, and lipase activity was significantly correlated with temperature. We propose that hydrographic conditions are an important factor controlling heterotrophic bacterial activity and export efficiency of organic carbon to the deep ocean, as are the sources and abundance of SPM produced in the euphotic zone via primary production.

  2. Insights into enzymatic thiamin catalysis


    Wikner, Christer


    Thiamin diphosphate, the biologically active form of vitamin B,, functions as a cofactor in various enzymes in the cell. The protein enhances the reactivity of the cofactor by binding it in a very specific manner. In this work, based upon information from the crystal structure, the mechanism of the thiamin dependent enzyme transketolase from yeast has been investigated by various methods. In enzymatic thiamin catalysis, the protein has three major tasks in the formation of a...

  3. Degradation of di(2-ethyl hexyl) phthalate by Fusarium culmorum: Kinetics, enzymatic activities and biodegradation pathway based on quantum chemical modelingpathway based on quantum chemical modeling. (United States)

    Ahuactzin-Pérez, Miriam; Tlecuitl-Beristain, Saúl; García-Dávila, Jorge; González-Pérez, Manuel; Gutiérrez-Ruíz, María Concepción; Sánchez, Carmen


    Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer widely used in the manufacture of plastics, and it is an environmental contaminant. The specific growth rate (μ), maximum biomass (Xmax), biodegradation constant of DEHP (k), half-life (t1/2) of DEHP biodegradation and removal efficiency of DEHP, esterase and laccase specific activities, and enzymatic yield parameters were evaluated for Fusarium culmorum grown on media containing glucose and different concentrations of DEHP (0, 500 and 1000mg/L). The greatest μ and the largest Xmax occurred in media supplemented with 1000mg of DEHP/L. F. culmorum degraded 95% of the highest amount of DEHP tested (1000mg/L) within 60h of growth. The k and t1/2 were 0.024h(-1) and 28h, respectively, for both DEHP concentrations. The removal efficiency of DEHP was 99.8% and 99.9% for 1000 and 500mg/L, respectively. Much higher specific esterase activity than specific laccase activity was observed in all media tested. The compounds of biodegradation of DEHP were identified by GC-MS. A DEHP biodegradation pathway by F. culmorum was proposed on the basis of the intermolecular flow of electrons of the identified intermediate compounds using quantum chemical modeling. DEHP was fully metabolized by F. culmorum with butanediol as the final product. This fungus offers great potential in bioremediation of environments polluted with DEHP.

  4. The role of the C-terminal region on the oligomeric state and enzymatic activity of Trypanosoma cruzi hypoxanthine phosphoribosyl transferase. (United States)

    Valsecchi, Wanda M; Cousido-Siah, Alexandra; Defelipe, Lucas A; Mitschler, André; Podjarny, Alberto; Santos, Javier; Delfino, José M


    Hypoxanthine phosphoribosyl transferase from Trypanosoma cruzi (TcHPRT) is a critical enzyme for the survival of the parasite. This work demonstrates that the full-length form in solution adopts a stable and enzymatically active tetrameric form, exhibiting large inter-subunit surfaces. Although this protein irreversibly aggregates during unfolding, oligomerization is reversible and can be modulated by low concentrations of urea. When the C-terminal region, which is predicted as a disordered stretch, is excised by proteolysis, TcHPRT adopts a dimeric state, suggesting that the C-terminal region acts as a main guide for the quaternary arrangement. These results are in agreement with X-ray crystallographic data presented in this work. On the other hand, the C-terminal region exhibits a modulatory role on the enzyme, as attested by the enhanced activity observed for the dimeric form. Bisphosphonates act as substrate-mimetics, uncovering long-range communications among the active sites. All in all, this work contributes to establish new ways applicable to the design of novel inhibitors that could eventually result in new drugs against parasitic diseases.

  5. Enzymatic activity of catechol 1,2-dioxygenase and catechol 2,3-dioxygenase produced by Gordonia polyisoprenivorans

    Directory of Open Access Journals (Sweden)

    Andréa Scaramal Silva


    Full Text Available This study aimed to evaluate the environmental conditions for enzyme activity of catechol 1,2-dioxygenase (C1,2O and catechol 2,3-dioxygenase (C2,3O produced by Gordonia polyisoprenivorans in cell-free and immobilized extracts. The optimum conditions of pH, temperature, time course and effect of ions for enzyme activity were determined. Peak activity of C1,2O occurred at pH 8.0. The isolate exhibited the highest activity of C2,3O at pH 7.0 and 8.0 for the cell-free extract and immobilized extract, respectively. This isolate exhibited important characteristics such as broad range of pH, temperature and time course for enzyme activity.

  6. Enzymatic Activity of Glutathione S-Transferase and Dental Fluorosis Among Children Receiving Two Different Levels of Naturally Fluoridated Water. (United States)

    Bonola-Gallardo, Irvin; Irigoyen-Camacho, María Esther; Vera-Robles, Liliana; Campero, Antonio; Gómez-Quiroz, Luis


    This study was conducted to measure the activity of the enzyme glutathione S-transferase (GST) in saliva and to compare the activity of this enzyme in children with and without dental fluorosis in communities with different concentrations of naturally fluoridated water. A total of 141 schoolchildren participated in this cross-sectional study. Children were selected from two communities: one with a low (0.4 ppm) and the other with a high (1.8 ppm) water fluoride concentration. Dental fluorosis was evaluated by applying the Thylstrup and Fejerskov Index (TFI) criteria. Stimulated saliva was obtained, and fluoride concentration and GST activity were measured. The GST activity was compared among children with different levels of dental fluorosis using multinomial logistic regression models and odds ratios (OR). The mean age of the children was 10.6 (±1.03) years. Approximately half of the children showed dental fluorosis (52.5 %). The average GST activity was 0.5678 (±0.1959) nmol/min/μg. A higher concentration of fluoride in the saliva was detected in children with a higher GST activity (p = 0.039). A multinomial logistic regression model used to evaluate the GST activity and the dental fluorosis score identified a strong association between TFI = 2-3 (OR = 15.44, p = 0.007) and TFI ≥ 4 (OR = 55.40, p = 0.026) and the GST activity level, compared with children showing TFI = 0-1, adjusted for age and sex. Schoolchildren with higher levels of dental fluorosis and a higher fluoride concentration in the saliva showed greater GST activity. The increased GST activity most likely was the result of the body's need to inactivate free radicals produced by exposure to fluoride.

  7. Pollution in the urban soils of Lianyungang, China, evaluated using a pollution index, mobility of heavy metals, and enzymatic activities. (United States)

    Li, Yu; Li, Hong-Guan; Liu, Fu-Cheng


    Soil samples from 16 urban sites in Lianyungang, China were collected and analyzed. A pollution index was used to assess the potential ecological risk of heavy metals and a sequential extraction procedure was used to evaluate the relative distribution of Cu, Zn, Pb, Cd, Cr, and As in exchangeable, carbonate, Fe/Mn oxide, organic/sulfide, and residual fractions. The mobility of heavy metals and urease (URE) activity, alkaline phosphatase (ALP) activity, and invertase (INV) activity of soils was determined. The results showed that the average concentrations of Cu, Zn, Pb, Cd, Cr, and As in Lianyungang soils were much higher than those in the coastal city soil background values of Jiangsu and China. Among the five studied regions (utilities, commercial, industrial, tourism, and roadside), the industrial region had the highest metal concentrations demonstrating that land use had a significant impact on the accumulation of heavy metals in Lianyungang soils. Compared to the other metals, Cd showed the highest ecological risk. According to chemical partitioning, Cu was associated with the organic/sulfides and Pb and Zn were mainly in the carbonate and the Fe/Mn oxide phase. The greatest amounts of Cd were found in exchangeable and carbonate fractions, while Cr and As were mainly in the residual fraction. Cd had the highest mobility of all metals, and the order of mobility (highest to lowest) of heavy metals in Lianyungang soils was Cd > Zn > Pb > Cu > As > Cr. Soil urease activity, alkaline phosphatase activity, and invertase activity varied considerably in different pollution degree sites. Soil enzyme activities had the lowest levels in roadside and industrial regions. Across all the soil data in the five regions, the total Cu, Zn, Pb, Cd, Cr, and As level was negatively correlated with urease activity, alkaline phosphatase activity, and invertase activity, but the relationship was not significant. In the industrial region, alkaline phosphatase activity had

  8. Distribution and enzymatic activity of heterotrophic bacteria decomposing selected macromolecular compounds in a Baltic Sea sandy beach (United States)

    Podgórska, B.; Mudryk, Z. J.


    The potential capability to decompose macromolecular compounds, and the level of extracellular enzyme activities were determined in heterotrophic bacteria isolated from a sandy beach in Sopot on the Southern Baltic Sea coast. Individual isolates were capable of hydrolysing a wide spectrum of organic macromolecular compounds. Lipids, gelatine, and DNA were hydrolyzed most efficiently. Only a very small percentage of strains were able to decompose cellulose, and no pectinolytic bacteria were found. Except for starch-hydrolysis, no significant differences in the intensity of organic compound decomposition were recorded between horizontal and vertical profiles of the studied beach. Of all the studied extracellular enzymes, alkaline phosphatase, esterase lipase, and leucine acrylaminidase were most active; in contrast, the activity α-fucosidase, α-galactosidase and β-glucouronidase was the weakest. The level of extracellular enzyme activity was similar in both sand layers.

  9. Effect of permethrin, anthracene and mixture exposure on shell components, enzymatic activities and proteins status in the Mediterranean clam Venerupis decussata. (United States)

    Sellami, Badreddine; Khazri, Abdelhafidh; Mezni, Amine; Louati, Héla; Dellali, Mohamed; Aissa, Patricia; Mahmoudi, Ezzeddine; Beyrem, Hamouda; Sheehan, David


    Anthracene (ANT) and permethrin (PER) are two of the more toxic compounds reaching the marine environment. This study aimed to determine the impact of these molecules on Venerupis decussata, an economically important species cultured on the Tunisian coast. Shell structure and its possible transformation upon exposure to the two contaminants were studied by X-ray diffraction and gravimetric analyses. Results revealed a phase transition in shell composition from aragonite to calcite after PER exposure, to a mixture of PER and ANT (Mix) but not for ANT alone. Catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GST) activities were determined in digestive gland and gills after exposure to ANT, PER and Mix to assess the impact of the contamination on the oxidative status of V. decussata. Enzyme activities increased in the digestive gland after PER treatment and in the gills after ANT treatment. PER exposure significantly reduced the levels of free thiols and increased levels of carbonylated proteins in the digestive gland, as compared to controls. In contrast, ANT exposure significantly reduced free thiols and increased the number of carbonylated proteins in the gills. Mix induced additive effects as measured by both enzymatic and proteomic approaches. The present study suggests that PER has a strong effect on shell structure; that PER and ANT exposure generate compound-dependent oxidative stress in the tissues of V. decussata and that a mixture of the two compounds has synergistic effects on biochemical response.

  10. Modifications on the hydrogen bond network by mutations of Escherichia coli copper efflux oxidase affect the process of proton transfer to dioxygen leading to alterations of enzymatic activities

    Energy Technology Data Exchange (ETDEWEB)

    Kajikawa, Takao; Kataoka, Kunishige [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan); Sakurai, Takeshi, E-mail: [Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa 920-1192 (Japan)


    Highlights: Black-Right-Pointing-Pointer Proton transfer pathway to dioxygen in CueO was identified. Black-Right-Pointing-Pointer Glu506 is the key amino acid to transport proton. Black-Right-Pointing-Pointer The Ala mutation at Glu506 formed a compensatory proton transfer pathway. Black-Right-Pointing-Pointer The Ile mutation at Glu506 shut down the hydrogen bond network. -- Abstract: CueO has a branched hydrogen bond network leading from the exterior of the protein molecule to the trinuclear copper center. This network transports protons in the four-electron reduction of dioxygen. We replaced the acidic Glu506 and Asp507 residues with the charged and uncharged amino acid residues. Peculiar changes in the enzyme activity of the mutants relative to the native enzyme indicate that an acidic amino acid residue at position 506 is essential for effective proton transport. The Ala mutation resulted in the formation of a compensatory hydrogen bond network with one or two extra water molecules. On the other hand, the Ile mutation resulted in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities of CueO. In contrast, the hydrogen bond network without the proton transport function was constructed by the Gln mutation. These results exerted on the hydrogen bond network in CueO are discussed in comparison with proton transfers in cytochrome oxidase.

  11. Postprandial incretin and islet hormone responses and dipeptidyl-peptidase 4 enzymatic activity in patients with maturity onset diabetes of the young

    DEFF Research Database (Denmark)

    Østoft, Signe Harring; Bagger, Jonatan Ising; Hansen, Torben;


    )), and dipeptidyl-peptidase 4 (DPP-4) enzymatic activity in patients with glucokinase (GCK)-diabetes (MODY2), hepatocyte nuclear factor 1α (HNF1A)-diabetes (MODY3), and in matched healthy individuals (CTRLs). Subjects and methods: Ten patients with GCK-diabetes (age: 43±5 years; BMI: 24±2 kg/m2; FPG: 7.1±0.3 mmol...... with a liquid test meal. Results: All groups exhibited similar baseline values of glucagon (7±1 (GCK-diabetes), 6±1 pmol/l (HNF1A-diabetes), 8±2 (CTRLs), P=0.787), but patients with HNF1A-diabetes exhibited postprandial hyperglucagonaemia (area under curve (AUC): 838±108 min×pmol/l) compared to CTRLs (182......±176 min×pmol/l, P=0.005) and tended to have a greater response than patients with GCK-diabetes (410±154 min×pmol/l, P=0.063). Similar peak concentrations and AUCs for plasma GIP and plasma GLP-1 were observed across the groups. Increased fasting DPP-4-activity was seen in patients with HNF1A-diabetes (17...

  12. Mechanistic and Structural Studies of Protein-Only RNase P Compared to Ribonucleoproteins Reveal the Two Faces of the Same Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Cédric Schelcher


    Full Text Available RNase P, the essential activity that performs the 5′ maturation of tRNA precursors, can be achieved either by ribonucleoproteins containing a ribozyme present in the three domains of life or by protein-only enzymes called protein-only RNase P (PRORP that occur in eukaryote nuclei and organelles. A fast growing list of studies has investigated three-dimensional structures and mode of action of PRORP proteins. Results suggest that similar to ribozymes, PRORP proteins have two main domains. A clear functional analogy can be drawn between the specificity domain of the RNase P ribozyme and PRORP pentatricopeptide repeat domain, and between the ribozyme catalytic domain and PRORP N4BP1, YacP-like Nuclease domain. Moreover, both types of enzymes appear to dock with the acceptor arm of tRNA precursors and make specific contacts with the corner of pre-tRNAs. While some clear differences can still be delineated between PRORP and ribonucleoprotein (RNP RNase P, the two types of enzymes seem to use, fundamentally, the same catalytic mechanism involving two metal ions. The occurrence of PRORP and RNP RNase P represents a remarkable example of convergent evolution. It might be the unique witness of an ongoing replacement of catalytic RNAs by proteins for enzymatic activities.

  13. Effect of ferrous chloride on biogas production and enzymatic activities during anaerobic fermentation of cow dung and Phragmites straw. (United States)

    Zhang, Huayong; Tian, Yonglan; Wang, Lijun; Mi, Xueyue; Chai, Yang


    The effect of ferrous (added as FeCl2) on the anaerobic co-digestion of Phragmites straw and cow dung was studied by investigating the biogas properties, pH values, organic matter degradation (COD) and enzyme activities (cellulase, protease and dehydrogenase) at different stages of mesophilic fermentation. The results showed that Fe(2+) addition increased the cumulative biogas yields by 18.1 % by extending the peak period with high daily biogas yields. Meanwhile, the methane (CH4) contents in the Fe(2+) added groups were generally higher than the control group before the 15th day. The pH values were not significantly impacted by Fe(2+) concentrations during the fermentation process. The COD concentrations, cellulase, protease and dehydrogenase activities varied with the added Fe(2+) concentrations and the stages of the fermentation process. At the beginning stage of fermentation (4th day), Fe(2+) addition increased the biogas production by improving the cellulase and dehydrogenase activities which caused a decline in COD. At the peak stage of fermentation (8th day), Fe(2+) addition enhanced the cellulase and protease activities, and resulted in lower COD contents than the control group. When the biogas yields decreased again (13th day), the COD contents varied similar with the protease and dehydrogenase activities, whilst cellulase activities were not sensitive to Fe(2+) concentrations. At the end of fermentation (26th day), Fe(2+) addition decreased the cellulase activities, led to lower COD contents and finally resulted the lower biogas yields than the control group. Taking the whole fermentation process into account, the promoting effect of Fe(2+) addition on biogas yields was mainly attributed to the extension of the gas production peak stage and the improvement of cellulase activities.

  14. Group III alcohol dehydrogenase from Pectobacterium atrosepticum: insights into enzymatic activity and organization of the metal ion-containing region. (United States)

    Elleuche, Skander; Fodor, Krisztian; von der Heyde, Amélie; Klippel, Barbara; Wilmanns, Matthias; Antranikian, Garabed


    NAD(P)(+)-dependent alcohol dehydrogenases (ADH) are widely distributed in all phyla. These proteins can be assigned to three nonhomologous groups of isozymes, with group III being highly diverse with regards to catalytic activity and primary structure. Members of group III ADHs share a conserved stretch of amino acid residues important for cofactor binding and metal ion coordination, while sequence identities for complete proteins are highly diverse (90 %). A putative group III ADH PaYqhD has been identified in BLAST analysis from the plant pathogenic enterobacterium Pectobacterium atrosepticum. The PaYqhD gene was expressed in the heterologous host Escherichia coli, and the recombinant protein was purified in a two-step purification procedure to homogeneity indicating an obligate dimerization of monomers. Four conserved amino acid residues involved in metal ion coordination were substituted with alanine, and their importance for catalytic activity was confirmed by circular dichroism spectrum determination, in vitro, and growth experiments. PaYqhD exhibits optimal activity at 40 °C with short carbon chain aldehyde compounds and NADPH as cofactor indicating the enzyme to be an aldehyde reductase. No oxidative activities towards alcoholic compounds were detectable. EDTA completely inhibited catalytic activity and was fully restored by the addition of Co(2+). Activity measurements together with sequence alignments and structure analysis confirmed that PaYqhD belongs to the butanol dehydrogenase-like enzymes within group III of ADHs.

  15. Syzygium jambos and Solanum guaraniticum Show Similar Antioxidant Properties but Induce Different Enzymatic Activities in the Brain of Rats

    Directory of Open Access Journals (Sweden)

    Maria Beatriz Moretto


    Full Text Available Syzygium jambos and Solanum guaraniticum are both employed in Brazil as medicinal plants, even though their potential toxicity is not well established and they are frequently misused. The aim of this study was investigate the effect of the aqueous leaf extracts of both plants on δ-aminolevulinate dehydratase (δ-ALA-D and acetylcholinesterase (AChE activities and the antioxidant action against oxidative damage induced by sodium nitroprusside in rats, using in vitro assays. In addition, the presence of gallic, caffeic and chlorogenic acids, as well as rutin, quercetin and kaempferol as bioactive compounds in the extracts was identified by HPLC and their levels quantified. The antioxidant activities of both extracts were assessed by their capabilities to scavenge nitric oxide and to inhibit lipid peroxidation. Only Syzygium jambos presented thiol-peroxidase-like activity. Although neither extract affected the AChE activity, the aqueous extract of Solanum guaraniticum inhibited brain δ-ALA-D activity, suggesting a possible impairment effect on the central nervous system. Our results showed that both extracts exhibited efficient free radical scavenger activity and are an interesting source of bioactive compounds, justifying their use in folk medicine, although Solanum guaraniticum extract could have neurotoxicity properties and we therefore suggest that its use should be restricted to ensure the health of the population.

  16. Recombinant production of enzymatically active male contraceptive drug target hTSSK2 - Localization of the TSKS domain phosphorylated by TSSK2. (United States)

    Shetty, Jagathpala; Sinville, Rondedrick; Shumilin, Igor A; Minor, Wladek; Zhang, Jianhai; Hawkinson, Jon E; Georg, Gunda I; Flickinger, Charles J; Herr, John C


    The testis-specific serine/threonine kinase 2 (TSSK2) has been proposed as a candidate male contraceptive target. Development of a selective inhibitor for this kinase first necessitates the production of highly purified, soluble human TSSK2 and its substrate, TSKS, with high yields and retention of biological activity for crystallography and compound screening. Strategies to produce full-length, soluble, biologically active hTSSK2 in baculovirus expression systems were tested and refined. Soluble preparations of TSSK2 were purified by immobilized-metal affinity chromatography (IMAC) followed by gel filtration chromatography. The biological activities of rec.hTSSK2 were verified by in vitro kinase and mobility shift assays using bacterially produced hTSKS (isoform 2), casein, glycogen synthase peptide (GS peptide) and various TSKS peptides as target substrates. Purified recombinant hTSSK2 showed robust kinase activity in the in vitro kinase assay by phosphorylating hTSKS isoform 2 and casein. The ATP Km values were similar for highly and partially purified fractions of hTSSK2 (2.2 and 2.7 μM, respectively). The broad spectrum kinase inhibitor staurosporine was a potent inhibitor of rec.hTSSK2 (IC50 = 20 nM). In vitro phosphorylation experiments carried out with TSKS (isoform 1) fragments revealed particularly strong phosphorylation of a recombinant N-terminal region representing aa 1-150 of TSKS, indicating that the N-terminus of human TSKS is phosphorylated by human TSSK2. Production of full-length enzymatically active recombinant TSSK2 kinase represents the achievement of a key benchmark for future discovery of TSSK inhibitors as male contraceptive agents.

  17. Antioxidant Activity of Enzymatic Gonad Hydrolysates from Jellyfish Rhopilema esculentum%海蜇生殖腺酶解物抗氧化活性的研究

    Institute of Scientific and Technical Information of China (English)

    郑杰; 于笛; 陈冲; 袁成玉


    为提高海蜇的综合利用率,利用木瓜蛋白酶酶解技术制备海蜇生殖腺酶解物。分析酶解物的氨基酸组成,并通过体外化学模拟体系评价酶解物与F e2+螯合前后的抗氧化活性。试验结果表明,海蜇生殖腺酶解物含有17种氨基酸,具有一定的Fe2+螯合能力和DPPH ·清除能力,其螯合率和清除率达到50%时的质量浓度分别为1.73 mg/mL 和22.20 mg/mL。同时,具有一定的还原能力,其在700 nm处吸光值达到0.5时的质量浓度为17.61 mg/mL。海蜇生殖腺酶解物与Fe2+螯合后,抗氧化能力显著增强,DPPH ·清除率达到50%和在700 nm处吸光值达到0.5时的质量浓度分别为0.15 mg/mL和0.86 mg/mL。%The enzymatic hydrolysates of gonad in jellyfish Rhopilema esculentum were prepared by papain ,and the amino acid composition and the antioxidant activities of the hydrolysates were analyzed by Fe2+-chelating assay ,1 ,1-diphenyl-2-picrylhydrazyl (DPPH · ) assay and reducing power assay to make full use of the jellyfish .The results showed that the hydrolysates from jellyfish gonad were composed of 17 amino acids ,and possessed Fe2+ -chelatin ability ,DPPH radical scavenging capacity with IC50 values of 1 . 73 and 22 .20 mg/mL , respectively , and reducing power with AC0 .5 value of 17 .61 mg/mL .The antioxidant activities of enzymatic hydrolysates ,chelated with Fe2+ ,were obviously improved ,with the IC50 and AC0 .5 values of 0 .15 mg/mL in DPPH radical scavenging capacity and 0 .86 mg/mL in reducing power .The findings provide theoretical support for the further exploitation of jellyfish gonad .

  18. Structural and Enzymatic Characterization of ABgp46, a Novel Phage Endolysin with Broad Anti-Gram-Negative Bacterial Activity (United States)

    Oliveira, Hugo; Vilas Boas, Diana; Mesnage, Stéphane; Kluskens, Leon D.; Lavigne, Rob; Sillankorva, Sanna; Secundo, Francesco; Azeredo, Joana


    The present study demonstrates the antibacterial potential of a phage endolysin against Gram-negative pathogens, particularly against multidrug resistant strains of Acinetobacter baumannii. We have cloned, heterologously expressed and characterized a novel endolysin (ABgp46) from Acinetobacter phage vb_AbaP_CEB1 and tested its antibacterial activity against several multidrug-resistant A. baumannii strains. LC-MS revealed that ABgp46 is an N-acetylmuramidase, that is also active over a broad pH range (4.0–10.0) and temperatures up to 50°C. Interestingly, ABgp46 has intrinsic and specific anti-A. baumannii activity, reducing multidrug resistant strains by up to 2 logs within 2 h. By combining ABgp46 with several organic acids that act as outer membrane permeabilizing agents, it is possible to increase and broaden antibacterial activity to include other Gram-negative bacterial pathogens. In the presence of citric and malic acid, ABgp46 reduces A. baumannii below the detection limit (>5 log) and more than 4 logs Pseudomonas aeruginosa and Salmonella typhimurium strains. Overall, this globular endolysin exhibits a broad and high activity against Gram-negative pathogens, that can be enhanced in presence of citric and malic acid, and be used in human and veterinary medicine. PMID:26955368


    Directory of Open Access Journals (Sweden)

    Mehdi Nadalian


    Full Text Available Poultry by-products are great economic sources that need to be exploited. Poultry skin could be utilized to extract protein particularly elastin, which is often incorporated in the production of functional food, cosmetic industry or medicine due to its antioxidative properties. In this study, water-soluble elastin was successfully extracted from broiler and spent hen skin and analysed for antioxidant activities including DPPH (1,1-diphenyl-2-picryl hydrazyl, ABTS and metal chelating activity. Antioxidant activity of elastin extracted from broiler skin hydrolysed by Alcalase (EBA and Elastase (EBE also elastin extracted from spent hen skin hydrolyzed by Alcalase (ESA and Elastease (ESE. The EBE, EBA, ESE and ESA had higher DPPH (16-30, 19-35, 29-48 and 31-50%, respectively, ABTS activity ( 73-79, 60-79, 67-79 and 72-79 %, respectively, and Fe2+chelating activity ( 65-69, 50-56, 71-77 and 62-70 %, respectively. This concluded that water-soluble elastin is a bioactive component that could potentially be used in the formulation of functional foods, nutraceuticals, cosmetic and pharmaceutical industry

  20. Modulation of Antioxidant Enzymatic Activities by Certain Antiepileptic Drugs (Valproic Acid, Oxcarbazepine, and Topiramate: Evidence in Humans and Experimental Models

    Directory of Open Access Journals (Sweden)

    Noemí Cárdenas-Rodríguez


    Full Text Available It is estimated that at least 100 million people worldwide will suffer from epilepsy at some point in their lives. This neurological disorder induces brain death due to the excessive liberation of glutamate, which activates the postsynaptic N-methyl-D-aspartic acid (NMDA receptors, which in turn cause the reuptake of intracellular calcium (excitotoxicity. This excitotoxicity elicits a series of events leading to nitric oxide synthase (NOS activation and the generation of reactive oxygen species (ROS. Several studies in experimental models and in humans have demonstrated that certain antiepileptic drugs (AEDs exhibit antioxidant effects by modulating the activity of various enzymes associated with this type of stress. Considering the above-mentioned data, we aimed to compile evidence elucidating how AEDs such as valproic acid (VPA, oxcarbazepine (OXC, and topiramate (TPM modulate oxidative stress.

  1. Modulation of antioxidant enzymatic activities by certain antiepileptic drugs (valproic acid, oxcarbazepine, and topiramate): evidence in humans and experimental models. (United States)

    Cárdenas-Rodríguez, Noemí; Coballase-Urrutia, Elvia; Rivera-Espinosa, Liliana; Romero-Toledo, Arantxa; Sampieri, Aristides; Ortega-Cuellar, Daniel; Montesinos-Correa, Hortencia; Floriano-Sánchez, Esaú; Carmona-Aparicio, Liliana


    It is estimated that at least 100 million people worldwide will suffer from epilepsy at some point in their lives. This neurological disorder induces brain death due to the excessive liberation of glutamate, which activates the postsynaptic N-methyl-D-aspartic acid (NMDA) receptors, which in turn cause the reuptake of intracellular calcium (excitotoxicity). This excitotoxicity elicits a series of events leading to nitric oxide synthase (NOS) activation and the generation of reactive oxygen species (ROS). Several studies in experimental models and in humans have demonstrated that certain antiepileptic drugs (AEDs) exhibit antioxidant effects by modulating the activity of various enzymes associated with this type of stress. Considering the above-mentioned data, we aimed to compile evidence elucidating how AEDs such as valproic acid (VPA), oxcarbazepine (OXC), and topiramate (TPM) modulate oxidative stress.

  2. Modulation of Antioxidant Enzymatic Activities by Certain Antiepileptic Drugs (Valproic Acid, Oxcarbazepine, and Topiramate): Evidence in Humans and Experimental Models (United States)

    Cárdenas-Rodríguez, Noemí; Coballase-Urrutia, Elvia; Rivera-Espinosa, Liliana; Romero-Toledo, Arantxa; Sampieri, Aristides III; Ortega-Cuellar, Daniel; Montesinos-Correa, Hortencia; Floriano-Sánchez, Esaú; Carmona-Aparicio, Liliana


    It is estimated that at least 100 million people worldwide will suffer from epilepsy at some point in their lives. This neurological disorder induces brain death due to the excessive liberation of glutamate, which activates the postsynaptic N-methyl-D-aspartic acid (NMDA) receptors, which in turn cause the reuptake of intracellular calcium (excitotoxicity). This excitotoxicity elicits a series of events leading to nitric oxide synthase (NOS) activation and the generation of reactive oxygen species (ROS). Several studies in experimental models and in humans have demonstrated that certain antiepileptic drugs (AEDs) exhibit antioxidant effects by modulating the activity of various enzymes associated with this type of stress. Considering the above-mentioned data, we aimed to compile evidence elucidating how AEDs such as valproic acid (VPA), oxcarbazepine (OXC), and topiramate (TPM) modulate oxidative stress. PMID:24454986


    Directory of Open Access Journals (Sweden)

    Ummay Honi


    Full Text Available Lactobacillus paracasei ssp. paracasei-1 was identified from traditional yogurts of Khulna region, Bangladesh and its enzyme and antibiotic resistance profiles were determined. A commercially available API Zym kit was employed to determine the activities of 19 different enzymes. We found that L. paracasei ssp. paracasei-1 showed strong activities for several enzymes, viz. leucine arylamidase, valine arylamidase, napthol-AS-BI-phosphohydrolase, β-galactosidase, α –Glucosidase, N-Acetyl- β- glucosaminidase while activities for other enzymes were absent. Antibiotic resistance profile was assessed by minimum inhibitory concentration (MIC test for 61 major antibiotics and 4 antifungal agents obtained from commercial sources in MRS Agar media. The strain generally showed resistance to gram negative spectrum antibiotic while it showed susceptibility towards β-lactam antibiotic to gram positive spectrum antibiotic. The findings provide the therapeutic basis of using L. paracasei ssp. paracasei-1 in finished food products.

  4. Microbial and enzymatic activity of soil contaminated with a mixture of diflufenican + mesosulfuron-methyl + iodosulfuron-methyl-sodium. (United States)

    Baćmaga, Małgorzata; Borowik, Agata; Kucharski, Jan; Tomkiel, Monika; Wyszkowska, Jadwiga


    The aim of this study was to determine the effect of three active substances, diflufenican, mesosulfuron-methyl and iodosulfuron-methyl-sodium, applied in combination, on soil microbial counts, the structure of soil microbial communities, activity of soil enzymes and their resistance to the tested product, the biochemical indicator of soil fertility, and spring wheat yield. Soil samples with the granulometric composition of sandy loam with pHKCl 7.0 were used in a pot experiment. The herbicide was applied to soil at seven doses: 0.057 (dose recommended by the manufacturer), 1.140, 2.280, 4.560, 9.120, 18.240 and 36.480 mg kg(-1) soil DM. Uncontaminated soil served as the control treatment. It was found that a mixture of the tested active substances increased the counts of total oligotrophic bacteria and spore-forming oligotrophic bacteria, organotrophic bacteria and actinomycetes, decreased the counts of Azotobacter and fungi, and modified the structure of soil microbial communities. The highest values of the colony development (CD) index and the ecophysiological (EP) index were observed in fungi and organotrophic bacteria, respectively. The herbicide applied in the recommended dose stimulated the activity of catalase, urease and acid phosphatase, but it had no effect on the activity of dehydrogenases, alkaline phosphatase, arylsulfatase and β-glucosidase. The highest dose of the analyzed substances (36.480 mg kg(-1)) significantly inhibited the activity of dehydrogenases, acid phosphatase, alkaline phosphatase and arylsulfatase. The values of the biochemical soil fertility indicator (BA21) decreased in response to high doses of the herbicide. Urease was most resistant and dehydrogenases were least resistant to soil contamination with a mixture of diflufenican + mesosulfuron-methyl + iodosulfuron-methyl-sodium. The analyzed herbicide had an adverse influence on spring wheat yield, and doses of 18.240 and 36.480 mg kg(-1) led to eventual death of plants.

  5. Enzymatic desulfurization of coal

    Energy Technology Data Exchange (ETDEWEB)

    Marquis, J.K. (Boston Univ., MA (United States). School of Medicine); Kitchell, J.P. (Holometrix, Inc., Cambridge, MA (United States))


    Our current efforts to develop clean coal technology emphasize the advantages of enzymatic desulfurization techniques and have specifically addressed the potential of using partially-purified extracellular microbial enzymes or commercially available enzymes. Our work is focused on the treatment of model'' organic sulfur compounds such as dibenzothiophene (DBT) and ethylphenylsulfide (EPS). Furthermore, we are designing experiments to facilitate the enzymatic process by means of a hydrated organic solvent matrix.

  6. Enzymatic activity of α-amylase in alimentary tract Spodoptera littoralis (Boisduval (Lepidoptera: Noctuidae: Characterization and Compartmentalization

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    Ali Darvishzadeh


    Full Text Available The Egyptian cotton leafworm, Spodoptera littoralis (Boisduval (Lepidoptera: Noctuidae damages a wide variety of crops in Middle East. Their hosts include cotton, alfalfa, eggplant, tomato, lettuce, bean and some ornamental crops. The intensive use of broad-spectrum insecticides against S. littoralis has led to the development of resistance to many registered pesticides use for its control. The purpose of the present study is biochemical characterization of digestive enzymes of this pest to gain a better understanding of the digestive physiology. The physiology and biochemistry of the insect digestive enzyme had an important role in the study of novel insecticidal strategies. The Egyptian cotton leafworm alimentary canal consists of a short foregut, a long midgut and a short hindgut. Application of pH indicators showed that alimentary canal was alkaline. Our results showed that activities of gut α-amylase were different in three parts of the insect gut. Also shown the greatest activity of α-amylase observed in the midgut followed by hindgut and foregut, respectively. However, there were not significant differences in activity of the enzyme in the midgut and hindgut. The optimal pH α-amylase in foregut, midgut and hindgut were 10.0. Zymogram analysis of different part of gut showed four bands in midgut, hind gut and two bands in foregut. Therefore, in midgut of S. littoralis, four isoenzymes were present. These results explain why more amylase activity was seen in these regions in the spectrophotometric assay.

  7. Influence of Tableting on Enzymatic Activity of Papain along with Determination of Its Percolation Threshold with Microcrystalline Cellulose. (United States)

    Sharma, Manu; Sharma, Vinay; Majumdar, Dipak K


    The binary mixture tablets of papain and microcrystalline cellulose (MCC), dicalcium phosphate dihydrate (DCP), carrageenan, tragacanth, and agar were prepared by direct compression. Carrageenan, tragacanth, and agar provided maximum protection to enzyme activity compared to MCC and DCP. However, stability studies indicated highest loss of enzyme activity with carrageenan, tragacanth, and agar. Therefore, compression behaviour of different binary mixtures of papain with MCC at different compaction pressures, that is, 40-280 MPa, was studied according to Heckel equation. The compressibility studies of binary mixtures indicated brittle behavior of papain. The application of percolation theory on the relationship between critical density as a function of enzyme activity and mixture composition revealed the presence of percolation threshold for binary mixture. Papain-MCC mixture composition showed significant percolation threshold at 18.48% (w/w) papain loading. Microcrystalline cellulose provided higher protection during stability study. However, higher concentrations of microcrystalline cellulose, probably as dominant particles, do not protect the enzyme with their plastic deformation. Below the percolation threshold, that is, 18.48% (w/w) papain amount in mixture with plastic excipient, activity loss increases strongly because of higher shearing forces during compaction due to system dominance of plastic particles. This mixture range should therefore be avoided to get robust formulation of papain.

  8. Characterization of an extracellular biofunctional alginate lyase from marine Microbulbifer sp. ALW1 and antioxidant activity of enzymatic hydrolysates. (United States)

    Zhu, Yanbing; Wu, Liyun; Chen, Yanhong; Ni, Hui; Xiao, Anfeng; Cai, Huinong


    A novel alginate-degrading marine bacterium Microbulbifer sp. ALW1 was isolated from rotten brown alga. An extracellular alginate lyase was purified to electrophoretic homogeneity and had a molecular mass of about 26.0 kDa determined by SDS-PAGE and size exclusion chromatography. This enzyme showed activities towards both polyguluronate and polymannuronate indicating its bifunctionality while with preference for the former substrate. Using sodium alginate as a substrate, strain ALW1 alginate lyase was optimally active at 45 °C and pH 7.0. It was stable at 25 °C, 30 °C, 35 °C and 40 °C, but not stable at 50 °C. This alginate lyase showed good stability over a broad pH range (5.0-9.0). The enzyme activity was increased to 5.1 times by adding NaCl to a final concentration of 0.5M. Strain ALW1 alginate lyase produced disaccharide (majority) and trisaccharide from alginate indicating that this enzyme could be a good tool for preparation of alginate oligosaccharides with low degree of polymerization (DP). The alginate oligosaccharides displayed the scavenging abilities towards radicals (DPPH, ABTS(+) and hydroxyl) and the reducing power. Therefore, the hydrolysates exhibited the antioxidant activity and had potential as a natural antioxidant.

  9. Magnetic Graphene Nanosheet-Based Microfluidic Device for Homogeneous Real-Time Electronic Monitoring of Pyrophosphatase Activity Using Enzymatic Hydrolysate-Induced Release of Copper Ion. (United States)

    Lin, Youxiu; Zhou, Qian; Li, Juan; Shu, Jian; Qiu, Zhenli; Lin, Yuping; Tang, Dianping


    A novel flow-through microfluidic device based on a magneto-controlled graphene sensing platform was designed for homogeneous electronic monitoring of pyrophosphatase (PPase) activity; enzymatic hydrolysate-induced release of inorganic copper ion (Cu(2+)) from the Cu(2+)-coordinated pyrophosphate ions (Cu(2+)-PPi) complex was assessed to determine enzyme activity. Magnetic graphene nanosheets (MGNS) functionalized with negatively charged Nafion were synthesized by using the wet-chemistry method. The Cu(2+)-PPi complexes were prepared on the basis of the coordination reaction between copper ion and inorganic pyrophosphate ions. Upon target PPase introduction into the detection system, the analyte initially hydrolyzed pyrophosphate ions into phosphate ions and released the electroactive copper ions from Cu(2+)-PPi complexes. The released copper ions could be readily captured through the negatively charged Nafion on the magnetic graphene nanosheets, which could be quantitatively monitored by using the stripping voltammetry on the flow-through detection cell with an external magnet. Under optimal conditions, the obtained electrochemical signal exhibited a high dependence on PPase activity within a dynamic range from 0.1 to 20 mU mL(-1) and allowed the detection at a concentration as low as 0.05 mU mL(-1). Coefficients of variation for reproducibility of the intra-assay and interassay were below 7.6 and 9.8%, respectively. The inhibition efficiency of sodium fluoride (NaF) also received good results in pyrophosphatase inhibitor screening research. In addition, the methodology afforded good specificity and selectivity, simplification, and low cost without the need of sample separations and multiple washing steps, thus representing a user-friendly protocol for practical utilization in a quantitative PPase activity.

  10. Cardiolipin hydrolysis by human phospholipases A2. The multiple enzymatic activities of human cytosolic phospholipase A2. (United States)

    Buckland, A G; Kinkaid, A R; Wilton, D C


    The ability of mammalian phospholipases A2 (PLA2) to hydrolyse cardiolipin (diphosphatidylglycerol) was monitored with a fluorescent displacement assay which allows the use of natural phospholipid substrates. The mammalian enzymes used were porcine pancreatic (Group I) secretory PLA2 (sPLA2), human non-pancreatic (Group II) sPLA2 and human cytosolic PLA2 (cPLA2). High activity was observed with porcine pancreas sPLA2 whereas the human sPLA2 demonstrated only minimal activity with this substrate. In comparison, sPLA2 from Naja naja venom (Group I) also showed only modest activity with this substrate. Since many lipases possess PLA1 activity, a representative enzyme from Rhizopus arrhizus was also assessed for its ability to hydrolyse cardiolipin which proved to be a good substrate for this fungal lipase. In all cases dilysocardiolipin was the major product while some monolyso intermediate was detected after chromatographic separation. Human cPLA2 was unable to hydrolyse cardiolipin at a significant rate, however, both monolysocardiolipin and dilysocardiolipin, which are prepared by the PLA2-catalysed hydrolysis of cardiolipin, were good substrates providing a further example of the extensive lysophospholipase activity of this enzyme. Moreover, cardiolipin that was initially hydrolysed in situ with either excess porcine pancreatic PLA2 or R. arrhizus lipase (PLA1) was subsequently hydrolysed by human cPLA2. One explanation of this result is that human cPLA2 is able to hydrolyse both 1-acyl and 2-acyl-lysophospholipids. (c) 1998 Elsevier Science B.V.

  11. Glutathione S-transferases in human renal cortex and neoplastic tissue: enzymatic activity, isoenzyme profile and immunohistochemical localization. (United States)

    Rodilla, V; Benzie, A A; Veitch, J M; Murray, G I; Rowe, J D; Hawksworth, G M


    1. Glutathione S-transferase (GST) activity in the cytosol of renal cortex and tumours from eight men and eight women was measured using 1-chloro-2,4-dinitrobenzene (CDNB) as a substrate. GST activities ranged from 685 to 2192 nmol/min/mg protein in cortex (median 1213) and from non-detectable (minimum 45) to 2424 nmol/min/mg protein in tumours (median 469). The activities in the tumours were lower than those in the normal cortices (p 0.05). 3. The age of the patients ranged from 42 to 81 years (median 62) and was not found to play a role in the levels of GST activity observed in cortex or in renal tumours from either sex. 4. Immunoblotting and immunohistochemical studies confirmed that GST-alpha was the predominant form expressed both in normal cortex and tumour and probably accounted for most of the GST activity present in these samples. GST-mu and GST-phi were expressed in both tumours and normal cortex and, while in some cases the level of expression in the cortices was higher than that found in the tumours, the reverse was also observed. Within the GST-mu class, GST M1/M2 was only detected in one sample (tumour), which showed the highest overall expression of GST-mu. GSTM3 was the predominant isoenzyme of the mu class in normal and tumour tissue, whereas GTM4 and GSTM5 were not detected. 5. These differences could have functional significance where xenobiotics or cytotoxic drugs are specific substrates for the different classes of GSTs.

  12. Diversity and extracellular enzymatic activities of yeasts isolated from King George Island, the sub-Antarctic region

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    Carrasco Mario


    Full Text Available Abstract Background Antarctica has been successfully colonized by microorganisms despite presenting adverse conditions for life such as low temperatures, high solar radiation, low nutrient availability and dryness. Although these “cold-loving” microorganisms are recognized as primarily responsible for nutrient and organic matter recycling/mineralization, the yeasts, in particular, remain poorly characterized and understood. The aim of this work was to study the yeast microbiota in soil and water samples collected on King George Island. Results A high number of yeast isolates was obtained from 34 soil and 14 water samples. Molecular analyses based on rDNA sequences revealed 22 yeast species belonging to 12 genera, with Mrakia and Cryptococcus genera containing the highest species diversity. The species Sporidiobolus salmonicolor was by far the most ubiquitous, being identified in 24 isolates from 13 different samples. Most of the yeasts were psychrotolerant and ranged widely in their ability to assimilate carbon sources (consuming from 1 to 27 of the 29 carbon sources tested. All species displayed at least 1 of the 8 extracellular enzyme activities tested. Lipase, amylase and esterase activity dominated, while chitinase and xylanase were less common. Two yeasts identified as Leuconeurospora sp. and Dioszegia fristingensis displayed 6 enzyme activities. Conclusions A high diversity of yeasts was isolated in this work including undescribed species and species not previously isolated from the Antarctic region, including Wickerhamomyces anomalus, which has not been isolated from cold regions in general. The diversity of extracellular enzyme activities, and hence the variety of compounds that the yeasts may degrade or transform, suggests an important nutrient recycling role of microorganisms in this region. These yeasts are of potential use in industrial applications requiring high enzyme activities at low temperatures.

  13. Enzymatic degradation of endomorphins. (United States)

    Janecka, Anna; Staniszewska, Renata; Gach, Katarzyna; Fichna, Jakub


    Centrally acting plant opiates, such as morphine, are the most frequently used analgesics for the relief of severe pain, even though their undesired side effects are serious limitation to their usefulness. The search for new therapeutics that could replace morphine has been mainly focused on the development of peptide analogs or peptidomimetics with high selectivity for one receptor type and high bioavailability, that is good blood-brain barrier permeability and enzymatic stability. Drugs, in order to be effective, must be able to reach the target tissue and to remain metabolically stable to produce the desired effects. The study of naturally occurring peptides provides a rational and powerful approach in the design of peptide therapeutics. Endogenous opioid peptides, endomorphin-1 and endomorphin-2, are two potent and highly selective mu-opioid receptor agonists, discovered only a decade ago, which display potent analgesic activity. However, extensive studies on the possible use of endomorphins as analgesics instead of morphine met with failure due to their instability. This review deals with the recent investigations that allowed determine degradation pathways of endomorphins in vitro and in vivo and propose modifications that will lead to more stable analogs.

  14. Enzymatic changes in phenylalanine ammonia-lyase, cinnamic-4-hydroxylase, capsaicin synthase, and peroxidase activities in capsicum under drought stress. (United States)

    Phimchan, Paongpetch; Chanthai, Saksit; Bosland, Paul W; Techawongstien, Suchila


    Penylalanine ammonia-lyase (PAL), cinnamic-4-hydroxylase (C4H), capsaicin synthase (CS), and peroxidase (POD) are involved in the capsaicinoid biosynthesis pathway and may be altered in cultivars with different pungency levels. This study clarified the action of these enzymes under drought stress for hot Capsicum cultivars with low, medium,and high pungency levels. At the flowering stage, control plants were watered at field capacity, whereas drought-induced plants were subjected to gradual drought stress. Under drought stress, PAL, C4H, CS, and POD enzyme activities increased as compared to the non-drought-stressed plants. A novel discovery was that PAL was the critical enzyme in capsaicinoid biosynthesis under drought stress because its activities and capsaicinoid increased across the different pungency levels of hot pepper cultivars examined.

  15. Direct Imaging by Cryo-TEM Shows Membrane Break-up by Phospholipase A2 Enzymatic Activity

    DEFF Research Database (Denmark)

    Callisen, Thomas Hønger; Talmon, Y.


    Phospholipid hydrolysis to free fatty acid and l-lyso-phospholipid by water-soluble phospholipase A(2) (PLA(2)) at the surface of lipid membranes exhibits a poorly understood transition from a low-activity lag phase to a burst regime of rapid hydrolysis. Understanding this kinetic phenomenon may...... hydrolysis of phospholipid vesicles with respect to changes in lipid composition and morphology. Our direct experimental results show that the initial reaction conditions are strongly perturbed during the course of hydrolysis, Most strikingly, cryo-TEM reveals that starting in the lag phase, vesicles become......-burst phenomenon reflects a cascade process. The PLA(2)-induced changes in lipid composition transform the morphology which in turn results in an acceleration of the rate of hydrolysis because of a strong coupling between the PLA(2) activity and the morphology of the lipid suspension....

  16. Ureases display biological effects independent of enzymatic activity: Is there a connection to diseases caused by urease-producing bacteria?

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    D. Olivera-Severo


    Full Text Available Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

  17. Ureases display biological effects independent of enzymatic activity: is there a connection to diseases caused by urease-producing bacteria? (United States)

    Olivera-Severo, D; Wassermann, G E; Carlini, C R


    Ureases are enzymes from plants, fungi and bacteria that catalyze the hydrolysis of urea to form ammonia and carbon dioxide. While fungal and plant ureases are homo-oligomers of 90-kDa subunits, bacterial ureases are multimers of two or three subunit complexes. We showed that some isoforms of jack bean urease, canatoxin and the classical urease, bind to glycoconjugates and induce platelet aggregation. Canatoxin also promotes release of histamine from mast cells, insulin from pancreatic cells and neurotransmitters from brain synaptosomes. In vivo it induces rat paw edema and neutrophil chemotaxis. These effects are independent of ureolytic activity and require activation of eicosanoid metabolism and calcium channels. Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach mucosa, causes gastric ulcers and cancer by a mechanism that is not understood. H. pylori produces factors that damage gastric epithelial cells, such as the vacuolating cytotoxin VacA, the cytotoxin-associated protein CagA, and a urease (up to 10% of bacterial protein) that neutralizes the acidic medium permitting its survival in the stomach. H. pylori whole cells or extracts of its water-soluble proteins promote inflammation, activate neutrophils and induce the release of cytokines. In this paper we review data from the literature suggesting that H. pylori urease displays many of the biological activities observed for jack bean ureases and show that bacterial ureases have a secretagogue effect modulated by eicosanoid metabolites through lipoxygenase pathways. These findings could be relevant to the elucidation of the role of urease in the pathogenesis of the gastrointestinal disease caused by H. pylori.

  18. Structural basis of enzymatic activity for the ferulic acid decarboxylase (FADase from Enterobacter sp. Px6-4.

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    Wen Gu

    Full Text Available Microbial ferulic acid decarboxylase (FADase catalyzes the transformation of ferulic acid to 4-hydroxy-3-methoxystyrene (4-vinylguaiacol via non-oxidative decarboxylation. Here we report the crystal structures of the Enterobacter sp. Px6-4 FADase and the enzyme in complex with substrate analogues. Our analyses revealed that FADase possessed a half-opened bottom β-barrel with the catalytic pocket located between the middle of the core β-barrel and the helical bottom. Its structure shared a high degree of similarity with members of the phenolic acid decarboxylase (PAD superfamily. Structural analysis revealed that FADase catalyzed reactions by an "open-closed" mechanism involving a pocket of 8 × 8 × 15 Å dimension on the surface of the enzyme. The active pocket could directly contact the solvent and allow the substrate to enter when induced by substrate analogues. Site-directed mutagenesis showed that the E134A mutation decreased the enzyme activity by more than 60%, and Y21A and Y27A mutations abolished the enzyme activity completely. The combined structural and mutagenesis results suggest that during decarboxylation of ferulic acid by FADase, Trp25 and Tyr27 are required for the entering and proper orientation of the substrate while Glu134 and Asn23 participate in proton transfer.

  19. The Enzymatic Activity and Molecular Characterization of a Secreted Subtilisin-Like Protease in Microsporum gypseum and Trichophyton vanbreuseghemii

    Directory of Open Access Journals (Sweden)

    H Moallaei


    Full Text Available "nBackground: Subtilisin -like proteases are the group of proteases including keratinases found in dermatophytes which de­graded keratin. Determination of the proteases activity of Trichophyton vanbreuseghemii isolates which were obtained from soil and clinical and soil isolates of Microsporum gypseum in Iran and characterization of their genome were aim of present study."nMethods: Ezymatic activity was determined by use of chromogenic substrates. The genes, which coded subtilisin-like pro­teases in above-mentioned dermatophytes, was identified and amplified by using specific primers in PCR."nResults:  The highest yield of enzyme production was observed in only one isolate of T. vanbreuseghemii Ir-84 whereas low enzyme activity was observed in M. gypseum isolates. Homology study of obtained nucleotide as well as amino acid sequences indicated different rates of homology with other subtilisin-like proteases genes in other pathogenic dermato­phytes."nConclusion: Intra-strain differences were observed in production of serine proteinases and molecular characterization of genes encoding such enzymes could be of great interest for studies on pathogenicity and other purposes.  

  20. Antioxidant activity of a novel synthetic hexa-peptide derived from an enzymatic hydrolysate of duck skin by-products. (United States)

    Lee, Seung-Jae; Cheong, Sun Hee; Kim, Yon-Suk; Hwang, Jin-Woo; Kwon, Hyuck-Ju; Kang, Seo-Hee; Moon, Sang-Ho; Jeon, Byong-Tae; Park, Pyo-Jam


    A peptide was synthesized on the basis of our previous study from solid phase peptide synthesis using ASP48S (Peptron Inc.) and identified by the reverse phase high-performance liquid chromatography (HPLC) using a Vydac Everest C18 column. The molecular mass of the peptide found to be 693.90 Da, and the amino acid sequences of the peptide was Trp-Tyr-Pro-Ala-Ala-Pro. The purpose of this study was to evaluate antioxidant effects of the peptide by electron spin resonance (ESR) spectrometer, and on t-BHP-induced liver cells damage in Chang cells. The antioxidative activity of the peptide was evaluated by measuring 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, alkyl and superoxide radical scavenging activity using an ESR spectrometer. The half maximal inhibitory concentration (IC50) value of the peptide for hydroxyl, DPPH, alkyl, and superoxide radical scavenging activity were 45.2, 18.5, 31.5, and 33.4 μM, respectively. In addition, the peptide inhibited productions of cell death against t-BHP-induced liver cell damage in Chang cells. It was presumed to be peptide involved in regulating the apoptosis-related gene expression in the cell environment. The present results indicate that the peptide substantially contributes to antioxidative properties in liver cells.

  1. Eggplant lipoxygenase (Solanum melongena): product characterization and effect of physicochemical properties of linoleic acid on the enzymatic activity. (United States)

    López-Nicolás, J M; Pérez-Gilabert, M; García-Carmona, F


    Lipoxygenase (LOX) from eggplant (Solanum melongena L. cv. Belleza negra) was partially purified, and the products and kinetics of the enzyme were studied. Linoleic acid (LA) was the best substrate for this enzyme. Product analysis by HPLC and GC/MS revealed that, at its pH optimum (pH 7.0), the enzyme converted LA almost totally into the 9-hydroperoxy isomer, whereas the 13-hydroperoxy isomer was only a minor product. At this pH, the enzyme had K(m) and V(max) values for LA of 1.4 microM and 2.2 micromol min(-1) (mg of protein)(-1), respectively, when the monomeric form of LA was used as substrate. The dependence of eggplant LOX activity on the physicochemical properties of LA was also studied. Experiments revealed that LA aggregates were used more efficiently than monomeric LA as substrate. The apparent substrate cooperativity observed may be due to the different activities exhibited toward monomers and aggregates. This result can be interpreted as a substrate-aggregation dependent activity.

  2. Microwave-assisted aqueous enzymatic extraction of oil from pumpkin seeds and evaluation of its physicochemical properties, fatty acid compositions and antioxidant activities. (United States)

    Jiao, Jiao; Li, Zhu-Gang; Gai, Qing-Yan; Li, Xiao-Juan; Wei, Fu-Yao; Fu, Yu-Jie; Ma, Wei


    Microwave-assisted aqueous enzymatic extraction (MAAEE) of pumpkin seed oil was performed in this study. An enzyme cocktail comprised of cellulase, pectinase and proteinase (w/w/w) was found to be the most effective in releasing oils. The highest oil recovery of 64.17% was achieved under optimal conditions of enzyme concentration (1.4%, w/w), temperature (44°C), time (66 min) and irradiation power (419W). Moreover, there were no significant variations in physicochemical properties of MAAEE-extracted oil (MAAEEO) and Soxhlet-extracted oil (SEO), but MAAEEO exhibited better oxidation stability. Additionally, MAAEEO had a higher content of linoleic acid (57.33%) than SEO (53.72%), and it showed stronger antioxidant activities with the IC50 values 123.93 and 152.84, mg/mL, according to DPPH radical scavenging assay and β-carotene/linoleic acid bleaching test. SEM results illustrated the destruction of cell walls and membranes by MAAEE. MAAEE is, therefore, a promising and environmental-friendly technique for oil extraction in the food industry.

  3. Degradation of di(2-ethyl hexyl) phthalate by Fusarium culmorum: Kinetics, enzymatic activities and biodegradation pathway based on quantum chemical modelingpathway based on quantum chemical modeling

    Energy Technology Data Exchange (ETDEWEB)

    Ahuactzin-Pérez, Miriam [Doctorado en Biología Experimental, Universidad Autónoma Metropolitana-Iztapalapa (UAM-I) (Mexico); Facultad de Agrobiología, Universidad Autónoma de Tlaxcala, Ixtacuixtla, Tlaxcala (Mexico); Tlecuitl-Beristain, Saúl; García-Dávila, Jorge [Universidad Politécnica de Tlaxcala, San Pedro Xalcatzinco, Tepeyanco, Tlaxcala CP 90180 (Mexico); González-Pérez, Manuel [Universidad Popular Autónoma del Estado de Puebla, Puebla CP 72410 (Mexico); Gutiérrez-Ruíz, María Concepción [Departamento de Ciencias de la Salud, Universidad Autónoma Metropolitana-Iztapalapa, D.F (Mexico); Sánchez, Carmen, E-mail: [Laboratory of Biotechnology, Research Centre for Biological Sciences, Universidad Autónoma de Tlaxcala, Ixtacuixtla, Tlaxcala CP. 90062 (Mexico)


    Di(2-ethylhexyl) phthalate (DEHP) is a plasticizer widely used in the manufacture of plastics, and it is an environmental contaminant. The specific growth rate (μ), maximum biomass (X{sub max}), biodegradation constant of DEHP (k), half-life (t{sub 1/2}) of DEHP biodegradation and removal efficiency of DEHP, esterase and laccase specific activities, and enzymatic yield parameters were evaluated for Fusarium culmorum grown on media containing glucose and different concentrations of DEHP (0, 500 and 1000 mg/L). The greatest μ and the largest X{sub max} occurred in media supplemented with 1000 mg of DEHP/L. F. culmorum degraded 95% of the highest amount of DEHP tested (1000 mg/L) within 60 h of growth. The k and t{sub 1/2} were 0.024 h{sup −1} and 28 h, respectively, for both DEHP concentrations. The removal efficiency of DEHP was 99.8% and 99.9% for 1000 and 500 mg/L, respectively. Much higher specific esterase activity than specific laccase activity was observed in all media tested. The compounds of biodegradation of DEHP were identified by GC–MS. A DEHP biodegradation pathway by F. culmorum was proposed on the basis of the intermolecular flow of electrons of the identified intermediate compounds using quantum chemical modeling. DEHP was fully metabolized by F. culmorum with butanediol as the final product. This fungus offers great potential in bioremediation of environments polluted with DEHP. - Highlights: • F. culmorum degraded 95% of DEHP (1000 mg/L) within 60 h. • Removal efficiency of DEHP was 99.8% and 99.9% for 1000 and 500 mg/L, respectively. • DEHP was fully metabolized by F. culmorum, with butanediol as the final product. • A DEHP biodegradation pathway was proposed using on quantum chemical modeling.

  4. Role of enzymatic activity in muscle damage and cytotoxicity induced by Bothrops asper Asp49 phospholipase A2 myotoxins: are there additional effector mechanisms involved?

    Directory of Open Access Journals (Sweden)

    Diana Mora-Obando


    Full Text Available Viperid venoms often contain mixtures of Asp49 and Lys49 PLA2 myotoxin isoforms, relevant to development of myonecrosis. Given their difference in catalytic activity, mechanistic studies on each type require highly purified samples. Studies on Asp49 PLA2s have shown that enzyme inactivation using p-bromophenacyl bromide (p-BPB drastically affects toxicity. However, based on the variable levels of residual toxicity observed in some studies, it has been suggested that effector mechanisms independent of catalysis may additionally be involved in the toxicity of these enzymes, possibly resembling those of the enzymatically inactive Lys49 myotoxins. A possibility that Lys49 isoforms could be present in Asp49 PLA2 preparations exists and, if undetected in previous studies, could explain the variable residual toxicity. This question is here addressed by using an enzyme preparation ascertained to be free of Lys49 myotoxins. In agreement with previous reports, inactivation of the catalytic activity of an Asp49 myotoxin preparation led to major inhibition of toxic effects in vitro and in vivo. The very low residual levels of myotoxicity (7% and cytotoxicity (4% observed can be attributed to the low, although detectable, enzyme remaining active after p-BPB treatment (2.7%, and would be difficult to reconcile with the proposed existence of additional catalytic-independent toxic mechanisms. These findings favor the concept that the effector mechanism of toxicity of Asp49 PLA2 myotoxins from viperids fundamentally relies on their ability to hydrolyze phospholipids, arguing against the proposal that membrane disruption may also be caused by additional mechanisms that are independent of catalysis.

  5. Evaluation of halofenozide against prey mosquito larvae Culex pipiens and the predator fish Gambusia affinis: impact on growth and enzymatic activities. (United States)

    Soltani, N; Chouahda, S; Smagghe, G


    Culex pipiens (Diptera: Culicidae) is the most widely distributed mosquito species in Algeria and many other countries in the world. Mosquitoes are generally controlled by conventional insecticides but these may pose strong secondary effects on the environment. In this context, the insect growth regulators (IGRs) have shown promise in controlling pest insects. Halofenozide (23% EC) is a novel IGRs belonging to the class of non-steroidal ecdysone agonists, and it was found toxic for larvae of C. pipiens. In addition biological methods constitute an alternative to chemical control. Several fish species have been tested against mosquitoes, and Gambusia affinis was found very efficient. In the present study we evaluated the impact of this new potent insecticide (halofenozide) on growth and metric indexes in the larvivorous fish G. affinis under laboratory conditions. In addition, the effects were evaluated on the enzymatic activities of acetyl cholinesterase (AChE) and glutathione S-transferase (GST). The insecticide was added in water at two concentrations (12.6 and 28.6 microg/L) corresponding to the LC50 and LC90 obtained against fourth instar larvae of C. pipiens, and adult females of G. affinis were exposed to halofenozide for 30 days. At different exposure times we measured the length and weight of fishes, the index of condition (K), the gonado-somatic ratio (GSR) and the hepato-somatic ratio (HSR). The results showed that halofenozide had no significant (p>0.05) effects on growth, metric indexes and AChE activities. However, treatment caused a significant induction (p<0.05) in GST activities at days 15 and 30 with the highest dose. Our results indicate that this ecdysteroid agonist presented only minor secondary effects on the non-target fish species, and so it has potential for controlling of mosquitoes in an integrated manner.

  6. Subcellular cadmium distribution and antioxidant enzymatic activities in the leaves of two castor (Ricinus communis L.) cultivars exhibit differences in Cd accumulation. (United States)

    Zhang, Hanzhi; Guo, Qingjun; Yang, Junxing; Shen, Jianxiu; Chen, Tongbin; Zhu, Guangxu; Chen, Hui; Shao, Chunyan


    The aims of this study were: (1) the study of cadmium (Cd) accumulation and toxicity in different castor cultivars (Ricinus communis L.); (2) to investigate changes in antioxidant enzymatic activities and the subcellular distribution of Cd in young and old leaves from two different castor cultivars, after exposure to two different Cd concentrations, and explore the underlying mechanism of Cd detoxification focusing on antioxidant enzymes and subcellular compartmentalization. The Cd concentration, toxicity, and subcellular distribution, as well as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities were measured in Zibo-3 and Zibo-9 cultivars after exposure to two different concentrations of Cd (2mg/L and 5mg/L) for 10 days. This research revealed Cd accumulation characteristics in castor are root>stem>young leaf>old leaf. Castor tolerance was Cd dose exposure and the cultivars themselves dependent. Investigation of subcellular Cd partitioning showed that Cd accumulated mainly in the heat stable protein (HSP) and cellular debris fractions, followed by the Cd rich granule (MRG), heat denatured protein (HDP), and organelle fractions. With increasing Cd concentration in nutrient solution, the decreased detoxified fractions (BDM) and the increased Cd-sensitive fractions (MSF) in young leaves may indicate the increased Cd toxicity in castor cultivars. The BDM-Cd fractions or MSF-Cd in old leaves may be linked with Cd tolerance of different cultivars of castor. The antioxidant enzymes that govern Cd detoxification were not found to be active in leaves. Taken together, these results indicate Cd tolerance and toxicity in castor can be explained by subcellular partitioning.

  7. Enzymatic removal of estrogenic activity of nonylphenol and octylphenol aqueous solutions by immobilized laccase from Trametes versicolor

    Energy Technology Data Exchange (ETDEWEB)

    Catapane, Maria [Institute of Genetics and Biophysics “ABT”, Via P. Castellino, 111, 80131 Naples (Italy); National Institute of Biostructures and Biosystems (INBB), Viale Medaglie d’Oro, 305, 00136 Rome (Italy); Nicolucci, Carla; Menale, Ciro; Mita, Luigi [National Institute of Biostructures and Biosystems (INBB), Viale Medaglie d’Oro, 305, 00136 Rome (Italy); Department of Experimental Medicine, Second University of Naples, Via S. M. di Costantinopoli, 16, 80138 Naples (Italy); Rossi, Sergio [Institute of Genetics and Biophysics “ABT”, Via P. Castellino, 111, 80131 Naples (Italy); Mita, Damiano G., E-mail: [Institute of Genetics and Biophysics “ABT”, Via P. Castellino, 111, 80131 Naples (Italy); National Institute of Biostructures and Biosystems (INBB), Viale Medaglie d’Oro, 305, 00136 Rome (Italy); Department of Experimental Medicine, Second University of Naples, Via S. M. di Costantinopoli, 16, 80138 Naples (Italy); Diano, Nadia [Institute of Genetics and Biophysics “ABT”, Via P. Castellino, 111, 80131 Naples (Italy); National Institute of Biostructures and Biosystems (INBB), Viale Medaglie d’Oro, 305, 00136 Rome (Italy); Department of Experimental Medicine, Second University of Naples, Via S. M. di Costantinopoli, 16, 80138 Naples (Italy)


    Highlights: ► Endocrine disruptors cause adverse effects in living organisms. ► Nonylphenol and Octylphenol are alkylphenols recognized as endocrine disruptors. ► It is necessary to remove or reduce their presence in the environment. ► Waters polluted by these pollutants have been bioremediated by immobilized laccase from Trametes versicolor. ► Laccase treated solutions were found to have lost any estrogenic activity. -- Abstract: A fluidized bed reactor, filled with laccase-based beads, has been employed to bioremediate aqueous solutions polluted by endocrine disruptors belonging to the alkylphenols (APs) class. In particular Octylphenol and Nonylphenol have been studied. The catalytic activity of free and immobilized laccase from Trametes versicolor has been characterized as a function of pH, temperature and substrate concentration in the reaction medium. In view of practical applications for each substrate concentration the removal efficiency (RE), the time to halve the initial concentration (τ{sub 50}), and the t{sub c=0}, i.e. the time to reach complete pollutant removal, have been calculated. The immobilized laccase exhibited a lower affinity for octylphenol (K{sub m} = 1.11 mM) than for Nonylphenol (K{sub m} = 0.72 mM), but all the other parameters of applicative interest resulted more significant for octylphenol. For example, the times to reach the complete removal of octylphenol compared to those for nonylphenol at the same concentration is shorter of about 15% (at low concentrations) up to 40% (at high concentrations). The study of cell proliferation with MPP89 cells, a human mesothelioma cell line, and the assay with the YES test indicated the loss of estrogenic activity of the APs solutions after laccase treatment.

  8. The C. elegans H3K27 Demethylase UTX-1 Is Essential for Normal Development, Independent of Its Enzymatic Activity

    DEFF Research Database (Denmark)

    Vandamme, Julien; Lettier, Gaëlle; Sidoli, Simone;


    specific for H3K27me2/3. We demonstrate that utx-1 is an essential gene that is required for correct embryonic and postembryonic development. Consistent with its homology to UTX, UTX-1 regulates global levels of H3K27me2/3 in C. elegans. Surprisingly, we found that the catalytic activity is not required......Epigenetic modifications influence gene expression and provide a unique mechanism for fine-tuning cellular differentiation and development in multicellular organisms. Here we report on the biological functions of UTX-1, the Caenorhabditis elegans homologue of mammalian UTX, a histone demethylase...

  9. Omission and Resupply of Nitrogen Affect Physiological and Enzymatic Activities and the Gene Expression of Eucalypt Clones

    Directory of Open Access Journals (Sweden)

    Loane Vaz Fernandes

    Full Text Available ABSTRACT: The mineral nutrient uptake of plants in the field occurs in pulses, due to variations in the substance concentrations at the root surface. The fluctuations in nutrient supply probably induce changes in the plant, which are to date unknown for Eucalyptus. This study evaluated these changes in plant growth, nutritional status, photosynthesis, and gene expression, which can serve as biomarkers of the nitrogen status, of four eucalypt clones exposed to N omission and resupply. A greenhouse experiment with four Eucalyptus clones was installed, and after initial growth exposed to N omission for 21 d, followed by N resupply in nutrient solution for 14 d. Nitrogen omission decreased the total N and photosynthetic pigments, net photosynthesis and photochemical dissipation, and increased enzyme activity especially in leaves and the gene expression in leaves and roots. Nitrogen resupply decreased these variations, indicating recovery. The total N concentration was highly and significantly correlated with net photosynthesis, enzyme activity, expression of genes GS2;1 and Gln1;3 in the leaves and AMT1;2 in the roots, contents of chlorophyll a and b, and photochemical energy dissipation. The enzymes GS and NR in the leaves and the genes AMT1;2, GS2;1 and Gln1;3 proved to be sensitive N indicators.

  10. Study of enzymatic activity and proline change in potted mini carnation (Dianthus caryophyllus L. flowers under ethylene stress

    Directory of Open Access Journals (Sweden)

    M. Karimi


    Full Text Available This study was performed to evaluate the role of ethylene stress, ethylene inhibitors and action treatments on longevity of potted-carnation flowers. For this purpose, first, potted-carnation flowers were pretreated with different concentrations of amino-oxyacetic acid (AOA, benzyladenine (BA and 1-methylcyclopropene (1-MCP. Then, ethephon (as stress treatment was sprayed on the pretreated flowers. Results showed that the lowest longevity of carnation flowers (5 and 6 days, respectively in both studied cultivars (‘Silver pink’ and ‘Lilac on purple’ was related to 30 mg/L ethephon. Maximum flower longevity (11.5 days was related to ‘Lilac on purple’ cultivar. The 0.6 µL/L of 1-MCP treatment, in both cultivars, was the most effective treatment in inhibition of ethephon on reduction of flowers longevity. Also, the lowest amount of ethylene production and the highest enzyme activity was observed when the flowers were pretreated by 0.6 µL/L of 1-MCP before the stress is applied. The highest proline accumulation was related to 30 mg/L ethephon treatment. Maximum α-amylase activity occurred in 30 mg/L ethephon, 10 and 20 mg/L BA and 50 mg/L AOA pretreatments.

  11. Optimization of Ultrasonic-Assisted Enzymatic Extraction Conditions for Improving Total Phenolic Content, Antioxidant and Antitumor Activities In Vitro from Trapa quadrispinosa Roxb. Residues. (United States)

    Li, Feng; Mao, Yi-Dan; Wang, Yi-Fan; Raza, Aun; Qiu, Li-Peng; Xu, Xiu-Quan


    Stems are the important residues of Trapa quadrispinosa Roxb., which are abundant in phenolic compounds. Ultrasonic-assisted enzymatic extraction (UAEE) is confirmed as a novel extraction technology with main advantages of enhancing extraction yield and physiological activities of the extracts from various plants. In this study, UAEE was applied to obtain the highest yield of phenolic content, strongest antioxidant, and antitumor activities and to optimize the extraction conditions using response surface methodology (RSM). The extracts from the stems of T. quadrispinosa were characterized by determination of their antioxidant activities through 2,2-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS), 1,1-Diphenyl-2-picrylhydrazxyl (DPPH) radical scavenging, total antioxidant capacity (TAC), ferric reducing antioxidant capacity (FRAC) methods and of their antitumor activity by MTT method. The selected key independent variables were cellulase concentration (X₁: 1.5%-2.5%), extraction time (X₂: 20-30 min) and extraction temperature (X₃: 40-60 °C). The optimal extraction conditions for total phenolic content (TPC) value of the extracts were determined as 1.74% cellulase concentration, 25.5 min ultrasonic extraction time and 49.0 °C ultrasonic temperature. Under these conditions, the highest TPC value of 53.6 ± 2.2 mg Gallic acid equivalent (GAE)/g dry weight (DW) was obtained, which agreed well with the predicted value (52.596 mg GAE/g·DW. Furthermore, the extracts obtained from UAEE presented highest antioxidant activities through ABTS, DPPH, TAC and FRAC methods were of 1.54 ± 0.09 mmol Trolox equivalent (TE)/g·DW; 1.45 ± 0.07 mmol·TE/g·DW; 45.2 ± 2.2 mg·GAE/g·DW; 50.4 ± 2.6 μmol FeSO₄ equivalent/g·DW and lowest IC50 values of 160.4 ± 11.6 μg/mL, 126.1 ± 10.8 μg/mL, and 178.3 ± 13.1 μg/mL against Hela, HepG-2 and U251 tumor cells, respectively. The results indicated that the UAEE was an efficient alternative to improve extraction yield

  12. Metabolism of Antarctic micronektonic crustacea across a summer ice-edge bloom: respiration, composition, and enzymatic activity (United States)

    Donnelly, Joseph; Kawall, Helena; Geiger, Stephen P.; Torres, Joseph J.


    The Antarctic marginal ice zone is an important oceanic front separating the pack-ice and open-water environments. During summer, the retreating pack ice creates a meltwater lens in the euphotic zone, allowing primary producers and microheterotrophs to flourish in a discrete bloom just seaward of the retreating ice edge that lasts about 60 days. The purpose of the present study was to see if the ice-edge bloom had a discernible effect on the metabolism and physiological condition of Antarctic micronekton similar to that observed in zooplankton species. We also wished to assess the importance of the summer season to species' life cycles. Two major data sets were collected on 25 species in the following taxonomic groups: amphipods, cephalopods, decapods, euphausiids, isopods, mysids, ostracods, and polychaetes. The first data set described the metabolic rates of individuals in areas of the marginal ice zone with widely different levels of chlorophyll biomass to investigate the effect of the ice-edge bloom on metabolism. Additionally, summer metabolic rates were compared with data from other seasons. The second data set detailed the levels of protein, water, ash, RNA and DNA, and the activities of metabolic enzymes (citrate synthase and malate dehydrogenase) to examine the efficacy of biochemical indices as predictive tools for metabolism. Results suggested that the mobility of the micronektonic species eliminated most direct effects of the bloom on metabolism. Individuals captured in very different productivity regimes showed few significant differences in the metabolic indicators listed above. Isolated cases of changes in body composition and enzyme activity, however, implied that longer-term effects of the bloom may be exhibited. Seasonal increases in metabolism from winter to summer were observed in the euphausiids Euphausia superba, E. triacantha, and Thysanoessa macrura and the amphipod Vibilia stebbingi. It was concluded that the seasonal shifts were indicative

  13. Isotopic and enzymatic analyses of planktonic nitrogen utilisation in the vicinity of Cape Sines (Portugal) during weak upwelling activity (United States)

    Slawyk, Gerd; Coste, Bernard; Collos, Yves; Rodier, Martine


    Using measurements of 15N uptake and activities of nitrate reductase and glutamine synthetase, the utilization of nitrogenous nutrients by microplankton in the Portuguese upwelling area was investigated. During this cruise the euphotic zone of coastal waters was in most cases bisected by a nitracline forming two layers. Total inorganic nitrogen uptake rates (NH 4+ + NO 3-) in the upper mixed and nitrate-impoverished layer ranged from 0.1 to 0.8 nM h -1 and were primarily supported by regenerated (ammonium) nitrogen (62-97%), whereas they varied between 0.9 and 10.4 nM h -1 in the deep nitrate-rich layer and were mainly driven by new (nitrate) nitrogen (52-82%). Depth profiles of Chl a-specific uptake rates for ammonium and nitrate paralleled those of absolute uptake rates, i.e. values of VNH 4+Chl were highest (up to 16.1 nmol μg -1 h -1) in nitrate-poor surface waters while values of VNO 3-Chl were maximum (up to 8.4 nmol μg -1 h -1)within the nitracline. This latter vertical ordering of planktonic nitrogen nutrition was consistent with an aged upwelling situation. However, applying several indices of cell metabolism and nutritional status, such as 15N uptake/enzyme activity, surge uptake internally controlled uptake, and V maxChl/K t ratios, we were able to demonstrate that the phytoplankton assemblages inhabiting the nutrient-impoverished upper layer still bore the signature of physically mediated nitrogen (nitrate) supply generated by active upwelling that had occurred during the week before our visit to the area. This signature was the most evident in samples from the station furthest inshore and faded with distance from shore as a result of the deepening of the nitrate isopleths (weakening of upwelling activity), which showed the same offshore trend. The appearance of nitrate-rich waters at the surface, after a strong pulse of upwelling favourable winds just before the end of the cruise, led to a five-fold increase in average (over the euphotic zone

  14. Effect of Eleutherine americana Merr. extract on enzymatic activity and enterotoxin production of Staphylococcus aureus in broth and cooked pork. (United States)

    Ifesan, Beatrice O T; Voravuthikunchai, Supayang P


    Crude ethanolic extract from the bulb of Eleutherine americana was investigated for its inhibitory activities against lipase and protease enzymes and enterotoxin production by Staphylococcus aureus. Eleven isolates that demonstrated high enzyme activity with three reference strains were selected to study the effect of extract on enzyme production. Exposure of the isolates to subminimal inhibitory concentrations, (1/2) minimum inhibitory concentration (MIC) (125 microg/mL), and (1/4)MIC (62.5 microg/mL) of the crude extract resulted in both partial and total inhibition of lipase and protease enzymes. About 15% of the 106 isolates were positive for enterotoxin production with staphylococcal enterotoxin A (11.3%), enterotoxin B (3.7%), and enterotoxin C (10.3%), and no enterotoxin D was produced. The production of staphylococcal enterotoxins A-D in the presence or absence of the crude extract was carried out. In the broth system, the extract reduced enterotoxin production at subminimal inhibitory concentrations compared with the control. At MIC, total enterotoxin inhibition was observed for enterotoxin C production, whereas synthesis of enterotoxins A, B, and D was totally eliminated at 2MIC. The food system study revealed that the extract could delay production of enterotoxins A, B, and C compared with the control. The extract at 2 mg/mL delayed production of toxins A and C for 8 and 4 h, while toxin B was not detected in the pork at 48 h. The ability of E. americana extract to inhibit lipase and protease enzymes and to delay enterotoxin production in food could present it as a novel food additive to combat the growth of S. aureus in food.

  15. High enzymatic activity preservation with carbon nanotubes incorporated in urease-lipid hybrid Langmuir-Blodgett films. (United States)

    Caseli, Luciano; Siqueira, José Roberto


    The search for optimized architectures, such as thin films, for the production of biosensors has been challenged in recent decades, and thus, the understanding of molecular interactions that occur at interfaces is essential to improve the construction of nanostructured devices. In this study, we investigated the possibility of using carbon nanotubes in hybrid Langmuir-Blodgett (LB) films of lipids and urease to improve the catalytic performance of the immobilized enzyme. The molecular interactions were first investigated at the air-water interface with the enzyme adsorbed from the aqueous subphase onto Langmuir monolayers of dimyristoylphosphatidic acid (DMPA). The transfer to solid supports as LB films and the subsequent incorporation of carbon nanotubes in the hybrid film permitted us to evaluate how these nanomaterials changed the physical properties of the ultrathin film. Colorimetric measurments indicated that the presence of nanotubes preserved and enhanced the enzyme activity of the film, even after 1 month. These results show that the use of such hybrid films is promising for the development of biosensors with an optimized performance.

  16. Flavonoid Interaction with a Chitinase from Grape Berry Skin: Protein Identification and Modulation of the Enzymatic Activity. (United States)

    Filippi, Antonio; Petrussa, Elisa; Rajcevic, Uros; Čurin Šerbec, Vladka; Passamonti, Sabina; Renzone, Giovanni; Scaloni, Andrea; Zancani, Marco; Vianello, Angelo; Braidot, Enrico


    In the present study, an antibody raised against a peptide sequence of rat bilitranslocase (anti-peptide Ab) was tested on microsomal proteins obtained from red grape berry skin. Previously, this antibody had demonstrated to recognize plant membrane proteins associated with flavonoid binding and transport. Immuno-proteomic assays identified a number of proteins reacting with this particular antibody, suggesting that the flavonoid binding and interaction may be extended not only to carriers of these molecules, but also to enzymes with very different functions. One of these proteins is a pathogenesis-related (PR) class IV chitinase, whose in vitro chitinolytic activity was modulated by two of the most representative flavonoids of grape, quercetin and catechin, as assessed by both spectrophotometric and fluorimetric assays in grape microsomes and commercial enzyme preparations. The effect of these flavonoids on the catalysis and its kinetic parameters was also evaluated, evidencing that they determine a hormetic dose-dependent response. These results highlight the importance of flavonoids not only as antioxidants or antimicrobial effectors, but also as modulators of plant growth and stress response. Implications of the present suggestion are here discussed in the light of environment and pesticide-reduction concerns.

  17. CuO nanoparticles incorporated in hierarchical MFI zeolite as highly active electrocatalyst for non-enzymatic glucose sensing. (United States)

    Dong, Junping; Tian, Taolei; Ren, Linxiao; Zhang, Yuan; Xu, Jiaqiang; Cheng, Xiaowei


    A hierarchical MFI zeolite, with typical micro/meso bimodal pore structures, was prepared by desilication method. CuO nanoparticles (NPs) were incorporated into the hierarchical MFI zeolite by impregnation method. CuO/hierarchical zeolite composites were characterized by X-ray diffraction, transmission electron microscopy and nitrogen sorption. It is shown that the CuO nanoparticles are mostly dispersed in the mesopores with remaining of the crystallinity and morphology of the host zeolite. CuO nanoparticles located in hierarchical zeolite exhibit the excellent electrocatalytic performances to oxidation of glucose in alkaline media. The electrocatalytic activity enhances with increasing the loading content of CuO from 5% to 15%. The composites were fabricated for nonenzyme glucose sensing. Under the optimal conditions, the sensor shows a wide linear range from 5×10(-7) to 1.84×10(-2) M with a low detection limit of 3.7×10(-7) M. The sensor also exhibits good repeatability, long-term stability as well as high selectivity against interfering species.

  18. Optimisation of enzymatic production of sulforaphane in broccoli sprouts and their total antioxidant activity at different growth and storage days. (United States)

    Tian, Ming; Xu, Xiaoyun; Hu, Hao; Liu, Yu; Pan, Siyi


    Sulforaphane, a type of isothiocyanate hydrolysed from glucosinolate, is a powerful anticancer compound naturally found in food especially in broccoli sprouts. Despite the function of sulforaphane has been extensively studied in recent years, little attention has been given to methods that can maximize the production of this compound in broccoli sprouts. The present study optimised the enzymolysis conditions for sulforaphane production in broccoli sprouts using response surface methodology. The maximum sulforaphane production (246.95 μg/g DW) was achieved using a solid-liquid ratio of 1:30, hydrolysis time of 1.5 h, ascorbic acid content of 3.95 mg/g DW sample, and temperature of 65 °C. The highest sulforaphane content in broccoli sprouts were 233.80 μg/g DW in 5-day-old sprouts and 1555.95 μg/g DW at day 4 of storage. The highest antioxidant activities were 37.22 U/min/g DW in 3-day-old sprouts and 35.08 U/min/g DW on 4th day of storage.

  19. Flavonoid Interaction with a Chitinase from Grape Berry Skin: Protein Identification and Modulation of the Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Antonio Filippi


    Full Text Available In the present study, an antibody raised against a peptide sequence of rat bilitranslocase (anti-peptide Ab was tested on microsomal proteins obtained from red grape berry skin. Previously, this antibody had demonstrated to recognize plant membrane proteins associated with flavonoid binding and transport. Immuno-proteomic assays identified a number of proteins reacting with this particular antibody, suggesting that the flavonoid binding and interaction may be extended not only to carriers of these molecules, but also to enzymes with very different functions. One of these proteins is a pathogenesis-related (PR class IV chitinase, whose in vitro chitinolytic activity was modulated by two of the most representative flavonoids of grape, quercetin and catechin, as assessed by both spectrophotometric and fluorimetric assays in grape microsomes and commercial enzyme preparations. The effect of these flavonoids on the catalysis and its kinetic parameters was also evaluated, evidencing that they determine a hormetic dose-dependent response. These results highlight the importance of flavonoids not only as antioxidants or antimicrobial effectors, but also as modulators of plant growth and stress response. Implications of the present suggestion are here discussed in the light of environment and pesticide-reduction concerns.

  20. Genome-Wide Identification, 3D Modeling, Expression and Enzymatic Activity Analysis of Cell Wall Invertase Gene Family from Cassava (Manihot esculenta Crantz

    Directory of Open Access Journals (Sweden)

    Yuan Yao


    Full Text Available The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6 were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a β-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(VD, in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a β-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6, and Val residue from the WECVD (MeCWINV3 and 4 are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker.

  1. Effect of permethrin, anthracene and mixture exposure on shell components, enzymatic activities and proteins status in the Mediterranean clam Venerupis decussata

    Energy Technology Data Exchange (ETDEWEB)

    Sellami, Badreddine, E-mail: [Laboratory of Environment Biomonitoring, Coastal Ecology Unit, Faculty of Sciences of Bizerta, University of Carthage, 7021 Zarzouna (Tunisia); Khazri, Abdelhafidh [Laboratory of Environment Biomonitoring, Coastal Ecology Unit, Faculty of Sciences of Bizerta, University of Carthage, 7021 Zarzouna (Tunisia); Mezni, Amine [Unit of Research 99/UR12-30, Department of Chemistry, Faculty of Sciences of Bizerte, 7021 Jarzouna (Tunisia); Louati, Héla; Dellali, Mohamed; Aissa, Patricia; Mahmoudi, Ezzeddine; Beyrem, Hamouda [Laboratory of Environment Biomonitoring, Coastal Ecology Unit, Faculty of Sciences of Bizerta, University of Carthage, 7021 Zarzouna (Tunisia); Sheehan, David, E-mail: [Environmental Research Institute and Department of Biochemistry, University College Cork, Western Gateway Building, Western Road, Cork (Ireland)


    Highlights: • We assessed toxicity of anthracene, permethrin and their mixture on clams. • Tissue and stressor-dependent changes were observed in biochemical responses. • Permethrin induces phase transition from aragonite to calcite in shell structure. • Interactive effects were observed on digestive gland and gill biomarkers. • Both approaches give new vision to risk assessment of organic pollution. - Abstract: Anthracene (ANT) and permethrin (PER) are two of the more toxic compounds reaching the marine environment. This study aimed to determine the impact of these molecules on Venerupis decussata, an economically important species cultured on the Tunisian coast. Shell structure and its possible transformation upon exposure to the two contaminants were studied by X-ray diffraction and gravimetric analyses. Results revealed a phase transition in shell composition from aragonite to calcite after PER exposure, to a mixture of PER and ANT (Mix) but not for ANT alone. Catalase (CAT), superoxide dismutase (SOD) and glutathione transferase (GST) activities were determined in digestive gland and gills after exposure to ANT, PER and Mix to assess the impact of the contamination on the oxidative status of V. decussata. Enzyme activities increased in the digestive gland after PER treatment and in the gills after ANT treatment. PER exposure significantly reduced the levels of free thiols and increased levels of carbonylated proteins in the digestive gland, as compared to controls. In contrast, ANT exposure significantly reduced free thiols and increased the number of carbonylated proteins in the gills. Mix induced additive effects as measured by both enzymatic and proteomic approaches. The present study suggests that PER has a strong effect on shell structure; that PER and ANT exposure generate compound-dependent oxidative stress in the tissues of V. decussata and that a mixture of the two compounds has synergistic effects on biochemical response.

  2. Genome-wide identification, 3D modeling, expression and enzymatic activity analysis of cell wall invertase gene family from cassava (Manihot esculenta Crantz). (United States)

    Yao, Yuan; Geng, Meng-Ting; Wu, Xiao-Hui; Liu, Jiao; Li, Rui-Mei; Hu, Xin-Wen; Guo, Jian-Chun


    The cell wall invertases play a crucial role on the sucrose metabolism in plant source and sink organs. In this research, six cell wall invertase genes (MeCWINV1-6) were cloned from cassava. All the MeCWINVs contain a putative signal peptide with a predicted extracellular location. The overall predicted structures of the MeCWINV1-6 are similar to AtcwINV1. Their N-terminus domain forms a β-propeller module and three conserved sequence domains (NDPNG, RDP and WECP(V)D), in which the catalytic residues are situated in these domains; while the C-terminus domain consists of a β-sandwich module. The predicted structure of Pro residue from the WECPD (MeCWINV1, 2, 5, and 6), and Val residue from the WECVD (MeCWINV3 and 4) are different. The activity of MeCWINV1 and 3 were higher than other MeCWINVs in leaves and tubers, which suggested that sucrose was mainly catalyzed by the MeCWINV1 and 3 in the apoplastic space of cassava source and sink organs. The transcriptional levels of all the MeCWINVs and their enzymatic activity were lower in tubers than in leaves at all the stages during the cassava tuber development. It suggested that the major role of the MeCWINVs was on the regulation of carbon exportation from source leaves, and the ratio of sucrose to hexose in the apoplasts; the role of these enzymes on the sucrose unloading to tuber was weaker.

  3. Solid lipid nanoparticles affect microbial colonization and enzymatic activity throughout the decomposition of alder leaves in freshwater microcosms. (United States)

    Sampaio, A C; Mendes, R J; Castro, P G; Silva, A M


    Solid lipid nanoparticles (SLNs) are used as carriers for drug delivery, and are high biocompatible and designed to endure in the host organism. Despite its current industrial production is low, many of these substances are available on the market, and much more are in the production pipeline. As a result, many of them will end in aquatic systems raising the question whether they can pose a risk to aquatic biota and the associated ecological processes. Microbial decomposers of plant litter, play a key role in forested streams being responsible for the energy flow between terrestrial and aquatic environments. Here, we investigated the effects of SLNs on alder leaf litter decomposition by aquatic microbes. Alder leaves were immersed in a stream of Northeast Portugal to allow microbial colonization before being exposed in microcosms of two types of SLNs at two concentrations for 42 days. Results showed that rates of leaf decomposition decreased with exposure to SLNs. Bacterial biomass was not inhibited by SLNs, and cultivable fungi densities remained constant (SLN-A) or increased (SLN-C) compared with control microcosms. The type and concentration of SLNs influenced differently the leaf colonization by fungi as well as fungal sporulation rate. These effects were accompanied by changes in the community extraenzymatic profile: the activities of alkaline phosphatase, acidic phosphatase, Naphthol-AS-BI-phosphohydrolase (P cycle) and lipases increased in the SLNs microcosms. This study provided the first evidence of the adverse effects of the release of SLNs to streams on leaf litter decomposition. Those effects seem to depend on the composition and concentration of SLNs, as well on the microbial target group, or enzyme. Thus, prior to massive industrial production of these nanomaterials, some measures should be taken to avoid environmental impact affecting the microbial communities responsible for detritus decomposition.

  4. Cathepsin G Induces Cell Aggregation of Human Breast Cancer MCF-7 Cells via a 2-Step Mechanism: Catalytic Site-Independent Binding to the Cell Surface and Enzymatic Activity-Dependent Induction of the Cell Aggregation

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    Riyo Morimoto-Kamata


    Full Text Available Neutrophils often invade various tumor tissues and affect tumor progression and metastasis. Cathepsin G (CG is a serine protease secreted from activated neutrophils. Previously, we have shown that CG induces the formation of E-cadherin-mediated multicellular spheroids of human breast cancer MCF-7 cells; however, the molecular mechanisms involved in this process are unknown. In this study, we investigated whether CG required its enzymatic activity to induce MCF-7 cell aggregation. The cell aggregation-inducing activity of CG was inhibited by pretreatment of CG with the serine protease inhibitors chymostatin and phenylmethylsulfonyl fluoride. In addition, an enzymatically inactive S195G (chymotrypsinogen numbering CG did not induce cell aggregation. Furthermore, CG specifically bound to the cell surface of MCF-7 cells via a catalytic site-independent mechanism because the binding was not affected by pretreatment of CG with serine protease inhibitors, and cell surface binding was also detected with S195G CG. Therefore, we propose that the CG-induced aggregation of MCF-7 cells occurs via a 2-step process, in which CG binds to the cell surface, independently of its catalytic site, and then induces cell aggregation, which is dependent on its enzymatic activity.


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    Tânia L. Montenegro STAMFORD


    Full Text Available O isolamento e a identificação de microrganismos produtores de enzimas de interesse comercial, utilizando tubérculos de jacatupé (Pachyrhizus erosus L. Urban, foi o objetivo principal deste trabalho. Isolaram-se microrganismos endofíticos e epifíticos identificados por observação micromorfológica. A avaliação da atividade enzimática das linhagens foi determinada pelo método de difusão em ágar. As sessenta e oito linhagens isoladas dos tubérculos de jacatupé foram cultivadas em meio sólido específico para amilase, lipase, protease e celulase por 96h a 280 C. Os microrganismos epifíticos encontrados foram Pithomyces (7,3%, Aspergillus (19,2%, Fusarium (5,9% e Trichoderma (5,8%, e os endofíticos foram Mucor (7,3%, Rhizopus (10,3%, Bacillus (19,0%, Staphylococcus (10,3% e Nocardiopsis (15%. As linhagens de Nocardiopsis sp. apresentaram atividade lipolítica superior à do padrão, porém a atividade amilolítica não apresentou diferença significativa comparada com o padrão. As linhagens de Mucor sp., Pithomyces sp. e Staphylococcus sp. produziram atividade proteolítica abaixo do padrão. Nenhum isolado apresentou atividade celulolítica.The isolation and identification of microorganisms that produce enzyme of commercial interest utilizing tubers of yam bean legume (Pachyrrizus erosus L. Urban was the main objective of this work. Endophytic and epiphytic microorganisms were isolated by micromorphologyc observation. The agar diffusion method was used to determine the enzymatic activity. Sixty-eight isolates from yam bean tubers were cultured at 280 C in solid medium specific to amylase, lipase, protease and cellulase for 96h. The epiphytic microorganisms Pithomyces (7,3%, Aspergillus (19,2%, Fusarium (5,9% and Trichoderma (5,8% and the endophytic microorganisms Mucor (7,3%, Rhizopus (10,3% Bacillus (19%, Staphylococcus (10,3% and Nocardiopsis (15% were isolated. Compared to the specific standard culture Nocardiopsis sp. showed

  6. Studies on gene structure, enzymatic activity and regulatory mechanism of acetohydroxy acid isomeroreductase from G2 pea

    Institute of Scientific and Technical Information of China (English)

    XU; Yunjian; (


    , P., Smith, J. K. et al., Regulation of tobacco acetolactate synthase gene expression, Plant Physiol., 1993, 102: 1009-1018.[21]Singh, B. K., Newhouse, K. E., Stidham, M. A. et al., Actohydroxyacid synthase-imidazolinone interaction, in Biosynthesis of Branched Chain Amino Acids (eds. Barak, Z., Chipman, D. M., Schloss, J. V.), Weinheim: VCH, 1990, 357-372.[22]Stidham, M. A., Singh, B. K., Imidazolinone-acetohydroxyacid synthase interactions, in The Imidazolinone Herbicides (eds. Shaner, L. L., O'Connor, S. L.), Boca Raton, F L: CRC Press, 1991, 71-90.[23]Szamosi, I. T., Shaner, D. L., Singh, B. K., Identification and characterization of a biodegradative form of threonine deghdratase in senescing tomato leaf, Plant Physiol., 1993, 101: 999-1004.[24]Hofgen, R., Laber, B., Schuttke, I. et al., Repression of acetolactate synthase activity through antisense inhibition: Molecular and biochemical analysis of transgenic potato (Solanum tuberosum L. cv Desiree) plants, Plant Physiol., 1995, 107: 469-477.

  7. Rabbit pulmonary angiotensin-converting enzyme: the NH2-terminal fragment with enzymatic activity and its formation from the native enzyme by NH4OH treatment. (United States)

    Iwata, K; Blacher, R; Soffer, R L; Lai, C Y


    The NH2-terminal sequence of 22 residues of rabbit lung angiotensin-converting enzyme has been determined as (NH2)Thr-Leu-Asp-Pro-Gly-Leu-Leu-Pro-Gly-Asp-Phe-Ala -Ala-Asp-Asn-Ala-Gly-Ala-Arg-Leu-Phe-Ala-. In the course of purification of the enzyme for structural analysis a protein of Mr = 82,000 with angiotensin-converting activity was separated from the major fraction containing the native enzyme (Mr = 140,000). This low-molecular-weight enzyme catalyzed the hydrolysis of the synthetic substrate Hip-His-Leu at a rate 23% of that with the native enzyme, and exhibited a similar Km value as well as behaviors towards various effectors of angiotensin-converting enzyme. Edman degradation of both the native and the 82K enzymes revealed that they contain identical amino acid sequences from the NH2-termini. This result and those of peptide mapping and carbohydrate and amino acid analyses indicate that the 82K enzyme is a fragment derived from the NH2-terminal portion of the native enzyme, and hence contains its catalytic site. Evidence has been obtained indicating that the active fragment was formed from the native enzyme during its elution from the antibody-affinity column with NH4OH: on treatment of the native enzyme (140K Mr) with 1 N NH4OH at room temperature, a cleavage occurred and two proteins with Mr = 82K and Mr = 62K were obtained. The 82K Mr fragment was found to be enzymatically active and to contain the same NH2-terminal sequence as the native enzyme. The other fragment (62K Mr) was devoid of the activity and was shown to derive from the COOH-terminal portion of the native enzyme by the peptide mapping and terminal analyses. Cleavage of a peptide bond with NH4OH is unusual and appears to be specific for the native angiotensin-converting enzyme from rabbit lung.

  8. Effect of aflatoxin B1 on growth and enzymatic activity of a native strain of Bacillus sp Efecto de la aflatoxina B1 sobre el crecimiento y actividad proteolítica de una cepa nativa de Bacillus sp

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    Márquez Edna Judith


    Full Text Available The effect of different aflatoxin B1 (AFAB1 concentrations on alkaline protease growth and enzymatic activity was evaluated; a native strain of alkalophilic Bacillus sp cultivated in CSL (Corn Steep Liquor was used. It was found that the effect of AFAB1 on the strain inhibited its growth and enzymatic activity to 1 ppm, showing that the strain is highly sensible to AFAB1, meaning that medium obtained f rom Colombian corn contaminated with this mycotoxin cannot be easily used. Concentrations less than 0.1 ppm did not affect growth and enzymatic activity. Key words: Bacillus, aflatoxin, alkaline proteases.Se evaluó el efecto de diferentes concentraciones de aflatoxina B1 (AFAB1 sobre el crecimiento y actividad enzimática de proteasas alcalinas de una cepa nativa de Bacillus sp Alcalofílico cultivada en LAM (Licor Agotado de Maíz. Se encontró que la cepa inhibe su crecimiento y actividad enzimática a 1 ppm, lo que demuestra una alta sensibilidad de la cepa evaluada a la AFAB1 e imposibilita utilizar fácilmente medios obtenidos de maíz nacional contaminado con esta micotoxina. Las concentraciones inferiores a 0.1 ppm no tienen ningún efecto sobre el crecimiento y la actividad enzimática. Palabras clave: Bacillus, aflatoxina, proteasas alcalinas.

  9. Signal peptide peptidase-mediated nuclear localization of heme oxygenase-1 promotes cancer cell proliferation and invasion independent of its enzymatic activity. (United States)

    Hsu, F-F; Yeh, C-T; Sun, Y-J; Chiang, M-T; Lan, W-M; Li, F-A; Lee, W-H; Chau, L-Y


    Heme oxygenase-1 (HO-1) is a heme-degrading enzyme anchored in the endoplasmic reticulum by a carboxyl-terminal transmembrane segment (TMS). HO-1 is highly expressed in various cancers and its nuclear localization is associated with the progression of some cancers. Nevertheless, the mechanism underlying HO-1 nuclear translocation and its pathological significance remain elusive. Here we show that the signal peptide peptidase (SPP) catalyzes the intramembrane cleavage of HO-1. Coexpression of HO-1 with wild-type SPP, but not a dominant-negative SPP, promoted the nuclear localization of HO-1 in cells. Mass spectrometry analysis of cytosolic HO-1 isolated from HeLa cells overexpressing HO-1 and SPP revealed two adjacent intramembrane cleavage sites located after S275 and F276 within the TMS. Mutations of S275F276 to A275L276 significantly hindered SPP-mediated HO-1 cleavage and nuclear localization. Nuclear HO-1 was detected in A549 and DU145 cancer cell lines expressing high levels of endogenous HO-1 and SPP. SPP knockdown or inhibition significantly reduced nuclear HO-1 localization in A549 and DU145 cells. The positive nuclear HO-1 stain was also evident in lung cancer tissues expressing high levels of HO-1 and SPP. Overexpression of a truncated HO-1 (t-HO-1) lacking the TMS in HeLa and H1299 cells promoted cell proliferation and migration/invasion. The effect of t-HO-1 was not affected by a mutation in the catalytic site. However, blockade of t-HO-1 nuclear localization abolished t-HO-1-mediated effect. The tumorigenic effect of t-HO-1 was also demonstrated in the mouse model. These findings disclose that SPP-mediated intramembrane cleavage of HO-1 promotes HO-1 nuclear localization and cancer progression independent of HO-1 enzymatic activity.

  10. CeO2 nanocrystallines ensemble-on-nitrogen-doped graphene nanocomposites: one-pot, rapid synthesis and excellent electrocatalytic activity for enzymatic biosensing. (United States)

    Du, Xiaojiao; Jiang, Ding; Chen, Saibo; Dai, Liming; Zhou, Lei; Hao, Nan; You, Tianyou; Mao, Hanping; Wang, Kun


    Ceria nanomaterials for heterogeneous catalysis have attracted much attention due to their excellent properties and have been extensively applied in recent years. But the poor electron conductivity and the aggregation behavior severely affect their electrocatalytic performances. In this paper, we prepared a novel catalyst based on CeO2 nanocrystallines (CeO2 NCs) ensemble-on-nitrogen-doped graphene (CeO2-NG) nanocomposites through a one-step heat-treatment without the need of the precursor. The results confirmed that the high dispersion of CeO2 NCs with the uniform size distribution of about 5nm on the surface of nitrogen-doped graphene (NG) sheets could be easily obtained via the one-step procedure and the resultant CeO2-NG nanocomposites were an excellent electrode material possessing outstanding electrochemical features for electron transfer. Luminol, an important electroactive substance, was further chosen to inspect the electrocatalytic properties of the as-prepared CeO2-NG nanocomposites. The studies showed that the presence of the NG in CeO2-NG nanocomposites could facilitate the electrochemical redox process of luminol. Compared with pristine CeO2 NCs, the synthesized CeO2-NG nanocomposites can enhance the electrochemiluminescence (ECL) intensity by 3.3-fold and decrease the onset ECL potential for about 72mV in the neutral condition. Employing above superiority, selecting cholesterol oxidase (ChOx) as the model oxidase, a facile ECL method for cholesterol detection with the CeO2-NG nanocomposites as the matrix to immobilize enzyme ChOx was developed. The results demonstrated CeO2-NG nanocomposites exhibited excellent performances in terms of sensitivity and catalytic activities, indicating that NG-based nanomaterials have great promise in electrocatalytic and enzymatic biosensing fields.

  11. Enzymatic hydrolysis of potato pulp

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    Mariusz Lesiecki


    Full Text Available Background. Potato pulp constitutes a complicated system of four types of polysaccharides: cellulose, hemicellulose, pectin and starch. Its composition makes it a potential and attractive raw material for the production of the second generation bioethanol. The aim of this research project was to assess the usefulness of commercial enzymatic preparations for the hydrolysis of potato pulp and to evaluate the effectiveness of hydrolysates obtained in this way as raw materials for ethanol fermentation. Material  and methods. Sterilised potato pulp was subjected to hydrolysis with commercial enzymatic preparations. The effectiveness of the preparations declared as active towards only one fraction of potato pulp (separate amylase, pectinase and cellulase activity and mixtures of these preparations was analysed. The monomers content in hydrolysates was determined using HPLC method. Results.  The application of amylolytic enzymes for potato pulp hydrolysis resulted in the release of only 18% of raw material with glucose as the dominant (77% constituent of the formed product. In addition, 16% galactose was also determined in it. The hydrolysis of the cellulose fraction yielded up to 35% raw material and the main constituents of the obtained hydrolysate were glucose (46% and arabinose (40%. Simultaneous application of amylolytic, cellulolytic and pectinolytic enzymes turned out to be the most effective way of carrying out the process as its efficiency in this case reached 90%. The obtained hydrolysate contained 63% glucose, 25% arabinose and 12% other simple substances. Conclusion. The application of commercial enzymatic preparations made it possible to perform potato pulp hydrolysis with 90% effectiveness. This was achieved by the application of a complex of amylolytic, cellulolytic and pectinolytic enzymes and the hydrolysate obtained in this way contained, primarily, glucose making it a viable substrate for ethanol fermentation.



    Reyna M., L.; Facultad de Química e ingeniería Química, Universidad Nacional Mayor de San Marcos. Lima-Perú; Robles, R.; Facultad de Química e ingeniería Química, Universidad Nacional Mayor de San Marcos. Lima-Perú; Reyes P., M.; Facultad de Química e ingeniería Química, Universidad Nacional Mayor de San Marcos. Lima-Perú; Mendoza R., Y.; Facultad de Química e ingeniería Química, Universidad Nacional Mayor de San Marcos. Lima-Perú; Romero D., J.; Facultad de Química e ingeniería Química, Universidad Nacional Mayor de San Marcos. Lima-Perú


    We obtained rate constant for enzymatic hydrolysis of barley at temperature of 70 ºC. Se ha llevado a cabo la hidrólisis enzimática de la cebada a 70 ºC y se determinó la constante cinética de reacción.

  13. Enzymatic synthesis of vanillin

    NARCIS (Netherlands)

    van den Heuvel, RHH; Fraaije, MW; Laane, C; van Berkel, WJH; Heuvel, Robert H.H. van den; Berkel, Willem J.H. van


    Due to increasing interest in natural vanillin, two enzymatic routes for the synthesis of vanillin were developed. The flavoprotein vanillyl alcohol oxidase (VAO) acts on a wide range of phenolic compounds and converts both creosol and vanillylamine to vanillin with high yield. The VAO-mediated conv

  14. Study on the effect of prostaglandin F2α treatment on semen characteristics and enzymatic activates of Awassi rams in breeding and non breeding seasons

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    Osama Ibrahim Azawi,


    non breeding season (52.34 ± 8.96 and 57.43 ± 19.9 vs. 117.02 ± 5.26 and 131.88 ± 5.01, respectively. Other enzymatic activities including ALT, AST, ACP and ALP showed no significant differences between Awassi rams treated with PGF2α and control groups in both breeding and non breeding seasons. In conclusion, PGF2αtreatment of Awassi rams improved sperm concentration and testicular volume

  15. Non-enzymatic modifications of prostaglandin H synthase 1 affect bifunctional enzyme activity - Implications for the sensitivity of blood platelets to acetylsalicylic acid. (United States)

    Kassassir, Hassan; Siewiera, Karolina; Talar, Marcin; Stec-Martyna, Emilia; Pawlowska, Zofia; Watala, Cezary


    Due to its ability to inhibit the blood platelet PGHS-1, acetylsalicylic acid (ASA, Aspirin(®)) is widely used as a preventive agent in atherothrombotic diseases. However, its beneficial effects seem to be lower in diabetic patients, suggesting that protein glycation may impair effective ASA-mediated acetylation process. On the other hand, it is proposed that ASA can prevent some of the late complications of diabetes by lowering the extent of glycation at protein free amino groups. The aim of this work was to evaluate the extents of non-enzymatic N-glycosylation (glycation) and acetylation of blood platelet PGHS-1 (COX-1) and the competition between glycation and acetylation was investigated in order to demonstrate how these two reactions may compete against platelet PGHS-1. When PGHS-1 was incubated with glycating/acetylating agents (glucose, Glu; 1,6-bisphosphofructose, 1,6-BPF; methylglyoxal, MGO, acetylsalicylic acid, ASA), the enzyme was modified in 13.4 ± 1.6, 5.3 ± 0.5, 10.7 ± 1.2 and 6.4 ± 1.1 mol/mol protein, respectively, and its activity was significantly reduced. The prior glycation/carbonylation of PGHS-1 with Glu, 1,6-BPF or MGO decreased the extent of acetylation from 6.4 ± 1.1 down to 2.5 ± 0.2, 3.6 ± 0.3 and 5.2 ± 0.2 mol/mol protein, respectively, but the enzyme still remained susceptible to the subsequent inhibition of its activity with ASA. When PGHS-1 was first acetylated with ASA and then incubated with glycating/carbonylating agents, we observed the following reductions in the enzyme modifications: from 13.4 ± 1.6 to 8.7 ± 0.6 mol/mol protein for Glu, from 5.3 ± 0.5 to 3.9 ± 0.3 mol/mol protein for 1,6-BPF and from 10.7 ± 1.2 to 7.5 ± 0.5 mol/mol protein for MGO, however subsequent glycation/carbonylation did not significantly affect PGHS-1 function. Overall, our outcomes allow to better understand the structural aspects of the chemical competition between glycation and acetylation of PGHS-1.

  16. Enzymatic production of cyclodextrins. (United States)

    Biwer, A; Antranikian, G; Heinzle, E


    Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly alpha-amylases. Due to its low solubility in water, separation and purification of beta-CD is relatively easy compared to alpha- and gamma-CD. In recent years more economic processes for gamma-CD and especially alpha-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of alpha-CD production processes is presented.

  17. Enzymatic desulfurization of coal

    Energy Technology Data Exchange (ETDEWEB)

    Boyer, Y.N.; Crooker, S.C.; Kitchell, J.P.; Nochur, S.V.


    The overall objective of this program was to investigate the feasibility of an enzymatic desulfurization process specifically intended for organic sulfur removal from coal. Toward that end, a series of specific objectives were defined: (1) establish the feasibility of (bio)oxidative pretreatment followed by biochemical sulfate cleavage for representative sulfur-containing model compounds and coals using commercially-available enzymes; (2) investigate the potential for the isolation and selective use of enzyme preparations from coal-utilizing microbial systems for desulfurization of sulfur-containing model compounds and coals; and (3) develop a conceptual design and economic analysis of a process for enzymatic removal of organic sulfur from coal. Within the scope of this program, it was proposed to carry out a portion of each of these efforts concurrently. (VC)

  18. A Single-Domain Llama Antibody Potently Inhibits the Enzymatic Activity of Botulinum Neurotoxin by Binding to the Non-Catalytic [alpha]-Exosite Binding Region

    Energy Technology Data Exchange (ETDEWEB)

    Dong, Jianbo; Thompson, Aaron A.; Fan, Yongfeng; Lou, Jianlong; Conrad, Fraser; Ho, Mengfei; Pires-Alves, Melissa; Wilson, Brenda A.; Stevens, Raymond C.; Marks, James D. (UIUC); (Scripps); (UCSF)


    Ingestion or inhalation of botulinum neurotoxin (BoNT) results in botulism, a severe and frequently fatal disease. Current treatments rely on antitoxins, which, while effective, cannot reverse symptoms once BoNT has entered the neuron. For treatments that can reverse intoxication, interest has focused on developing inhibitors of the enzymatic BoNT light chain (BoNT Lc). Such inhibitors typically mimic substrate and bind in or around the substrate cleavage pocket. To explore the full range of binding sites for serotype A light chain (BoNT/A Lc) inhibitors, we created a library of non-immune llama single-domain VHH (camelid heavy-chain variable region derived from heavy-chain-only antibody) antibodies displayed on the surface of the yeast Saccharomyces cerevisiae. Library selection on BoNT/A Lc yielded 15 yeast-displayed VHH with equilibrium dissociation constants (K{sub d}) from 230 to 0.03 nM measured by flow cytometry. Eight of 15 VHH inhibited the cleavage of substrate SNAP25 (synaptosome-associated protein of 25,000 Da) by BoNT/A Lc. The most potent VHH (Aa1) had a solution K{sub d} for BoNT/A Lc of 1.47 x 10{sup -10} M and an IC{sub 50} (50% inhibitory concentration) of 4.7 x 10{sup -10} M and was resistant to heat denaturation and reducing conditions. To understand the mechanism by which Aa1 inhibited catalysis, we solved the X-ray crystal structure of the BoNT/A Lc-Aa1 VHH complex at 2.6 {angstrom} resolution. The structure reveals that the Aa1 VHH binds in the {alpha}-exosite of the BoNT/A Lc, far from the active site for catalysis. The study validates the utility of non-immune llama VHH libraries as a source of enzyme inhibitors and identifies the BoNT/A Lc {alpha}-exosite as a target for inhibitor development.

  19. Enzymatic treatment of estrogens and estrogen glucuronide

    Institute of Scientific and Technical Information of China (English)

    Takaaki Tanaka; Toshiyuki Tamura; Yuuichi Ishizaki; Akito Kawasaki; Tomokazu Kawase; Masahiro Teraguchi; Masayuki Taniguchi


    Natural and synthetic estrogens from sewage treatment systems are suspected to influence the reproductive health of the animals in the rivers.In this article we investigated the enzymatic treatment of three estrogens (estrone,17β-estradiol,and 17α-ethynyletstradiol) by a fungal laccase which oxidize phenolic compounds with dissolved oxygen.The elimination of the estrogenic activities by enzymatic oxidation was demonstrated by medaka vitellogenin assay.In addition,we developed an enzymatic treatment system comprised of β-D-glucuronidase and the laccase for 17β-estradiol 3-(β-D-glucuronide) degradation.The two enzymes eliminated 17β-estradiol 3-(β-D-glucuronide) and the intermediate,17β-estradiol,efficiently.

  20. A quantitative analysis for the ADP-ribosylation activity of pertussis toxin: an enzymatic-HPLC coupled assay applicable to formulated whole cell and acellular pertussis vaccine products. (United States)

    Cyr, T; Menzies, A J; Calver, J; Whitehouse, L W


    The majority of the biological effects of pertussis toxin (PT) are the result of a toxin-catalyzed transfer of an adenosine diphosphate-ribose (ADP-ribose) moiety from NAD(+)to the alpha-subunits of a subset of signal-transducing guanine-nucleotide-binding proteins (G-proteins). This generally leads to an uncoupling of the modified G-protein from the corresponding receptor and the loss of effector regulation. This assay is based on the PT S1 subunit enzymatic transfer of ADP-ribose from NAD to the cysteine moiety of a fluorescent tagged synthetic peptide homologous to the 20 amino acid residue carboxyl-terminal sequence of the alpha-subunit of the G(i3)protein. The tagged peptide and the ADP-ribosylated product were characterized by HPLC/MS and MS/MS for structure confirmation. Quantitation of this characterized ADP-ribosylated fluorescently tagged peptide was by HPLC fluorescence using Standard Addition methodology. The assay was linear over a five hr incubation period at 20 degrees C at PT concentrations between 0.0625 and 4.0 microg/ml and the sensitivity of the assay could be increased several fold by increasing the incubation time to 24 h. Purified S1 subunit of PT exhibited 68.1+/-10.1% of the activity of the intact toxin on a molar basis, whereas the pertussis toxin B oligomer, the genetically engineered toxoid, (PT-9K/129G), and several of the other components of the Bordetella pertussis organism possessed little (<0.6%) or no detectable ribosylation activity. Commonly used pertussis vaccine reference materials, US PV Lot #11, BRP PV 66/303, and BRP PV 88/522, were assayed by this method against Bordetella pertussis Toxin Standard 90/518 and demonstrated to contain, respectively, 0.323+/-0.007, 0.682+/-0.045, and 0.757+/-0.006 microg PT/ml (Mean+/-SEM) or in terms of microg/vial: 3.63, 4.09 and 4.54, respectively. A survey of several multivalent pertussis vaccine products formulated with both whole cell as well as acellular components indicated that

  1. 高血压患者体内脯氨酸寡肽酶酶学活性的临床研究%Evaluation of prolyl oligopeptidase enzymatic activity in hypertensive patients

    Institute of Scientific and Technical Information of China (English)

    曾彦; 王东; 许晶晶; 张宇清


    Objective To determine the enzymatic activity of prolyl oligopeptidase (POP)in hypertensive patients and explore the relations between the changes of POP enzymatic activity and clinical features.Methods Forty-five pa-tients with grade 1 hypertension,26 patients with grade 2 hypertension,24 patients with grade 3 hypertension and 27 normotensives were included in the present study,who were selected from Fuwai Hospital from 1st August,2012 to 1st June,2014.All the participants underwent physical and echocardiographic examinations.Meanwhile,fasting blood was collected to determine the POP enzymatic activity and routine biochemical indicators.Results Compared with the con-trols,POP enzymatic activities were significantly reduced in hypertensive patients (grade 1 hypertention 1.40±0.56 U, grade 2 hypertention 1.13±0.60 U,grade 3 hypertention 1.10±0.58 U vs the controls 1.82±0.47 U,all P <0.01), and this reduction was more evident in patients with grade 2 or 3 hypertention as compared to ones with grade 1 hyper-tention(P <0.05).Pearson correlation analysis showed that POP enzymatic activity was significantly associated with systolic blood pressure,diastolic blood pressure,triglyceride,high density lipoproteins and left ventricular mass index(P<0.05).Conclusion POP enzymatic activity was reduced with the increasing blood pressure.%目的:检测高血压患者体内脯氨酸寡肽酶(prolyl oligopeptidase,POP)酶学活性变化并探讨这一变化与各临床指标的相关性。方法选取2012年8月1日至2014年6月1日间就诊于北京阜外心血管病医院的高血压1级患者45例、高血压2级患者26例,高血压3级患者24例及正常血压受试者27例。所有参与者均行体格及超声心动图检查,并抽取静脉血以测定 POP 酶学活性以及相关生化指标。采用 Pearson 线性相关分析确定参数之间的变化及关系。结果与对照组相比,高血压患者 POP 酶学活性减低(1级高血压1.40±0

  2. Enzymatic biotransformation of terpenes as bioactive agents. (United States)

    Sultana, Nighat; Saify, Zafar Saeed


    The plant-derived terpenoids are considered to be the most potent anticancer, anti-inflammatory and anticarcinogenic compounds known. Enzymatic biotransformation is a very useful approach to expand the chemical diversity of natural products. Recent enzymatic biotransformation studies on terpenoids have resulted in the isolation of novel compounds. 14-hydroxy methyl caryophyllene oxide produced from caryophyllene oxide showed a potent inhibitory activity against the butyryl cholinesterase enzyme, and was found to be more potent than parent caryophyllene oxide. The metabolites 3β,7β-dihydroxy-11-oxo-olean-12-en-30-oic acid, betulin, betulonic acid, argentatin A, incanilin, 18β glycyrrhetinic acid, 3,11-dioxo-olean-12-en-30-oic acid produced from 18β glycyrrhetinic acid were screened against the enzyme lipoxygenase. 3,11-Dioxo-olean-12-en-30-oic acid, was found to be more active than the parent compound. The metabolites 3β-hydroxy sclareol 18α-hydroxy sclareol, 6α,18α-dihydroxy sclareol, 11S,18α-dihydroxy sclareol, and 1β-hydroxy sclareol and 11S,18α-dihydroxy sclareol produced from sclareol were screened for antibacterial activity. 1β-Hydroxy sclareol was found to be more active than parent sclareol. There are several reports on natural product enzymatic biotransformation, but few have been conducted on terpenes. This review summarizes the classification, advantages and agents of enzymatic transformation and examines the potential role of new enzymatically transformed terpenoids and their derivatives in the chemoprevention and treatment of other diseases.

  3. Apoptosis induction in human breast cancer (MCF-7) cells by a novel venom L-amino acid oxidase (Rusvinoxidase) is independent of its enzymatic activity and is accompanied by caspase-7 activation and reactive oxygen species production. (United States)

    Mukherjee, Ashis K; Saviola, Anthony J; Burns, Patrick D; Mackessy, Stephen P


    We report the elucidation of a mechanism of apoptosis induction in breast cancer (MCF-7) cells by an L-amino acid oxidase (LAAO), Rusvinoxidase, purified from the venom of Daboia russelii russelii. Peptide mass fingerprinting analysis of Rusvinoxidase, an acidic monomeric glycoprotein with a mass of ~57 kDa, confirmed its identity as snake venom LAAO. The enzymatic activity of Rusvinoxidase was completely abolished after two cycles of freezing and thawing; however, its cytotoxicity toward MCF-7 cells remained unaffected. Dose- and time-dependent induction of apoptosis by Rusvinoxidase on MCF-7 cells was evident from changes in cell morphology, cell membrane integrity, shrinkage of cells and apoptotic body formation accompanied by DNA fragmentation. Rusvinoxidase induced apoptosis in MCF-7 cells by both the extrinsic (death-receptor) and intrinsic (mitochondrial) signaling pathways. The former pathway of apoptosis operated through activation of caspase-8 that subsequently activated caspase-7 but not caspase-3. Rusvinoxidase-induced intrinsic pathway of apoptosis was accompanied by a time-dependent depolarization of the mitochondrial membrane through the generation of reactive oxygen species, followed by a decrease in cellular glutathione content and catalase activity, and down-regulation of expression of anti-apoptotic proteins Bcl-XL and heat-shock proteins (HSP-90 and HSP-70). Rusvinoxidase treatment resulted in increase of the pro-apoptotic protein Bax, subsequently leading to the release of cytochrome c from mitochondria to the cytosol and activating caspase-9, which in turn stimulated effector caspase-7. Rusvinoxidase at a dose of 4 mg/kg was non-toxic in mice, indicating that it may be useful as a model for the development of peptide-based anticancer drugs.

  4. Enzymatic cascade bioreactor (United States)

    Simmons, Blake A.; Volponi, Joanne V.; Ingersoll, David; Walker, Andrew


    Disclosed is an apparatus and method for continuously converting sucrose to .beta.-D-glucose. The method comprises a three stage enzymatic reactor in which an aqueous solution of sucrose is first converted into a solution of fructose and .alpha.-D-glucose by passing it through a porous, packed column containing an inert media on which invertase is immobilized. This solution is then sent through a second packed column containing glucose isomerase and finally a third packed column containing mutarotase. Solution temperature and pH are adjusted to maximize glucose output.

  5. Enzymatic Modification of Sphingomyelin

    DEFF Research Database (Denmark)

    Zhang, Long; Hellgren, Lars; Xu, Xuebing

    Due to its major role in maintaining the water-retaining properties of the epidermis, ceramide is of great commercial potential in cosmetic and pharmaceuticals such as hair and skin care products. Currently, chemical synthesis of ceramide is a costly process, and developments of alternative cost......-efficient, high yield production methods are of great interest. In the present study, the potential of producing ceramide through enzymatic hydrolysis of sphingomyelin have been studied. Sphingomyelin (SM) is a ubiquitous membrane-lipid and dairy products or by-products is a rich source of sphingomyelin....... In present study, we have optimized the production of ceramide from sphingomyelin using Phospholipase C from Clostridium perfringens....

  6. Thermal-induced conformational changes in the product release area drive the enzymatic activity of xylanases 10B: Crystal structure, conformational stability and functional characterization of the xylanase 10B from Thermotoga petrophila RKU-1

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Camila Ramos; Meza, Andreia Navarro [Laboratorio Nacional de Biociencias (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Hoffmam, Zaira Bruna; Silva, Junio Cota; Alvarez, Thabata Maria; Ruller, Roberto [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Giesel, Guilherme Menegon; Verli, Hugo [Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, Porto Alegre, RS (Brazil); Squina, Fabio Marcio [Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol (CTBE), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil); Prade, Rolf Alexander [Department of Microbiology and Molecular Genetics, Oklahoma State University, Stillwater, OK (United States); Murakami, Mario Tyago, E-mail: [Laboratorio Nacional de Biociencias (LNBio), Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP (Brazil)


    Research highlights: {yields} The hyperthermostable xylanase 10B from Thermotoga petrophila RKU-1 produces exclusively xylobiose at the optimum temperature. {yields} Circular dichroism spectroscopy suggests a coupling effect of temperature-induced structural changes with its enzymatic behavior. {yields} Crystallographic and molecular dynamics studies indicate that conformational changes in the product release area modulate the enzyme action mode. -- Abstract: Endo-xylanases play a key role in the depolymerization of xylan and recently, they have attracted much attention owing to their potential applications on biofuels and paper industries. In this work, we have investigated the molecular basis for the action mode of xylanases 10B at high temperatures using biochemical, biophysical and crystallographic methods. The crystal structure of xylanase 10B from hyperthermophilic bacterium Thermotoga petrophila RKU-1 (TpXyl10B) has been solved in the native state and in complex with xylobiose. The complex crystal structure showed a classical binding mode shared among other xylanases, which encompasses the -1 and -2 subsites. Interestingly, TpXyl10B displayed a temperature-dependent action mode producing xylobiose and xylotriose at 20 {sup o}C, and exclusively xylobiose at 90 {sup o}C as assessed by capillary zone electrophoresis. Moreover, circular dichroism spectroscopy suggested a coupling effect of temperature-induced structural changes with this particular enzymatic behavior. Molecular dynamics simulations supported the CD analysis suggesting that an open conformational state adopted by the catalytic loop (Trp297-Lys326) provokes significant modifications in the product release area (+1,+2 and +3 subsites), which drives the enzymatic activity to the specific release of xylobiose at high temperatures.

  7. Enzymatic Hydrolysis of Lignocelluloses

    DEFF Research Database (Denmark)

    Kolasa, Marta; Ahring, Birgitte Kiær; Lübeck, Peter Stephensen;


    Lignocellulosic materials form a huge part of the plant biomass from agricultural and forestry wastes. They consist of three major components: cellulose, hemicellulose and lignin. Cellulose, the main constituent of plant cell wall, is a polymer of D–glucopyranose units linked by β-1,4-glucosidic...... bonds. Cellulose can be degraded to simple sugar components by means of enzymatic hydrolysis. However, due to its complex, crystalline structure it is difficult to break it down and the cooperative action of a variety of cellulolytic enzymes is necessary. Fungi are known to have potential in production...... using suppression subtractive hybridization and a targeted genomic screening approach using degenerate PCR. Suppression subtractive hybridization facilitates identification of genes encoding cellulolytic enzymes that are expressed when cultivating a fungal strain in medium with cellulose as the carbon...

  8. Changes in enzymatic activities and microbial properties in vermicompost of water hyacinth as affected by pre-composting and fungal inoculation: a comparative study of ergosterol and chitin for estimating fungal biomass. (United States)

    Pramanik, P


    In this experiment, three different fungal species, viz. Trichoderma viridae, Aspergillus niger and Phanerochaete chrysosporium, were inoculated in 7 day and 15 day partially decomposed water hyacinth to study their effect on enzymatic activities, microbial respiration and fungal biomass of the final stabilized product. The results suggested that increasing the duration of pre-composting from 7 days to 15 days did not show any significant effect on the activities of hydrolytic enzymes. Inoculation of fungi significantly (P vermicomposts. Inoculation of P. chrysosporium in initial organic waste registered the highest chitin content in vermicompost. A comparison of fungal biomass and chitin content revealed a conversion factor of 2.628 with a standard deviation of 0.318. Due to significant correlation (r = 0.864), this conversion factor allows for the calculation of fungal biomass from chitin, which is comparatively more stable than ergosterol.

  9. The diversity and extracellular enzymatic activities of yeasts isolated from water tanks of Vriesea minarum, an endangered bromeliad species in Brazil, and the description of Occultifur brasiliensis f.a., sp. nov. (United States)

    Gomes, Fátima C O; Safar, Silvana V B; Marques, Andrea R; Medeiros, Adriana O; Santos, Ana Raquel O; Carvalho, Cláudia; Lachance, Marc-André; Sampaio, José Paulo; Rosa, Carlos A


    The diversity of yeast species collected from the bromeliad tanks of Vriesea minarum, an endangered bromeliad species, and their ability to produce extracellular enzymes were studied. Water samples were collected from 30 tanks of bromeliads living in a rupestrian field site located at Serrada Piedade, Minas Gerais state, Brazil, during both the dry and rainy seasons. Thirty-six species were isolated, representing 22 basidiomycetous and 14 ascomycetous species. Occultifur sp., Cryptococcus podzolicus and Cryptococcus sp. 1 were the prevalent basidiomycetous species. The yeast-like fungus from the order Myriangiales, Candida silvae and Aureobasidium pullulans were the most frequent ascomycetous species. The diversity of the yeast communities obtained between seasons was not significantly different, but the yeast composition per bromeliad was different between seasons. These results suggest that there is significant spatial heterogeneity in the composition of populations of the yeast communities within bromeliad tanks, independent of the season. Among the 352 yeast isolates tested, 282 showed at least one enzymatic activity. Protease activity was the most widely expressed extracellular enzymatic activity, followed by xylanase, amylase, pectinase and cellulase activities. These enzymes may increase the carbon and nitrogen availability for the microbial food web in the bromeliad tank of V. minarum. Sequence analyses revealed the existence of 10 new species, indicating that bromeliad tanks are important sources of new yeasts. The novel species Occultifur brasiliensis, f.a., sp. nov., is proposed to accommodate the most frequently isolated yeast associated with V. minarum. The type strain of O. brasiliensis, f.a., sp. nov. is UFMG-CM-Y375(T) (= CBS 12687(T)). The Mycobank number is MB 809816.

  10. Combined enzymatic starch hydrolysis

    Energy Technology Data Exchange (ETDEWEB)

    Nebesny, E.


    From researches so far there comes out that glucoamylase AMG 300 L and pullulanase Promozyme 200 L when used in quantities the same as in preparation of Dextrozyme 225/75 L Novo at an action on liquified starch by means of /alpha/-amylase after 48 h of saccharification already (similarly like Dextrozyme) are able to get up to 98 DE. Chromatographic analysis proved that glucoamylase AMG 300 L Novo and succouring it pullulanase Promozyme 200 L are working most effectively when both enzymes are added to the liquified starch medium simultaneously. From this comes out that pullulanase hydrolyzes better /alpha/-1,6 bonds in lowmolecular dextrins than in oligosaccharides G/sub 4/ to G/sub 7/ formed at previous action of glucoamylase. At an optimum ratio of glucoamylase and pullulanase in relation to the dissolved starch after 8 h of the hydrolysis there are neither iso-sugars (isomaltose, panose), no oligosaccharides higher than G/sub 5/ and no dextrins. At the solution of the starch by /alpha/-amylase and its hydrolysis by enzymatic preparation Fungamyl 800 L Novo, at doses 0.02-0.8% to d.s. of starch, already after 8 h the reaction of hydrolysis contents of 36-62% maltose in dry substance of hydrolyzates are reached with only traces of glucose. (orig.).

  11. Enzymatic hydrolysis of poly(ethylene furanoate). (United States)

    Pellis, Alessandro; Haernvall, Karolina; Pichler, Christian M; Ghazaryan, Gagik; Breinbauer, Rolf; Guebitz, Georg M


    The urgency of producing new environmentally-friendly polyesters strongly enhanced the development of bio-based poly(ethylene furanoate) (PEF) as an alternative to plastics like poly(ethylene terephthalate) (PET) for applications that include food packaging, personal and home care containers and thermoforming equipment. In this study, PEF powders of various molecular weights (6, 10 and 40kDa) were synthetized and their susceptibility to enzymatic hydrolysis was investigated for the first time. According to LC/TOF-MS analysis, cutinase 1 from Thermobifida cellulosilytica liberated both 2,5-furandicarboxylic acid and oligomers of up to DP4. The enzyme preferentially hydrolyzed PEF with higher molecular weights but was active on all tested substrates. Mild enzymatic hydrolysis of PEF has a potential both for surface functionalization and monomers recycling.

  12. An approach for extraction of kernel oil from Pinus pumila using homogenate-circulating ultrasound in combination with an aqueous enzymatic process and evaluation of its antioxidant activity. (United States)

    Chen, Fengli; Zhang, Qiang; Gu, Huiyan; Yang, Lei


    In this study, a novel approach involving homogenate-circulating ultrasound in combination with aqueous enzymatic extraction (H-CUAEE) was developed for extraction of kernel oil from Pinus pumila. Following comparison of enzyme types and concentrations, an enzyme mixture consisting of cellulase, pectinase and hemicellulase (1:1:1, w/w/w) at a concentration of 2.5% was selected and applied for effective oil extraction and release. Several variables potentially influencing extraction yields, namely, homogenization time, incubation temperature, incubation time, mark-space ratio of ultrasound irradiation, ultrasound irradiation power, liquid-solid ratio, pH and stirring rate, were optimized by Plackett-Burman design. Among the eight variables, incubation temperature, incubation time and liquid-solid ratio were statistically significant and were further optimized by Box-Behnken design to predict optimum extraction conditions and ascertain operability ranges for maximum extraction yield. Under optimum operating conditions, extraction yields of P. pumila kernel oil were 31.89±1.12% with a Δ5-unsaturated polymethylene interrupted fatty acid content of 20.07% and an unsaturated fatty acid content of 93.47%. Our study results indicate that the proposed H-CUAEE process has enormous potential for efficient and environmentally friendly extraction of edible oils.

  13. Enzymatic hydration activity assessed by selective spectrophotometric detection of alcohols: a novel screening assay using oleate hydratase as a model enzyme. (United States)

    Hiseni, Aida; Medici, Rosario; Arends, Isabel W C E; Otten, Linda G


    Hydroxy fatty acids (HFAs) are high-added-value compounds, which are incorporated in polymers, lubricants, emulsifiers and stabilizers and have potential medicinal use. In nature, HFAs are regio-specifically synthesized by several enzymes, including P450 monooxygenases, lipoxygenases, hydratases, 12-hydroxylases, and diol synthases. The growing demand for HFAs warrants the development of simple and efficient analytical methods that enable high-throughput detection of the hydroxylated product in the presence of its unsaturated precursor. Herein a novel high-throughput assay for the detection of alcohols is described using oleate hydratase (OHase, EC from Elizabethkingia meningoseptica as the model enzyme. The developed assay is based on the selective spectrophotometric detection of alkyl nitrites formed upon the reaction between the hydroxyl group and nitrous acid. The assay proved to discriminate between unsaturated fatty acids as well as small cyclic and acyclic unsaturated alkenes and their corresponding alcohols. Lower detection limits were 1.5-3 mM with excellent Z'-factors. Enzymatic reactions using OHase with oleic acid resulted in somewhat lower Z-factors for various enzyme preparations. This small scale assay can enable fast discovery of new microorganisms or improved enzymes from mutant libraries and will be useful for biocatalytic strategies involving fatty acid (de)hydrating enzymes.

  14. Homogeneous, Heterogeneous, and Enzymatic Catalysis. (United States)

    Oyama, S. Ted; Somorjai, Gabor A.


    Discusses three areas of catalysis: homegeneous, heterogeneous, and enzymatic. Explains fundamentals and economic impact of catalysis. Lists and discusses common industrial catalysts. Provides a list of 107 references. (MVL)

  15. Humanized-Single Domain Antibodies (VH/VHH that Bound Specifically to Naja kaouthia Phospholipase A2 and Neutralized the Enzymatic Activity

    Directory of Open Access Journals (Sweden)

    Wanpen Chaicumpa


    Full Text Available Naja kaouthia (monocled cobra venom contains many isoforms of secreted phospholipase A2 (sPLA2. The PLA2 exerts several pharmacologic and toxic effects in the snake bitten subject, dependent or independent on the enzymatic activity. N. kaouthia venom appeared in two protein profiles, P3 and P5, after fractionating the venom by ion exchange column chromatography. In this study, phage clones displaying humanized-camel single domain antibodies (VH/VHH that bound specifically to the P3 and P5 were selected from a humanized-camel VH/VHH phage display library. Two phagemid transfected E. coli clones (P3-1 and P3-3 produced humanized-VHH, while another clone (P3-7 produced humanized-VH. At the optimal venom:antibody ratio, the VH/VHH purified from the E. coli homogenates neutralized PLA2 enzyme activity comparable to the horse immune serum against the N. kaouthia holo-venom. Homology modeling and molecular docking revealed that the VH/VHH covered the areas around the PLA2 catalytic groove and inserted their Complementarity Determining Regions (CDRs into the enzymatic cleft. It is envisaged that the VH/VHH would ameliorate/abrogate the principal toxicity of the venom PLA2 (membrane phospholipid catabolism leading to cellular and subcellular membrane damage which consequently causes hemolysis, hemorrhage, and dermo-/myo-necrosis, if they were used for passive immunotherapy of the cobra bitten victim. The speculation needs further investigations.

  16. Alteração da atividade enzimática em organismos aquáticos por poluentes de origem agrícola: uma abordagem geral e sobre a suscetibilidade da fosfatase ácida Alteration of enzymatic activity in aquatic organisms by agricultural pollutants: a general approach and the susceptibility of the acid phosphatase

    Directory of Open Access Journals (Sweden)

    Claudio Martín Jonsson


    Full Text Available The input of agrochemicals in the aquatic compartment can results in biochemical injuries for living organisms. In this context, the knowledge of alterations of enzymatic activities due the presence of agriculture pollutants contributes for the elucidation of the mechanisms of toxicity, implementation of economic methods for monitoring purposes and establishment of maximum allowed concentrations. In the present work, the above considerations are discussed, and data concerning changes in enzymatic function by pesticides and fertilizer contaminants are reviewed. Also, we focused on the acid phosphatase due its susceptibility to several pollutants and diversity in cellular functions.

  17. Lactose hydrolysis in an enzymatic membrane reactor

    Energy Technology Data Exchange (ETDEWEB)

    Mertens, B.; Huyghebaert, A.


    The enzymatic hydrolysis of lactose in whey permeate with subsequent recuperation of Saccharomyces lactis lactase by means of ultrafiltration was investigated. In whey permeate, S. lactis lactase shows maximal activity at pH 6.5; the optimal temperature was found to be 45/sup 0/C and is limited by strong thermal inactivation beyond this temperature. High activity combined with acceptable thermal inactivation (< 10% after 5 h incubation) was established at 30/sup 0/C. S. lactis lactase also displays considerable activity at low temperature (5/sup 0/C). Enzyme stability is reduced drastically by demineralisation: addition of low concentrations of manganese ions (10/sup -3/ M) considerably enhances stability. Using a DDS Lab-Unit 35 fitted with GR61PP polysulphon membranes (cut-off: 20.000), pilot scale experiments were carried out (pH 6.5; 30/sup 0/C) in which whey permeate was hydrolyzed to a degree of hydrolysis of 82% minimum. Enzyme recuperation amounted to 96.5% per batch, all enzyme activity loss being due to thermal inactivation. Microbiological examination of the enzymatic membrane reactor showed that growth of mcicroorganisms can largely be suppressed by working at lower temperature (5/sup 0/C). Eventually, 50 ppm H/sub 2/O/sub 2/ or sterile filtration will adequately solve microbiological problems without affecting enzyme activity.

  18. Structural perspective on enzymatic halogenation. (United States)

    Blasiak, Leah C; Drennan, Catherine L


    Simple halogen substituents frequently afford key structural features that account for the potency and selectivity of natural products, including antibiotics and hormones. For example, when a single chlorine atom on the antibiotic vancomycin is replaced by hydrogen, the resulting antibacterial activity decreases by up to 70% ( Harris , C. M. ; Kannan , R. ; Kopecka , H. ; Harris , T. M. J. Am. Chem. Soc. 1985 , 107 , 6652 - 6658 ). This Account analyzes how structure underlies mechanism in halogenases, the molecular machines designed by nature to incorporate halogens into diverse substrates. Traditional synthetic methods of integrating halogens into complex molecules are often complicated by a lack of specificity and regioselectivity. Nature, however, has developed a variety of elegant mechanisms for halogenating specific substrates with both regio- and stereoselectivity. An improved understanding of the biological routes toward halogenation could lead to the development of novel synthetic methods for the creation of new compounds with enhanced functions. Already, researchers have co-opted a fluorinase from the microorganism Streptomyces cattleya to produce (18)F-labeled molecules for use in positron emission tomography (PET) ( Deng , H. ; Cobb , S. L. ; Gee , A. D. ; Lockhart , A. ; Martarello , L. ; McGlinchey , R. P. ; O'Hagan , D. ; Onega , M. Chem. Commun. 2006 , 652 - 654 ). Therefore, the discovery and characterization of naturally occurring enzymatic halogenation mechanisms has become an active area of research. The catalogue of known halogenating enzymes has expanded from the familiar haloperoxidases to include oxygen-dependent enzymes and fluorinases. Recently, the discovery of a nucleophilic halogenase that catalyzes chlorinations has expanded the repertoire of biological halogenation chemistry ( Dong , C. ; Huang , F. ; Deng , H. ; Schaffrath , C. ; Spencer , J. B. ; O'Hagan , D. ; Naismith , J. H. Nature 2004 , 427 , 561 - 565 ). Structural

  19. Enzymatic and bacterial conversions during sourdough fermentation. (United States)

    Gänzle, Michael G


    Enzymatic and microbial conversion of flour components during bread making determines bread quality. Metabolism of sourdough microbiota and the activity of cereal enzymes are interdependent. Acidification, oxygen consumption, and thiols accumulation by microbial metabolism modulate the activity of cereal enzymes. In turn, cereal enzymes provide substrates for bacterial growth. This review highlights the role of cereal enzymes and the metabolism of lactic acid bacteria in conversion of carbohydrates, proteins, phenolic compounds and lipids. Heterofermentative lactic acid bacteria prevailing in wheat and rye sourdoughs preferentially metabolise sucrose and maltose; the latter is released by cereal enzymes during fermentation. Sucrose supports formation of acetate by heterofermentative lactobacilli, and the formation of exopolysaccharides. The release of maltose and glucose by cereal enzymes during fermentation determines the exopolysaccharide yield in sourdough fermentations. Proteolysis is dependent on cereal proteases. Peptidase activities of sourdough lactic acid bacteria determine the accumulation of (bioactive) peptides, amino acids, and amino acid metabolites in dough and bread. Enzymatic conversion and microbial metabolism of phenolic compounds is relevant in sorghum and millet containing high levels of phenolic compounds. The presence of phenolic compounds with antimicrobial activity in sorghum selects for fermentation microbiota that are resistant to the phenolic compounds.

  20. Active packaging use to inhibit enzymatic browning of apples/ Uso de embalagem ativa na inibição do escurecimento enzimático de maçãs

    Directory of Open Access Journals (Sweden)

    Giulliano Amaral Viana


    Full Text Available The enzymatic browning is the most limiting factor of fruits and vegetables shelf-life. The objective of this study was to evaluate the effect of an active packaging incorporated with anti-oxidant agents to inhibit apple’s enzymatic browning. Cellulosic films were incorporated with cysteine and sulphite and used to cover apples divided in halves. Browning inhibition was measured by polyphenoloxidase activity and colour analysis (CIE Lab colour system. Low concentration of sulphite (1% showed efficient browning inhibition and higher concentration of cysteine (15% was necessary to reach the same results. Treatments containing cysteine and sulphite resulted in brighter apples and less browning compared with control. The quantity of sulphite released to apples was lower than the limit allowed by legislation, decreasing, in this way, the levels of additives ingested by the consumer. In this study, the effectiveness of active packaging in providing product conservation was confirmed by the inhibition of browning in apples.O escurecimento enzimático é um dos fatores mais limitantes da vida de prateleira de frutas e vegetais. O objetivo desse estudo foi avaliar o efeito do uso de embalagem ativa incorporada com agentes antioxidantes na inibição do escurecimento enzimático de maçãs. Os filmes foram produzidos a base de polímero celulósico e incorporados com sulfito e cisteína para recobrimento de maçãs divididas ao meio. Foi avaliada a inibição do escurecimento através da atividade da polifenoloxidase e pela análise de cor (sistema CIE Lab. Baixas concentrações de sulfito (1% mostraram-se eficientes na inibição do escurecimento das maçãs e altas concentrações de cisteína (15% foram necessárias para a obtenção do mesmo resultado. Os tratamentos tanto com sulfito quanto com cisteína, comparados com os tratamentos controle, proporcionaram maior brilho às maçãs e menor escurecimento. O teor de sulfito liberado para a ma

  1. The dependence of the discharge of nitrous oxide by ordinary chernozem steppe of the Central-Chernozem Region of Russia from the content of humus, nitrogen and enzymatic activity (United States)

    Avksentev, Alexey; Negrobova, Elena; Kramareva, Tatiana; Moiseeva, Evgenya


    The dependence of the discharge of nitrous oxide by ordinary chernozem steppe of the Central-Chernozem Region of Russia from the content of humus, nitrogen and enzymatic activity Alexey Avksentev, Elena Negrobova, Tatiana Kramareva, Evgenya Moiseeva 394000 Voronezh, Universitetskaya square, 1 Voronezh State University Nitrous oxide is emitted by soil as a result of microbiological processes, ranks third in the list of aggressive greenhouse gas after carbon dioxide and methane. Nitrous oxide is formed during nitrification and denitrification of ammonia that enters the soil during microbial decomposition of complex organic compounds. Denitrification can be direct and indirect. In the microbiological process of recovery of nitrates involved of the organic substance. In aerobic conditions microorganisms denitrificator behave like normal saprotrophs and oxidize organic matter in the act of breathing oxygen. Thus, they operate at different times two enzyme systems: the electron transport chain with an oxygen acceptor in aerobic and restoration of nitrates under anaerobic conditions. Investigation of the emission of nitrous oxide by ordinary Chernozem steppe of the Central-Chernozem Region showed that it depends on the type of cenosis and the content of available forms of nitrogen. Natural ecosystems emit nitrous oxide more than the soil of arable land. The dependence of the emission of nitrous oxide from the humus content shows positive trend, but the aggregation of data, significant differences are not detected. Research shows that nitrous oxide emissions are seasonal. So the autumn season is characterized by nitrous oxide emissions than spring. Enzymatic processes are an important link in the biological cycle of elements and, consequently, participate in the process of decomposition of organic matter, nitrification and other processes. Analysis of the data on enzyme activity of ordinary Chernozem and the intensity of emission of N20 shows a clear relationship between

  2. Angiotensin-I converting enzyme inhibitory and antioxidant activity of bioactive peptides produced by enzymatic hydrolysis of skin from grass carp (Ctenopharyngodon idella)

    DEFF Research Database (Denmark)

    Yi, Jierong; De Gobba, Cristian; Skibsted, Leif Horsfelt


    Grass carp skin pieces were homogenized in water and hydrolyzed by Alcalase®, collagenase, proteinase K, and/or trypsin at their optimum conditions. Samples were taken at various degrees of hydrolysis and were evaluated for antioxidant, antimicrobial, and angiotensin-converting enzyme inhibitory...... activities. Alcalase and collagenase completely hydrolyzed the skin with different rates, and released peptides with antioxidant and angiotensin-converting enzyme-inhibitory activity. These activities increased linearly with increasing degrees of hydrolysis. Subsequent incubation of the collagenase...... hydrolysates with trypsin slightly increased the antioxidant activity. Proteinase K, although only partially hydrolyzing the skin, also catalyzed the release of peptides with antioxidant and angiotensin-converting enzyme-inhibitory activities. These results show that skin by-products from grass carp can...

  3. Glycolic acid inhibits enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper snake venom: insights from docking and molecular modeling. (United States)

    Pereañez, Jaime Andrés; Patiño, Arley Camilo; Rey-Suarez, Paola; Núñez, Vitelbina; Henao Castañeda, Isabel Cristina; Rucavado, Alexandra


    Glycolic acid (GA) (2-Hydroxyethanoic acid) is widely used as chemical peeling agent in Dermatology and, more recently, as a therapeutic and cosmetic compound in the field of skin care and disease treatment. In this work we tested the inhibitory ability of glycolic acid on the enzymatic, hemorrhagic and edema-inducing activities of BaP1, a P-I metalloproteinase from Bothrops asper venom, which induces a variety of toxic actions. Glycolic acid inhibited the proteolytic activity of BaP1 on azocasein, with an IC₅₀ of 1.67 mM. The compound was also effective at inhibiting the hemorrhagic activity of BaP1 in skin and muscle in experiments involving preincubation of enzyme and inhibitor prior to injection. When BaP1 was injected i.m. and then, at the same site, different concentrations of glycolic acid were administered at either 0 or 5 min, 7 mM solutions of the inhibitor partially abrogated hemorrhagic activity when administered at 0 min. Moreover, glycolic acid inhibited, in a concentration-dependent manner, edema-forming activity of BaP1 in the footpad. In order to have insights on the mode of action of glycolic acid, UV-vis and intrinsic fluorescence studies were performed. Results of these assays suggest that glycolic acid interacts directly with BaP1 and chelates the Zn²⁺ ion at the active site. These findings were supported by molecular docking results, which suggested that glycolic acid forms hydrogen bonds with residues Glu143, Arg110 and Ala111 of the enzyme. Additionally, molecular modeling results suggest that the inhibitor chelates Zn²⁺, with a distance of 3.58 Å, and may occupy part of substrate binding cleft of BaP1. Our results suggest that glycolic acid is a candidate for the development of inhibitors to be used in snakebite envenomation.

  4. A Comparison between the Effects of Albendazole and Meben¬ dazole on the Enzymatic Activity of Excretory / Secretory Prod-ucts of Echinococcus granulosus Protoscoleces in Vitro

    Directory of Open Access Journals (Sweden)

    Seyed Jafar ADNANI SADATI


    Full Text Available Background: Hydatid cysts are formed in human body can be treated clinically by surgery or drugs such as albendazole (ABZ and mebendazole (MBZ. The purpose of this study was comparing the effects of ABZ and MBZ on glutathione-S-transferase, alkaline phosphatase and protease enzymes activities in protoscoleces of hydatid cyst. Methods: The culture supernatants containing the parasite Excretory / Secretory (E/S products were collected every 12 h for 72 h. The E/S products of treated samples with 1µg/ml ABZ and MBZ and the control one were collected and after centrifugation then protein concentrations were measured according to Bradford method. GST, ALP and protease activities of E/S products were assessed photometrically.Results: The mean of GST specific activity level in treated protoscoleces with ABZ and MBZ and in control group were obtained 69.44, 132.83 and 225.47U/mg/protein/ml respectively. The mean ALP activity level in treated protoscoleces with ABZ and MBZ and in control group were detected 19.22, 22.27 and 27.85 U/mg/protein/ml respectively. The protease activity level in treated protoscoleces with ABZ and MBZ were not detected. While the mean of protease activity level in control group was 7.61U/mg/proteins. Statistical analysis showed the significant difference between protein concentrations, the specific activities of GST, ALP and protease enzymes in treated protoscoleces in comparison with control group (P<0.05. Also, the significant difference were seen between specific activities of GST and ALP enzymes in treated protoscoleces with ABZ in comparison with treated group with MBZ (P<0.05.Conclusion: ABZ is more effective on the enzymes activities (GST and ALP as compared with MBZ. Keywords: Hydatid cyst protoscoleces, Albendazole, Mebandazole, Protease, Glutathione S-Transferase, Alkaline phosphatase

  5. Mechanistic insight into sono-enzymatic degradation of organic pollutants with kinetic and thermodynamic analysis. (United States)

    Malani, Ritesh S; Khanna, Swati; Chakma, Sankar; Moholkar, Vijayanand S


    In this paper, we have attempted to get a physical insight into process of sono-enzymatic treatment for degradation of recalcitrant organic pollutants. Decolourization of an azo dye has been used as model reaction with different experimental protocols that alter characteristics of ultrasound and cavitation phenomena in the system. Experimental data is analyzed to determine kinetic and thermodynamic parameters of decolorization process. The trends observed in kinetic and thermodynamic parameters of decolourization are essentially manifestations of the dominating mechanism of the decolorization of the textile dye (or nature of prevalent chemical reaction in the system), viz. either molecular reaction due to enzyme or radical reaction due to transient cavitation. The activation energy for sonochemical protocol is negative, which indicates instantaneity of the radical reactions. The frequency factor is also low, which is attributed to high instability of radicals. For enzymatic and sono-enzymatic protocols, activation energy is positive with higher frequency factor. Enthalpy change for sonochemical protocol is negative, while that for enzymatic and sono-enzymatic protocols is positive. The net entropy change for sonochemical protocol is more negative than enzymatic or sono-enzymatic protocol due to differences in prevalent chemical mechanism of dye decolorization. Due to inverse variations of frequency factor and activation energy, marginal rise in reaction kinetics is seen for sono-enzymatic protocol, as compared to enzymatic treatment alone. Due to inverse variations of enthalpy and entropy change, net Gibbs energy change in all experimental protocols shows little variation indicating synergism of the mechanism of ultrasound and enzyme.

  6. [Effect of population density on enzymatic activity of antioxidative and phenol oxidase systems of imagoes and nymphs of the marble cockroach Nauphoeta cinerea]. (United States)

    Murzagulov, G S; Saltykova, E S; Gaĭfullina, L R; Nikolenko, A G


    The work deals with effect of density of population on functional activity of components pf protective system of adult individuals and nymphs of the marble cockroach. The resistance of individuals has been noted to decrease both at individual maintenance and under conditions of overpopulation. Changes in activities of enzymes of antioxidative and phenoloxidase systems are studied ion the insect hemolymph and intestine. Possible consequences of isolation and overpopulation are discussed both for stability and for individual development.

  7. Study on Correlation Between Effect of Winter Jujube Storage and Related Enzymatic Activity%冬枣贮藏效果与相关酶活性的相关研究

    Institute of Scientific and Technical Information of China (English)

    胡波; 谢志兵; 姚国新; 李国元


    Using winter jujube as experimental materials ,the correlation analysis ,path analysis ,and grey correlation analysis on related enzymatic activity and fruit firmness under low temperature stor‐age are studied .The analytical results show that the degree of correlation between the related enzy‐matic activity and the firmness are PG ,SOD ,CAT ,PME and POD ,respectively .And the contribution rates of fruit firmness are PG ,CAT ,SOD ,PME ,and POD ,respectively .While the degree of associa‐tion with fruit firmness are PME ,SOD ,PG ,CAT ,and POD ,respectively .Based upon the above com‐prehensive analyses ,the PG activity ,the CAT activity and the SOD activity are the major enzymes dur‐ing fruit storage ,which are important to fruit softening and senescence .%以鄂北冬枣为材料,对果实采后低温贮藏过程中相关酶活性与果实硬度进行相关分析、通径分析和灰色关联分析。分析结果表明,各相关酶活性与硬度的相关程度依次为PG、SOD、CAT、PME、POD ,与果实硬度的贡献率大小依次为 PG、CAT、SOD、PME、POD ,与果实硬度的关联度顺序为 PME、SOD、PG、CAT、POD。综合分析结果表明,PG活性、CAT活性、SOD活性为果实贮藏过程中主要的酶,在贮藏过程中对果实软化衰老起重要作用。

  8. Analysis on enzymatic browning in pine needles

    Energy Technology Data Exchange (ETDEWEB)

    Kong, K.H.; Park, H.J.; Choi, S.S.; Cho, S.H. [Chung-Ang University, Seoul (Korea); Kim, Y.T. [Aoyama Gakuin University, Tokyo (Japan)


    Tyrosinases are related to the enzymatic browning of plants and attract the major scientific interest for the prevention of it. Three tyrosinase isozymes (P{sub 1}, P{sub 2} and P{sub 3}) from pine needles were purified to homogeneity and characterized the factors that affect their activities. The L-ascorbic acid and {beta}-mercaptoethanol notably inhibited the enzymatic activities of the three isozymes. The sodium diethyldithiocarbamate was a competitive inhibitor of isozymes with the K{sub i} values of P{sub 1}(0.30 mM), P{sub 2}(0.015 mM) and P{sub 3}(0.019 mM), respectively. Their enzyme activities were however, increased by the addition of most metal ions. The optimum pH for the three isozymes was 9.0{approx}9.5 and the optimum temperatures ranged from 55 to 60{sup o} C using L-DOPA as substrate. 15 refs., 3 figs., 2 tabs.

  9. Bioluminescence methods for enzymatic determinations (United States)

    Bostick, William D.; Denton, Mark S.; Dinsmore, Stanley R.


    An enzymatic method for continuous, on-line and rapid detection of diagnostically useful biomarkers, which are symptomatic of disease or trauma-related tissue damage, is disclosed. The method is characterized by operability on authentic samples of complex biological fluids which contain the biomarkers.

  10. Hepatic monooxygenase (CYP1A and CYP3A) and UDPGT enzymatic activities as biomarkers for long-term carbofuran exposure in tench (Tinca tinca L). (United States)

    Hernández-Moreno, David; Soler-Rodríguez, Francisco; Míguez-Santiyán, M Prado; Pérez-López, Marcos


    The effect of a long-term exposure of tenchs to different concentrations (10 and 100 micro g/L) of the pesticide carbofuran has been evaluated. Microsomal hepatic cytochrome P450 subfamily 1A (CYP1A) and 3A (CYP3A) activities, as well as the phase II enzyme uridine diphospho-glucuronosyltransferase (UDPGT) activity were evaluated as adequate biomarkers of fish exposure to environmentally relevant concentrations of the pesticide carbofuran in freshwater ecosystems. A clear time-dependent inhibition of both CYP1A and UDPGT activities was observed in fish exposed to the highest dose of carbofuran with respect to controls, whereas in the case of CYP3A activity, values of exposed animals did not show a clear pattern of alteration during the experiment. The results of the present study demonstrated that hepatic CYP1A and UDPGT activities from tench could be considered as sensitive biomarkers for carbamate pesticides in polluted water, thus allowing future and ecologically relevant biomonitoring studies with this species.

  11. A novel intestinal trans-factor (NF-LPH1) interacts with the lactase-phlorizin hydrolase promoter and co-varies with the enzymatic activity

    DEFF Research Database (Denmark)

    Troelsen, J T; Norén, O; Sjöström, H


    The promoter of the pig lactase-phlorizin hydrolase was cloned and showed to be functional in the human intestinal cell line Caco2. The proximal promoter was analyzed for binding of nuclear proteins from small intestine and liver. DNase I footprinting and electrophoretic mobility shift assays show......, that an intestinal nuclear factor (NF-LPH1) binds to a sequence (-40 to -54) located close to the TATA-box. Enterocytes from newborn pigs with high lactase activity contain high amounts of NF-LPH1, whereas enterocytes from adult pigs with low lactase activity contain low amounts of NF-LPH1. The liver does...... not contain lactase activity, and NF-LPH1 is not present in liver nuclear extracts in detectable amounts. This indicates that NF-LPH1 is involved in the decline of lactase at weaning and may be of importance for the molecular explanation of hypolactasia in humans. It was demonstrated by transfection of two...

  12. Expression and enzymatic activity of phenylalanine ammonia-lyase and p-coumarate 3-hydroxylase in mango (Mangifera indica 'Ataulfo') during ripening. (United States)

    Palafox-Carlos, H; Contreras-Vergara, C A; Muhlia-Almazán, A; Islas-Osuna, M A; González-Aguilar, G A


    Phenylalanine ammonia lyase (PAL) and p-coumarate 3-hydroxylase (C3H) are key enzymes in the phenylpropanoid pathway. The relative expression of PAL and C3H was evaluated in mango fruit cultivar 'Ataulfo' in four ripening stages (RS1, RS2, RS3, and RS4) by quantitative polymerase chain reaction. In addition, enzyme activity of PAL and C3H was determined in mango fruits during ripening. The PAL levels were downregulated at the RS2 and RS3 stages, while C3H levels were upregulated in fruits only at RS3. The enzyme activity of PAL followed a pattern that was different from that of the PAL expression, thus suggesting regulation at several levels. For C3H, a regulation at the transcriptional level is suggested because a similar pattern was revealed by its activity and transcript level. In this study, the complexity of secondary metabolite biosynthesis regulation is emphasized because PAL and C3H enzymes are involved in the biosynthesis of several secondary metabolites that are active during all mango ripening stages.

  13. Soil microbiological properties and enzymatic activities of long-term post-fire recovery in dry and semiarid Aleppo pine (Pinus halepensis M.) forest stands (United States)

    Hedo, J.; Lucas-Borja, M. E.; Wic, C.; Andrés-Abellán, M.; de Las Heras, J.


    Wildfires affecting forest ecosystems and post-fire silvicultural treatments may cause considerable changes in soil properties. The capacity of different microbial groups to recolonise soil after disturbances is crucial for proper soil functioning. The aim of this work was to investigate some microbial soil properties and enzyme activities in semiarid and dry Aleppo pine (Pinus halepensis M.) forest stands. Different plots affected by a wildfire event 17 years ago without or with post-fire silvicultural treatments 5 years after the fire event were selected. A mature Aleppo pine stand, unaffected by wildfire and not thinned was used as a control. Physicochemical soil properties (soil texture, pH, carbonates, organic matter, electrical conductivity, total N and P), soil enzymes (urease, phosphatase, β-glucosidase and dehydrogenase activities), soil respiration and soil microbial biomass carbon were analysed in the selected forests areas and plots. The main finding was that long time after this fire event produces no differences in the microbiological soil properties and enzyme activities of soil after comparing burned and thinned, burned and not thinned, and mature plots. Moreover, significant site variation was generally seen in soil enzyme activities and microbiological parameters. We conclude that total vegetation recovery normalises post-fire soil microbial parameters, and that wildfire and post-fire silvicultural treatments are not significant factors affecting soil properties after 17 years.

  14. Enzymatic activities and arbuscular mycorrhizal colonization of Plantago lanceolata and Plantago major in a soil root zone under heavy metal stress. (United States)

    Gucwa-Przepióra, Ewa; Nadgórska-Socha, Aleksandra; Fojcik, Barbara; Chmura, Damian


    The objectives of the present field study were to examine the soil enzyme activities in the soil root zones of Plantago lanceolata and Plantago major in different heavy metal contaminated stands. Moreover, the investigations concerned the intensity of root endophytic colonization and metal bioaccumulation in roots and shoots. The investigated Plantago species exhibited an excluder strategy, accumulating higher metal content in the roots than in the shoots. The heavy metal accumulation levels found in the two plantain species in this study were comparable to other plants suggested as phytostabilizers; therefore, the selected Plantago species may be applied in the phytostabilization of heavy metal contaminated areas. The lower level of soil enzymes (dehydrogenase, urease, acid, and alkaline phosphatase) as well as the higher bioavailability of metals in the root zone soil of the two plantain species were found in an area affected by smelting activity, where organic matter content in the soil was also the smallest. Mycorrhizal colonization on both species in the contaminated area was similar to colonization in non-contaminated stands. However, the lowest arbuscule occurrence and an absence of dark septate endophytes were found in the area affected by the smelting activity. It corresponded with the lowest plant cover observed in this stand. The assessment of enzyme activity, mycorrhizal colonization, and the chemical and physical properties of soils proved to be sensitive to differences between sites and between Plantago species.

  15. Effect of ageing and ischemia on enzymatic activities linked to Krebs' cycle, electron transfer chain, glutamate and aminoacids metabolism of free and intrasynaptic mitochondria of cerebral cortex. (United States)

    Villa, Roberto Federico; Gorini, Antonella; Hoyer, Siegfried


    The effect of ageing and the relationships between the catalytic properties of enzymes linked to Krebs' cycle, electron transfer chain, glutamate and aminoacid metabolism of cerebral cortex, a functional area very sensitive to both age and ischemia, were studied on mitochondria of adult and aged rats, after complete ischemia of 15 minutes duration. The maximum rate (Vmax) of the following enzyme activities: citrate synthase, malate dehydrogenase, succinate dehydrogenase for Krebs' cycle; NADH-cytochrome c reductase as total (integrated activity of Complex I-III), rotenone sensitive (Complex I) and cytochrome oxidase (Complex IV) for electron transfer chain; glutamate dehydrogenase, glutamate-oxaloacetate-and glutamate-pyruvate transaminases for glutamate metabolism were assayed in non-synaptic, perikaryal mitochondria and in two populations of intra-synaptic mitochondria, i.e., the light and heavy mitochondrial fraction. The results indicate that in normal, steady-state cerebral cortex, the value of the same enzyme activity markedly differs according (a) to the different populations of mitochondria, i.e., non-synaptic or intra-synaptic light and heavy, (b) and respect to ageing. After 15 min of complete ischemia, the enzyme activities of mitochondria located near the nucleus (perikaryal mitochondria) and in synaptic structures (intra-synaptic mitochondria) of the cerebral tissue were substantially modified by ischemia. Non-synaptic mitochondria seem to be more affected by ischemia in adult and particularly in aged animals than the intra-synaptic light and heavy mitochondria. The observed modifications in enzyme activities reflect the metabolic state of the tissue at each specific experimental condition, as shown by comparative evaluation with respect to the content of energy-linked metabolites and substrates. The derangements in enzyme activities due to ischemia is greater in aged than in adult animals and especially the non-synaptic and the intra-synaptic light

  16. Enzymatic activation of double-targeted 5'-O-L-valyl-decitabine prodrug by biphenyl hydrolase-like protein and its molecular design basis. (United States)

    Tao, Wenhui; Zhao, Dongyang; Sun, Mengchi; Li, Meng; Zhang, Xiangyu; He, Zhonggui; Sun, Yinghua; Sun, Jin


    A primary focus of this research was to explore the activation process and mechanism of decitabine (5-aza-2'-deoxycytidine, DAC) prodrug. Recently, it has been reported that biphenyl hydrolase-like protein (BPHL) can play an important role in the activation of some amino acid nucleoside prodrugs with a general preference for hydrophobic amino acids and 5'-esters. Therefore, we put forward a bold hypothesis that this novel enzyme may be primarily responsible for the activation process of DAC prodrug as well. 5'-O-L-valyl-decitabine (L-val-DAC) was synthesized before and can be transported across biological membranes by the oligopeptide transporter (PEPT1), granting it much greater utility in vivo. In this report, L-val-DAC was found to be a good substrate of BPHL protein (K m 0.59 mM; k cat/K m 553.69 mM(-1) s(-1)). After intestinal absorption, L-val-DAC was rapidly and almost completely hydrolyzed to DAC and L-valine. The catalysis was mainly mediated by the BPHL hydrolase and resulted in the intestinal first-pass effect of L-val-DAC after oral administration in Sprague-Dawley rats with cannulated jugular and portal veins. The structural insights using computational molecular docking showed that BPHL had a unique binding mode for L-val-DAC. As a fundamental basis, the simulation was employed to explain the catalytic mechanism in molecular level. In conclusion, BPHL was at least one of the primary candidate enzymes for L-val-DAC prodrug activation. This promising double-targeted prodrug approach have more advantages than the traditional targeted designs due to its higher transport and more predictable activation, thereby leading to a favorable property for oral delivery.

  17. Kinetic modelling of enzymatic starch hydrolysis

    NARCIS (Netherlands)

    Bednarska, K.A.


    Kinetic modelling of enzymatic starch hydrolysis – a summary K.A. Bednarska The dissertation entitled ‘Kinetic modelling of enzymatic starch hydrolysis’ describes the enzymatic hydrolysis and kinetic modelling of liquefaction and saccharification of wheat starch. A

  18. Enzymatic modification of starch

    DEFF Research Database (Denmark)

    Jensen, Susanne Langgård

    In the food industry approaches for using bioengineering are investigated as alternatives to conventional chemical and physical starch modification techniques in development of starches with specific properties. Enzyme-assisted post-harvest modification is an interesting approach to this, since...... branching connecting larger chain segments. In case of high BE activity this transfer happened prior to hydration and phase separation. The starch substrates thereby became locked in their granular structure and blocked furher access of BE. Transferase-based modification of starch has today almost...... exclusively been conducted on gelatinized starch. This study provides a new concept for transferase-based modification of starches in granular state....

  19. Enzymatic synthesis of farnesyl laurate in organic solvent: initial water activity, kinetics mechanism, optimization of continuous operation using packed bed reactor and mass transfer studies. (United States)

    Rahman, N K; Kamaruddin, A H; Uzir, M H


    The influence of water activity and water content was investigated with farnesyl laurate synthesis catalyzed by Lipozyme RM IM. Lipozyme RM IM activity depended strongly on initial water activity value. The best results were achieved for a reaction medium with an initial water activity of 0.11 since it gives the best conversion value of 96.80%. The rate constants obtained in the kinetics study using Ping-Pong-Bi-Bi and Ordered-Bi-Bi mechanisms with dead-end complex inhibition of lauric acid were compared. The corresponding parameters were found to obey the Ordered-Bi-Bi mechanism with dead-end complex inhibition of lauric acid. Kinetic parameters were calculated based on this model as follows: V (max) = 5.80 mmol l(-1) min(-1) g enzyme(-1), K (m,A) = 0.70 mmol l(-1) g enzyme(-1), K (m,B) = 115.48 mmol l(-1) g enzyme(-1), K (i) = 11.25 mmol l(-1) g enzyme(-1). The optimum conditions for the esterification of farnesol with lauric acid in a continuous packed bed reactor were found as the following: 18.18 cm packed bed height and 0.9 ml/min substrate flow rate. The optimum molar conversion of lauric acid to farnesyl laurate was 98.07 ± 0.82%. The effect of mass transfer in the packed bed reactor has also been studied using two models for cases of reaction limited and mass transfer limited. A very good agreement between the mass transfer limited model and the experimental data obtained indicating that the esterification in a packed bed reactor was mass transfer limited.

  20. Production Response and Digestive Enzymatic Activity of the Pacific White Shrimp Litopenaeus vannamei (Boone, 1931 Intensively Pregrown in Microbial Heterotrophic and Autotrophic-Based Systems

    Directory of Open Access Journals (Sweden)

    Manuel J. Becerra-Dórame


    Full Text Available Shrimp postlarvae were reared into different microcosm systems without water exchange; a traditional system based on simple fertilization to improve microalgae concentration (control, an autotrophic system (AS based on the promotion of biofloc and biofilm by the addition of fertilizer and artificial substrates and a heterotrophic system (HS based on the promotion of heterotrophic bacteria by the addition of nitrogenous and carbonaceous sources and artificial substrates. Better growth performance and survival were registered in shrimp from the AS and HS compared to the control. Feed conversion ratios were below 0.7 for all treatments, but AS and HS were significantly lower than the control. Regarding digestive performance, no significant differences were observed for trypsin, amylase and lipase activities among AS and control shrimp; however, shrimp from HS showed a higher trypsin and amylase activities, suggesting a higher digestive activity caused by the presence of microbial bioflocs. The presence of biofilm and bioflocs composed by either autotrophic or heterotrophic organisms in combination with formulated feed improved the growth performance and survival of shrimp. Apparently, such combination fits the nutritional requirements of shrimp.

  1. Reconstruction of enzymatic activity from split genes encoding glyphosate-tolerant EPSPS protein of Psedomonas fluorescens G2 strain by intein mediated protein complementation

    Institute of Scientific and Technical Information of China (English)

    DUN Baoqing; ZHAO Zhonglin; LIANG Aimin; HOU Songna; XU Ming-Qun; LIN Min; LU Wei; ZHANG Wei; PING Shuzhen; WANG Xujing; CHEN Ming; XU Yuquan; JIN Dan; WANG Jin


    A mutagenesis library was constructed using GPS-LS system to insert a random 5 aa into the 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoded by aroA gene. Active EPSPS proteins were identified by the ability to rescue growth of aroA-deleted mutant ER2799 on M9 minimal media.12 unique sites, which can tolerate a 5-aa insertion,were identified. In all of the 12 sites, only F295/T296site was found to split the G2-EPSPS properly by co-transformation of plasmids into E. coli ER2799.The G2-EPSPS gene was then divided into N-terminal and C-terminal from F295/T296 site which were fused to the N-terminal and C-terminal of Ssp. DnaE intein, respectively, creating two plasmids pMEPSN295IN and pKEPSc296Ic. Co-transformation of plasmids, pMEPSN295IN and pKEPSc296Ic, rescued growth of ER2799 in M9 minimal media, indicating that the intein splicing domains were bringing the EPSPS fragments together to generate activity. Reconsituted activity of splitted G2-EPSPS enzyme was 4.48 U/mg.

  2. Production response and digestive enzymatic activity of the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) intensively pregrown in microbial heterotrophic and autotrophic-based systems. (United States)

    Becerra-Dórame, Manuel J; Martínez-Porchas, Marcel; Martínez-Córdova, Luis R; Rivas-Vega, Martha E; Lopez-Elias, José A; Porchas-Cornejo, Marco A


    Shrimp postlarvae were reared into different microcosm systems without water exchange; a traditional system based on simple fertilization to improve microalgae concentration (control), an autotrophic system (AS) based on the promotion of biofloc and biofilm by the addition of fertilizer and artificial substrates and a heterotrophic system (HS) based on the promotion of heterotrophic bacteria by the addition of nitrogenous and carbonaceous sources and artificial substrates. Better growth performance and survival were registered in shrimp from the AS and HS compared to the control. Feed conversion ratios were below 0.7 for all treatments, but AS and HS were significantly lower than the control. Regarding digestive performance, no significant differences were observed for trypsin, amylase and lipase activities among AS and control shrimp; however, shrimp from HS showed a higher trypsin and amylase activities, suggesting a higher digestive activity caused by the presence of microbial bioflocs. The presence of biofilm and bioflocs composed by either autotrophic or heterotrophic organisms in combination with formulated feed improved the growth performance and survival of shrimp. Apparently, such combination fits the nutritional requirements of shrimp.

  3. Polyphenolic content and antioxidant properties of Moringa oleifera leaf extracts and enzymatic activity of liver from goats supplemented with Moringa oleifera leaves/sunflower seed cake. (United States)

    Moyo, B; Oyedemi, S; Masika, P J; Muchenje, V


    The study investigated antioxidant potency of Moringa oleifera leaves in different in vitro systems using standard phytochemical methods. The antioxidative effect on the activities of superoxide dismutase (SOD), catalase (CAT), lipid peroxidation (LPO) and reduced glutathione (GSH) were investigated in goats supplemented with M. oleifera (MOL) or sunflower seed cake (SC). The acetone extract had higher concentrations of total flavonoids (295.01 ± 1.89 QE/g) followed by flavonols (132.74 ± 0.83 QE/g), phenolics (120.33 ± 0.76 TE/g) and then proanthocyanidins (32.59 ± 0.50 CE/g) than the aqueous extract. The reducing power of both solvent extracts showed strong antioxidant activity in a concentration dependent manner. The acetone extract depicted higher percentage inhibition against DPPH, ABTS and nitric oxide radicals which were comparable with reference standard antioxidants (vitamin C and BHT). MOL increased the antioxidant activity of GSH (186%), SOD (97.8%) and catalase (0.177%). Lipid peroxidation was significantly reduced by MOL. The present study suggests that M. oleifera could be a potential source of compounds with strong antioxidant potential.

  4. A novel familial mutation in the PCSK1 gene that alters the oxyanion hole residue of proprotein convertase 1/3 and impairs its enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Michael Wilschanski

    Full Text Available Four siblings presented with congenital diarrhea and various endocrinopathies. Exome sequencing and homozygosity mapping identified five regions, comprising 337 protein-coding genes that were shared by three affected siblings. Exome sequencing identified a novel homozygous N309K mutation in the proprotein convertase subtilisin/kexin type 1 (PCSK1 gene, encoding the neuroendocrine convertase 1 precursor (PC1/3 which was recently reported as a cause of Congenital Diarrhea Disorder (CDD. The PCSK1 mutation affected the oxyanion hole transition state-stabilizing amino acid within the active site, which is critical for appropriate proprotein maturation and enzyme activity. Unexpectedly, the N309K mutant protein exhibited normal, though slowed, prodomain removal and was secreted from both HEK293 and Neuro2A cells. However, the secreted enzyme showed no catalytic activity, and was not processed into the 66 kDa form. We conclude that the N309K enzyme is able to cleave its own propeptide but is catalytically inert against in trans substrates, and that this variant accounts for the enteric and systemic endocrinopathies seen in this large consanguineous kindred.

  5. Avaliação do tempo de secagem e da atividade de óxido-redutases de yacon (Smallanthus sonchifolius sob tratamento químico Drying evaluation time and yacon (Smallanthus sonchifolius enzymatic activity inhibition under chemical treatment

    Directory of Open Access Journals (Sweden)

    Vivianne Montarroyos Padilha


    project aimed to evaluate the use of chemical agents in yacon processing to obtain flour in a way that inhibits enzymatic darkening of the product besides determining the enzymatic activity in these treatments. Samples of yacon without chemical inhibitions, yacon treated with 1.0g 100g-1 calcium chloride for 30 seconds and yacon treated with 0.5g 100g-1 potassium metabisulfite for 5 minutes were dried at 55oC in a ventilated greenhouse and the proportions of humidity and drying curves were determined. The peroxidase activities and polyphenol oxidase enzymes were checked before and after being dried with an enzymatic darkening possible biochemist marker of this tubercle. Regarding humidity Parameter all the three treatment were equivalent, but treatment 2 (calcium chloride reduced the humidity in lower time. Before and after the thermal treatment the enzymatic activity was higher in treatment 3 (potassium metabisulfite. The thermal action did not inhibit completely polyphenol oxidase and peroxidase. The treatment with calcium chloride at 1.0g 100g-1 for 30 minutes to obtain yacon flour was the one with better result despite the fact that it did not inhibit completely peroxidase and polyphenol oxidase enzymes activity, offering a better drying time better material of row firmness , facilitating the process for obtaining the meal.

  6. 15-Deoxy-delta(12,14)-prostaglandin J(2) inhibits IL-1beta-induced IKK enzymatic activity and IkappaBalpha degradation in rat chondrocytes through a PPARgamma-independent pathway. (United States)

    Boyault, Sandrine; Bianchi, Arnaud; Moulin, David; Morin, Sylvie; Francois, Mathias; Netter, Patrick; Terlain, Bernard; Bordji, Karim


    Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands have been shown to inhibit the effects of proinflammatory cytokines such as interleukin-1beta (IL-1beta). This cytokine plays a key role in articular pathophysiologies by inducing the production of inflammatory mediators such as nitric oxide (NO) and prostaglandin E(2) (PGE(2)). We previously demonstrated that 15d-PGJ(2) was more potent than troglitazone to counteract IL-1beta effects on chondrocytes. Here, we studied the action of 15d-PGJ(2) on intracellular targets in nuclear factor-kappaB (NF-kappaB) signalling pathway in IL-1beta treated rat chondrocytes. We found that 15d-PGJ(2) decreased inhibitor kappaBalpha (IkappaBalpha) degradation but not its phosphorylation by specifically inhibiting IkappaB kinase beta (IKKbeta), but not IKKalpha, enzymatic activity. We further evaluated the involvement of PPARgamma in the anti-inflammatory action of its ligands. In chondrocytes overexpressing functional PPARgamma protein, 15d-PGJ(2) pre-treatment inhibited inducible NO synthase and COX-2 mRNA expression, nitrite and PGE(2) production, p65 translocation and NF-kappaB activation. Troglitazone or rosiglitazone pre-treatment had no effect. 15d-PGJ(2) exhibited the same effect in chondrocytes overexpressing mutated PPARgamma protein. These results suggest that 15d-PGJ(2) exerts its anti-inflammatory effect in rat chondrocytes by a PPARgamma-independent mechanism, which can be conferred to a partial inhibition of IkappaBalpha degradation.

  7. 冰温贮藏对绿芦笋品质及酶活性的影响%Effect of ice-temperature preservation on quality and enzymatic activity of Green Asparagus

    Institute of Scientific and Technical Information of China (English)

    宋秀香; 鲁晓翔; 陈绍慧; 李江阔


    To 4℃ refrigerated storage for comparison,the effect of ice-temperature preservation on quality and enzymatic activity of Green Asparagus was investigated.The results showed that the storage life of Green asparagus was increased 14d,and its organoleptic quality was improved by ice-temperature preservation.While the firmness and tincture of Green asparagus was kept,the decrease of the V c content was slowed down,and its commodity value was improved by ice-temperature preservation.The activity of PPO and POD of Green asparagus was increased,and the activity of PAL was decreased under ice-temperature.So the protection ability of Green asparagus was enhanced,and the aging of Green asparagus was delayed.The control effect of ice-temperature preservation on respiration and ethylene production was unconspicuous.%以4℃冷藏作为对照,研究了冰温贮藏对绿芦笋品质及酶活性的影响.结果表明:冰温贮藏延长了绿芦笋贮藏期14d,提高其感官品质;同时,冰温贮藏保持了绿芦笋的硬度和色泽,减缓了Vc含量的降低,提高了其商品价值;冰温条件下绿芦笋多酚氧化酶(PPO)和过氧化物酶(POD)的活性提高,苯丙氨酸解氨酶(PAL)活性降低,增强了自身保护能力,延缓绿芦笋衰老;但冰温贮藏对绿芦笋呼吸作用和乙烯生成量的抑制效果不明显.

  8. An active site mutant of Escherichia coli cyclopropane fatty acid synthase forms new non-natural fatty acids providing insights on the mechanism of the enzymatic reaction. (United States)

    E, Guangqi; Drujon, Thierry; Correia, Isabelle; Ploux, Olivier; Guianvarc'h, Dominique


    We have produced and purified an active site mutant of the Escherichia coli cyclopropane fatty acid synthase (CFAS) by replacing the strictly conserved G236 within cyclopropane synthases, by a glutamate residue, which corresponds to E146 of the homologous mycolic acid methyltransferase, Hma, producing hydroxymethyl mycolic acids. The G236E CFAS mutant had less than 1% of the in vitro activity of the wild type enzyme. We expressed the G236E CFAS mutant in an E. coli (DE3) strain in which the chromosomal cfa gene had been deleted. After extraction of phospholipids and conversion into the corresponding fatty acid methyl esters (FAMEs), we observed the formation of cyclopropanated FAMEs suggesting that the mutant retained some of the normal activity in vivo. However, we also observed the formation of new C17 methyl-branched unsaturated FAMEs whose structures were determined using GC/MS and NMR analyses. The double bond was located at different positions 8, 9 or 10, and the methyl group at position 10 or 9. Thus, this new FAMEs are likely arising from a 16:1 acyl chain of a phospholipid that had been transformed by the G236E CFAS mutant in vivo. The reaction catalyzed by this G236E CFAS mutant thus starts by the methylation of the unsaturated acyl chain at position 10 or 9 yielding a carbocation at position 9 or 10 respectively. It follows then two competing steps, a normal cyclopropanation or hydride shift/elimination events giving different combinations of alkenes. This study not only provides further evidence that cyclopropane synthases (CSs) form a carbocationic intermediate but also opens the way to CSs engineering for the synthesis of non-natural fatty acids.

  9. Extraction and inhibition of enzymatic activity of botulinum neurotoxins/A1, /A2, and /A3 by a panel of monoclonal anti-BoNT/A antibodies.

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    Suzanne R Kalb

    Full Text Available Botulinum neurotoxins (BoNTs are extremely potent toxins that are capable of causing death or respiratory failure leading to long-term intensive care. Treatment includes serotype-specific antitoxins, which must be administered early in the course of the intoxication. Rapidly determining human exposure to BoNT is an important public health goal. In previous work, our laboratory focused on developing Endopep-MS, a mass spectrometry-based endopeptidase method for detecting and differentiating BoNT/A-G serotypes in buffer and BoNT/A, /B, /E, and /F in clinical samples. We have previously reported the effectiveness of antibody-capture to purify and concentrate BoNTs from complex matrices, such as clinical samples. Because some antibodies inhibit or neutralize the activity of BoNT, the choice of antibody with which to extract the toxin is critical. In this work, we evaluated a panel of 16 anti-BoNT/A monoclonal antibodies (mAbs for their ability to inhibit the in vitro activity of BoNT/A1, /A2, and /A3 complex as well as the recombinant LC of A1. We also evaluated the same antibody panel for the ability to extract BoNT/A1, /A2, and /A3. Among the mAbs, there were significant differences in extraction efficiency, ability to extract BoNT/A subtypes, and inhibitory effect on BoNT catalytic activity. The mAbs binding the C-terminal portion of the BoNT/A heavy chain had optimal properties for use in the Endopep-MS assay.

  10. Crystal Structure of Full-length Mycobacterium tuberculosis H37Rv Glycogen Branching Enzyme; Insights of N-Terminal [beta]-Sandwich in Sustrate Specifity and Enzymatic Activity

    Energy Technology Data Exchange (ETDEWEB)

    Pal, Kuntal; Kumar, Shiva; Sharma, Shikha; Garg, Saurabh Kumar; Alam, Mohammad Suhail; Xu, H. Eric; Agrawal, Pushpa; Swaminathan, Kunchithapadam (NU Sinapore); (Van Andel); (IMT-India)


    The open reading frame Rv1326c of Mycobacterium tuberculosis (Mtb) H37Rv encodes for an {alpha}-1,4-glucan branching enzyme (MtbGlgB, EC, Uniprot entry Q10625). This enzyme belongs to glycoside hydrolase (GH) family 13 and catalyzes the branching of a linear glucose chain during glycogenesis by cleaving a 1 {yields} 4 bond and making a new 1 {yields} 6 bond. Here, we show the crystal structure of full-length MtbGlgB (MtbGlgBWT) at 2.33-{angstrom} resolution. MtbGlgBWT contains four domains: N1 {beta}-sandwich, N2 {beta}-sandwich, a central ({beta}/{alpha}){sub 8} domain that houses the catalytic site, and a C-terminal {beta}-sandwich. We have assayed the amylase activity with amylose and starch as substrates and the glycogen branching activity using amylose as a substrate for MtbGlgBWT and the N1 domain-deleted (the first 108 residues deleted) Mtb{Delta}108GlgB protein. The N1 {beta}-sandwich, which is formed by the first 105 amino acids and superimposes well with the N2 {beta}-sandwich, is shown to have an influence in substrate binding in the amylase assay. Also, we have checked and shown that several GH13 family inhibitors are ineffective against MtbGlgBWT and Mtb{Delta}108GlgB. We propose a two-step reaction mechanism, for the amylase activity (1 {yields} 4 bond breakage) and isomerization (1 {yields} 6 bond formation), which occurs in the same catalytic pocket. The structural and functional properties of MtbGlgB and Mtb{Delta}108GlgB are compared with those of the N-terminal 112-amino acid-deleted Escherichia coli GlgB (EC{Delta}112GlgB).

  11. Isolated and combined exposure to ammonia and nitrite in giant freshwater pawn (Macrobrachium rosenbergii): effects on the oxidative stress, antioxidant enzymatic activities and apoptosis in haemocytes. (United States)

    Zhang, Yufan; Ye, Chaoxia; Wang, Anli; Zhu, Xuan; Chen, Changhong; Xian, Jianan; Sun, Zhenzhu


    The residual contaminators such as ammonia and nitrite are widely considered as relevant sources of aquatic environmental pollutants, posing a great threat to shrimp survival. To study the toxicological effects of ammonia and nitrite exposure on the innate immune response in invertebrates, we investigated the oxidative stress and apoptosis in haemocytes of freshwater prawn (Macrobrachium rosenbergii) under isolated and combined exposure to ammonia and nitrite in order to provide useful information about adult prawn immune responses. M. rosenbergii (13.44 ± 2.75 g) were exposed to 0, 5, and 25 mg/L total ammonia-N (TAN) and 0, 5, and 20 mg/L nitrite-N for 24 h. All ammonia concentrations were combined with all nitrite concentrations, making a total of nine treatments studied. Following the exposure treatment, antioxidant enzyme activity, reactive oxygen species (ROS) generation, nitric oxide (NO) generation, and apoptotic cell ratio of haemocytes were measured using flow cytometry. Results indicated that ROS generation was sensitive to the combined effect of ammonia and nitrite, which subsequently affected the Cu-Zn SOD activity. In addition, CAT showed the highest activity at 5 mg/L TAN while GPx decreased at 5 mg/L TAN and returned towards baseline at 25 mg/L. NO generation synchronized with the apoptotic cell ratio in haemocytes, indicating that NO production was closely associated with programmed cell death. Both NO production and apoptotic ratios significantly decreased following 25 mg/L TAN, which may be due to the antagonistic regulation of NO and GPx. We hypothesized that the toxicological effect of nitrite exhibited less change in physiological changes compared to that of ammonia, because of the high tolerance to nitrite exposure in mature M. rosenbergii and/or the competitive effects of chloride ions. Taken together, these results showed that ammonia and nitrite caused a series of combined oxidative stress and apoptosis in M. rosenbergi, but further

  12. Modulation of the Microenvironment Surrounding the Active Site of Penicillin G Acylase Immobilized on Acrylic Carriers Improves the Enzymatic Synthesis of Cephalosporins

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    Paolo Bonomi


    Full Text Available The catalytic properties of penicillin G acylase (PGA from Escherichia coli in kinetically controlled synthesis of β-lactam antibiotics are negatively affected upon immobilization on hydrophobic acrylic carriers. Two strategies have been here pursued to improve the synthetic performance of PGA immobilized on epoxy-activated acrylic carriers. First, an aldehyde-based spacer was inserted on the carrier surface by glutaraldehyde activation (immobilization yield = 50%. The resulting 3-fold higher synthesis/hydrolysis ratio (vs/vh1 = 9.7 ± 0.7 and 10.9 ± 0.7 for Eupergit® C and Sepabeads® EC-EP, respectively with respect to the unmodified support (vs/vh1 = 3.3 ± 0.4 was ascribed to a facilitated diffusion of substrates and products as a result of the increased distance between the enzyme and the carrier surface. A second series of catalysts was prepared by direct immobilization of PGA on epoxy-activated acrylic carriers (Eupergit® C, followed by quenching of oxiranes not involved in the binding with the protein with different nucleophiles (amino acids, amines, amino alcohols, thiols and amino thiols. In most cases, this derivatization increased the synthesis/hydrolysis ratio with respect to the non derivatized carrier. Particularly, post-immobilization treatment with cysteine resulted in about 2.5-fold higher vs/vh1 compared to the untreated biocatalyst, although the immobilization yield decreased from 70% (untreated Eupergit® C to 20%. Glutaraldehyde- and cysteine-treated Eupergit® C catalyzed the synthesis of cefazolin in 88% (±0.9 and 87% (±1.6 conversion, respectively, whereas untreated Eupergit® C afforded this antibiotic in 79% (±1.2 conversion.

  13. Desempenho e atividades enzimáticas em girinos de rã-touro Performance and enzymatic activities of the bullfrog tadpoles

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    José Teixeira de Seixas Filho


    Full Text Available O objetivo deste trabalho foi avaliar o desempenho e as atividades das enzimas digestivas de girinos de rã-touro, alimentados com ração comercial durante 60 dias, com um girino por litro, em caixas com 30 L de água. Os parâmetros medidos quinzenalmente foram: sobrevivência, comprimento, peso final, consumo de ração, e a atividade de amilase, lipase e tripsina. O maior crescimento dos girinos foi do 15º ao 45º dia - 20,61 mm. Durante os primeiros 15 dias, os girinos apresentaram a maior taxa de crescimento específico, 16,73% por dia. Do 45º ao 60º dia, os girinos apresentaram o maior ganho de peso, 3,98±0,73 g, o maior consumo de ração, 14,40±1,14 g, e a melhor conversão alimentar, 3,67:1,0±0,18. Houve aumento significativo da atividade enzimática depois do 30º dia. Observaram-se relações entre as atividades específicas das enzimas e os distintos estágios de desenvolvimento dos girinos.The objective of this study was to evaluate the performance and the digestive enzyme activity in bullfrog tadpoles, fed with commercial ration during 60 days, at the density of one tadpole per liter in 30 L boxes. The parameters measured fortnightly were: survival, length, weight, ration consumption, and amylase, lipase and trypsin activities. There larger growth of the tadpoles occurred from the 15th to the 45th day - 20.61 mm. During the first 15 days, the tadpoles had the largest specific growth rate, 16.7% per day. From the 45th to the 60th day, the tadpoles had the largest weight gain, 3.98±0.73 g, the largest ration consumption, 14.40±1.14 g and the best feed conversion, 3.67:1.0±0.18. There was significant increase in enzyme activities after the 30th day. Relationships were observed between the specific enzyme activities and the different development phases of the tadpoles.

  14. Gene cloning, characterization and expression and enzymatic activities related to trehalose metabolism during diapause of the onion maggot Delia antiqua (Diptera: Anthomyiidae). (United States)

    Guo, Qiang; Hao, You-Jin; Li, Yuan; Zhang, Yu-Juan; Ren, Shuang; Si, Feng-Ling; Chen, Bin


    Trehalose represents the main hemolymph sugar in many insects, and it functions in energy metabolism and protection in extreme environmental conditions. To gain an insight into trehalose functions in Delia antiqua diapausing pupae, genes encoding trehalose-6-phosphate synthase (TPS), trehalose-6-phosphatase (TPP) and trehalase (TRE) were identified and characterized. Analysis of the deduced amino acid sequences indicated that these genes were highly similar to each homolog from Diptera insects. Gene expressions and their enzyme activities were also investigated. The differential expressions of TPS and TPP shared very similar trends for summer and winter diapausing pupae. Their enzyme activities were consistent with the gene expressions. Trehalose concentrations in summer- and winter-diapausing pupae were lower at the initial phase (4.37-5.09μg/mg) but increased gradually and peaked in the maintenance phase (10.59-14.36μg/mg); the concentrations then declined in the quiescence phase. We speculated that a higher trehalose content during the maintenance stage may contribute to protein and/or biological membrane stabilization in winter or to desiccation resistance in the summertime. Diapause termination requires a decrease in the trehalose concentration to promote pupal-adult development. The glucose content also varied during the diapausing processes. Our results provide an overview of the differential expression levels of trehalose metabolic enzymes, confirming the important roles of trehalose in diapausing pupae of the onion maggot. Further work remains to explore its actual functions.

  15. Ultrasensitive electrochemical immunoassay for DNA methyltransferase activity and inhibitor screening based on methyl binding domain protein of MeCP2 and enzymatic signal amplification. (United States)

    Yin, Huanshun; Zhou, Yunlei; Xu, Zhenning; Wang, Mo; Ai, Shiyun


    In this work, we fabricated a novel electrochemical immunosensor for detection of DNA methylation, analysis of DNA MTase activity and screening of MTase inhibitor. The immunosensor was on the basis of methyl binding domain protein of MeCP2 as DNA CpG methylation recognization unit, anti-His tag antibody as "immuno-bridge" and horseradish peroxidase labeled immuneglobulin G functionalized gold nanoparticles (AuNPs-IgG-HRP) as signal amplification unit. In the presence of M. SssI MTase, the symmetrical sequence of 5'-CCGG-3' was methylated and then recognized by MeCP2 protein. By the immunoreactions, anti-His tag antibody and AuNPs-IgG-HRP was captured on the electrode surface successively. Under the catalysis effect of HRP towards hydroquinone oxidized by H2O2, the electrochemical reduction signal of benzoquinone was used to analyze M. SssI MTase activity. The electrochemical reduction signal demonstrated a wide linear relationship with M. SssI concentration ranging from 0.05 unit/mL to 90 unit/mL, achieving a detection limit of 0.017 unit/mL (S/N=3). The most important advantages of this method were its high sensitivity and good selectivity, which enabled the detection of even one-base mismatched sequence. In addition, we also verified that the developed method could be applied for screening the inhibitors of DNA MTase and for developing new anticancer drugs.

  16. Anti-inflammatory activity of a sulfated polysaccharide isolated from an enzymatic digest of brown seaweed Sargassum horneri in RAW 264.7 cells (United States)

    Sanjeewa, Kalu Kapuge Asanka; Fernando, Ilekkuttige Priyan Shanura; Kim, Eun-A; Jee, Youngheun


    BACKGROUND/OBJECTIVES Sargassum horneri is an edible brown alga that grows in the subtidal zone as an annual species along the coasts of South Korea, China, and Japan. Recently, an extreme amount of S. horneri moved into the coasts of Jeju Island from the east coast of China, which made huge economic and environmental loss to the Jeju Island. Thus, utilization of this biomass becomes a big issue with the local authorities. Therefore, the present study was performed to evaluate the anti-inflammatory potential of crude polysaccharides (CPs) extracted from S. horneri China strain in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. MATERIALS/METHODS CPs were precipitated from S. horneri digests prepared by enzyme assistant extraction using four food-grade enzymes (AMG, Celluclast, Viscozyme, and Alcalase). The production levels of nitric oxide (NO) and pro-inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-1β were measured by Griess assay and enzyme-linked immunosorbent assay, respectively. The levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), nuclear factor (NF)-κB, and mitogen-activated protein kinases (MAPKs) were measured by using western blot. The IR spectrums of the CPs were recorded using a fourier transform infrared spectroscopy (FT-IR) spectrometer. RESULTS The polysaccharides from the Celluclast enzyme digest (CCP) showed the highest inhibition of NO production in LPS-stimulated RAW 264.7 cells (IC50 value: 95.7 µg/mL). Also, CCP dose-dependently down-regulated the protein expression levels of iNOS and COX-2 as well as the production of inflammatory cytokines, including TNF-α and IL-1β, compared to the only LPS-treated cells. In addition, CCP inhibited the activation of NF-κB p50 and p65 and the phosphorylation of MAPKs, including p38 and extracellular signal-regulated kinase, in LPS-stimulated RAW 264.7 cells. Furthermore, FT-IR analysis showed that the FT-IR spectrum of CCP is similar

  17. Enzymatic chlorination and bromination. (United States)

    van Pée, Karl-Heinz


    Our knowledge about the enzymes catalyzing the incorporation of halide ions during the biosynthesis of halometabolites has increased tremendously during the last 15 years. Between 1960 and 1995, haloperoxidases were the only halogenating enzymes known. However, absolute proof for the connection of haloperoxidases to the biosynthesis of halometabolites is still missing. In 1997, FADH(2)-dependent halogenases were identified as the type of halogenating enzymes responsible for the incorporation of chloride and bromide atoms into aromatic and aliphatic compounds activated for electrophilic attack. FADH(2)-dependent halogenases are two-component systems consisting of a flavin reductase providing the FADH(2) required by the halogenase. Elucidation of the three-dimensional structure of FADH(2)-dependent halogenases led to the understanding of the reaction mechanism, which involves the formation of hypohalous acids. Unactivated carbon atoms were found to be halogenated by nonheme iron, α-ketoglutarate- and O(2)-dependent halogenases. The reaction mechanism of this type of halogenase was shown to involve the formation of a substrate radical. These two types of halogenating enzymes, together with the much less common fluorinases, are the major types of halogenating enzymes. However, the existence of other types of halogenating enzymes, yet not detected, cannot be completely ruled out. Here, we describe the detection, purification, characterization, and reaction mechanisms of flavin-dependent halogenases and of nonheme iron, α-ketoglutarate- and O(2)-dependent halogenases.

  18. Enzymatic pretreatment of lignocellulosic wastes to improve biogas production. (United States)

    Ziemiński, K; Romanowska, I; Kowalska, M


    The effect of enzymatic pretreatment of sugar beet pulp and spent hops prior to methane fermentation was determined in this study. These industrial residues were subjected to enzymatic digestion before anaerobic fermentation because of high fiber content (of 85.1% dry matter (DM) and 57.7% DM in sugar beet pulp and spent hops, respectively). Their 24h hydrolysis with a mix of enzymatic preparations Celustar XL and Agropect pomace (3:1, v/v), with endoglucanase, xylanase and pectinase activities, was most effective. Reducing sugars concentrations in hydrolysates of sugar beet pulp and spent hops were by 88.9% and 59.4% higher compared to undigested materials. The highest yield of biogas was obtained from the enzymatic hydrolysate of sugar beet pulp (183.39 mL/d from 1g COD at fermenter loading with organic matter of 5.43 g COD/L × d). Fermentation of sugar beet pulp gave 19% less biogas. Methane fermentation of spent hops hydrolysate yielded 121.47 mL/d biogas from 1g COD (at 6.02 g COD/L × d, 13% more than from spent hops). These results provide evidence that suitable enzymatic pretreatment of lignocellulosic wastes improve biogas yield from anaerobic fermentation.

  19. Recent Advances in Enzymatic Fuel Cells: Experiments and Modeling

    Directory of Open Access Journals (Sweden)

    Ivan Ivanov


    Full Text Available Enzymatic fuel cells convert the chemical energy of biofuels into electrical energy. Unlike traditional fuel cell types, which are mainly based on metal catalysts, the enzymatic fuel cells employ enzymes as catalysts. This fuel cell type can be used as an implantable power source for a variety of medical devices used in modern medicine to administer drugs, treat ailments and monitor bodily functions. Some advantages in comparison to conventional fuel cells include a simple fuel cell design and lower cost of the main fuel cell components, however they suffer from severe kinetic limitations mainly due to inefficiency in electron transfer between the enzyme and the electrode surface. In this review article, the major research activities concerned with the enzymatic fuel cells (anode and cathode development, system design, modeling by highlighting the current problems (low cell voltage, low current density, stability will be presented.

  20. Enzymatic Breakdown of Type II Collagen in the Human Vitreous

    NARCIS (Netherlands)

    van Deemter, Marielle; Pas, Hendri H.; Kuijer, Roel; van der Worp, Roelofje J.; Hooymans, Johanna M. M.; Los, Leonoor I.


    PURPOSE. To investigate whether enzymatic collagen breakdown is an active process in the human vitreous. METHODS. Human donor eyes were used for immunohistochemistry to detect the possible presence of the matrix metalloproteinase (MMP)-induced type II collagen breakdown product col2-3/4C-short in th

  1. Renal failure affects the enzymatic activities of the three first steps in hepatic heme biosynthesis in the acute intermittent porphyria mouse.

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    Carmen Unzu

    Full Text Available Chronic kidney disease is a long-term complication in acute intermittent porphyria (AIP. The pathophysiological significance of hepatic overproduction of the porphyrin precursors aminolevulinate acid (ALA and porphobilinogen (PBG in chronic kidney disease is unclear. We have investigated the effect of repetitive acute attacks on renal function and the effect of total or five-sixth nephrectomy causing renal insufficiency on hepatic heme synthesis in the porphobilinogen deaminase (PBGD-deficient (AIP mouse. Phenobarbital challenge in the AIP-mice increased urinary porphyrin precursor excretion. Successive attacks throughout 14 weeks led to minor renal lesions with no impact on renal function. In the liver of wild type and AIP mice, 5/6 nephrectomy enhanced transcription of the first and rate-limiting ALA synthase. As a consequence, urinary PBG excretion increased in AIP mice. The PBG/ALA ratio increased from 1 in sham operated AIP animals to over 5 (males and over 13 (females in the 5/6 nephrectomized mice. Total nephrectomy caused a rapid decrease in PBGD activity without changes in enzyme protein level in the AIP mice but not in the wild type animals. In conclusion, high concentration of porphyrin precursors had little impact on renal function. However, progressive renal insufficiency aggravates porphyria attacks and increases the PBG/ALA ratio, which should be considered a warning sign for potentially life-threatening impairment in AIP patients with signs of renal failure.

  2. Direct electrochemistry and enzymatic activity of hemoglobin in positively charged colloid Au nanoparticles and hemoglobin layer-by-layer self-assembly films

    Institute of Scientific and Technical Information of China (English)

    YUAN; Ruo; CAO; ShuRui; CHAI; YaQin; GAO; FengXian; ZHAO; Qing; TANG; MingYu; TONG; ZhongQiang; XIE; Yi


    Alternate adsorption of positively charged colloid-Au nanoparticles (nano-Au(Ξ) and negatively charged hemoglobin (Hb) on L-cysteine (L-cys) modified gold electrode resulted in the assembly of {Hb/nano-Au(Ξ)}n layer-by-layer films/L-cys modified gold electrode. The nano-Au(Ξ) was characterized by transmission electron micrograph (TEM) and microelectrophoresis. The modified electrode interface morphology was characterized by electrochemical impedance spectroscopy (EIS), atomic force microscopy (AFM), cyclic voltammograms (CV) and chronoamperometry. Direct electron transfer between hemoglobin and gold electrodes was studied, and the apparent Michaelis-Menten constant (Kappm) of the modified electrode was evaluated to be 0.10 mmol·L-1. Moreover, the higher activity of proteins in the nano-Au(Ξ)films could be retained compared with the electropolymerization membrane, since the proteins in nano-Au(Ξ) films retained their near-native structure. Direct electron transfer between hemoglobin and electrode and electrochemically catalyzed reduction of hydrogen peroxide on a modified electrode was studied, and the linear range was from 2.1×10-8 to 1.2 ×10-3 mol·L-1 (r = 0.994) with a detection limit of 1.1×10?8 mol·L-1 H2O2.

  3. Antioxidant Enzymatic Activities and Gene Expression Associated with Heat Tolerance in the Stems and Roots of Two Cucurbit Species (“Cucurbita maxima” and “Cucurbita moschata”) and Their Interspecific Inbred Line “Maxchata” (United States)

    Ara, Neelam; Nakkanong, Korakot; Lv, Wenhui; Yang, Jinghua; Hu, Zhongyuan; Zhang, Mingfang


    The elucidation of heat tolerance mechanisms is required to combat the challenges of global warming. This study aimed to determine the antioxidant enzyme responses to heat stress, at the enzymatic activity and gene expression levels, and to investigate the antioxidative alterations associated with heat tolerance in the stems and roots of squashes using three genotypes differing in heat tolerance. Plants of heat-tolerant “C. moschata”, thermolabile “C. maxima” and moderately heat-tolerant interspecific inbred line “Maxchata” genotypes were exposed to moderate (37 °C) and severe (42 °C) heat shocks. “C. moschata” exhibited comparatively little oxidative damage, with the lowest hydrogen peroxide (H2O2), superoxide (O2−) and malondialdehyde (MDA) contents in the roots compared to stems, followed by “Maxchata”. The enzyme activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) were found to be increased with heat stress in tolerant genotypes. The significant inductions of FeSOD, MnSOD, APX2, CAT1 and CAT3 isoforms in tolerant genotypes suggested their participation in heat tolerance. The differential isoform patterns of SOD, APX and CAT between stems and roots also indicated their tissue specificity. Furthermore, despite the sequence similarity of the studied antioxidant genes among “C. maxima” and “Maxchata”, most of these genes were highly induced under heat stress in “Maxchata”, which contributed to its heat tolerance. This phenomenon also indicated the involvement of other unknown genetic and/or epigenetic factors in controlling the expression of these antioxidant genes in squashes, which demands further exploration. PMID:24336062

  4. Antioxidant Enzymatic Activities and Gene Expression Associated with Heat Tolerance in the Stems and Roots of Two Cucurbit Species (“Cucurbita maxima” and “Cucurbita moschata” and Their Interspecific Inbred Line “Maxchata”

    Directory of Open Access Journals (Sweden)

    Neelam Ara


    Full Text Available The elucidation of heat tolerance mechanisms is required to combat the challenges of global warming. This study aimed to determine the antioxidant enzyme responses to heat stress, at the enzymatic activity and gene expression levels, and to investigate the antioxidative alterations associated with heat tolerance in the stems and roots of squashes using three genotypes differing in heat tolerance. Plants of heat-tolerant “C. moschata”, thermolabile “C. maxima” and moderately heat-tolerant interspecific inbred line “Maxchata” genotypes were exposed to moderate (37 °C and severe (42 °C heat shocks. “C. moschata” exhibited comparatively little oxidative damage, with the lowest hydrogen peroxide (H2O2, superoxide (O2− and malondialdehyde (MDA contents in the roots compared to stems, followed by “Maxchata”. The enzyme activities of superoxide dismutase (SOD, ascorbate peroxidase (APX, catalase (CAT and peroxidase (POD were found to be increased with heat stress in tolerant genotypes. The significant inductions of FeSOD, MnSOD, APX2, CAT1 and CAT3 isoforms in tolerant genotypes suggested their participation in heat tolerance. The differential isoform patterns of SOD, APX and CAT between stems and roots also indicated their tissue specificity. Furthermore, despite the sequence similarity of the studied antioxidant genes among “C. maxima” and “Maxchata”, most of these genes were highly induced under heat stress in “Maxchata”, which contributed to its heat tolerance. This phenomenon also indicated the involvement of other unknown genetic and/or epigenetic factors in controlling the expression of these antioxidant genes in squashes, which demands further exploration.

  5. Antioxidant enzymatic activities and gene expression associated with heat tolerance in the stems and roots of two cucurbit species ("Cucurbita maxima" and "Cucurbita moschata") and their interspecific inbred line "Maxchata". (United States)

    Ara, Neelam; Nakkanong, Korakot; Lv, Wenhui; Yang, Jinghua; Hu, Zhongyuan; Zhang, Mingfang


    The elucidation of heat tolerance mechanisms is required to combat the challenges of global warming. This study aimed to determine the antioxidant enzyme responses to heat stress, at the enzymatic activity and gene expression levels, and to investigate the antioxidative alterations associated with heat tolerance in the stems and roots of squashes using three genotypes differing in heat tolerance. Plants of heat-tolerant "C. moschata", thermolabile "C. maxima" and moderately heat-tolerant interspecific inbred line "Maxchata" genotypes were exposed to moderate (37 °C) and severe (42 °C) heat shocks. "C. moschata" exhibited comparatively little oxidative damage, with the lowest hydrogen peroxide (H2O2), superoxide (O2(-)) and malondialdehyde (MDA) contents in the roots compared to stems, followed by "Maxchata". The enzyme activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT) and peroxidase (POD) were found to be increased with heat stress in tolerant genotypes. The significant inductions of FeSOD, MnSOD, APX2, CAT1 and CAT3 isoforms in tolerant genotypes suggested their participation in heat tolerance. The differential isoform patterns of SOD, APX and CAT between stems and roots also indicated their tissue specificity. Furthermore, despite the sequence similarity of the studied antioxidant genes among "C. maxima" and "Maxchata", most of these genes were highly induced under heat stress in "Maxchata", which contributed to its heat tolerance. This phenomenon also indicated the involvement of other unknown genetic and/or epigenetic factors in controlling the expression of these antioxidant genes in squashes, which demands further exploration.

  6. Structural basis of enzymatic benzene ring reduction. (United States)

    Weinert, Tobias; Huwiler, Simona G; Kung, Johannes W; Weidenweber, Sina; Hellwig, Petra; Stärk, Hans-Joachim; Biskup, Till; Weber, Stefan; Cotelesage, Julien J H; George, Graham N; Ermler, Ulrich; Boll, Matthias


    In chemical synthesis, the widely used Birch reduction of aromatic compounds to cyclic dienes requires alkali metals in ammonia as extremely low-potential electron donors. An analogous reaction is catalyzed by benzoyl-coenzyme A reductases (BCRs) that have a key role in the globally important bacterial degradation of aromatic compounds at anoxic sites. Because of the lack of structural information, the catalytic mechanism of enzymatic benzene ring reduction remained obscure. Here, we present the structural characterization of a dearomatizing BCR containing an unprecedented tungsten cofactor that transfers electrons to the benzene ring in an aprotic cavity. Substrate binding induces proton transfer from the bulk solvent to the active site by expelling a Zn(2+) that is crucial for active site encapsulation. Our results shed light on the structural basis of an electron transfer process at the negative redox potential limit in biology. They open the door for biological or biomimetic alternatives to a basic chemical synthetic tool.

  7. The Enzymatic Antioxidant System of Human Spermatozoa

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    Cristian O’Flaherty


    Full Text Available The ejaculated spermatozoon, as an aerobic cell, must fight against toxic levels of reactive oxygen species (ROS generated by its own metabolism but also by other sources such as abnormal spermatozoa, chemicals and toxicants, or the presence of leukocytes in semen. Mammalian spermatozoa are extremely sensitive to oxidative stress, a condition occurring when there is a net increase in ROS levels within the cell. Opportunely, this specialized cell has a battery of antioxidant enzymes (superoxide dismutase, peroxiredoxins, thioredoxins, thioredoxins reductases, and glutathione s-transferases working in concert to assure normal sperm function. Any impairment of the antioxidant enzymatic activities will promote severe oxidative damage which is observed as plasma membrane lipid peroxidation, oxidation of structural proteins and enzymes, and oxidation of DNA bases that lead to abnormal sperm function. Altogether, these damages occurring in spermatozoa are associated with male infertility. The present review contains a description of the enzymatic antioxidant system of the human spermatozoon and a reevaluation of the role of its different components and highlights the necessity of sufficient supply of reducing agents (NADPH and reduced glutathione to guarantee normal sperm function.

  8. Oxygen-glucose deprivation increases the enzymatic activity and the microvesicle-mediated release of ectonucleotidases in the cells composing the blood-brain barrier. (United States)

    Ceruti, Stefania; Colombo, Laura; Magni, Giulia; Viganò, Francesca; Boccazzi, Marta; Deli, Mária A; Sperlágh, Beáta; Abbracchio, Maria P; Kittel, Agnes


    The blood-brain barrier (BBB), the dynamic interface between the nervous tissue and the blood, is composed by endothelial cells, pericytes and astrocytes. Extracellular nucleotides and nucleosides and their receptors (the purinergic system) constitute a widely diffused signaling system involved in many pathophysiological processes. However, the role of this system in controlling BBB functions is still largely unknown. By using cultures of these three cell types grown separately and a BBB in vitro model consisting of triple co-cultures, we studied for the first time the expression and distribution of the ecto-enzymes nucleoside triphosphate diphosphohydrolases (NTPDases, the enzymes which hydrolyze extracellular nucleotides) under control and ischemic (oxygen-glucose deprivation in vitro; OGD) conditions. NTPDase1 was detected in all three cell types, whereas NTPDase2 was expressed by astrocytes and pericytes and, to a lesser extent, by endothelial cells. Endothelial cells were extremely susceptible to cell death when OGD was applied to mimic in vitro the cytotoxicity induced by ischemia, whereas astrocytes and pericytes were more resistant. A semi-quantitative assay highlighted markedly increased e-ATPase activity following exposure to OGD in all three cell types, either when grown separately or when co-cultured together to resemble the composition of the BBB. Moreover, electron microscopy analysis showed that both endothelial cells and astrocytes shed microvesicles containing NTPDases from their membrane, which may suggest a novel mechanism to increase the breakdown of ATP released to toxic levels by damaged BBB cells. We hypothesize that this phenomenon could have a protective and/or modulatory effect for brain parenchymal cells. This in vitro model is therefore useful to study the role of extracellular nucleotides in modulating BBB responses to ischemic events, and to develop new effective purinergic-based approaches for brain ischemia.

  9. Evolution of Enzymatic Activities in the Enolase Superfamily: Stereochemically Distinct Mechanisms in Two Families of cis,cis-Muconate Lactonizing Enzymes

    Energy Technology Data Exchange (ETDEWEB)

    Sakai, A.; Fedorov, A; Fedorov, E; Schnoes, A; Glasner, M; Burley, S; Babbitt, P; Almo, S; Gerlt, J


    The mechanistically diverse enolase superfamily is a paradigm for elucidating Nature's strategies for divergent evolution of enzyme function. Each of the different reactions catalyzed by members of the superfamily is initiated by abstraction of the a-proton of a carboxylate substrate that is coordinated to an essential Mg2+. The muconate lactonizing enzyme (MLE) from Pseudomonas putida, a member of a family that catalyzes the syn-cycloisomerization of cis,cis-muconate to (4S)-muconolactone in the e-ketoadipate pathway, has provided critical insights into the structural bases for evolution of function within the superfamily. A second, divergent family of homologous MLEs that catalyzes anti-cycloisomerization has been identified. Structures of members of both families liganded with the common (4S)-muconolactone product (syn, Pseudomonas fluorescens, gi 70731221; anti, Mycobacterium smegmatis, gi 118470554) document that the conserved Lys at the end of the second e-strand in the (e/a)7e-barrel domain serves as the acid catalyst in both reactions. The different stereochemical courses (syn and anti) result from different structural strategies for determining substrate specificity: although the distal carboxylate group of the cis,cis-muconate substrate attacks the same face of the proximal double bond, opposite faces of the resulting enolate anion intermediate are presented to the conserved Lys acid catalyst. The discovery of two families of homologous, but stereochemically distinct, MLEs likely provides an example of 'pseudoconvergent' evolution of the same function from different homologous progenitors within the enolase superfamily, in which different spatial arrangements of active site functional groups and substrate specificity determinants support catalysis of the same reaction.

  10. Application of photocatalytic cadmium sulfide nanoparticles to detection of enzymatic activities of glucose oxidase and glutathione reductase using oxidation of 3,3′,5,5′-tetramethylbenzidine

    Energy Technology Data Exchange (ETDEWEB)

    Grinyte, Ruta; Garai-Ibabe, Gaizka; Saa, Laura; Pavlov, Valeri, E-mail:


    Highlights: • The light-powered nanosensor fabricated by enzymatic reactions was reported. • The sensor use energy of photons for oxidation of chromogenic enzymatic substrates. • Enzymatic assays for glucose oxidase and glutathione reductase were developed. - Abstract: It was found out that semiconductor CdS nanoparticles (NPs) are able to catalyze photooxidation of the well known chromogenic enzymatic substrate 3,3′,5,5′-tetramethylbenzidine (TMB) by oxygen. The photocatalytical oxidation of TMB does not require hydrogen peroxide and its rate is directly proportional to the quantity of CdS NPs produced in situ through the interaction of Cd{sup 2+} and S{sup 2−} ions in an aqueous medium. This phenomenon was applied to development of colorimetric sensitive assays for glucose oxidase and glutathione reductase based on enzymatic generation of CdS NPs acting as light-powered catalysts. Sensitivity of the developed chromogenic assays was of the same order of magnitude or even better than that of relevant fluorogenic assays. The present approach opens the possibility for the design of simple and sensitive colorimetric assays for a number of enzymes using inexpensive and available TMB as a universal chromogenic compound.

  11. Black-dot ringworm caused by Trichophyton tonsurans and analysis of its extracellular enzymatic activity%断发毛癣菌致黑点癣一例及菌体胞外酶活性分析

    Institute of Scientific and Technical Information of China (English)

    张瑞峰; 冉玉平; 代亚玲


    目的 报道1例发生在3岁女孩的由断发毛癣菌所致黑点癣.方法 取病发标本作真菌直接镜检和培养,对培养菌株进行形态学鉴定、生化鉴定、分子生物学鉴定.分析本菌株胞外酶活性.结果 KOH涂片可见发干内充满孢子,典型菌落呈灰白绒毛样质地,小培养见蜈蚣样侧生棒状小分生孢子,尿素酶实验阳性.扩增真菌rDNA的ITS区得到687 bp的片段,测序后比对与基因库中多株断发毛癣菌同源性100%.胞外酶活性分析见碱性磷酸酶、酸性磷酸酶、酯酶、β-葡萄糖苷酶、白氨酸芳胺酶、N-乙酰-葡萄糖胺酶、α-甘露糖苷酶活性较高.结论 根据真菌形态学特征、生化特性及DNA序列分析,鉴定本致病菌株为断发毛癣菌.诊断为黑点癣.%Objective To report a case of black-dot ringworm caused by Trichophyton tonsurans in a 3-year-old girl. Methods Lesional hair was obtained from the patient and subjected to direct microscopic examination as well as culture. Subsequently, the isolate underwent morphological, biochemical and molecular biology identification. The extracellular enzymatic activity of the isolate was analyzed. Results Microscopy revealed that the hair shaft was filled with fungal spores. Typical colony of the isolate was grayish-white with downy appearance. Slide culture showed centipede-like, lateral, rod-shaped microconidia. Urease test was positive. The amplification of ribosomal DNA (rDNA) ITS domains by PCR produced a 687 bp-sized fragment which had a 100% homology with the sequences of several Trichophyton tonsurans strains in the GenBank database. The extracellular enzymatic activity analysis showed an increase in the activity of alkaline phos-phatase, acid phosphatase, esterase (C4), β-glucosidase, leucine arylamidase, N-acetyl-β-glucosaminidase and a-mannosidase. Conclusions The pathogenic fungus is identified as Trichophyton tonsurans based on morphological and biochemical features as well as

  12. Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies

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    Kalb Suzanne R


    Full Text Available Abstract Background Botulism is caused by botulinum neurotoxins (BoNTs, extremely toxic proteins which can induce respiratory failure leading to long-term intensive care or death. Treatment for botulism includes administration of antitoxins, which must be administered early in the course of the intoxication; therefore, rapid determination of human exposure to BoNT is an important public health goal. In previous work, our laboratory reported on Endopep-MS, a mass spectrometry-based activity method for detecting and differentiating BoNT/A, /B, /E, and /F in clinical samples. We also demonstrated that antibody-capture is effective for purification and concentration of BoNTs from complex matrices such as clinical samples. However, some antibodies inhibit or neutralize the enzymatic activity of BoNT, so the choice of antibody for toxin extraction is critical. Results In this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs for their ability to inhibit the in vitro activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the in vitro light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs. Conclusions In addition to determining in vitro inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.

  13. Method to Improve Enzymatic Activity during Beer Brewing%改善啤酒酿造过程酶活力方法的研究

    Institute of Scientific and Technical Information of China (English)

    刘乃侨; 孙丽华


    以国外大麦Gairdner为原料,分别用麦芽厂所用的空白水、pH值4.0盐酸溶液、pH值4.0硫酸溶液、100 mg/L钙离子溶液、1μg/L赤霉素溶液使大麦浸渍、发芽来达到降低绿麦芽内部pH值的目的,实验结果表明,虽然硫酸、盐酸2种强酸物质具备降低大麦发芽内部pH值的能力,但其食品安全性差、对设备腐蚀大,而用钙离子和赤霉素溶液培养大麦发芽,虽然在大麦发芽过程中对pH值的变化影响不是很稳定,但均能使大麦发芽结束点即绿麦芽的pH值降低,所以对于后期啤酒的酿造均能够使淀粉酶、蛋白酶等活力近于最适pH值状态,为在啤酒酿造过程中减少外源添加酸的使用量,或为温和型弱酸或酸性中草药等物质的添加提供条件.%The aim of the experiment was to reduce pH value inside green malt during barley germination using Gaird-ner barley as raw material, soaking respectively with fresh water used in the malt factory, pH 4. 0 hydrochloric acid solution, pH 4.0 sulphuric acid solution, 100 mg/L calcium ion solution, and 1 u.g/L gibberellin solution. The results showed that although strong acids of sulphuric acid and hydrochloric acid possesses the capabilities to reduce pH value inside the barley during the germination, yet the food security is poor, and they corrode the facilities greatly, though while using calcium ion and/or gibberellin solution to cultivate barley to germinate had unstable pH changes during the barley germination, but they all could reduce pH value when reaching the end of barley germination I. E. Green malt. Therefore, at the late stage of beer brewing it could reach closely to the most suitable pH value for the activities of amylase and protease, to reduce the adding amount of acid during the beer brewing process, or provide conditions of adding mild weak acids and/or acidic Chinese medical herbs, and other materials.

  14. Differential activity and structure of highly similar peroxidases. Spectroscopic, crystallographic, and enzymatic analyses of lignifying Arabidopsis thaliana peroxidase A2 and horseradish peroxidase A2. (United States)

    Nielsen, K L; Indiani, C; Henriksen, A; Feis, A; Becucci, M; Gajhede, M; Smulevich, G; Welinder, K G


    Anionic Arabidopsis thaliana peroxidase ATP A2 was expressed in Escherichia coli and used as a model for the 95% identical commercially available horseradish peroxidase HRP A2. The crystal structure of ATP A2 at 1.45 A resolution at 100 K showed a water molecule only 2.1 A from heme iron [Ostergaard, L., et al. (2000) Plant Mol. Biol. 44, 231-243], whereas spectroscopic studies of HRP A2 in solution at room temperature [Feis, A., et al. (1998) J. Raman Spectrosc. 29, 933-938] showed five-coordinated heme iron, which is common in peroxidases. Presented here, the X-ray crystallographic, single-crystal, and solution resonance Raman studies at room temperature confirmed that the sixth coordination position of heme iron of ATP A2 is essentially vacant. Furthermore, electronic absorption and resonance Raman spectroscopy showed that the heme environments of recombinant ATP A2 and glycosylated plant HRP A2 are indistinguishable at neutral and alkaline pH, from room temperature to 12 K, and are highly flexible compared with other plant peroxidases. Ostergaard et al. (2000) also demonstrated that ATP A2 expression and lignin formation coincide in Arabidopsis tissues, and docking of lignin precursors into the substrate binding site of ATP A2 predicted that coniferyl and p-coumaryl alcohols were good substrates. In contrast, the additional methoxy group of the sinapyl moiety gave rise to steric hindrance, not only in A2 type peroxidases but also in all peroxidases. We confirm these predictions for ATP A2, HRP A2, and HRP C. The specific activity of ATP A2 was lower than that of HRP A2 (pH 4-8), although a steady-state study at pH 5 demonstrated very little difference in their rate constants for reaction with H2O2 (k1 = 1.0 microM(-1) x s(-1). The oxidation of coniferyl alcohol, ferulic, p-coumaric, and sinapic acids by HRP A2, and ATP A2, however, gave modest but significantly different k3 rate constants of 8.7 +/- 0.3, 4.0 +/- 0.2, 0.70 +/- 0.03, and 0.04 +/- 0.2 microM(-1) x

  15. Parameters optimization for enzymatic assays using experimental design

    Directory of Open Access Journals (Sweden)

    J. F. M. Burkert


    Full Text Available The conditions for maximization enzymatic activity were determined using experimental design and inulinase from Kluyveromyces marxianus ATCC 16045. The effects of substrate concentration (sucrose and inulin, pH and temperature on inulinase activity were verified using four factorial design and surface response analysis. Using sucrose as substrate. It has bean shown that the effects sucrose on enzymatic activity is not statistically significant and the best condition for the highest activity (110 U/mL was achieved with temperature between 60°C and 68°C and pH between 4.5 and 5.0. Using inulin as substrate it was verified that temperature is the only variable statistically significant and the maximum activity was 7.3 U/mL at temperature between 50°C and 51°C.

  16. 酶法水解花生蛋白及产物抗氧化活性的研究%Enzymatic Hydrolysis of Peanut Protein and Its Hydrolysate of Antioxidant Activity

    Institute of Scientific and Technical Information of China (English)

    罗世龙; 张榴萍; 刘锦


    Taking extracted peanut protein from peanut meal as raw material, variations of degree of hydrolysis, soluble nitrogen content in trichloroacetic acid, and antioxidant activity at different time were studied by using Alcalase, Protamex, and Papain, respectively. Results showed that degree of hydrolysis of enzymatic hydrolysis product stabilized after increased with extension of hydrolysis time. Soluble nitrogen content in trichloroacetic acid increased first and then decreased. And superoxide anion free radical scavenging capacity increased first and then slightly decreased. There were not directly related between antioxidant properties of different hydrolysis products from different enzyme types and their hydrolysis ability.%本文以花生粕中提取的花生蛋白为原料,分别使用Alcalase、 Protamex和Papain酶解,研究不同酶解时间酶解产物的水解度、三氯乙酸中可溶性氮含量和抗氧化活性的变化规律。结果表明:随着酶解时间的延长, Alcalase、 Protamex和Papain酶解花生蛋白的酶解产物的水解度先逐渐增加后趋于稳定,三氯乙酸中可溶性氮含量先增加后减小,超氧阴离子自由基清除能力先增加后有小幅降低。不同酶的酶解产物的抗氧化性能与其水解能力并不直接相关。

  17. Cloning and expression of the enzymatic region of Streptococcal hyaluronidase

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    Nafiseh Al-Sadat Mirjamali


    Full Text Available Objective(s: Streptococcus pyogenes produces extracellular hyaluronidase enzyme. This enzyme is directly associated with the spread of the organism during infection. The objective of the present study was to clone and express the nucleotide sequence of the enzyme which is involved in hyaluronidase enzymatic activity. Materials and Methods: The enzymatic region of hyaluronidase gene was detected by bioinformatics method. The PCR method was used to amplify enzymatic region of hyaluronidase gene from chromosomal DNA of Streptococcus pyogenes. The eluted product was cloned into the prokaryotic expression vector pET32a which was digested by BamHI and HindIII restriction endonuclease enzymes. The target protein was expressed in the Escherichia coli. The bacteria including pET32a-hylA (hylA is abbreviation of Streptococcus pyogenes hyaluronidase gene and hylA is abbreviation of Streptococcus pyogenes hyaluronidase protein plasmids were induced by IPTG and analyzed by SDS-PAGE. The enzymatic evaluation and antigenicity was finally studied. Results: Enzymes digestion analysis, sequencing results showed that the target gene (1296 base pair was inserted correctly into the recombinant vector. The expressed protein (65 KDa was purified successfully via affinity chromatography. Data also indicated that enzymatic region of hyaluronidase protein from Streptococcus pyogenes was recognized in all 5 patient’s sera. Conclusion: In general, it is possible to produce the enzymatic regions of the Streptococcus pyogenes hyaluronidase in E. coli. The antigenic property of the produced protein is well retained. Considering the product's domestic demand and also low efficiency of production and pathogenicity of Streptococcus species, it is possible to produce it as recombinant product.

  18. Mechanobiocatalysis: Modulating Enzymatic Activity with Mechanical Force (United States)


    novel polymeric materials that are designed to resist mechanical degradation. This ambition project sought to develop molecular scaffolds that...were dependent on the applied force. Recent efforts have focused on new classes of polymeric materials that effectively resist mechanical degradation...photophysical properties that were dependent on the applied force. Recent efforts have focused on new classes of polymeric materials that effectively resist


    Directory of Open Access Journals (Sweden)

    Wilberg K. Q.


    Full Text Available This paper presents an enzymatic process for sorbitol and gluconic acid production using cells of Zymomonas mobilis permeabilized with CTAB. Equimolar solutions of glucose and fructose (from 96.0 to 422.2 g/L were used. In a batch reactor, conversions of 97% were attained after 15 to 20 hours of reaction. The effect of the initial concentration of the substrates was evaluated in experiments using 20% more and 20% less glucose than fructose. It was observed that the reaction performed with more fructose reached completion faster and with a higher value of conversion

  20. Papain Degrades Tight Junction Proteins of Human Keratinocytes In Vitro and Sensitizes C57BL/6 Mice via the Skin Independent of its Enzymatic Activity or TLR4 Activation. (United States)

    Stremnitzer, Caroline; Manzano-Szalai, Krisztina; Willensdorfer, Anna; Starkl, Philipp; Pieper, Mario; König, Peter; Mildner, Michael; Tschachler, Erwin; Reichart, Ursula; Jensen-Jarolim, Erika


    Papain is commonly used in food, pharmaceutical, textile, and cosmetic industries and is known to induce occupational allergic asthma. We have previously shown that the papain-like cysteine protease Dermatophagoides pteronyssinus 1 from house dust mite exhibits percutaneous sensitization potential. We aimed here to investigate the potential of papain itself in epicutaneous sensitization. The effects of papain on tight junction (TJ) proteins were tested in vitro in human primary keratinocytes. Using C57BL/6 wild-type and Toll-like receptor 4 (TLR4)-deficient mice, we analyzed the sensitization potential of papain, its effects on the skin barrier, and immune cell recruitment. Our results show that papain affects the skin barrier by increasing transepidermal water loss, degrading TJ proteins and inducing vasodilation. When topically applied, papain exhibited a high epicutaneous inflammatory potential by recruiting neutrophils, mast cells, and CD3-positive cells and by induction of a TH2-biased antibody response. However, its high potency for specific sensitization via the skin was TLR4 independent and, in spite of its capacity to degrade epidermal TJ proteins, does not rely on its enzymatic function. From our data, we conclude that papain has all features to act as a strong allergen via the skin.

  1. Real-time ESI-MS of enzymatic conversion: impact of organic solvents and multiplexing. (United States)

    Scheerle, Romy K; Grassmann, Johanna; Letzel, Thomas


    Different enzymatic assays were characterized systematically by real-time electrospray ionization mass spectrometry (ESI-MS) in the presence of organic solvents as well as in multiplex approaches and in a combination of both. Typically, biological enzymatic reactions are studied in aqueous solutions, since most enzymes show their full activity solely in aqueous solutions. However, in recent years, the use of organic solvents in combination with enzymatic reactions has gained increasing interest due to biotechnological advantages in chemical synthesis, development of online coupled setups screening for enzyme regulatory compounds, advantages regarding mass spectrometric detection and others. In the current study, the influence of several common organic solvents (methanol, ethanol, isopropanol, acetone, acetonitrile) on enzymatic activity (hen egg white lysozyme, chitinase, α-chymotrypsin, elastase from human neutrophils and porcine pancreas, acetylcholinesterase) was tested. Moreover, multiplexing is a promising approach enabling fast and cost-efficient screening methods, e.g. for determination of inhibitors in complex mixtures or in the field of biomedical research. Although in multiplexed setups the enzymatic activity may be affected by the presence of other substrates and/or enzymes, the expected advantages possibly will predominate. To investigate those effects, we measured multiple enzymatic assays simultaneously. For all conducted measurements, the conversion rate of the substrate(s) was calculated, which reflects the enzymatic activity. The results provide an overview about the susceptibility of the selected enzymes towards diverse factors and a reference point for many applications in analytical chemistry and biotechnology.


    Directory of Open Access Journals (Sweden)

    Sofia-Paulina BALAURE


    Full Text Available This study highlights the problem of soil pollution as the result of human activities. Soil pollutans may be either chemicals or biological in nature. microbial enzymatic activities are often proposed as indicators of environmental stress. The soil samples were submitted by chemical, microbiological and enzymatic analyses. Chemical analyses were been made for determinating the heavy metals. Heavy metals from the forest soil were represented by Cu, Zn, Mn, Ni, Pb, Cd and Cr. To evaluate the concentration in heavy metals from the filtrate, we used a acetylene-nitrous oxide flame atomic absorption spectrophotometry. Potential dehydrogenase activity, the only indicator of the possible sources of pollution, excluded the presence of either chemical or biological pollution. The number of bacteria involved in the biogeochemical cycle of nitrogen in the analyzed soil indicated a high efficiency regarding the mineralization of the organic residues of plant and animal origin.

  3. Ultrasound assisted enzymatic depolymerization of aqueous guar gum solution. (United States)

    Prajapat, Amrutlal L; Subhedar, Preeti B; Gogate, Parag R


    The present work investigates the effectiveness of application of low intensity ultrasonic irradiation for the intensification of enzymatic depolymerization of aqueous guar gum solution. The extent of depolymerization of guar gum has been analyzed in terms of intrinsic viscosity reduction. The effect of ultrasonic irradiation on the kinetic and thermodynamic parameters related to the enzyme activity as well as the intrinsic viscosity reduction of guar gum using enzymatic approach has been evaluated. The kinetic rate constant has been found to increase with an increase in the temperature and cellulase loading. It has been observed that application of ultrasound not only enhances the extent of depolymerization but also reduces the time of depolymerization as compared to conventional enzymatic degradation technique. In the presence of cellulase enzyme, the maximum extent of depolymerization of guar gum has been observed at 60 W of ultrasonic rated power and ultrasonic treatment time of 30 min. The effect of ultrasound on the kinetic and thermodynamic parameters as well as the molecular structure of cellulase enzyme was evaluated with the help of the chemical reaction kinetics model and fluorescence spectroscopy. Application of ultrasound resulted in a reduction in the thermodynamic parameters of activation energy (Ea), enthalpy (ΔH), entropy (ΔS) and free energy (ΔG) by 47%, 50%, 65% and 1.97%, respectively. The changes in the chemical structure of guar gum treated using ultrasound assisted enzymatic approach in comparison to the native guar gum were also characterized by FTIR. The results revealed that enzymatic depolymerization of guar gum resulted in a polysaccharide with low degree of polymerization, viscosity and consistency index without any change in the core chemical structure which could make it useful for incorporation in food products.

  4. Synthesis of monoacylglycerols by enzymatic methods

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    Bradić Milena R.


    Full Text Available Monoacylglycerols are non-ionic surfactants widely used in the food industry. They are also important in cosmetic and pharmaceutical industries as drug carriers and for the consistency improvements in creams and lotions. Current process for their production is based on the glycerolysis of natural fats and oils in the presence of inorganic catalysts at temperatures higher than 220 oC. The major drawbacks of this process include high-energy consumption, low yield, and poor product quality. The use of lipases for the monoacylglycerols production offers environmental advantages and a reduction in energy consumption. Besides, the same surfactants prepared by the enzymatic synthesis may be labeled as “natural”. Recent progress in the application of highly-stable lipases in the organic solvents offers the possibility of employing various methods to the enzyme-catalyzed synthesis of monoacylglycerols, such as selective hydrolysis of fats and oils using 1,3-regiospecific lipases, the esterification of glycerol with fatty acids and the glycerolysis of fats or oils. In this review, different reaction systems such as aqueous-organic two-phase systems, microemulsions and reverse micelles systems, anhydrous organic solvents, solvent-free systems with free or immobilized lipases, as well as the use of two-phase membrane reactor systems are presented. We discuss some of the key factors, such as the control of water content, removing of the products from reaction system, and the effects of solvent on the lipase activity and selectivity, that must be addressed in order to obtain an efficient reaction system with high yields of monoacylglycerols. Engineering of the enzymatic monoacylglycerols synthesis processes requires also optimization of other factors as: molar ratio of substrates, temperature, type of lipase immobilization and supports (if any, reactor design and operating regime.

  5. Characterizing Enzymatic Deposition for Microelectrode Neurotransmitter Detection

    Energy Technology Data Exchange (ETDEWEB)

    Hosein, W. K. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Yorita, A. M. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States); Tolosa, V. M. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)


    The enzyme immobilization process, one step in creating an enzymatic biosensor, was characterized and analyzed as a function of its physical properties. The neural glutamic biosensor is a flexible device, effectively minimizing trauma to the area of implantation. The Multielectrode Array (MEA) is composed primarily of a proprietary polymer which has been successfully implanted into human subjects in recent years. This polymer allows the device the pliability that other devices normally lack, though this poses some challenges to implantation. The electrodes are made of Platinum (Pt), and can range in number from eight to thirty two electrodes per device. These electrodes are electroplated with a semipermeable polymer layer to improve selectivity of the electrode to the neurotransmitter of interest, in this case glutamate. A signal is created from the interaction of glutamate in the brain with the glutamate oxidase (GluOx) which is immobilized on the surface of the electrode by using crosslinking chemistry in conjunction with glutaraldehyde and Bovine Serum Albumin (BSA). The glutamate is oxidized by glutamate oxidase, producing α-ketoglutarate and hydrogen peroxide (H2O2) as a by-product. The production of H2O2 is crucial for detection of the presence of the glutamate within the enzymatic coating, as it diffuses through the enzyme layer and oxidizes at the surface of the electrode. This oxidation is detectable by measurable change in the current using amperometry. Hence, the MEA allows for in vivo monitoring of neurotransmitter activity in real time. The sensitivity of the sensor to these neurotransmitters is dependent on the thickness of the layer, which is investigated in these experiments in order to optimize the efficacy of the device to detecting the substrate, once implanted.


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    Full Text Available The yield, productivity and cost for the enzymatic hydrolysis of cellulose to glucoseare crucial for the production of second generation ethanol. In the first study wehave evaluated the activity of several commercial cellulolytic enzymes and a crudeextract of a local strain of Trichoderma viride. The load used was 15 U ofcellulase/gram cellulose and 90 U of cellobiase/gram cellulose. The hydrolysis wascarried out at 50oC and pH 4,8 for 96 hours. The best cellulose hydrolysis yield of58% was obtained with the cocktail formed of crude cellulases from T. virideCMIT3.5 combined with Novozyme 188. This cocktail was used in the second study,when alkaline-steam pretreated wheat straw and corn stover where hydrolyzed at pH4,8 for 96 hours. The temperature was set at 50oC and 40oC. The hydrolysis at lowertemperature was tested for a future experiment of simultaneous hydrolysis andfermentation. An enzymatic assay using glucose-6-phosphate dehydrogenase wasused to determine exclusively glucose, instead of wide-range sugar DNS assay.Reporting to 100 grams of wet pretreated biomass, the following results wereobtained: 14.4 g% glucose for corn stover at 50oC and 13,0 g% at 40oC; 13,1 g%glucose for wheat straw at 50oC and 10.3 g% at 40oC. Considering that wheat strawcontain 36.6% glucose-based carbohydrates, the hydrolysis yields are between39.3% and 28.1%. Further studies, concerning the optimal parameters for cellulasecocktail will be made.


    Directory of Open Access Journals (Sweden)



    Full Text Available The yield, productivity and cost for the enzymatic hydrolysis of cellulose to glucose are crucial for the production of second generation ethanol. In the first study we have evaluated the activity of several commercial cellulolytic enzymes and a crude extract of a local strain of Trichoderma viride. The load used was 15 U of cellulase/gram cellulose and 90 U of cellobiase/gram cellulose. The hydrolysis was carried out at 50oC and pH 4,8 for 96 hours. The best cellulose hydrolysis yield of 58% was obtained with the cocktail formed of crude cellulases from T. viride CMIT3.5 combined with Novozyme 188. This cocktail was used in the second study, when alkaline-steam pretreated wheat straw and corn stover where hydrolyzed at pH 4,8 for 96 hours. The temperature was set at 50oC and 40oC. The hydrolysis at lower temperature was tested for a future experiment of simultaneous hydrolysis and fermentation. An enzymatic assay using glucose-6-phosphate dehydrogenase was used to determine exclusively glucose, instead of wide-range sugar DNS assay. Reporting to 100 grams of wet pretreated biomass, the following results were obtained: 14.4 g% glucose for corn stover at 50oC and 13,0 g% at 40oC; 13,1 g% glucose for wheat straw at 50oC and 10.3 g% at 40oC. Considering that wheat straw contain 36.6% glucose-based carbohydrates, the hydrolysis yields are between 39.3% and 28.1%. Further studies, concerning the optimal parameters for cellulase cocktail will be made.

  8. 磷对铝胁迫下荞麦根际土壤铝形态和酶活性的影响%Effects of phosphorus on aluminum forms and soil enzymatic activities of buckwheat rhizosphere under aluminum stress

    Institute of Scientific and Technical Information of China (English)

    邢承华; 朱美红; 张淑娜; 李方; 蔡妙珍; 汪增基


    Two different Al tolerence buckwheat that were Neimeng(Al tolerant) and Jiangxi (Al sensitive) were compared under aluminum stress. The effects of P on buckwheat growth, Al forms and soil enzymatic activities of root rhizosphere microecology under Al toxicity were studied by using methods of soil culture. The results revealed that the biomass of Neimeng and Jiangxi buck-wheat supplied with 0.2 g·kg~(-1) P and 0.4 g·kg~(-1) Al were 67.9% and 21.2% higher than that supplied without P. P could ameliorate the inhibition of Al on root elongation, enhance the root biomass and root-to-shoot ratio. P and Al interaction significantly decreased both exchangeable Al (ExAl), while increased hydroxyl Al (HyAl) and organically complexed Al (OrAl) content of rhizosphere soil that were low toxicity. Changes of soil enzyme activities in Rhizosphere was complexity. Catalase activities showed positive correlation with P. 0.2 g·kg~(-1) P was the most convenient concentration for catalase activities. These results indicated that P application may alle-viate Al toxicity by decreasing ExAl content and enhancing catalase activity of rhizosphere soil.%采用土培法,以耐铝性明显差异的两个荞麦Fagopyrum esculentum基因型“江西养麦”(耐性)和“内蒙荞麦”(敏感)为材料,研究铝胁迫下磷对荞麦生长和根际土壤铝形态、土壤酶活性的影响.结果表明,0.4 g·kg~(-1)铝配施0.2 g·kg~(-1)磷的内蒙和江西荞麦根系生物量分别比不施磷组增加了67.9%和21.2%,磷能显著缓解铝对荞麦根系生长的抑制,提高根系生物量和根冠比.磷铝互作下根际土壤的交换态铝含量显著降低,毒性较小的吸附态羟基铝和络合态铝含量显著增加.根际土壤酶活性变化复杂,过氧化氢酶活性与磷质量分数呈正相关,w_p=0.2 g·kg~(-1)对铝胁迫下荞麦根际土壤转化酶活性最有利.说明施磷降低铝胁迫根际土壤的交换态铝含量,提高土壤过氧化氢酶活性,减缓


    Directory of Open Access Journals (Sweden)

    A. Vaisi-Raygani


    Full Text Available The etiopathogenesis of dementia in Alzheimer's disease (AD is still unclear. However, long-term oxidative stress is believed to be one of the major contributing factors in progression of neuronal degeneration and decline of cognitive function in AD. In order to assess the presence of oxidative stress in AD, we examined the enzymatic activities of the erythrocyte Cu-Zn superoxide dismutase (Cu-Zn SOD, glutathione peroxidase (GSH-Px, catalase (CAT, and plasma level of total antioxidant status (TAS in AD and control groups (age and sex-matched. The results showed that the Cu-Zn SOD activity was significantly higher and the level of GSH-Px and TAS activities were significantly lower in AD subjects than that in the control group (2111±324 U/grHb, 43.7±11.6 U/grHb, and 1.17 ±0.23 mmol/L compared with 1371±211 U/gHb; t= -2.17, p=0.036, 56.3±9.5 U/gHb; t=3.8, p=0.014, and 1.54±0.2 mmol/L; t=11.18, P<0.001, respectively. While, the erythrocyte CAT activity was lower in AD subjects compared to the control group, the difference was not statistically significant (t=1.3, P=0.15. These findings support the idea that the oxidative stress plays an important role in the pathogenesis underlying AD neurodegeneration. In addition, the enzymatic activity of the erythrocyte Cu-Zn SOD and GSH-Px and the plasma level of TAS can be used as a measure of the oxidative stress and a marker for pathological changes in the brain of patients with AD.

  10. 对硝基苯酚法对雅致放射毛霉脂肪酶特性的研究%Determination on lipase enzymatic characteristics and activity of Actinomucor elegans by pNP method

    Institute of Scientific and Technical Information of China (English)

    李蓓; 李晓晖; 衣杰荣


    以不同碳链长度的对硝基苯酚酯为底物对雅致放射毛霉(Actinomucor elegans AS3.27)脂肪酶的酶学特性和其发酵的腐乳在发酵过程中脂肪酶活力的变化进行研究,结果表明:分别以对硝基苯酚正辛酸酯(pNPC)和对硝基苯酚棕榈酸酯(pNPP)为反应底物所测得的脂肪酶酶学特性有所不同。以pNPC和pNPP为反应底物时,雅致放射毛霉脂肪酶的最适作用温度分别为30、35℃,最适作用pH为7.5、7.0,最适盐浓度都为0.20mol/L。以两种底物分别测定雅致放射毛霉接种的腐乳在发酵过程中的脂肪酶活力,均显示在后期发酵过程中脂肪酶活力呈现出不断下降的趋势,然而在第20~25d脂肪酶活力呈现出小幅度的反复。使用碱滴定法以聚乙二醇-橄榄油为底物进行验证,得到相同的趋势。%Actinomucorr elegans AS 3.27 lipase activities after cultivation and during sufu mature process were determined by pNP method with substrates of different carbon length.Differences in enzymatic characteristics were observed with different substrates-pNPC and pNPP.The optimum temperature was 30,35℃,and the optimum pH was 7.5,7.0 for pNPC and pNPP,respectively.While the optimum concentrations of NaCl were the same(0.20mol/L).No significant differences in lipase activity of sufu during the mature process existed between pNPC and pNPP method.The lipase activity of sufu decreased gradually during the ripening process,but during the period of 20 ~ 25 days,activity recurrent by a small margin.Simultaneously,the titrimetric method(substrate:PVA-olive oil)was applied to verify.The same trend was proved.

  11. Effects of cadmium on enzymatic and non-enzymatic antioxidative defences of rice (Oryza sativa L.). (United States)

    Yu, Fangming; Liu, Kehui; Li, Mingshun; Zhou, Zhenming; Deng, Hua; Chen, Bin


    The effects of 60-d cadmium (Cd) exposure on enzymatic and non-enzymatic antioxidative system of Oryza sativa L. seedlings at tillering stage were studied using soil culture experiment. Research findings showed that chlorophyll content of Oryza sativa L. declined with the increase in soil metal concentration. Cd pollution induced the antioxidant stress by inducing O2(-1) and H2O2, which increased in plants; at the same time, MDA as the final product of peroxidation of membrane lipids, accumulated in plant. The antioxidant enzyme system was initiated under the Cd exposure, i.e. almost all the activities of superoxide dismutase (SOD), peroxidase, catalase, glutathione peroxidase, and ascorbate peroxidase were elevated both in leaves and roots. The non-protein thiols including phytochelatins and glutathione to scavenge toxic free radicals caused by Cd stress was also studied. The contents of phytochelatins and glutathione were about 3.12-6.65-fold and 3.27-10.73-fold in leaves, against control; and the corresponding values were about 3.53-9.37-fold and 1.41-5.11-fold in roots, accordingly.

  12. Pre and Postharvest Enzymatic Activity in Gulupa (Passiflora edulis Sims Fruits from the Colombian Lower Montane Rain Forest / Actividad Enzimática Precosecha y Poscosecha en Frutos de Gulupa (Passiflora edulis Sims, en Condiciones del Bosque Húmedo Mo

    Directory of Open Access Journals (Sweden)

    Germán Franco


    Full Text Available “High-Andean fruits” are deemed important because oftheir potential for domestic consumption and exportation. Among them, gulupa (Passiflora edulis Sims is an exotic fruit of good acceptance in European markets. However, the technological support associated with the crop is incipient and its short shelf life leads to rapid deterioration of the product. This fact makes it necessary to investigate the physical, physiological and biochemical processes that characterize fruit ripening, in order to take actions to ensure that it arrives in its best possible condition to the consumer. In this context, the current study aimed at identifying enzymatic activity in gulupa fruits during pre and postharvest. Plant materialfrom the Colombian Gene Bank (administered by Corpoica was used. Fruits of known age were periodically harvested to determine the activity of the enzymes α-amylase, polygalacturonase (PG, pectinmethylesterase (PME and polyphenol oxidase (PPO through destructive samplings. It was found that α-amylase and PG are linked to the increase of soluble solids, which favors the sweet taste of the fruit. In turn, the low activity of PPO enables agroindustrial processing without severe fruit browning. / Los “frutales alto-andinos”, se consideran importantes por su potencial de consumo nacional y exportación. Entre ellos está la gulupa (Passiflora edulis Sims, reconocida como un frutal exótico de buena aceptación en mercados europeos. Sin embargo, el respaldo tecnológico asociado al cultivo, es incipiente y su corta vida poscosecha conduce al rápido deterioro del producto. Esto hace necesario plantear estudios de los procesos físicos, fisiológicos y bioquímicos que caracterizan la maduración, con el fin de procurar que el fruto llegue en las mejores condiciones de calidad a los consumidores. El estudio tuvo como objetivo conocer la actividad enzimática en los frutos de gulupa en precosecha y en poscosecha, con el

  13. Quantitative, functional and biochemical alterations in the peritoneal cells of mice exposed to whole-body gamma-irradiation. 1. Changes in cellular protein, adherence properties and enzymatic activities associated with platelet-activating factor formation and inactivation

    Energy Technology Data Exchange (ETDEWEB)

    Steel, L.K.; Hughes, H.N.; Walden, T.L. Jr.


    Changes in total number, differentials, cell protein, adherence properties, acetyl-CoA transferase and acetylhydrolase activities, prostaglandin E/sub 2/ and leukotriene C/sub 4/ production, as well as Ca/sup 2+/ ionophore A23187 stimulation were examined in resident peritoneal cells isolated from mice 2 h to 10 days postexposure to a single dose (7, 10 or 12 Gy) of gamma-radiation. Radiation dose-related reductions in macrophage and lymphocyte numbers and increases in cellular protein and capacity to adhere to plastic surfaces were evident. In vivo irradiation also elevated the activities of acetyltransferase and acetyl-CoA hydrolase (catalysing platelet-activating factor biosynthesis and inactivation, respectively) in adherent and nonadherent peritoneal cells, particularly 3-4 days postexposure. Blood plasma from irradiated animals did not reflect the increased cellular acetyl-hydrolase activity. Prostaglandin E/sub 2/ and leukotriene C/sub 4/ synthesis were elevated postexposure, suggesting increased substrate (arachidonate) availability and increased cyclooxygenase and lipoxygenase activities. Ionophore stimulation of enzyme activities and eicosanoid release also differed in irradiated peritoneal cells.

  14. Enzymatic Reactions in Microfluidic Devices (United States)

    Ristenpart, W. D.; Wan, J.; Stone, H. A.


    We establish simple scaling laws for enzymatic reactions in microfluidic devices, and we demonstrate that kinetic parameters obtained conventionally using multiple stop-flow experiments may instead be extracted from a single microfluidic experiment. Introduction of an enzyme and substrate species in different arms of a Y-shaped channel allows the two species to diffuse across the parallel streamlines and to begin reacting. Measurements of the product concentration versus distance down the channel provide information about the kinetics of the reaction. In the limit where the enzyme is much larger (and thus less diffusive) than the substrate, we show that near the entrance the total amount of product (P) formed varies as a power law in the distance x down the channel. For reactions that follow standard Michaelis-Menten kinetics, the power law takes the form P˜(Vmax/Km) x^5/2, where Vmax and Km are the maximum reaction rate and Michaelis constant respectively. If a large excess of substrate is used, then Km is identified by measuring Vmax far downstream where the different species are completely mixed by diffusion. Numerical simulations and experiments using the bioluminescent reaction between luciferase and ATP as a model system are both shown to accord with the model. We discuss the implications for significant savings in the amount of time and enzyme required for determination of kinetic parameters.

  15. Enzymatic biodiesel synthesis. Key factors affecting efficiency of the process

    Energy Technology Data Exchange (ETDEWEB)

    Szczesna Antczak, Miroslawa; Kubiak, Aneta; Antczak, Tadeusz; Bielecki, Stanislaw [Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz (Poland)


    Chemical processes of biodiesel production are energy-consuming and generate undesirable by-products such as soaps and polymeric pigments that retard separation of pure methyl or ethyl esters of fatty acids from glycerol and di- and monoacylglycerols. Enzymatic, lipase-catalyzed biodiesel synthesis has no such drawbacks. Comprehension of the latter process and an appreciable progress in production of robust preparations of lipases may soon result in the replacement of chemical catalysts with enzymes in biodiesel synthesis. Engineering of enzymatic biodiesel synthesis processes requires optimization of such factors as: molar ratio of substrates (triacylglycerols: alcohol), temperature, type of organic solvent (if any) and water activity. All of them are correlated with properties of lipase preparation. This paper reports on the interplay between the crucial parameters of the lipase-catalyzed reactions carried out in non-aqueous systems and the yield of biodiesel synthesis. (author)

  16. Rational design of functional and tunable oscillating enzymatic networks (United States)

    Semenov, Sergey N.; Wong, Albert S. Y.; van der Made, R. Martijn; Postma, Sjoerd G. J.; Groen, Joost; van Roekel, Hendrik W. H.; de Greef, Tom F. A.; Huck, Wilhelm T. S.


    Life is sustained by complex systems operating far from equilibrium and consisting of a multitude of enzymatic reaction networks. The operating principles of biology's regulatory networks are known, but the in vitro assembly of out-of-equilibrium enzymatic reaction networks has proved challenging, limiting the development of synthetic systems showing autonomous behaviour. Here, we present a strategy for the rational design of programmable functional reaction networks that exhibit dynamic behaviour. We demonstrate that a network built around autoactivation and delayed negative feedback of the enzyme trypsin is capable of producing sustained oscillating concentrations of active trypsin for over 65 h. Other functions, such as amplification, analog-to-digital conversion and periodic control over equilibrium systems, are obtained by linking multiple network modules in microfluidic flow reactors. The methodology developed here provides a general framework to construct dissipative, tunable and robust (bio)chemical reaction networks.

  17. Electrochemical, Chemical and Enzymatic Oxidations of Phenothiazines

    NARCIS (Netherlands)

    Blankert, B.; Hayen, H.; Leeuwen, van S.M.; Karst, U.; Bodoki, E.; Lotrean, S.; Sandulescu, R.; Mora Diaz, N.; Dominguez, O.; Arcos, J.; Kauffmann, J.-M.


    The oxidation of several phenothiazine drugs (phenothiazine, promethazine hydrochloride, promazine hydrochloride, trimeprazine hydrochloride and ethopropazine hydrochloride) has been carried out in aqueous acidic media by electrochemical, chemical and enzymatic methods. The chemical oxidation was pe


    Many immunotoxic compounds, such as benzene and other organic solvents, pesticides, mycotoxins and polycyclic aromatic hydrocarbons, can alter immune function only after undergoing enzyme-mediated reactions within various tissues. In the review that follows, the role of enzymatic...

  19. Nanostructured Metal Oxides Based Enzymatic Electrochemical Biosensors


    Ansari, Anees A.; Alhoshan, M.; M. S. AlSalhi; Aldwayyan, A.S.


    The unique electrocatalytic properties of the metal oxides and the ease of metal oxide nanostructured fabrication make them extremely interesting materials for electrochemical enzymatic biosensor applications. The application of nanostructured metal oxides in such sensing devices has taken off rapidly and will surely continue to expand. This article provides a review on current research status of electrochemical enzymatic biosensors based on various new types of nanostructured metal oxides su...

  20. Enzymatic biodiesel production: Technical and economical considerations

    DEFF Research Database (Denmark)

    Munk Nielsen, Per; Brask, Jesper; Fjerbæk, Lene


    It is well documented in the literature that enzymatic processing of oils and fats for biodiesel is technically feasible. However, with very few exceptions, enzyme technology is not currently used in commercial-scale biodiesel production. This is mainly due to non-optimized process design and a l...... that enzyme catalysts can become cost effective compared to chemical processing. This work reviews the enzymatic processing of oils and fats into biodiesel with focus on process design and economy....

  1. Preparation and Enzymatic Degradation of Porous Crosslinked Polylactides of Biomass Origin

    Directory of Open Access Journals (Sweden)

    Yuya Kido


    Full Text Available To understand the enzymatic degradation behavior of crosslinked polylactide (PLA, the preparation and enzymatic degradation of both thermoplastic (linear and crosslinked PLAs that have pore structures with different dimensions were carried out. The porous structures of the linear PLA samples were of micro and nanoporous nature, and prepared by batch foaming with supercritical CO2 and compared with the porous structures of crosslinked PLA (Lait-X created by the salt leaching method. The surface and cross-sectional morphologies of the porous structures were investigated by using scanning electron microscopy. The morphological analysis of porous Lait-X showed a rapid loss of physical features within 120 h of exposure to proteinase-K enzymatic degradation at 37 °C. Due to the higher affinity for water, enhanced enzymatic activity as compared to the linear PLA porous structures in the micro and nanoporous range was observed.

  2. 泥蚶血细胞的形态结构特征及部分免疫功能%Morphological, structural characteristics and phagocytic and enzymatic activities of haemocytes in blood clam Tegillarca granosa

    Institute of Scientific and Technical Information of China (English)

    朱泽闻; 徐立蒲; 吴信忠; 张振国; 吴刘记; 娄绘芳


    Light,electron microscopic and flow cytometer studies were conducted to characterize morphology and structure and phagocytosis of haemocytes in blood clam Tegillarca granosa. Based on cell size, and microstructure and granular types of cell, blood clam haemocytes were divided into red granulocyte (89. 67% ) ,basophil granulocyte (7. 05% ) and hyalinocyte (3. 28% ) respectively under light microscopy. The haemocyte densities in healthy blood clam haemolymph were assessed (3. 29 ±0. 82) × 106 cell/mL. Three kinds of haemocytes were respectively recognized by transmission electron microscope: granulocyte type Ⅰ , granulocyte type Ⅱ and agranulocyte. It was observed under scanning electron microscopy that haemocytes appear smooth-surface and some of them have knot-like projections in cell surface. APIZYM assays on enzymatic activities of 19 enzymes in haemolymph were performed and, of these, 13 and 10 enzymes were detected respectively in serum and cell. There were marked increases in the levels of ALP,ACP,ELIP(C8) and AGLA in serum and haemocytes after being stimulated by zymosan. Observation of phagocytosis in vitro revealed that there was phagocytic response in red granulocyte to zymosan and there was no significant phagocytic response in basophil granulocyte and hyalinocyte. It was observed that basophilic granulocyte accumulation and hyalinocyte adsorption after being stimulated by Aeromonas hydrophila. The study of environmental stress factors on phagocytosis activity revealed that the temperature and salinity have significant influence on phagocytosis activity. The phagocytosis percentage of blood clam reached the highest values with optimum growth temperature( 13 -30 ℃ )and salinity(20.0 -26.2) × 10-12.%利用光镜、透射电镜、扫描电镜及流式细胞仪技术对泥蚶血细胞形态结构以及吞噬活性进行了研究.根据细胞大小、形态结构、颗粒特点,光镜下可将血细胞区分为红色颗粒细胞(占89.67%)、嗜

  3. Study on Extraction, Limited Enzymatic Hydrolysis and Antioxidant Activity of Tea Dregs Protein Complexes%茶渣蛋白的提取、有限酶解及抗氧化性质研究

    Institute of Scientific and Technical Information of China (English)

    赵立娜; 汪少芸; 饶平凡; 江勇; 黄家炽; 周绍迁


    Objective:There are abundant protein in tea dregs, this study is to develop a new, green, natural an-tioxidants by utilizing waste tea dregs. Methods:Tea dregs protein was extracted by a mixed solution system,then antioxi-dant activity of these proteins in vitro and liposome -meat system after limited enzymatic hydrolysis was ' studied, and comparison with the traditional synthetic antioxidants VC, BHT was engaged. Results:the optimum condition of protein extraction was as follows: ratio of material to solvent 1:40, the extraction time 60 min, the NaOH concentration 0.10 mol/L, the extraction temperature 90 ℃, and one time extraction. Under this condition with pH3.0-4.0, the maximum extracting rate of protein complexes was 76%. By the screening of the hydrolysis of TP with complex protease, the technology was selected as temperature 50 ℃, pH 7.0, ratio of protease to substrate 6 000 U/g, substrateconcentration 1.5%, hydrolysis time 20 min. The DH of tea dregs protein after limited hydrolysis was determined to be 9%. When the concentration of peptide complexes was 0.1 mg/mL, the radical eliminating ratio on DPPH was 90.30%, and the eliminating ratio on hydroxyl radical was 65.18% when the concentration of peptide complex was 1.0 mg/mL The antioxidant activity was more than VC, but less than BHT in LA system. Conclusion: With high security and strong antioxidant activity, tea dregs protein peptide complexes has the potential to replace synthetic antioxidants.%目的:茶渣含有丰富的蛋白质,本研究旨在利用废弃荼渣,开发一种新型、绿色、天然的抗氧化剂.方法:利用碱溶酸沉法提取茶渣蛋白,酶法水解后对荼渣蛋白多肽复合物的体外抗氧化性进行研究,并与传统的合成抗氧化剂VC、BHT进行抗氧化效果的比较.结果:荼渣蛋白提取的最佳工艺为固液比1∶40(W/V),提取时间60min,碱浓度0.1 mol/L,提取温度90℃,最佳提取次数1次,最佳沉淀pH范围3.0~4.0,在以上条件

  4. Cometabolic Enzymatic Transformation of Organic Micropollutants under Methanogenic Conditions. (United States)

    Gonzalez-Gil, Lorena; Carballa, Marta; Lema, Juan M


    Anaerobic digestion (AD) has been shown to have the biological potential to decrease concentrations of several organic micropollutants (OMPs) in sewage sludge. However, the mechanisms and factors behind these biotransformations, which are essential for elucidating the possible transformation products and to foster the complete removal of OMPs via operational strategies, remain unclear. Therefore, this study investigated the transformation mechanisms of 20 OMPs during the methanogenic step of AD with a focus on the role of acetate kinase (AK), which is a key enzyme in methane production. The results from lab-scale methanogenic reactors showed that this step accounts for much of the reported OMP biotransformation in AD. Furthermore, enzymatic assays confirmed that AK transforms galaxolide, naproxen, nonylphenol, octylphenol, ibuprofen, diclofenac, bisphenol A, and triclosan. Except for galaxolide, for which further studies are required to refine conclusions, the OMP's chemical structure was a determinant for AK action because only compounds that contain a carboxyl or hydroxyl group and have moderate steric hindrance were enzymatically transformed, likely by phosphorylation. For these seven compounds, this enzymatic mechanism accounts for 10-90% of the measured methanogenic biotransformation, suggesting that other active enzymes of the AD process are also involved in OMP biotransformation.

  5. Enzymatic hydrolysis of protein:mechanism and kinetic model

    Institute of Scientific and Technical Information of China (English)

    Qi Wei; He Zhimin


    The bioreaction mechanism and kinetic behavior of protein enzymatic hydrolysis for preparing active peptides were investigated to model and characterize the enzymatic hydrolysis curves.Taking into account single-substrate hydrolysis,enzyme inactivation and substrate or product inhibition,the reaction mechanism could be deduced from a series of experimental results carried out in a stirred tank reactor at different substrate concentrations,enzyme concentrations and temperatures based on M-M equation.An exponential equation dh/dt = aexp(-bh) was also established,where parameters a and b have different expressions according to different reaction mechanisms,and different values for different reaction systems.For BSA-trypsin model system,the regressive results agree with the experimental data,i.e.the average relative error was only 4.73%,and the reaction constants were determined as Km = 0.0748 g/L,Ks = 7.961 g/L,kd = 9.358/min,k2 =38.439/min,Ea= 64.826 kJ/mol,Ed= 80.031 kJ/mol in accordance with the proposed kinetic mode.The whole set of exponential kinetic equations can be used to model the bioreaction process of protein enzymatic hydrolysis,to calculate the thermodynamic and kinetic constants,and to optimize the operating parameters for bioreactor design.

  6. Preparation of Enzymatic Hydrolysate of R-phycoerythrin from Porphyra yezoensis and Its Antioxidant and Tumor Cell Proliferation Inhibiting Activities%条斑紫菜R-藻红蛋白酶解物的制备及其抗氧化和肿瘤细胞增殖抑制活性

    Institute of Scientific and Technical Information of China (English)

    方勇; 杨方美; 赵殿峰; 杨文建; 赵立艳; 辛志宏; 马宁; 施瑛; 胡秋辉


    [目的]研究木瓜蛋白酶对条斑紫菜R-藻红蛋白的抗氧化性和肿瘤增殖抑制活性的影响.[方法]采用超声波破壁法提取条斑紫菜R-藻红蛋白,用DEAE柱层析法纯化后进行木瓜蛋白酶酶解,通过正交试验设计,以还原力A700为考察指标确定酶解反应的最佳工艺参数,进一步测定获得的R-藻红蛋白及其酶解物的还原力、清除羟自由基能力和对人肉瘤细胞U2O及人肝癌细胞HepG-2的肿瘤增殖抑制活性.[结果]R-藻红蛋白的最佳酶解工艺条件为:木瓜蛋白酶添加量25000 U·g-1,pH 7.0,温度50℃,酶解时间4h.在此条件下,R-藻红蛋白酶解物还原力为0.573,较未酶解的R-藻红蛋白提高了2.35倍;清除羟自由基能力为51.03%,较未酶解的R-藻红蛋白活性提高了3.22倍.随着浓度的增加,R-藻红蛋白及其酶解物对人肉瘤细胞U2O和人肝癌细胞HepG-2抑制生长作用增强.R-藻红蛋白对人肉瘤细胞U2O抑制作用的IC50值为2431.32 μg·mL-1,其酶解物IC50值降低为1271.46μg·mL-1.R-藻红蛋白对肝癌细胞HepG-2抑制作用的IC50值为1593.61 μg·mL-1,其酶解物IC50值降低为512.05 μg·mL-1.[结论]经木瓜蛋白酶酶解后,R-藻红蛋白酶解物具有较强的抗氧化和抑瘤活性.酶解技术可作为进一步提高R-藻红蛋白生物活性的有效手段.%[Objective] The effect of enzymatic hydrolysis by papain on antioxidant and tumor cell proliferation inhibiting activities of R-phycoerythrin from Porphyra yezoensis was investigated. [Method] R-phycoerythrin of Porphyra yezoensis was extracted by ultrasonic cell wall breaking, and then purified by DEAE column chromatography. Orthogonal design was employed to obtain the optimal condition of enzymatic hydrolysis by determination of reducing powder A700 of their enzymatic hydrolysates. Subsequently, hydroxyl radical-scavenging ability and proliferation inhibiting activities of human sarcoma cancer cell U2O and liver cancer cell HepG-2

  7. 鳄鱼血蛋白酶解产物抗氧化特性和血管紧张素转化酶抑制活性研究%Study on the antioxidant properties and angiotensin-converting enzyme inhibitory activity of crocodile blood protein enzymatic hydrolysate

    Institute of Scientific and Technical Information of China (English)

    黄和平; 陈孙福; 罗永康


    研究了鳄鱼血蛋白酶解产物的抗氧化特性和对血管紧张素转化酶( ACE)的抑制活性。利用木瓜蛋白酶酶解鳄鱼血浆蛋白和血球蛋白,用分光光度法测定了酶解产物的抗氧化能力和用高效液相色谱( HPLC)测定其ACE的抑制率。结果显示:鳄鱼血浆和血球蛋白酶解产物的亚铁离子螯合能力差异性不显著( P>0.05);在0~5 mg/mL的浓度范围内,血球蛋白酶解产物清除ABTS自由基的能力大于血浆蛋白酶解产物,且在浓度为1 mg/mL时,两者清除ABTS自由基的能力差异性极显著( P<0.01);血浆蛋白酶解产物清除DPPH自由基的能力在0~5 mg/mL的浓度范围内随着蛋白浓度的增加而升高,血球蛋白酶解产物在蛋白浓度为4 mg/mL处达到最大清除率,之后下降;在0~20 mg/mL的浓度范围内,两种酶解产物的还原力随着蛋白浓度的提高显著升高,但两者还原力的差异性不显著( P>0.05);鳄鱼血浆和血球蛋白酶解产物对ACE具有良好的抑制力,其最大抑制率可分别达到75.56%和86.42%。研究表明,鳄鱼血蛋白酶解产物在体外具有抗氧化和抑制ACE的活性。%The antioxidant properties and angiotensin-converting enzyme ( ACE) inhibitory activity of enzymatic hydrolysate from crocodile blood protein were analyzed. The crocodile plasma and blood cell protein were hydrolyzed by papain, and then antioxidant properties and ACE inhibition rate of enzymatic hydrolysate were measured by spectrophotometer and HPLC. Results showed that there were no statistical significance ( P>0.05) between the enzymatic hydrolysate of crocodile plasma and blood cell protein; the ABTS radical-scavenging ability of enzymatic hydrolysate from blood cell protein was higher than that from crocodile plasma protein from 0 to 5 mg/mL of protein concentration, and there were statistical significance at P0.05) between them ; crocodile plasma

  8. Automatic single- and multi-label enzymatic function prediction by machine learning (United States)

    Paragios, Nikos


    The number of protein structures in the PDB database has been increasing more than 15-fold since 1999. The creation of computational models predicting enzymatic function is of major importance since such models provide the means to better understand the behavior of newly discovered enzymes when catalyzing chemical reactions. Until now, single-label classification has been widely performed for predicting enzymatic function limiting the application to enzymes performing unique reactions and introducing errors when multi-functional enzymes are examined. Indeed, some enzymes may be performing different reactions and can hence be directly associated with multiple enzymatic functions. In the present work, we propose a multi-label enzymatic function classification scheme that combines structural and amino acid sequence information. We investigate two fusion approaches (in the feature level and decision level) and assess the methodology for general enzymatic function prediction indicated by the first digit of the enzyme commission (EC) code (six main classes) on 40,034 enzymes from the PDB database. The proposed single-label and multi-label models predict correctly the actual functional activities in 97.8% and 95.5% (based on Hamming-loss) of the cases, respectively. Also the multi-label model predicts all possible enzymatic reactions in 85.4% of the multi-labeled enzymes when the number of reactions is unknown. Code and datasets are available at

  9. Automatic single- and multi-label enzymatic function prediction by machine learning

    Directory of Open Access Journals (Sweden)

    Shervine Amidi


    Full Text Available The number of protein structures in the PDB database has been increasing more than 15-fold since 1999. The creation of computational models predicting enzymatic function is of major importance since such models provide the means to better understand the behavior of newly discovered enzymes when catalyzing chemical reactions. Until now, single-label classification has been widely performed for predicting enzymatic function limiting the application to enzymes performing unique reactions and introducing errors when multi-functional enzymes are examined. Indeed, some enzymes may be performing different reactions and can hence be directly associated with multiple enzymatic functions. In the present work, we propose a multi-label enzymatic function classification scheme that combines structural and amino acid sequence information. We investigate two fusion approaches (in the feature level and decision level and assess the methodology for general enzymatic function prediction indicated by the first digit of the enzyme commission (EC code (six main classes on 40,034 enzymes from the PDB database. The proposed single-label and multi-label models predict correctly the actual functional activities in 97.8% and 95.5% (based on Hamming-loss of the cases, respectively. Also the multi-label model predicts all possible enzymatic reactions in 85.4% of the multi-labeled enzymes when the number of reactions is unknown. Code and datasets are available at

  10. Enzymatic exploration of catalase from a nanoparticle producing and biodecolorizing algae Shewanella xiamenensis BC01. (United States)

    Ng, I-Son; Xu, Fangxin; Zhang, Xia; Ye, Chiming


    Shewanella xiamenensis (SXM) was found to produce nanoparticles (NPs) under aerobic condition. The oxidoreductase enzymatic activities including of catalase, manganese peroxidase, laccase, NADH dehydrogenase, flavin reductase, azoreductase and Fe reductase are first investigated. Catalase showed the greatest enzymatic activity among all oxidoreductases in SXM, which with strong activities in multiple substrates of ABTS, guaiacol and 2,6-DMP. The optimum temperature, pH, concentrations of H2O2 and 2,6-DMP for this enzyme were found to be 65 °C, pH 4.0, 128.7 mM and 10 mM, respectively. Finally, from the kinetic parameters and structure simulation of catalase, implied that SXM would potentially apply in bioremediation, microbe fuel cells (MFCs) and nano-biotechnology based on its distinguished enzymatic system.

  11. 发色底物法在酶促反应初速度内测定α1抗胰蛋白酶的活性%Detection ofα1 antitrypsin activity by chromogenic substrate assay with initial veloci-ty of enzymatic reaction

    Institute of Scientific and Technical Information of China (English)

    张晋超; 赵雄; 吕茂民; 尹惠琼; 王延琳; 章金刚


    Objective To detect the activity of α1 antitrypsin(AAT) with initial velocity of enzymatic reaction in order to detect the activity of samples in the process of separating and purifying plasma protein ,chromogenic substrate assay was optimized.Methods The effect of trypsin concentration and reaction time on enzymatic reaction was acquired by the kinetic monitoring mode of the microplate reader .Initial velocity was calculated to confirm the largest concentration of trypsin which was saturated by substrate .AAT was incubated with trypsin and absorbance produced by enzymatic reaction of remaining trypsin and substrate could reflect the activity of AAT .A standard curve was established with △D fitting with the activity of AAT standard.The activity of related samples was detected and the precision and accuracy of the method were evaluated . Results Trypsin concentration was 0.0625 mg/ml.Within 20 minutes, enzymatic reaction was with initial velocity .The range of the standard curve was 200-1200 IU/ml.Correlation coefficient was more than 0.99.The activity of Cohn Ⅳ, samples of pre-processing and elution were (720.59 ±18.63), (601.84 ±19.18),and (568.09 ±24.83)IU/ml, respec-tively.The relative standard deviation was less than 10%. Sample recovery rate was 90%-110%.Conclusion The optimized chromogenic substrate assay greatly improves accuracy and precision .The method can be used for the detec-tion of AAT activity of samples in laboratories and workshops .%目的:优化发色底物法,使其在酶促反应初速度内测定α1抗胰蛋白酶( AAT)的活性并用于血浆蛋白纯化过程中各样品活性的检测。方法采用酶标仪动态监测模式观察酶浓度和反应时间对酶促反应的影响;计算初速度并确定被底物饱和的最大酶浓度。将AAT与胰蛋白酶孵育,剩余靶酶和底物作用产生的光密度可反映AAT的活性。通过△D与AAT标准品活性进行拟合建立标准曲线,测定相关样品的活

  12. Enzymatic production of polysaccharides from gum tragacanth

    DEFF Research Database (Denmark)


    Plant polysaccharides, relating to the field of natural probiotic components, can comprise structures similar to human milk oligosaccharides. A method for enzymatic hydrolysis of gum tragacanth from the bush-like legumes of the genus Astragalus, using a combination of pectin hydrolases and a xylo......Plant polysaccharides, relating to the field of natural probiotic components, can comprise structures similar to human milk oligosaccharides. A method for enzymatic hydrolysis of gum tragacanth from the bush-like legumes of the genus Astragalus, using a combination of pectin hydrolases...... and a xylogalacturonan hydrolase, is described. Fractions with different oligo- and/or polysaccharide compositions and structure are separated according to molecular weight....

  13. Sugar ester surfactants: enzymatic synthesis and applications in food industry. (United States)

    Neta, Nair S; Teixeira, José A; Rodrigues, Lígia R


    Sugar esters are non-ionic surfactants that can be synthesized in a single enzymatic reaction step using lipases. The stability and efficiency of lipases under unusual conditions and using non-conventional media can be significantly improved through immobilization and protein engineering. Also, the development of de novo enzymes has seen a significant increase lately under the scope of the new field of synthetic biology. Depending on the esterification degree and the nature of fatty acid and/or sugar, a range of sugar esters can be synthesized. Due to their surface activity and emulsifying capacity, sugar esters are promising for applications in food industry.

  14. Organic synthesis - applications in enzymatic studies, catalysis and surface modification

    DEFF Research Database (Denmark)

    Viart, Helene Marie-France

    as liposomes and the enzymatic activity was studied. Hydrolysis (or absence of hydrolysis) was monitored by MALDI-TOF-MS. The results observedin these experiments are compared to MD predictions and confirm them. The second chapter deals with surface functionalization of liposomes. The copper mediated [3...... and a functionality covalently linked to a coupling partner (such as small molecule, peptide, etc.), we investigated the efficiency of the reaction depending on the position of the functional groups (whether on the liposome or on the coupling partner). Our results indicate that the reaction is most efficient when...

  15. Detecting platform for phenolic compounds-characteristic of enzymatic electrode (United States)

    Cabaj, Joanna; Chyla, Antoni; Jędrychowska, Agnieszka; Olech, Kamila; Sołoducho, Jadwiga


    We report here on simple and universal method for the highly efficient, electrolytic immobilization of tyrosinase (from Agaricus bisporus), for amperometric biosensing purposes. Tyrosinase has been successfully deposited on the surface of thin, ordered films of copolymerized derivative of thiophene (3-methylthiophene/3-thiopheneacetic acid/bis(ethylenedioxythiophene)diphenylamine). The tyrosinase retains well its activity well within the fabricated copolymer matrix. Reduction peaks, observed in cyclic voltammetry at 0.125 to +0.07 V, were attributed to the reduction of enzymatically liberated quinone species. Considering the fact, that immobilization strategy showed high efficiency, obtained results suggest that the method for phenoloxidase immobilization has a great potential for fabrication of bioelectronics' devices.

  16. Kinetics of enzymatic hydrolysis of methyl ricinoleate

    Directory of Open Access Journals (Sweden)

    Neeharika, T. S.V.R.


    Full Text Available Ricinoleic acid is an unsaturated hydroxy fatty acid that naturally occurs in castor oil in proportions of up to 85–90%. Ricinoleic acid is a potential raw material and finds several applications in coatings, lubricant formulations and pharmaceutical areas. Enzymatic hydrolysis of castor oil is preferred over conventional hydrolysis for the preparation of ricinoleic acid to avoid estolide formation. A kinetics analysis of the enzymatic hydrolysis of Methyl Ricinoleate in the presence of Candida antarctica Lipase B was carried out in this study by varying reaction temperature (40–60 °C and enzyme concentration (2–5%. The optimal conditions were found to be 6 h reaction time, temperature 60°C, buffer to methyl ricinoleate ratio 2:1(v/w and 4% enzyme concentration to achieve a maximum conversion of 98.5%. A first order reversible reaction kinetic model was proposed to describe this reaction and a good agreement was observed between the experimental data and the model values. The effect of temperature on the forward reaction rate constant was determined by fitting data to the Arrhenius equation. The activation energy for forward reaction was found to be 14.69 KJ·mol−1.El ácido ricinoleico es un hidroxiácido insaturado que se produce naturalmente en el aceite de ricino en proporciones de hasta el 85–90%. El ácido ricinoleico es una materia prima con gran potencial y tiene aplicaciones en revestimientos, formulaciones lubricantes y en áreas farmacéuticas. Para la preparación del ácido ricinoleico se prefiere la hidrólisis enzimática del aceite de ricino a la hidrólisis convencional, para evitar la formación de estólidos. En este estudio se llevó a cabo la cinética de la hidrólisis enzimática del ricinoleato de metilo en presencia de lipasa de Candida antarctica B mediante la variación de la temperatura de reacción (40–60 °C y la concentración de la enzima (2–5%. Las condiciones óptimas de la reacción para

  17. Bacterial and enzymatic bioassays for toxicity testing in the environment

    Energy Technology Data Exchange (ETDEWEB)

    Bitton, G.; Koopman, B. (Department of Environmental Engineering Sciences, University of Florida, Gainesville (United States))


    Microbioassays using bacteria or enzymes are increasingly applied to measure chemical toxicity in the environment. Attractive features of these assays may include low cost, rapid response to toxicants, high sample throughput, modest laboratory equipment and space requirements, low sample volume, portability, and reproducible responses. Enzymatic tests rely on measurement of either enzyme activity or enzyme biosynthesis. Dehydrogenases are the enzymes most used in toxicity testing. Assay of dehydrogenase activity is conveniently carried out using oxidoreduction dyes such as tetrazolium salts. Other enzyme activity tests utilize ATPases, esterases, phosphatases, urease, luciferase, beta-galactosidase, protease, amylase, or beta-glucosidase. Recently, the inhibition of enzyme (beta-galactosidase, tryptophanase, alpha-glucosidase) biosynthesis has been explored as a basis for toxicity testing. Enzyme biosynthesis was found to be generally more sensitive to organic chemicals than enzyme activity.107 references.

  18. Intramolecular epistasis and the evolution of a new enzymatic function.

    Directory of Open Access Journals (Sweden)

    Sajid Noor

    Full Text Available Atrazine chlorohydrolase (AtzA and its close relative melamine deaminase (TriA differ by just nine amino acid substitutions but have distinct catalytic activities. Together, they offer an informative model system to study the molecular processes that underpin the emergence of new enzymatic function. Here we have constructed the potential evolutionary trajectories between AtzA and TriA, and characterized the catalytic activities and biophysical properties of the intermediates along those trajectories. The order in which the nine amino acid substitutions that separate the enzymes could be introduced to either enzyme, while maintaining significant catalytic activity, was dictated by epistatic interactions, principally between three amino acids within the active site: namely, S331C, N328D and F84L. The mechanistic basis for the epistatic relationships is consistent with a model for the catalytic mechanisms in which protonation is required for hydrolysis of melamine, but not atrazine.

  19. In-vitro antioxidant and antibacterial properties of fermentatively and enzymatically prepared chicken liver protein hydrolysates. (United States)

    Chakka, Ashok Kumar; Elias, Mercy; Jini, R; Sakhare, P Z; Bhaskar, N


    Protein hydrolysates were prepared from chicken liver using fermentation and