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)

A new, weakly hydrophobic, high-performance liquid chromatography column has been developed for the separation of native proteins based on their relative hydrophobicities. Starting with a covalently bound, hydrophilic polyamine matrix, packing materials were synthesized through acylation with anhydrides and acid chlorides of increasing chain length to obtain increasingly hydrophobic surfaces. Proteins in aqueous buffers were induced to bind hydrophobically to the columns by the use of high salt concentrations in the mobile phase. Elution was achieved by decreasing the ionic strength of the solvent in a linear gradient. A mixture of cytochrome c, conalbumin, and beta-glucosidase was used as a standard to test the resolving power of newly synthesized columns. On a 4-cm butyrate column, baseline resolution was achieved in 20 min with a gradient of 3.0 mu sodium sulfate in 0.1 M potassium phosphate buffer, pH 7.0, to water. The static loading capacity for each column was determined using a hemoglobin binding assay. Capacities normally ranged between 150 and 180 mg of hemoglobin per gram of support. Since proteins are not denatured in hydrophobic interaction chromatography, enzymes eluted from the column retained enzymatic activity. Samples of alpha-amylase and beta-glucosidase ranging in size from 10 to 200 micrograms were recovered from the butyrate column with greater than 92% enzymatic activity in all cases. In a single trial, the enzyme citrate synthase was recovered from the benzoate column with 92% retention of enzymatic activity.
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PMID:High-performance hydrophobic interaction chromatography of proteins. 642 67

The interaction of alcohols in the hydrolysis of aryl beta-D-glucopyranosides and aryl beta-D-xylopyranosides by beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) from Stachybotrys atra has been investigated. The results constitute support for the presence of a glycosyl-enzyme intermediate, formed during the first step (glycosylation) of the proposed two-step mechanism. Transfer of the glycosyl group to an alcohol, with the formation of an alkyl glycopyranoside, can take place in parallel to the transfer to a water molecule (second or deglycosylation step). The alcohol binds to the free enzyme and to the glycosyl-enzyme intermediate. The glycosyl-enzyme-alcohol complex undergoes hydrolysis in addition to the alcoholysis. For aryl beta-D-glucopyranosides the deglycosylation step is rate-limiting. For aryl beta-D-xylopyranosides two kinds of substrate behaviour can be observed. Depending on the substituent group on the phenyl ring, either both steps are rate-controlling or the first step is rate-limiting. Electron-withdrawing substituents increase the rate at which the substrate aglycon group is released.
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PMID:Effects of alcohols on hydrolysis catalyzed by beta-D-glucosidase from Stachybotrys atra. 679 76

1. In the presence of a high concentration of p-nitrophenyl beta-D-glucopyranoside (donor) the rates of production of p-nitrophenol and a transglucosylation product (1-glyceryl beta-D-glucopyranoside) increased, whereas the rate of production of glucose decreased with increasing concentration of glycerol in reactions catalysed by the high-molecular-weight beta-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) obtained from culture filtrates of Botryodiplodia theobromae Pat. 2. When [donor] greater than Km the rate of production of p-nitrophenol was higher in the presence of glycerol than in its absence, whereas when [donor] less than Km the rate of production of p-nitrophenol was lower in the presence of glycerol than in its absence. 3. Glycerol increased both the Michaelis constant (Km) and maximum velocity (Vmax.), whereas dioxan increased Km but decreased Vmax. 4. Up to 1 mM-AgNO3 had no effect on enzyme activity. 5. A 2H-solvent-isotope-effect [Vmax. (H2O)/V max. (2H2O)] value of 1.40 +/- 0.05 was found at pH (or p2H) 5.8 6. alpha-2H-kinetic isotope-effect (kappa H/kappa 2H) values of 1.03 +/- 0.01 and 1.05 +/- 0.01 were found in the absence and presence of glycerol respectively. 7. Although maltose was a non-competitive inhibitor of beta-glucosidase activity, the ratio of velocity in the presence of glycerol to that in its absence increased, after an initial decline, with increasing concentration of maltose. 8. These results are discussed in terms of a mechanism involving a solvent-separated glucosyl cation-carboxylate ion-pair, which has greater affinity for alcoholic glucosyl acceptors, and an intimate ion-pair, which has greater affinity for water as a glucosyl acceptor and which could collapse reversibly and rapidly into a preponderance of an unreactive covalent glucosyl-enzyme.
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PMID:The beta-glucosidase from Botryodiplodia theobromae. Mechanism of enzyme action. 680 33

The contribution deals with histochemical localization of alpha-glucosidase, beta-glucosidase, beta-galactosidase and beta-glucuronidase in the mesencephalon of fresh water turtle. These enzymes demonstrate strong activity in all the myelinated fibers. Neuronal elements of nucleus ruber, nucleus isthmi, torus semicircularis, third and fourth cranial nerve nuclei, nucleus profundus mesencephali etc. demonstrate variable activity. Interestingly enough, there is parallel localization of all these enzymes in nuclei and tracts of mesencephalon. Further, the pattern of phospholipids and neutral lipids localization is almost identical to that of glycosidases. Since lipids and carbohydrates are rich source of energy in the central nervous system, and these enzymes are involved in their breakdown, their possible role in nerve cells and fibers of mesencephalon of turtle has been discussed.
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PMID:Significance of glycosidases in lipid and carbohydrate metabolism II. Studies in the mesencephalon of fresh water turtle (Lissemys punctata). 681 15

Beta-N-Acetylglucosaminidase has been purified from an acetone extract of Aspergillus niger. The protein has a Mr = 149,000. It contains neither Mn2+, Zn2+, nor cysteine and exhibits no cation requirement for activity. Isoelectric focusing separates two isozymes; the major isoenzyme has a pI = 4.4. Both isozymes exhibit beta-N-acetylgalactosaminidase and beta-glucosidase, as well as glucosaminidase activity. The mechanism of action of this enzyme has been studied in detail using a variety of substrate structure/activity and kinetic experiments. Rate data plotted versus pH depends on the following ionization constants, respectively: for pKm, 2.95; for log Kcat, 7.6; and for log kcat/Km, 2.95 and 8.25. The kcat value of H2O/D2O for p-nitrophenyl-beta-N-acetylglucosaminide hydrolysis is 1.27 at pH 4.6 and 1.00 at pH 7.0. The rho value for the hydrolysis of para-substituted phenylglucosaminides is +0.36; rho for the hydrolysis of fluoro-substituted N-acetyl derivatives is -1.41. Two sulfur-containing substrate analogues, the 1-thioglucosaminide, and the N-thioacetyl derivative, exhibit either no or little substrate activity. The hydrolysis of the 2,4-dinitrophenyl-glucosaminide is not biphasic as indicated by stopped flow kinetic studies. These several results are interpreted to show that: 1) enzymatic nucleophilic catalysis is not employed by beta-N-acetylglucosaminidase; 2) the glycosidic oxygen is protonated very early in the reaction, perhaps even in the Michaelis complex; 3) the acetamido oxygen provides anchimeric assistance to hydrolysis via charge stabilization of the oxocarbonium ion (or via oxazoline formation); 4) additional charge stabilization is provided by an enzymic anion, perhaps a side chain carboxylate group. The role of the acetamido group is discussed and comparisons are made between lysozyme, beta-galactosidase, and beta-N-acetylglucosaminidase.
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PMID:Purification, properties, kinetics, and mechanism of beta-N-acetylglucosamidase from Aspergillus niger. 744 May 73

Five calystegins were extracted from the roots of Physalis alkekengi var. francheti (Solanaceae) with hot water and purified to homogeneity by the combination of a variety of ion-exchange column chromatographies. Their structures have been determined from the 1H- and 13C-NMR spectral data, and two of the compounds were identified as calystegins A3 and B2, which have been isolated from the roots of Calystegia sepium (Convolvulaceae). Two of the remaining three were found to be 1 alpha, 3 alpha, 4 beta-trihydroxy-nor-tropane and 1 alpha, 2 alpha, 3 alpha, 4 beta-tetrahydroxy-nor-tropane and given the trivial name calystegins A5 and B3, respectively. The last calystegin was assigned as 1 alpha, 2 beta, 3 alpha, 6 alpha-tetrahydroxy-nor-tropane, which was the same as the relative configuration proposed in the literature for calystegin B1 isolated from C. sepium. However, the 13C-NMR spectral data for the compound from C. sepium differed substantially from our results. From a personal communication with the authors of the original paper on calystegins, it was clarified that the 13C-NMR chemical shifts of calystegin B1 in the original paper had been erroneous. Since their corrected 13C-NMR data of calystegin B1 and its 1H-NMR chemical shifts in the original paper are very close to our present data, we concluded that both compounds from C. sepium and P. alkekengi are identical. Calystegin B2 has been known to be a potent competitive inhibitor of almond beta-glucosidase (Ki = 1.2 microM) and coffee bean alpha-galactosidase (Ki = 0.86 microM). In this study calystegin B1 (1 alpha, 2 beta, 3 alpha, 6 alpha-tetrahydroxy-nor-tropane) proved to be a potent competitive inhibitor of almond beta-glucosidase (Ki = 1.9 microM) and bovine liver beta-galactosidase (Ki = 1.6 microM), but not an inhibitor of alpha-galactosidases. Calystegin A3 was found to be a weaker inhibitor compared to calystegin B2 but with the same inhibitory spectrum. Calystegin A5, a 2-deoxy derivative of calystegin B2, showed no activity against any glycosidases tested. Since calystegin B3, a 2-epimer of calystegin B2, also exhibited only a weak inhibitory activity, it was concluded that the equatorially oriented OH group at C2 is the essential feature for recognition and strong binding by the active site of glycosidases.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Calystegins of Physalis alkekengi var. francheti (Solanaceae). Structure determination and their glycosidase inhibitory activities. 774 59

The broad-specificity cyanogenic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) from Manihot esculenta Crantz (cassava) was kinetically characterized in mixed substrate systems and with the transition-state analogue glucono(1-5)lactone and a series of 1-thio substrate analogues. The results indicate a common catalytic and a common sugar binding site in the enzyme for all of the investigated substrates. Kinetic parameters of the hydrolysis of linamarin and p-nitrophenyl beta-D-glucopyranoside were determined over the pH range 3.5-9.0. The pH-dependence curves gave apparent pK values of 4.5 (4.6) and 7.1 (7.3) for the free enzyme, while values of 4.5 (3.7) and 9.3 were obtained for the enzyme-substrate complexes, using either linamarin or p-nitrophenyl beta-D-glucopyranoside as the substrate. Kinetic analysis of the modification indicated that one molecule of water-soluble carbodiimide or Woodward's reagent K is required to bind to the enzyme for inactivation. The enzyme was protected against inactivation by the competitive inhibitors p-nitrothiophenyl beta-D-glucopyranoside, beta-D-glucopyranosylamine, and glucono(1-5)lactone. Spectrophotometric analysis at 340 nm showed that from the three carboxylate groups modified by Woodward's reagent K essentially one was protected by p-nitrothiophenyl beta-D-glucopyranoside. During modification Vmax decreased to 30% of that of the unmodified enzyme and Km remained unchanged. The pH dependence of inactivation showed the involvement of a group with a pK value of 4.6, indicating the modification of a carboxyl residue essential for activity. Treatment of the enzyme with the histidine-group-specific reagent diethylpyrocarbonate resulted in 80% loss of enzyme activity, in biphasic kinetics. A treatment with 0.5 M hydroxylamine at pH 7.0 regenerated 92% of the original enzyme activity. The presence of the competitive inhibitor beta-D-glucopyranosylamine protected the enzyme against inactivation, preventing the modification of one histidine residue. Statistical analysis of the residual fractional activity against the number of modified residues indicated that the modification of one histidine is responsible for 40-50% of the inactivation. The pH dependence of the inactivation gave a pK value of 7.0 for the histidine group upon which the activity depends. During modification, Vmax decreased to 30% and Km decreased to 50% of the original values.
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PMID:Investigation of the active site of the cyanogenic beta-D-glucosidase (linamarase) from Manihot esculenta Crantz (cassava). I. Evidence for an essential carboxylate and a reactive histidine residue in a single catalytic center. 794 86

A thermo-reversible poly(N-isopropylacrylamide) poly(NIPAAm) oligomer with a carboxyl functional end group has been synthesized by radical polymerization using beta-mercaptopropionic acid as a chain transfer reagent. This polymer has been conjugated to an enzyme, beta-D-glucosidase, to form a thermo-reversible water soluble-insoluble polymer-enzyme conjugate. This conjugate can be used for separation, recovery and recycle of an enzyme simply by applying small temperature changes to the reaction medium. In contrast to the random polymer-enzyme conjugates reported in the literature, in this study the enzyme is coupled to each polymer chain by a single end attachment. These preliminary studies show that the conjugated enzyme exhibits very high retention of activity (> 90%) compared to the native enzyme and shows improved thermal stability.
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PMID:Synthesis of carboxylated poly(NIPAAm) oligomers and their application to form thermo-reversible polymer-enzyme conjugates. 802 32

The outer membrane (OM) of Fibrobacter succinogenes was isolated by a combination of salt, sucrose, and water washes from whole cells grown on either glucose or cellulose. The cytoplasmic membrane (CM) was isolated from OM-depleted cells after disruption with a French press. The OM and membrane vesicles isolated from the extracellular culture fluid of cellulose-grown cells had a higher density, much lower succinate dehydrogenase activity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles different from those of the CM. The OM from both glucose- and cellulose-grown cells and the extracellular membrane vesicles from cellulose-grown cultures exhibited higher endoglucanase, xylanase, and acetylesterase activities than the CM and other cell fractions. Endoglucanase 2 was absent from the isolated OM fractions of glucose- and cellulose-grown cells and from the extracellular membrane vesicles of cellulose-grown cells but was present in the CM and intracellular glycogen granule fractions, while endoglucanase 3 was enriched in the OM. Cellobiosidase was located primarily in the periplasm as previously reported, while cellobiase was mainly present in the glycogen granule fraction of glucose-grown cells and in a nongranular glycogen and CM complex in cellulose-grown cells. The cellobiase was not eluted from glycogen granules by cellobiose, maltose, and maltotriose nor from either the granules or the cell membranes by nondenaturing detergents but was eluted from both glycogen granules and cell membranes by high concentrations of salts. The eluted cellobiase rebound almost quantitatively when diluted and mixed with purified glycogen granules but exhibited a low affinity for Avicel cellulose. Thus, we have documented a method for isolation of OM from F. succinogenes, identified the OM origin of the extracellular membrane vesicles, and located glycanases and cellobiase in membrane and glycogen fractions.
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PMID:Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase. 822 22

A series of water-insoluble acrylic polymers containing disaccharide side groups were synthesized and evaluated in vitro. A cellobiose-derived monomer, 4-O-beta-D-glucopyranosyl-1-methacrylamido-1-deoxy-D-glucitol, was prepared and copolymerized with methacrylic acid. Two different modes of polymerization were used to give two products, P-1 and P-2. A homopolymer, P-3, was also synthesized using the same method as P-2. The degradation of the disaccharide side groups in these polymers and the monomer was evaluated by incubation with beta-glucosidase and measurement of the amount of glucose cleaved. It was found that the degradation rate increased in those polymers possessing lower contents of the disaccharide side groups (i.e. higher content of methacrylic acid). Scanning electron microscopy (SEM) observations of cross-sectioned slabs of P-1 visualized the degradation of the polymer. The enzymatic reaction caused a porous structure to be formed. The increased porosity may be used for the specific release of drugs into organs that contain large amounts of beta-glucosidases, such as the human colon.
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PMID:Enzymatic cleavage of disaccharide side groups in insoluble synthetic polymers: a new method for specific delivery of drugs to the colon. 832 19


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