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)

Genomic DNA fragments encoding beta-glucosidase activities of the thermophilic actinomycete Microbispora bispora were cloned into Escherichia coli. Transformants expressing beta-glucosidase activity were selected by their ability to hydrolyze the fluorogenic substrate 4-methylumbelliferyl-beta-D-glucoside. Two genes encoding beta-glucosidase activity were isolated and distinguished by restriction analysis, Southern hybridization, and the substrate specificities of the encoded enzymes. One gene, bglB, encoded a beta-glucosidase that was expressed intracellularly in E. coli. It exhibited a molecular mass of approximately 52,000 Da by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) and 51,280 Da by nondenaturing gradient PAGE, a pI of 4.6, and temperature and pH optima of 60 degrees C and 6.2, respectively. Cloned BglB showed greater activity against cellobiose than against aryl-beta-D-glucosides and was thermostable, retaining about 70% of its activity after 48 h at 60 degrees C. BglB activity is activated two- to threefold in the presence of 2 to 5% (0.1 to 0.3 M) glucose. The DNA sequence of the 2.2-kb insert carrying bglB has been determined. An open reading frame which codes for a protein of 473 amino acids with a predicted molecular mass of 52,227 Da showed significant homology (40 to 47% identity) with beta-glucosidases from glycosal hydrolase family 1.
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PMID:Cloning, characterization, and nucleotide sequence of a gene encoding Microbispora bispora BglB, a thermostable beta-glucosidase expressed in Escherichia coli. 148 72

A genomic library of Pseudomonas fluorescens subsp. cellulosa DNA constructed in pUC18 and expressed in Escherichia coli was screened for recombinants expressing 4-methylumbelliferyl beta-D-glucoside hydrolysing activity (MUGase). A single MUGase-positive clone was isolated. The MUGase hydrolysed cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose to glucose, by sequentially cleaving glucose residues from the non-reducing end of the cello-oligosaccharides. The Km values for cellobiose and cellohexaose hydrolysis were 1.2 mM and 28 microM respectively. The enzyme exhibited no activity against soluble or insoluble cellulose, xylan and xylobiose. Thus the MUGase is classified as a 1,4-beta-D-glucan glucohydrolase (EC 3.2.1.74) and is designated 1,4-beta-D-glucan glucohydrolase D (CELD). When expressed by E. coli, CELD was located in the cell-envelope fraction; a significant proportion of the native enzyme was also associated with the cell envelope when synthesized by its endogenous host. The nucleotide sequence of the gene, celD, which encodes CELD, revealed an open reading frame of 2607 bp, encoding a protein of M(r) 92,000. The deduced primary structure of CELD was confirmed by the M(r) of CELD (85,000) expressed by E. coli and P. fluorescens subsp. cellulosa, and by the experimentally determined N-terminus of the enzyme purified from E. coli, which showed identity with residues 52-67 of the celD translated sequence. The structure of the N-terminal region of full-length CELD was similar to the signal peptides of P. fluorescens subsp. cellulosa plant-cell-wall hydrolases. Deletion of the N-terminal 47 residues of CELD solubilized MUGase activity in E. coli. CELD exhibited sequence similarity with beta-glucosidase B of Clostridium thermocellum, particularly in the vicinity of the active-site aspartate residue, but did not display structural similarity with the mature forms of cellulases and xylanases expressed by P. fluorescens subsp. cellulosa.
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PMID:Characterization of the gene celD and its encoded product 1,4-beta-D-glucan glucohydrolase D from Pseudomonas fluorescens subsp. cellulosa. 149 31

Piromyces sp. strain E2, an anaerobic fungus isolated from an Indian elephant (hindgut fermenter) was tested for its ability to ferment a range of substrates. The fungus was able to use bagasse, cellobiose, cellulose, fructose, glucose, lactose, mannose, starch, wheat bran, wheat straw, xylan and xylose. Formate and acetate were the main fermentation products after growth on these substrates. The amount of carbon found in the fermentation products of cultures, in which substrate digestion was complete averaged 88.5 mM, or 59% of the carbon offered as substrate. No growth was observed on other substrates tested. Lactose, starch, cellobiose and filter paper cellulose were good inducers of cellulolytic and xylanolytic enzymes. Cellulolytic and xylanolytic enzymes were produced constitutively by Piromyces strain E2, although enzyme activities were generally lower after growth on glucose and other soluble sugars. Complex substrates (bagasse, wheat bran, and wheat straw) were good inducers for xylanolytic enzymes but not for cellulolytic enzymes. The extracellular protein banding pattern after SDS-PAGE was therefore only slightly affected by the growth substrate. Identical beta-glucosidase and endoglucanase activity patterns were found after growth on different substrates. This indicated that differences in enzyme activities were not the result of secretion of different sets of isoenzymes although it remains possible that the relative amount of each isoenzyme produced is influenced by the growth substrate.
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PMID:Production of cellulolytic and xylanolytic enzymes during growth of the anaerobic fungus Piromyces sp. on different substrates. 152 5

The beta-glucosidase encoded by the bglA gene from Bacillus polymyxa was overproduced in Escherichia coli by using a plasmid in which bglA is under control of the lacI promoter. Induction with isopropyl-beta-D-thiogalactopyranoside allowed an increase in the specific activity of the enzyme of about 100 times the basal level of gene expression. The enzyme was purified by a two-step procedure involving salting out with ammonium sulfate and ion-exchange chromatography with DEAE-cellulose. Fractions of beta-glucosidase activity recovered by this procedure, after electrophoresis in an acrylamide gel and staining with silver nitrate, yielded a single band of protein. This band was shown by a zymogram to correspond to beta-glucosidase activity. The purified protein showed an apparent molecular mass of 50 kDa and an isoelectric point of 4.6, values in agreement with those expected from the nucleotide sequence of the gene. Km values of the enzyme, with either cellobiose or p-nitrophenyl-beta-D-glucoside as the substrate, were determined. It was shown that the enzyme is competitively inhibited by glucose. The effects of different metallic ions and other agents were studied. Hg2+ was strongly inhibitory, while none of the other cations tested had any significant effect. Ethanol did not show the stimulating effect observed with other beta-glucosidases. The mechanism of enzyme action was investigated. High-pressure liquid chromatography analysis with cellobiose as the substrate confirmed previous data revealing the formation of two products, glucose and another, unidentified, compound. Results presented here indicate that this compound is cellotriose formed by transglycosylation.
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PMID:Purification and characterization of a Bacillus polymyxa beta-glucosidase expressed in Escherichia coli. 156 36

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.
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PMID:Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei. 159 51

Four anaerobic fungi were grown on filter paper cellulose and monitored over a 7-8 days period for substrate utilisation, fermentation products, and secretion of cellulolytic and xylanolytic enzymes. Two of the fungi (N1 and N2) were Neocallimastix species isolated from a ruminant (sheep) and the other two fungi were Piromyces species (E2 and R1) isolated from an Indian Elephant and an Indian Rhinoceros, respectively. The tested anaerobic fungi degraded the filter paper cellulose almost completely and estimated cellulose digestion rates were 0.25, 0.13, 0.21 and 0.18 g.l-1.h-1 for strains E2, N1, N2, R1, respectively. All strains secreted cellulolytic and xylanolytic enzymes, including endoglucanase, exoglucanase, beta-glucosidase and xylanase. Strain E2 secreted the highest levels of enzymes in a relatively short time. The product formation on avicel by enzymes secreted by the four fungi was studied. Both in the presence and absence of glucurono-1,5-delta-lactone, a specific inhibitor of beta-glucosidase, mainly glucose was formed but no cellobiose. Therefore the exoglucanase secreted by the four fungi is probably a glucohydrolase.
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PMID:Fermentation of cellulose and production of cellulolytic and xylanolytic enzymes by anaerobic fungi from ruminant and non-ruminant herbivores. 179 36

A HindIII fragment of R. albus DNA encoding beta-glucosidase was cloned into E. coli. The DNA sequence (3158 bp) was determined, and the longest potential encoding sequence consisted of 2,841 bp (947 amino acids with the calculated molecular weight of 104,276. The deduced NH2-terminal amino acid sequence from the first (methionine) to the twentieth (glycine) was identical to that of the purified enzyme, suggesting that the gene for beta-glucosidase does not encode a signal peptide. The enzyme purified from the culture supernatant of the transformant had a molecular weight of 120,000 and its maximum activity was revealed at pH 6.5 and 30 degrees C. Reducing reagents activated the enzyme, whereas the sulfhydryl group-blocking reagents and reaction products (glucose) inhibited the activity. Hydrolyzates of celloorigomers contained glucose as a major product, indicating that the enzyme acts as beta-glucosidase. The enzyme from the transformant revealed similar properties to that from R. albus, and both enzyme proteins were immunologically the same to each other, indicating that the cloned gene encodes beta-glucosidase from R. albus.
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PMID:Cloning of a beta-glucosidase gene from Ruminococcus albus and its expression in Escherichia coli. 180 3

A beta-glucosidase with cellobiase activity was purified to homogeneity from the culture filtrate of the mushroom Termtomyces clypeatus. The enzyme had optimum activity at pH 5.0 and temperature 65 degrees C and was stable up to 60 degrees C and within pH 2-10. Among the substrates tested, p-nitrophenyl-beta-D-glucopyranoside and cellobiose were hydrolysed best by the enzyme. Km and Vm values for these substrates were 0.5, 1.25 mM and 95, 91 mumol/min per mg, respectively. The enzyme had low activity towards gentiobiose, salicin and beta-methyl-D-glucoside. Glucose and cellobiose inhibited the beta-D-glucosidase (PNPGase) activity competitively with Ki of 1.7 and 1.9 mM, respectively. Molecular mass of the native enzyme was approximated to be 450 kDa by HPLC, whereas sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated a molecular mass of 110 kDa. The high molecular weight enzyme protein was present both intracellularly and extracellularly from the very early growth phase. The enzyme had a pI of 4.5 and appeared to be a glycoprotein.
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PMID:Purification and characterisation of a beta-glucosidase (cellobiase) from a mushroom Termitomyces clypeatus. 190 Jan 98

Combined kinetic, ultracentrifugation and X-ray-crystallographic studies have characterized the effect of the beta-glucosidase inhibitor gluconohydroximo-1,5-lactone on the catalytic and structural properties of glycogen phosphorylase. In the direction of glycogen synthesis, gluconohydroximo-1,5-lactone was found to competitively inhibit both the b (Ki 0.92 mM) and the alpha form of the enzyme (Ki 0.76 mM) with respect to glucose 1-phosphate in synergism with caffeine. In the direction of glycogen breakdown, gluconohydroximo-1,5-lactone was found to inhibit phosphorylase b in a non-competitive mode with respect to phosphate, and no synergism with caffeine could be demonstrated. Ultracentrifugation and crystallization experiments demonstrated that gluconohydroximo-1,5-lactone was able to induce dissociation of tetrameric phosphorylase alpha and stabilization of the dimeric T-state conformation. A crystallographic binding study with 100 mM-gluconohydroximo-1,5-lactone at 0.24 nm (2.4 A) resolution showed a major peak at the catalytic site, and no significant conformational changes were observed. Analysis of the electron-density map indicated that the ligand adopts a chair conformation. The results are discussed with reference to the ability of the catalytic site of the enzyme to distinguish between two or more conformations of the glucopyranose ring.
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PMID:The binding of D-gluconohydroximo-1,5-lactone to glycogen phosphorylase. Kinetic, ultracentrifugation and crystallographic studies. 190 Sep 87

Glucocerebrosidase, the lysosomal enzyme that is deficient in patients with Gaucher's disease, hydrolyses non-physiological aryl beta-D-glucosides and glucocerebroside, its substrate in vivo. We document that 2,3,-di-O-tetradecyl-1-O-(beta-D-glucopyranosyl)-sn-glycerol (2,3,-di-14:0-beta-Glc-DAG) inhibits human placental glucocerebrosidase activity in vitro (Ki 0.18 mM), and the nature of inhibition is typical of a mixed-type pattern. Furthermore, 2,3-di-14:0-beta-Glc-DAG was shown to be an excellent substrate for the lysosomal beta-glucosidase (Km 0.15 mM; Vmax. 19.8 units/mg) when compared with the natural substrate glucocerebroside (Km 0.080 mM; Vmax. 10.4 units/mg). The observations that (i) glucocerebrosidase-catalysed hydrolysis of 2,3-di-14:0-beta-Glc-DAG is inhibited by conduritol B epoxide and glucosylsphingosine, and (ii) spleen and brain extracts from patients with Gaucher's disease are unable to hydrolyse 2,3-di-14:O-beta-Glc-DAG demonstrate that the same active site on the enzyme is responsible for catalysing the hydrolysis of 4-methylumbelliferyl beta-D-glucopyranoside, glucocerebroside and 2,3-di-14:O-beta-Glc-DAG. With the aid of computer modelling we have established that the oxygen atoms in 2,3-DAG-Glc at the C-1, C-4*, C-5* (the ring oxygen in glucose) and C-2 positions correspond topologically to the oxygens at C-1, C-4* and C-5* and the nitrogen atom attached to C-2 respectively in glucocerebroside (* signifies a carbon atom in glucose); furthermore, all of the distances with respect to overlap of corresponding heteroatoms range from 0.02 A to 0.77 A (0.002-0.077 nm). A root-mean-square deviation of 0.31 A (0.031 nm) was obtained when the energy-minimized structures of 2,3-di-14:O-beta-Glc-DAG and glucocerebroside were compared using the latter four heteroatom co-ordinates.
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PMID:2,3-di-O-tetradecyl-1-O-(beta-D-glucopyranosyl)-sn-glycerol is a substrate for human glucocerebrosidase. 190 Sep 89


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