Induction of β-D-Glucosidase inStreptomyces granaticolor

beta-D-glucosidase in Streptomyces granaticolor is an inducible enzyme. Methyl-beta-D-glucoside or cellobiose, added to a glycerol-containing medium, are most suitable inducers. The activity of beta-D-glucosidase in a culture fully induced by cellobiose is 50 times higher than the basal level of the enzyme. beta-D-glucosidase is an intracellular enzyme, whose inducibility differ with culture age and reaches its maximum in a 10-h-old mycelium. The enzyme synthesis begins 2 h after the addition of the induced and reaches its maximum after a 10-h-induction.     

Physical and kinetic properties of a plasma-membrane-bound beta-D-glucosidase (cellobiase) from midgut cells of an insect (Rhynchosciara americana larva).

The midgut caecal cells from Rhynchosciara americana larvae possess a plasma-membrane-bound beta-D-glucosidase (cellobiase, EC 3.2.1.21), which is recovered (75-95%) in soluble form both after treatment with Triton X-100 and after treatment with papain. The Triton X-100-solubilized beta-D-glucosidase displays Mr106000 and pI 5.4, whereas the papain-released beta-D-glucosidase shows Mr65000 and pI 4.7. Thermal inactivations of the detergent-solubilized and the papain-released forms of beta-D-glucosidase both follow apparent first-order kinetics with similar half-lives. The papain-released beta-D-glucosidase, after being purified by density-gradient centrifugation, hydrolyses beta-D-glucosides, beta-D-galactosides and beta-D-fucosides at the same active site, as inferred from experiments of competition between substrates. The beta-D-glucosidase seems to operate in accordance with rapid-equilibrium kinetics, since the Km (0.61 mM) for the enzyme is constant over a wide range of pH. The hydrolysis of the beta-D-glucosidic bond catalysed by the beta-D-glucosidase occurs without inversion of configuration, delta-gluconolactone is a strong (Ki 0.5 microM) inhibitor of the enzyme and substituents in the substrate aglycone affect the catalytic constant of the reaction. These data support the assumption that the mechanism of the reaction catalysed by the beta-D-glucosidase involves the intermediary formation of a carbonium ion, rather than a glucosyl-enzyme intermediate.     

Properties and application of immobilized beta-D-glucosidase coentrapped with Zymomonas mobilis in calcium alginate

The enzyme beta-D-glucosidase has been immobilized on concanavalin A-Sepharose to give a maximum loading of 2050 units/g dry weight of support material. The immobilized beta-D-glucosidase was also entrapped within calcium alginate gel spheres with apparently only 35% retention of activity when assayed with 10mM cellobiose. However, it was discovered that, unlike the immobilized enzyme, the entrapped immobilized enzyme was not subject to substrate inhibition up to 100mM cellobiose, suggesting that a concentration gradient of cellobiose existed between the bulk solution and the interior of the gel sphere. Thus, the activity of the entrapped immobilized enzyme was almost twice as high as that of the immobilized enzyme when assayed with 100mM cellobiose. Concanavalin A-Sepharose-immobilized beta-D-glucosidase and the bacterium Zymomonas mobilis coimmobilized in calcium alginate gel spheres converted cellobiose to ethanol in both batch and continuous-flow fermentation systems.     

Kinetic studies on the broad-specificity beta-D-glucosidase from pig kidney.

A broad-specificity beta-D-glucosidase from pig kidney cortex was isolated and purified to homogeneity by a rapid purification procedure. The pI (5.14 +/- 0.05), Mr (59,000 +/- 2000) and specific activities with several p-nitrophenyl glycosides (galactopyranoside, glucopyranoside, arabinopyranoside, xylopyranoside) were comparable with those published previously for cytoplasmic beta-D-glucosidase from other sources and organs. Mixed-substrate experiments and inhibition studies with glucono-(1----5)-lactone revealed that a single active centre, containing one catalytic site and one saccharide-binding site, was responsible for the splitting of all four synthetic substrates. Inhibition experiments with substrate analogues demonstrated that (i) the major binding determinant of the glycosides was the aglycone moiety, (ii) an anionic side chain of the enzyme (probably a carboxy group) interacted with the glycosidic linkages and (iii) the properties of the aglycone significantly influenced the binding of the carbohydrate moiety. The inhibition constants of the p-nitrothiophenyl derivatives were in good agreement with the Km values of the corresponding substrates. Therefore the Michaelis constants could be regarded as true equilibrium constants (Ks). The 'three-point-attachment model' of the substrate splitting, proposed by Daniels [(1983) Ph.D. Dissertation, University of Pittsburgh] for the analogous liver enzyme, was applicable for beta-D-glucosidase from pig kidney too. The possible nature of the 'attachments' is discussed.     

Purification and properties of beta-D-glucosidase (linamarase) from the butter bean, Phaseolus lunatus

A beta-D-glucosidase (linamarase) was purified 11,700-fold from the butter bean, Phaseolus lunatus L., by means of successive procedures including extraction, ammonium sulfate fractionation, acetone treatment, and chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-200. The final preparation gave a single protein band on both disc polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. In spite of its electrophoretic purity, the final enzyme preparation showed four glycosidase activities; beta-D-glucosidase, beta-D-galactosidase, beta-D-fucosidase, and beta-D-xylosidase. The molecular weight of the enzyme was determined to be 124,000 +/- 9,000 by Sephadex G-200 gel filtration, and 59,000 +/- 2,400 by SDS-disc gel electrophoresis. The enzyme showed a pH optimum in the range of 5.1 to 6.0 with p-nitrophenyl beta-D-glucoside, 4-methylumbelliferyl beta-D-glucoside, and linamarin. Among natural substrates containing a beta-glucosyl terminal, linamarin, prunasin, and salicin were hydrolyzed by the enzyme from butter beans, but amygdalin, cellobiose, gentiobiose, and laminarin were hardly hydrolyzed.     

Purification and characterization of a strictly specific beta-D-fucosidase from Aspergillus phoenicis.

Although beta-D-fucosidase (beta-D-fucohydrolase, EC 3.2.1.38) has been isolated from various sources, all those enzymes were associated with a high activity of beta-D-galactosidase and/or beta-D-glucosidase. We have purified a specific beta-D-fucosidase in electrophoretically homogeneous form from crude extracts of Aspergillus phoenicis by polyethyleneglycol 8000-phosphate buffer aqueous two-phase separation, and successive chromatography on DEAE-Sephadex A-50, hydroxyapatite, and Sephadex G-100 columns. The molecular weight of the enzyme was estimated to be 57,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 50,000 to 60,000 by gel filtration on Sephadex G-100. The enzyme showed optimum activity at pH 6.0 and 40 degrees C; it was stable in the pH range 5.5-6.5 and below 35 degrees C. The Km and the Vmax values for pNP-beta -D-fucoside were 2.4 mM, and 12.8 mumol.min-1.mg-1, respectively. The enzyme was strongly inhibited by sulfhydryl group reagents, p-chloromercuribenzoate, n-ethylmaleimide, and iodoacetate. It was also inhibited by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, diethyl pyrocarbonate, and N-bromosuccinimide. Thus, -SH and -COOH groups and histidyl and tryptophyl residues were essential for enzyme activity. The purified beta-D-fucosidase showed high specificity toward p-nitrophenyl-beta-D-fucoside. The enzyme was inhibited by D-fucose and D-fucono-gamma-lactone, but not by D-galactose, D-galactono-gamma-lactone, D-glucose, or D-glucono-gamma-lactone; the latter compounds are specific inhibitors of beta-D-galactosidase and beta-D-glucosidase, respectively. Thus, this enzyme is the most strictly specific beta-D-fucosidase when compared with those previously reported.     

Enzymatic glucosylation of hydrophobic alcohols in organic medium by the reverse hydrolysis reaction using almond-beta-D-glucosidase

Alkyl beta-D-glucosides were synthesized from D-glucose and alcohols by reverse hydrolysis using the commercially available almond beta-D-glucosidase in 9:1 (v/v) acetonitrile-water medium. The main characteristics of this enzyme-catalyzed glucosylation were established by using 2-hydroxybenzyl alcohol. The reaction is entirely regio- and stereoselective. The solvent plays a fundamental role because, by decreasing the water concentration in the medium, the shift of the reaction equilibrium toward synthesis is realized without using an excessive amount of alcohol. Nevertheless, a minimum amount of water is necessary to maintain the enzyme activity. In contrast to the use of the enzyme in aqueous medium, the pH of the added water in acetonitrile did not influence the synthesis. Using this procedure, we have conducted systematic glucosylation of numerous alcohols and we have investigated enzyme specificity and alcohol reactivity. The enzyme has a pronounced affinity for the alcohols containing a phenyl group, and enantioselectivity for the aglycon is obtained with 1-phenylethyl alcohol. Moreover, by using almond beta-D-glucosidase it was also possible to synthesize alkyl beta-D-galactosides. (c) 1995 John Wiley & Sons, Inc.     

Purification and characterisation of a beta-glucosidase (cellobiase) from a mushroom Termitomyces clypeatus

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.     

The extracellular acid phosphatase of the mosquito-parasitizing fungus Lagenidium giganteum

The mosquito-parasitizing fungus Lagenidium giganteum secreted a soluble acid phosphatase and beta-D-glucosidase into the growth medium. The acid phosphatase was isolated and purified to single component, and some of its physicochemical properties were determined. The enzyme exhibited a pH optimum of 5.6 in phthalate buffer with p-nitrophenyl phosphate and was temperature-inactivated at 55 degrees C. Enzyme activity seems to be limited to phenyl-phosphate substrates. A molecular weight of 42,800 was found and the amino acid content was also determined. A Km for p-nitrophenyl phosphate of 1.6 x 10(-7) M was found. The possible involvement of the enzyme in the infective process was discussed.     

Multiple carbohydrate-cleaving specificities in human acidic and neutral glycosidases.

The common identity of human acidic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) and beta-D-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) as one enzyme and that of acidic beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), beta-D-fucosidase (no allotted EC number) and alpha-L-arabinosidase (alpha-L-arabinofuranoside arabinohydrolase, EC 3.2.1.55) as another enzyme is indicated by similar binding patterns of glycosidase activities of each enzyme to various lectins. by similar ratios between their intra- and extracellular levels in normal and I-cell fibroblasts and by their deficiencies in liver tissues from patients with Gaucher disease and GM1 gangliosidosis, respectively. A third enzyme, neutral beta-D-galactosidase, purified to homogeneity from human liver has been shown to possess all these five glycosidase activities at neutral pH. These neutral enzymic activities were not bound by any of the lectins examined and found to be reduced in liver and spleen of a patient with neutral beta-D-galactosidase deficiency. An additional form of beta-D-xylosidase with optimal activity at pH 7.4 was bound by the fucose-binding lectin from Ulex eurpaeus while no binding was observed for the acidic (pH 4.8) and neutral (pH 7.0) beta-D-xylosidase activities of the multiple glycosidase enzymes.     

Beta-D-glycosidase activities of Humicola grisea: biochemical and kinetic characterization of a multifunctional enzyme

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.     

Partial purification and characterization of Naegleria fowleri beta-glucosidase

Naegleria fowleri cells, grown axenically, contain high levels of beta-D-glucosidase which catalyzes the hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside (4MUGlc) (Km, 0.9 mM), octyl-beta-D-glucoside (Km, 0.17 mM), and p-nitrophenyl-beta-D-glucopyranoside at relative rates of 1.00, 2.88, and 1.16, respectively (substrate concentration, 3.0 mM). When the amebae are subjected to freeze-thawing, sonication, and centrifugation (100,000 g, 1 h), 85% of the beta-glucosidase activity appears in the supernatant fraction. The beta-glucosidase was purified 40-fold (34% yield) using a combination of chromatographic steps involving DE-52 cellulose, concanavalin A-Sepharose, and hydroxylapatite followed by isoelectric focusing. The predominant soluble beta-D-galactosidase activity in the Naegleria extract copurifies with the beta-D-glucosidase; the two activities have the same isoelectric point (pI, 6.9), similar heat stabilities, are both inhibited by lactobionic acid (Ki, 0.40 mM), and exhibit optima at pH 4.5, indicating that they are probably the same enzyme. The Naegleria beta-D-glucosidase has an apparent molecular weight of 66,000, a Stokes radius of 25 A, and a sedimentation coefficient of 4.2S. The beta-glucosidase is not inhibited by conduritol beta-epoxide or galactosylsphingosine but is completely inhibited by 1.25 mM bromo conduritol beta-epoxide. The latter compound, when present in the growth medium, inhibits the growth of the organism and profoundly alters its ultrastructure, the main effect being the apparent inhibition of cytokinesis and the generation of multinucleate cells. The issue of the role of the beta-glucosidase in the metabolism of the ameba and its possible role in pathogenic mechanisms are discussed.     

Metabolites of marine organisms as regulators of O-glycosylhydrolases

The ability of metabolites contained in culture liquid of 62 strains of marine fungi to affect the activity of two digestive enzymes of marine mollusks--endo-1,3-beta-D-glucanase of Spisula sachalinensis and beta-D-glucosidase of Littorina kurila--was studied. It was found that 66 and 71% of specimens activated, 18 and 7% inhibited, and 16 and 22% did not affect the activity of endo-1,3-beta-D-glucanase and beta-D-glucosidase, respectively. It is demonstrated that the metabolites of brown algae and marine sponges can be used for a targeted regulation of enzyme biosynthesis by marine fungi. The protein inhibitor of endo-1,3-beta-D-glucanases isolated from the brown alga Laminaria cichorioides blocked the biosynthesis of almost all O-glycosylhydrolases in five strains of marine fungi studied. The presence in culture medium of halistanol sulfate from the marine sponge of the family Halichondriidae either did not affect or activated the biosynthesis of enzymes involved in carbohydrate metabolism by marine fungi.     

A heterogeneous beta-D-glucosidase activator from monkey parotid gland and its use as an affinity ligand in the purification of human salivary beta-D-glucosidase

We have isolated a heat-stable, low molecular weight activator peptide(s) from monkey parotid gland that specifically activated human salivary beta-D-glucosidase. This activator appeared to be heterogeneous on Sephadex G-25 gel filtration and polyacrylamide gel electrophoresis under non-denaturing conditions. About 45% of the human salivary beta-glucosidase could bind to the activator immobilised on Sepharose and be eluted by Cutscum. The purified enzyme was nearly homogeneous, with a subunit Mr of 46,000 as revealed by SDS-gel electrophoresis and silver staining.     

A stepwise approach to the understanding of extracellular enzyme activity in soil. I. Effect of electrostatic interactions on the conformation of a beta-D-glucosidase adsorbed on different mineral surfaces

The enzymatic activity of sweet almond beta-D-glucosidase adsorbed on various mineral surfaces was studied. Our aim was to elucidate the mechanism responsible for the observed changes in catalytic activity. The results of the investigation are discussed with reference to the hypotheses generally proposed to explain the well-documented shift in optimal pH of the activity of adsorbed enzymes. By separate determinations of enzymatic activity in a mineral suspension and of its supernatant solution, and comparison with a control without mineral added, we obtained accurate measurements of the catalytic activity of the adsorbed enzyme alone. Different pH profiles of activity profiles were found when the enzyme was adsorbed onto montmorillonite, kaolinite and goethite. The activity profiles, were also found to vary with ionic strength, the pH at which enzyme adsorbed onto the mineral surface, and in the case of goethite, on the nature of the anions in the buffer. Our observations cannot be adequately explained by assuming a more acidic microenvironment at the mineral surface. We postulate that on some mineral surfaces a conformational change is induced in the adsorbed protein, which reduces its catalytic activity. We contend that such conformational changes are due primarily to electrostatic forces.     

Dynamic interactions between enzyme activity and the microstructured environment

A new approach for the study of an enzyme's relationship with its own reaction medium has been developed. One technique of micellar enzymology is the use of pseudohomogeneous systems composed of surfactant/water/organic solvent. In such systems, the physicochemical properties and textures of the medium depend on the relative ratios of the different components. Enzymes are catalytically active in such systems and up to the present have been studied in different microenvironments, such as micelles, microemulsions and lyotropic liquid crystals. Our purpose was to develop a system in which the enzyme could, by its activity, modify one of the components in such a way that the relative ratios among them changed sufficiently to produce a transition from one phase domain to another. The three components, water (or glucose in water), octanol and octyl-beta-D-glucoside, form a classical ternary water/oil/surfactant system. The relevant phase diagram shows different macroheterogeneous phases and microstructured domains. The enzyme beta-D-glucosidase hydrolyses octyl-beta-D-glucoside to form glucose and octanol. The enzyme was found to change the relative ratios of water (or glucose in water), octanol and octyl-beta-D-glucoside in such a manner that the physicochemical structure of the medium was modified. At the beginning of the reaction beta-D-glucosidase was present in a micellar solution of octyl-beta-D-glucoside in water. As the enzymatic reaction proceeded, the medium became biphasic. One of the two phases was the micellar solution of octyl beta-D-glucoside in water, while the other phase was either a microemulsion or a liquid crystalline phase. In addition the enzyme, through its catalytic activity, was able to modify the physiocochemical properties of the reaction medium.     

A stepwise approach to the understanding of extracellular enzyme activity in soil. II. Competitive effects on the adsorption of a beta-D-glucosidase in mixed mineral or organo-mineral systems

A previous study has shown the effect of individual mineral surfaces on the activity of sweet almond beta-D-glucosidase. We now consider more complex situations likely to occur in soil, such as adsorption onto mixtures of different mineral surfaces, and the effect on enzyme activity of mineral surfaces with organic coatings. The effect of the order of addition of the minerals to enzyme suggests that the rate of adsorption is limited by the diffusion of the protein towards the interface and is not influenced by the magnitude of attractive forces between the protein and the surface. Adsorption is found to be quasi-irreversible. A study of the effect of artificial coatings of montmorillonite on enzyme activity led to the conclusion that an exchange of the enzyme with molecules of the coating occurs. This exchange is dependent upon the adsorption energy of the molecules of the coating and the electric charge of beta-D-glucosidase. This model is used for the interpretation of the effect of natural clay-humic complexes on enzyme activity.     

Copper and zinc fractions affecting microorganisms in long-term sludge-amended soils

The influences of Zn and Cu on soil enzyme activities (acid phosphatase, alkaline phosphatase, arylsulfatase, cellulase, dehydrogenase, protease (z-FLase), urease, beta-D-glucosidase and beta-D-fructofuranosidase (invertase)) and microbial biomass carbon were investigated in agricultural soils amended with municipal sewage sludge or compost since 1978. The trace metals in the soils were fractionated using a sequential extraction method. Long-term application of the sewage sludge and composts caused accumulations of Cu and Zn in the soils, ranging from 140 to 144 and from 216 to 292 mg kg(-1), respectively. The percentage of Cu was highest in the NaOH- and HNO3-extractable fractions (44-51% and 38-46%, respectively), while the percentage of Zn was highest in the HNO3- and EDTA-extractable fractions (65-83% and 11-32%, respectively). Although the percentage of the bioavailable fractions (sum of KNO3 + H2O-, NaOH-, and EDTA-extractable amounts) of Cu (53-64%) was higher than that of Zn (15-37%), the percentage of the most labile fractions (KNO3 + H2O) of Zn (2.1-5.9%) was larger than that of Cu (1.1-2.4%). The size of the microbial biomass carbon increased with the application of sewage sludge or compost. For some enzymes, however, the ratio of the enzyme activity to microbial biomass was lower in the soils amended with sewage sludge or compost than that in the control soil. The soil enzyme activities were more adversely affected by Zn than by Cu. From a multiple regression analysis, it was found that dehydrogenase, urease, and beta-D-glucosidase activities were reduced by the KNO3 + H2O-extractable fraction of Zn in the soils. These microbial activities seem to be sensitive to Zn stress, indicating the possibility that they might be useful bioindicators for evaluation of the toxic effects of Zn on microorganisms in the soils.     

Changes in glycosidase activities during galactoglucomannan oligosaccharide inhibition of auxin induced growth

The inhibition of 2,4-D-induced elongation growth by galactoglucomannan oligosaccharides (GGMOs) in pea stem segments (Pisum sativum L. cv. Tyrkys) after 18 h of incubation results in changes of extracellular, intracellular and cell wall glycosidase activities (beta-D-glucosidase, beta-D-mannosidase, beta-D-galactosidase, beta-D-xylosidase, alpha-D-galactosidase, and alpha-L-arabinosidase). GGMOs lowered the glycosidase activities in the extracellular fraction, while in the cell wall fractions their activities were markedly increased. The intracellular enzyme alpha-d-galactosidase increased while the beta-d-galactosidase decreased in activity in response to the GGMO treatment. Extracellular enzymes showed low values of activities in comparison with intracellular and cell wall glycosidases. It is evident that GGMOs can alter auxin induced elongation and glycosidase activities in different compartments of the cell, however, the mode and site of their action remains unclear.     

The common identity of five glycosidases in human liver.

An enzyme has been isolated from human liver by DEAE-cellulose chromatography and has been shown by competitive substrate inhibition to be capable of hydrolysing synthetic beta-D-galactosides, beta-D-glucosides, beta-D-fucosides, beta-D-xylosides, and alpha-L-arabinosides. Another form of alpha-L-arabinosidase activity elutes with the major beta-D-galactosidase component on DEAE-chromatography, but has a different identity on the basis of its stability at 4 degrees C. Liver samples from patients with Gaucher's disease are deficient in beta-D-fucosidase as well as beta-D-glucosidase activity.