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)

beta-Glucosidase activity was measured in control subjects and in five patients with neuropathic Gaucher's disease. In three patients with Gaucher's disease, methylumbelliferyl- and p-nitrophenyl-beta-d-glucopyranoside (4MU- and PNP-beta-glucosidase) activity was almost normal in the liver but markedly reduced in the spleen and fibroblasts. In the other patients with Gaucher's disease 4MU- and PNP-beta-glucosidase activity was also very much reduced in the liver, spleen, and fibroblasts. DEAE-cellulose column chromatography with a chloride gradient elution of the liver extract from a control subject and from two patients with Gaucher's disease, exhibiting normal 4MU- and PNP-beta-glucosidase activity, revealed the presence of two peaks of 4MU- and PNP-beta-glucosidase activity (fractions 1 and 2). pH activity curves of beta-glucosidases and Km measured with 4MU-beta-glucoside in fractions 1 and 2 from patients with Gaucher's liver were identical to those from the control liver. However, fractions 1 and 2 from infantile Gaucher's liver exhibited no activity measured with glucocerebroside whereas those from juvenile Gaucher's liver showed a considerable activity. Glucocerebroside was greatly accumulated in the liver, even though an almost normal activity of 4MU-beta-glucosidase was detected in three of the five patients studied.
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PMID:Neuropathic Gaucher's disease with normal 4-methylumbelliferyl-beta-glucosidase activity in the liver. 87 Aug 71

A beta-D-glycosidase activity was purified from mycelium of Humicola grisea var. thermoidea grown on avicel as the main carbon source. The purified enzyme was a glycoprotein and migrated as a single polypeptide band on polyacrylamide gel electrophoresis under native or denaturing conditions. The apparent molecular weight of the enzyme was estimated to be 55 kDa by gel filtration and SDS-PAGE. The enzyme was active against o-nitrophenyl beta-D-galactoside; p-nitrophenyl beta-D-glucoside, p-nitrophenyl beta-D-fucoside, lactose and cellobiose, PNP fucoside (synthetic substrate) and cellobiose (natural substrate) being the best utilized. A comparison of the properties of beta-D-galactosidase, beta-D-glucosidase and beta-D-fucosidase showed that three activities exhibited similar pH and temperature optima and the same thermostability. The hydrolysis rate of substrate mixtures suggests that the enzyme possesses a common catalytic site for all the substrates assayed.
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PMID:Beta-D-glycosidase activities of Humicola grisea: biochemical and kinetic characterization of a multifunctional enzyme. 210 72

In this report we attempt to explain the discrepancy between beta-glucosidase (EC 3.2.1.21) activity in Candida albicans as measured by commercial kits and that found in an experimental assay. beta-Glucosidase activity in American and Israeli isolates of C. albicans was evaluated with the API ZYM and YeastIdent systems (Analytab Products) and with experimental biochemical assays. Activity was found with whole cells and cell extracts of isolates from both sources. The greatest beta-glucosidase activity was found at pH 5.0 and with p-nitrophenyl-beta-glucopyranoside (PNP-BDG) as the substrate. In assays with beta-naphthyl-beta-D-glucopyranoside and 6-bromo-2-naphthyl-beta-D-glucopyranoside (6-Br-2-naphthyl-BDG), no enzyme activity was detected in whole cells and only limited activity was found in cell extracts of isolates from both sources. In studies with PNP-BDG at pH 5.0 and 7.5, 29 to 38% less activity was found at both pHs with American whole cells, and minor activity (20%) was found at pH 7.5 with isolates from both sources. Because assays with PNP-BDG in cell extracts of isolates from both sources showed no significant differences in activity, the more limited beta-glucosidase activity in American whole cells was most likely due to less efficient transport. Because the API ZYM system uses 6-Br-2-naphthyl-BDG as the substrate and because the substrate is buffered at pH 7.5 in the API YeastIdent kit, both systems appear to be of limited value for the detection of beta-glucosidase activity in C. albicans.
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PMID:beta-Glucosidase in Candida albicans and its application in yeast identification. 310 12

A novel substrate, beta-2-chloro-4-nitrophenylmaltopentaoside (beta CNPG5), was used for the enzyme-coupled determination of alpha-amylase in biological fluids. It was hydrolyzed specifically by alpha-amylase to about 90% producing beta-2-chloro-4-nitrophenylmaltoside (beta CNPG2) and maltotriose. Under the assay conditions, the absorption of 2-chloro-4-nitrophenol (CNP) generated by the secondary reaction of alpha-glucosidase and beta-glucosidase as auxiliary enzymes is about twice the absorption of 4-nitrophenol (PNP), which is the end product currently measured in some alpha-amylase assay methods. The sensitivity of the assay using beta CNPG5 was thus much higher than that using 4-nitrophenyl-maltopentaoside (PNPG5) as substrate. The absorption of CNP did not fluctuate with temperature or with pH between 6.8 and 7.2, which are the conditions normally used for determination of amylase activity in biological fluids.
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PMID:Determination of alpha-amylase in biological fluids using a new substrate (beta-2-chloro-4-nitrophenyl-maltopentaoside). 387 77

Glycosidase activities with starch and PNP-glycosides as substrates can be detected in the cotyledons, leaves, pods and teguments of Vicia faba beans. The mainly hydrolize alpha- and beta-glucosides, alpha- and beta-galactosides as well as starch. The amylase activity arises at very early stages of seed maturation and then rapidly decreases, whereas glycosidases increase up to the 30% of the beans dry weight before decreasing. The beta-glucosidase might play a role in regulating the metabolism of possibly haemolitic glycosides (i.e. vicine, convicine and L-DOPA glucoside) thus explaining the presence of this enzyme.
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PMID:[Genetic improvement in Vicia and favism. II. Glycosidase activity]. 729 14

The coding sequence of the mature cyanogenic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) of Manihot esculenta Crantz (cassava) was cloned into the vector pGEX-2T and expressed in Escherichia coli. The bacterial chaperonin GroEL [Braig, Otwinowski, Hedge, Boisvert, Joachimiak, Horwich and Sigler (1994) Nature (London) 371, 578-586] was found to be tightly associated with the fusion protein and co-purified with it. In the presence of excess MgATP, release and folding of the fusion beta-glucosidase were demonstrated by a fast increase in both linamarase and p-nitrophenyl-beta-D-glucopyranosidase activity at a low protein concentration. A slow endogenous folding process was also detected by activity measurements. Michaelis constants (Km) and the ratio between the maximal velocities and efficiency constants (Vmax., Vmax./Km) for the hydrolysis of the natural substrate, linamarin, and p-nitrophenyl beta-D-glucopyranoside (PNP-Glc) by the recombinant protein were found to be almost identical with those of the native glycosylated plant enzyme [Keresztessy, Kiss and Hughes (1994) Arch. Biochem. Biophys. 314, 142-152]. Molecular dissociation constants for the free enzyme (pK(E)1, pK(E)2) obtained with linamarin and PNP-Glc, and the enzyme substrate complexes (pK(ES)1, pK(ES)2) were also in accordance with that of the original protein. The reactive substrate analogue N-bromoacetyl beta-D-glucosylamine inactivated the fusion enzyme according to pseudo-first-order kinetics with first-order rate constant (k1=0.007 min-1) and apparent inhibition constants (k1=20 mM) comparable with those of the plant protein [Keresztessy, Kiss and Hughes (1994) Arch. Biochem. Biophys. 315, 323-330]. In comparison with the native glycosylated plant protein, the recombinant protein was, however, found to be extremely sensitive to proteolysis and misfolding.
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PMID:Co-purification from Escherichia coli of a plant beta-glucosidase-glutathione S-transferase fusion protein and the bacterial chaperonin GroEL. 866 Mar 8

The gene for thermostable 1,3-beta-glucosidase BglB was cloned from the chromosome of Thermotoga neapolitana and its primary sequence was determined. The purified recombinant beta-glucosidase B had a monomer molecular mass of 81 kDa in accordance with the amino acid sequence predicted from the nucleotide sequence of clone pTT51. It was a member of glycosylhydrolase family 3 and belonged to enzyme class EC 3.2.1.21. beta-Glucosidase B had a specific activity of 255 U mg-1 on 4-nitrophenyl(PNP)-beta-glucoside at the optima of pH (5.5) and temperature (90 degrees C), and K(m) values of 0.1, 10 and 50 mM for PNP-beta-glucoside, laminaribiose and cellobiose, respectively. The gene bglB was located immediately upstream of the laminarinase gene lamA. Both genes were transcribed from the same DNA strand and were not separated by a palindromic transcription terminator. The two purified enzymes 1,3-beta-glucosidase BglB (laminaribiase) and 1,3-beta-glucanase LamA (laminarinase) were together capable of completely degrading laminarin to glucose.
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PMID:Thermotoga neapolitana bglB gene, upstream of lamA, encodes a highly thermostable beta-glucosidase that is a laminaribiase. 938 32

Glycosynthases are nucleophile mutants of retaining glycosidases that catalyze the glycosylation of sugar acceptors using glycosyl fluoride donors, thereby synthesizing oligosaccharides. The 'original' glycosynthase, derived from Agrobacterium sp. beta-glucosidase (Abg) by mutating the nucleophile glutamate to alanine (E358A), synthesizes oligosaccharides in yields exceeding 90% [Mackenzie, L.F., Wang, Q., Warren, R.A.J. and Withers, S.G. (1998) J. Am. Chem. Soc. 120, 5583-5584]. This mutant has now been re-cloned with a His(6)-tag into a pET-29b(+) vector, allowing gram scale production and single step chromatographic purification. A dramatic, 24-fold, improvement in synthetic rates has also been achieved by substituting the nucleophile with serine, resulting in improved product yields, reduced reaction times and an enhanced synthetic repertoire. Thus poor acceptors for Abg E358A, such as PNP-GlcNAc, are successfully glycosylated by E358S, allowing the synthesis of PNP-beta-LacNAc. The increased glycosylation activity of Abg E358S likely originates from a stabilizing interaction between the Ser hydroxyl group and the departing anomeric fluorine of the alpha-glycosyl fluoride.
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PMID:The E358S mutant of Agrobacterium sp. beta-glucosidase is a greatly improved glycosynthase. 1064 8

The coding sequence of the mature cyanogenic beta-glucosidase (beta-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (K(m)=1.06 mM) and the synthetic p-nitrophenyl beta-D-glucopyranoside (PNP-Glc; K(m)=0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substrate complexes (pK(E)(1)=4.4-4.8, pK(E)(2)=6.7-7.2, pK(ES)(1)=3.9-4.4, pK(ES)(2)=8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased K(m) for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta-glucosidase.
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PMID:Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis. 1113 81