Home About us Contact
|
Home About us Contact | ||
|
|
||
Enzyme Extracts (enzyme + extract)
Selected AbstractsEvaluation of In Vitro Apparent Protein Digestibility by Shrimp Using Gut Enzyme ExtractsJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 3 2009Joe M Fox Knowledge of apparent protein digestibility (APD) is required for optimization of feed formulae for the production of marine penaeid shrimp. The purpose of this study was to evaluate an in vitro method for determining APD in marine penaeid shrimp using gut enzyme extracts. A high correlation (r2 = 0.95) was shown between single-ingredient APD values for fish meal diets using in vivo methodology and those derived from in vitro testing of ingredients. A second study showed positive correlation (r2 = 0.71) between in vitro APD of selected purified and semipurified ingredients and their reported in vivo APDs. This correlation was much higher for purified ingredients (r2 = 0.93) versus less-refined ingredients (r2 = 0.24). A third trial compared in vitro APD at three different enzyme extract pH values and showed that for most protein sources, APD was significantly highest (P < 0.05) at pH = 7.0 and lower at pH = 6.1 or 7.9, indicating a neutral pH optimum for this methodology. [source] AN ESTEROLYTIC ACTIVITY FROM A WILD EDIBLE MUSHROOM, LYCOPERDON PERLATUMJOURNAL OF FOOD BIOCHEMISTRY, Issue 4 2009AHMET COLAK ABSTRACT Lycoperdon perlatum Pers. (Lycoperdaceae, Agaricales, Agaricomycetidae, Agaricomycetes, Basidiomycota, Fungi) was evaluated for its esterolytic potential. Native electrophoresis of the crude extracts showed four bands having Rf values of 0.34, 0.39, 0.52 and 0.59. The esterase showed the highest activity toward a short-chain substrate, p -nitrophenyl acetate. Optimum reaction conditions for L. perlatum crude extract were attained at pH 8.0 and 40C. Esterolytic activity of enzyme extract was stimulated in the presence of Mn2+, Fe2+, Ca2+ and Zn2+ in the reaction mixture. The enzyme activity was stimulated by incubation at pH 6.0 but retained 77% of its original activity at its optimum pH after 24 h. Thermal inactivation was displayed after incubation for 20 min at various temperatures above 30C. At 1 mM final concentration, 2-mercaptoethanol, dithiothreitol, ethylenediamine tetraacetic acid and p -methylphenyl sulfonylfluoride inhibited the esterolytic reaction. These results support that the crude L. perlatum extract possesses an esterolytic activity having properties similar to other esterases. PRACTICAL APPLICATIONS Esterases catalyzing the cleavage and formation of ester bonds are known ,/,-hydrolases (EC 3.1.1.X). Esterases are used for the synthesis of flavor esters for the food industry, modification of triglycerides for fat and oil industry and resolution of racemic mixtures used for the synthesis of fine chemicals for the pharmaceutical industry. Therefore, the search for new enzyme sources is important for the development of new enzymes and applications. [source] Immunolocalization of 1,3-,-Glucanases Secreted by Gaeumannomyces graminis var. tritici in Infected Wheat RootsJOURNAL OF PHYTOPATHOLOGY, Issue 5 2010Yongting Yu Abstract The distribution of extracellular 1,3-,-glucanase secreted by Gaeumannomyces graminis var. tritici (Ggt) was investigated in situ in inoculated wheat roots by immunogold labelling and transmission electron microscopy. Antiserum was prepared by subcutaneously injecting rabbits with purified 1,3-,-glucanase secreted by the pathogenic fungus. A specific antibody of 1,3-,-glucanase, anti-GluGgt, was purified and characterized. Double immunodiffusion tests revealed that the antiserum was specific for 1,3-,-glucanase of Ggt, but not for 1,3-,-glucanase from wheat plants. Native polyacrylamide gel electrophoresis of the purified and crude enzyme extract and immunoblotting showed that the antibody was monospecific for 1,3-,-glucanase in fungal extracellular protein populations. After incubation of ultrathin sections of pathogen-infected wheat roots with anti-1,3-,-glucanase antibody and the secondary antibody, deposition of gold particles occurred over hyphal cells and the host tissue. Hyphal cell walls and septa as well as membranous structures showed regular labelling with gold particles, while few gold particles were detected over the cytoplasm and other organelles such as mitochondria and vacuoles. In host tissues, cell walls in contact with the hyphae usually exhibited a few gold particles, whereas host cytoplasm and cell walls distant from the hyphae were free of labelling. Furthermore, over lignitubers in the infected host cells labelling with gold particles was detected. No gold particles were found over sections of non-inoculated wheat roots. The results indicate that 1,3-,-glucanase secreted by Ggt may be involved in pathogenesis of the take-all fungus through degradation of callose in postinfectionally formed cell wall appositions, such as lignitubers. [source] Evaluation of In Vitro Apparent Protein Digestibility by Shrimp Using Gut Enzyme ExtractsJOURNAL OF THE WORLD AQUACULTURE SOCIETY, Issue 3 2009Joe M Fox Knowledge of apparent protein digestibility (APD) is required for optimization of feed formulae for the production of marine penaeid shrimp. The purpose of this study was to evaluate an in vitro method for determining APD in marine penaeid shrimp using gut enzyme extracts. A high correlation (r2 = 0.95) was shown between single-ingredient APD values for fish meal diets using in vivo methodology and those derived from in vitro testing of ingredients. A second study showed positive correlation (r2 = 0.71) between in vitro APD of selected purified and semipurified ingredients and their reported in vivo APDs. This correlation was much higher for purified ingredients (r2 = 0.93) versus less-refined ingredients (r2 = 0.24). A third trial compared in vitro APD at three different enzyme extract pH values and showed that for most protein sources, APD was significantly highest (P < 0.05) at pH = 7.0 and lower at pH = 6.1 or 7.9, indicating a neutral pH optimum for this methodology. [source] ,-Glucosidase and peroxidase stability in crude enzyme extracts from green beans of Vanilla planifolia AndrewsPHYTOCHEMICAL ANALYSIS, Issue 3 2001Mark J. W. Dignum Abstract The extraction method for ,-glucosidase from green vanilla beans has been studied. The effect of storage of green beans and protein extracts on ,-glucosidase and peroxidase activity was investigated: the best method, resulting in the highest enzyme activities, particularly for glucosidase, was through extraction of very fresh green beans in the presence of BisTris propane buffer at pH 8. The best method for storage of the extracts was at ,80°C after addition of 15% glycerol, when over 90% of initial activity was still present. Peroxidase activity did not change in frozen beans or in frozen extracts. Copyright © 2001 John Wiley & Sons, Ltd. [source] Regulation of nitrate reductase by nitric oxide in Chinese cabbage pakchoi (Brassica chinensis L.)PLANT CELL & ENVIRONMENT, Issue 2 2008SHAOTING DU ABSTRACT Nitrate reductase (NR), a committed enzyme in nitrate assimilation, involves generation of nitric oxide (NO) in plants. Here we show that the NR activity was significantly enhanced by the addition of NO donors sodium nitroprusside (SNP) and NONOate (diethylamine NONOate sodium) to the culturing solution, whereas it was decreased by NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO). Interestingly, both NO gas and SNP directly enhanced but cPTIO inhibited the NR activities of crude enzyme extracts and purified NR enzyme. The cPTIO terminated the interaction between NR-generated NO and the NR itself. Furthermore, the NR protein content was not affected by the SNP treatment. The investigation of the partial reactions catalysed by purified NR using various electron donors and acceptors indicated that the haem and molybdenum centres in NR were the two sites activated by NO. The results suggest that the activation of NR activity by NO is regulated at the post-translational level, probably via a direct interaction mechanism. Accordingly, the concentration of nitrate both in leaves and roots was decreased after 2 weeks of cultivation with SNP. The present study identifies a new mechanism of NR regulation and nitrate assimilation, which provides important new insights into the complex regulation of N-metabolism in plants. [source] Digestive peptidases and proteinases in the midgut gland of the pink shrimp Farfantepenaeus paulensis (Crustacea, Decapoda, Penaeidae)AQUACULTURE RESEARCH, Issue 7 2009Diego Souza Buarque Abstract Proteases from the midgut gland of the Farfantepenaeus paulensis juveniles were assessed. Enzyme activity was determined using protease substrates and inhibitors. The effect of pH, temperature and calcium on proteolytic activity was assayed. Caseinolytic activity was analysed in substrate-sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Trypsin, chymotrypsin and leucine aminopeptidase activity was detected. Proteolytic activity was strongly inhibited by the specific trypsin inhibitors. Tosyl-phenylalanine chloromethyl ketone inhibited 59.3% of chymotrypsin activity. The greatest trypsin-like activity occurred at pH 8.0 and 45 °C. Chymotrypsin-like activity reached maximal values at alkaline pH (7.2,9.0) and 55 °C. CaCl2 did not increase trypsin-like activity, but rather inhibited it at concentrations of 30 (20%), 50 (30%) and 100 mM (50%). The substrate-SDS-PAGE zymogram revealed eight proteinase bands. Two possibly thermal-resistant (85 °C, 30 min) chymotrypsin isoforms were found, which were inhibited by phenyl-methyl-sulphonyl-fluoride. Aminopeptidase activity of enzyme extracts (Arg, Leu, Lys, Phe and Val) and the recommended concentrations of these essential amino acids in penaeid shrimp diets were positively correlated (P<0.05). Beause protein digestion involves the combined action of different enzymes, adequate knowledge of shrimp digestion and enzyme characteristics is required for the assessment of the digestive potential of different feed sources and development of in vitro digestibility protocols. [source] Identification and partial characterization of selected proteolytic enzymes in the digestive system of giant freshwater Prawn Macrobrachium rosenbergii (De Man) PostlarvaeAQUACULTURE RESEARCH, Issue 5 2009Mohamed Ayaz Hasan Chisty Abstract Biochemical assays and substrate SDS-PAGE were conducted to partially characterize and identify various types of proteases present in the digestive tract of PL15 giant freshwater prawn (Macrobrachium rosenbergii). Casein hydrolytic assay of the enzyme extracts showed major proteolytic activities at pH 3.0, 6.0 and 9.0, while assay of preincubated enzyme extracts with phenylmethylsulphonyl fluoride (PMSF), a serine protease inhibitor produced a 33.17% reduction in alkaline protease activity. When specific inhibitors tosyl-lysine chloromethyl ketone and tosyl-phenylalanine chloromethyl ketone were used, they resulted in a reduction in activity of proteases in the enzyme extracts by 82.41% and 55.03%, respectively, confirming the presence of trypsin and chymotrypsin, while ethylenediamine tetraacetic acid produced protease activity reduction in 33.92% showing the presence of metalloproteases in the digestive tract of the prawn. Further characterization of the alkaline proteases using SDS-PAGE technique, after incubating the extract in the presence or absence of specific inhibitors, produced six bands corresponding to molecular masses of between 13.48 and 136.1 kDa; two trypsin bands of 13.48 and 36.4 kDa, three chymotrypsin bands in the range of 23.0,73.4 kDa and one for metalloprotease of 136.1 kDa, all of which were identified from a zymogram. This study suggests that protein digestion in M. rosenbergii is initiated by an acid protease followed by a combination of action of alkaline proteases: trypsin, chymotrypsin and metalloproteases. [source] | ||||||||||||||||