Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.21 (beta-glucosidase)
3,280 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Since At2g25630 is an intronless gene with a premature stop codon, its cDNA encoding the predicted mature beta-glucosidase isoenzyme was synthesized from the previously isolated Arabidopsis thaliana genomic DNA. The stop codon was converted to a sense codon by site-directed mutagenesis. The native and mutated cDNA sequences were separately cloned into the vector pPICZalphaB and expressed in Pichia pastoris. Only the cells transformed with mutated cDNA-vector construct produced the active protein. The mutated recombinant beta-glucosidase isoenzyme was chromatographically purified to apparent homogeneity. The molecular mass of the protein is estimated as ca. 60 kD by SDS-PAGE. The pH optimum of activity is 5.6, and it is fairly stable in the pH range of 5.0-8.5. The purified recombinant beta-glucosidase is effectively active on para-/ortho-nitrophenyl-beta-D-glucopyranosides (p-/o-NPG) and 4-methylumbelliferyl-beta-D-glucopyranoside (4-MUG) with K(m) values of 1.9, 2.1, 0.78 mM and k(cat) values of 114, 106, 327 nkat/mg, respectively. It also exhibits different levels of activity against para-/ortho-nitrophenyl-beta-D-fucopyranosides (p-/o-NPF), amygdalin, prunasin, cellobiose, gentiobiose, and salicin. The enzyme is competitively inhibited by gluconolactone and p-nitrophenyl-1-thio-beta-D-glucopyranoside with p-NPG, o-NPG, and 4-MUG as substrates. The enzyme is found to be very tolerant to glucose inhibition. The catalytic role of nucleophilic glutamic acid in the motif YITENG of beta-glucosidases and mutated recombinant enzyme is discussed.
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PMID:A pseudo-beta-glucosidase in Arabidopsis thaliana: correction by site-directed mutagenesis, heterologous expression, purification, and characterization. 1877 38

An extracellular protein exhibiting beta-xylosidase activity was purified from the culture filtrate of a filamentous fungus, Aspergillus japonicus strain MU-2, grown on oat spelt xylan. The purified enzyme was a monomeric glycoprotein with an apparent M(r) of 113.2 kDa as estimated by SDS-PAGE. beta-Xylosidase activity was optimal at pH 4.0 and 70 degrees C. The enzyme also showed beta-glucosidase and alpha-l-arabinofuranosidase activities. The genomic DNA and cDNA encoding this protein were cloned and sequenced. Southern blot analysis indicated that the beta-xylosidase gene (xylA) was present as a single copy in the genome. An open reading frame, consisting of 2412 bp, was not interrupted by introns, and it encoded a presumed signal peptide of 17 amino acids and a mature protein of 787 amino acids. The deduced amino acid sequence of the xylA gene product showed a high degree of identity (69%) to the primary structure of the Aspergillus niger beta-xylosidase XlnD that belongs to the glycoside hydrolase family 3. Moreover, the xylA gene was functionally expressed in the yeast Pichia pastoris.
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PMID:Purification and properties of an extracellular beta-xylosidase from Aspergillus japonicus and sequence analysis of the encoding gene. 1900 Jun 18

Microorganisms that colonize plants require a number of hydrolytic enzymes to help degrade the cell wall. The maize endophyte Acremonium zeae was surveyed for production of extracellular enzymes that hydrolyze cellulose and hemicellulose. The most prominent enzyme activity in cell-free culture medium from A. zeae NRRL 6415 was xylanase, with a specific activity of 60 U/mg from cultures grown on crude corn fiber. Zymogram analysis following SDS-PAGE indicated six functional xylanase polypeptides of the following masses: 51, 44, 34, 29, 23, and 20 kDa. Xylosidase (0.39 U/mg), arabinofuranosidase (1.2 U/mg), endoglucanase (2.3 U/mg), cellobiohydrolase (1.3 U/mg), and beta-glucosidase (0.85 U/mg) activities were also detected. Although apparently possessing a full complement of hemicellulolytic activities, cell-free culture supernatants prepared from A. zeae required an exogenously added xylosidase to release more than 90% of the xylose and 80% of the arabinose from corn cob and wheat arabinoxylans. The hydrolytic enzymes from A. zeae may be suitable for application in the bioconversion of lignocellulosic biomass into fermentable sugars.
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PMID:Extracellular hemicellulolytic enzymes from the maize endophyte Acremonium zeae. 1918 10

A molecular display technology that uses the displayed proteins on cell surfaces has many applications in microbiology and molecular biology. Here, we describe the resistance of displayed proteins to proteases using simulated gastric fluid (SGF), which included pepsin at pH 2. The displayed beta-glucosidase resisted pepsin digestion compared with secreted, free beta-glucosidase. In SDS-PAGE and Western blotting analysis, the secreted beta-glucosidase was immediately digested within 1 min following SGF treatment, although the displayed beta-glucosidase was stable for more than 60 min following SGF treatment. In addition, the residual activity of secreted beta-glucosidase was completely destroyed after 10 min SGF treatment. However, displayed beta-glucosidase retained 14% of its residual activity following the same treatment. These results clearly show that cell surface display technology using enzymes can reveal the protease resistance of a protein of interest under various conditions.
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PMID:Evaluation of cell surface-displayed protein stability against simulated gastric fluid. 1943 Sep 13

A multienzyme complex, cellulosome, of the facultatively anaerobic bacterium, Paenibacillus curdlanolyticus B-6 was produced on microcrystalline cellulose (Avicel) under aerobic conditions. During growth on Avicel, the bacterial cells were found to be capable of adhesion to Avicel by scanning electron microscopic (SEM) analysis. The multienzyme complex of P. curdlanolyticus B-6 was isolated from the crude enzyme preparation by gel filtration chromatography on Sephacryl S-300 and affinity purification on cellulose. The isolated multienzyme complex was able to bind to both Avicel and insoluble xylan and consists of cellulolytic and xylanolytic enzymes such as avicelase, carboxymethyl cellulase (CMCase), cellobiohydrolase, beta-glucosidase, xylanase, beta-xylosidase and alpha-l-arabinofuranosidase. The molecular mass of the complex was estimated to be 1600 kDa. It composed of at least 12 proteins on SDS-PAGE and 10 CMCases and 11 xylanases on zymograms. The isolated multienzyme complex could degrade the raw lignocellulosic substances effectively.
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PMID:Isolation and characterization of a multienzyme complex (cellulosome) of the Paenibacillus curdlanolyticus B-6 grown on Avicel under aerobic conditions. 1944 36

The study of digestive enzymes, especially in important pests like Chilo suppressalis Walker (Lepidoptera: Pyralidae), which are a key constraint on rice production in a wide area of the globe and also in Iran, could be a successful procedure in the development of a safe and useful control strategy. Glycosidase are a type of digestive enzymes which have a critical role in the final stages of carbohydrate digestion; they hydrolyze alpha-D-(1,4)-glucose linkage such as p-nitrophenyl-alpha-D-glucoside in di and oligosaccharide components. Laboratory reared 4th instar larvae were randomly selected; midgut and salivary gland were removed by dissection under a stereo microscope and glucosidase activities were assayed by Ferreira and Terra's procedures. The activities of alpha- and beta-glucosidase in the midgut and salivary gland were 0.009, 0.0063, 0.005 and 0.003 micromol/min/mg protein, respectively. The optimal pH and temperature for enzyme activity were determined to be 9 and 45 degrees C for the glucosidases measured, values which are in agreement with other reports, especially in lepidopteran insects, which give values between 8-12 and 20-50 degrees C. The enzyme activity increased with the addition of NaCl, MgCl(2) and CaCl(2) and decreased due to the use of different concentrations of KCl, Urea, EDTA, SDS and Urea both in midgut and the salivary glands. Control of pests by using resistant varieties is one of the most important practices that are dependent on inhibitors in plants. Hence, characterization of digestive enzymes, especially the effect of inhibitors on enzyme activity, could be useful, on the one hand for a better understanding of enzyme roles in the nutrition physiology of insects, and on the other hand to reach safe and useful controls of insect pests.
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PMID:Enzymatic properties of alpha- and beta-glocusidases extracted from midgut and salivary glands of rice striped stem borer, Chilo suppressalis Walker (Lepidoptera: Pyralidae). 1952 3

A recombinant beta-glucosidase from Caldicellulosiruptor saccharolyticus DSM 8903 with a specific activity of 13 U/mg was purified by heat treatment and His-Trap affinity chromatography and identified as a single 54 kDa band on SDS-PAGE. The molecular mass of the native enzyme was 108 kDa as a dimer by gel filtration. beta-Glucosidase showed optimum activity at pH 5.5 and 70 degrees C for p-nitrophenyl (pNP)-beta-d-glucopyranoside. The half-lives of the enzyme at 60, 70, and 80 degrees C were 250, 24.3, and 0.4 h, respectively. The enzyme exhibited catalytic efficiency and specific activity for pNP-beta-d-fucopyranoside, pNP-beta-d-glucopyranoside, and pNP-beta-d-galactopyranoside in decreasing order among aryl-beta-glycosides, but not for aryl-alpha-glycosides. Cello-oligosaccharides from n = 2 to 5 as substrates using 4 mM each sugar and 3 U/mg of enzyme were completely hydrolyzed to glucose at 70 degrees C within 16 h.
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PMID:Characterization of a recombinant beta-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus. 1957 89

A beta-glucosidase was purified from the digestive fluid of the palm weevil Rhynchophorus palmarum L. (Coleoptera: Curculionidae) by chromatography on anion-exchange, gel filtration, and hydrophobic interaction columns. The preparation was shown to be homogeneous on polyacrylamide gels, beta-glucosidase is a monomeric protein with a molecular weight of 58 kDa based on its mobility in SDS-PAGE and 60 kDa based on gel filtration. Maximal beta-glucosidase activity occurred at 55 degrees C and pH 5.0. The purified beta-glucosidase was stable at 37 degrees C and its pH stability was in the range of 5.0-6.0. The enzyme readily hydrolyzed p-nitrophenyl-beta-D-glucoside, cellobiose, cellodextrins and required strictly beta-gluco configuration for activity. It cleaved glucose-glucose beta-(1-4) linkages better than beta-(1-2), beta-(1-3) and beta-(1-6) linkages. The catalytic efficiency (K(cat)/K(M)) values for p-nitrophenyl-beta-D-glucoside and cellobiose were respectively 240.48 mM(-1)s(-1) and 134.80 mM(-1)s(-1). Beta-glucosidase was capable of catalysing transglucosylation reactions. The yield of glucosylation of 2-phenylethanol (20 %), catalysed by the beta-glucosidase in the presence of cellobiose as glucosyl donor, is lower than those reported previously with conventional sources of beta-glucosidases. In addition, the optimum pH is different for the hydrolysis (pH 5.0) and transglucosylation reactions (pH 6.6).
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PMID:Purification and biochemical characterization of a specific beta-glucosidase from the digestive fluid of larvae of the palm weevil, Rhynchophorus palmarum. 1961 Dec 39

Myrosinases (EC 3.2.1.147) are beta-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1-TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS-PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation-inhibition responses towards ascorbic acid with maximal activity around 0.7-1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-beta-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.
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PMID:Myrosinases from root and leaves of Arabidopsis thaliana have different catalytic properties. 1970 94

beta-Glucosidase [EC 3.2.1.21] hydrolyzes cellobiose or cello-oligosaccharides into glucose during cellulose digestion in termites. SDS-PAGE and zymogram analyses of the digestive system in the higher termite Nasutitermes takasagoensis revealed that beta-glucosidase activity is localized in the salivary glands and midgut as dimeric glycoproteins. Degenerate PCR using primers based on the N-terminal amino acid sequences of the salivary beta-glucosidase resulted in cDNA fragments of 1.7 kb, encoding 489 amino acids with a sequence similar to glycosyl hydrolase family 1. Moreover, these primers amplified cDNA fragments from the midgut, and the deduced amino acid sequences are 87-91% identical to those of the salivary beta-glucosidases. Successful expression of the cDNAs in Escherichia coli implies that these sequences also encode functional beta-glucosidases. These results indicate that beta-glucosidases that primarily contribute to the digestive process of N. takasagoensis are produced in the midgut. Reverse transcription-PCR analysis indicated the site-specific expression of beta-glucosidase mRNAs in the salivary glands and midgut. These results suggest that termites have developed the ability to produce beta-glucosidases in the midgut, as is the case for endo-beta-1,4-glucanase, in which the site of expression has shifted from the salivary glands of lower termites to the midgut of higher termites.
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PMID:Digestive beta-glucosidases from the wood-feeding higher termite, Nasutitermes takasagoensis: intestinal distribution, molecular characterization, and alteration in sites of expression. 1994 57


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