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PubMed1994

Production, Purification, and Properties of a Thermostable beta-Glucosidase from a Color Variant Strain of Aureobasidium pullulans.

A color variant strain of Aureobasidium pullulans (NRRL Y-12974) produced beta-glucosidase activity when grown in liquid culture on a variety of carbon sources, such as cellobiose, xylose, arabinose, lactose, sucrose, maltose, glucose, xylitol, xylan, cellulose, starch, and pullulan. An extracellular beta-glucosidase was purified 129-fold to homogeneity from the cell-free culture broth of the organism grown on corn bran. The purification protocol included ammonium sulfate treatment, CM Bio-Gel A agarose column chromatography, and gel filtrations on Bio-Gel A-0.5m and Sephacryl S-200. The beta-glucosidase was a glycoprotein with native molecular weight of 340,000 and was composed of two subunits with molecular weights of about 165,000. The enzyme displayed optimal activity at 75 degrees C and pH 4.5 and had a specific activity of 315 mumol . min . mg of protein under these conditions. The purified beta-glucosidase was active against p-nitrophenyl-beta-d-glucoside, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose, with K(m) values of 1.17, 1.00, 0.34, 0.36, 0.64, 0.68, and 1.65 mM, respectively. The enzyme activity was competitively inhibited by glucose (K(i) = 5.65 mM), while fructose, arabinose, galactose, mannose, and xylose (each at 56 mM) and sucrose and lactose (each at 29 mM) were not inhibitory. The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol (7.5%, vol/vol) stimulated the initial enzyme activity by 15%. Glucose production was enhanced by 7.9% when microcrystalline cellulose (2%, wt/vol) was treated for 48 h with a commercial cellulase preparation (5 U/ml) that was supplemented with the purified beta-glucosidase (0.21 U/ml) from A. pullulans.

Journal Article↗

PubMed1998

Assimilation of cellooligosaccharides by a cell surface-engineered yeast expressing beta-glucosidase and carboxymethylcellulase from aspergillus aculeatus

Since Saccharomyces cerevisiae lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies. The genes encoding FI-carboxymethylcellulase (CMCase) and beta-glucosidase from the fungus Aspergillus aculeatus were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin and introduced into S. cerevisiae. The delivery of CMCase and beta-glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from Rhizopus oryzae for beta-glucosidase, respectively. The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase and beta-glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant. The display of CMCase and beta-glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy. The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source. The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography. This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides. This is the first step in the assimilation of cellulosic materials by S. cerevisiae expressing heterologous cellulase genes.

Journal Article↗

PubMed1999

Production and distribution of endoglucanase, cellobiohydrolase, and beta-glucosidase components of the cellulolytic system of Volvariella volvacea, the edible straw mushroom.

The edible straw mushroom, Volvariella volvacea, produces a multicomponent enzyme system consisting of endo-1,4-beta-glucanase, cellobiohydrolase, and beta-glucosidase for the conversion of cellulose to glucose. The highest levels of endoglucanase and cellobiohydrolase were recorded in cultures containing microcrystalline cellulose (Avicel) or filter paper, while lower but detectable levels of activity were also produced on carboxymethyl cellulose, cotton wool, xylitol, or salicin. Biochemical analyses of different culture fractions in cultures exhibiting peak enzyme production revealed that most of the endoglucase was present either in the culture filtrate (45.8% of the total) or associated with the insoluble pellet fraction remaining after centrifugation of homogenized mycelia (32.6%). Cellobiohydrolase exhibited a similar distribution pattern, with 58.9% of the total enzyme present in culture filtrates and 31.0% associated with the pellet fraction. Conversely, most beta-glucosidase activity (63.9% of the total) was present in extracts of fungal mycelia whereas only 9.4% was detected in culture filtrates. The endoglucanase and beta-glucosidase distribution patterns were confirmed by confocal laser scanning microscopy combined with immunolabelling. Endoglucanase was shown to be largely cell wall associated or located extracellularly, with the highest concentrations being present in a region 1 to 2 microm wide immediately adjacent to the outer surface of (and possibly including) the hyphal wall and extending 60 to 70 microm from the hyphal tip. Immunofluorescence patterns indicated little if any intracellular endoglucanase. Most beta-glucosidase was located intracellularly in the apical area extending 60 to 70 microm below the hyphal tip, although enzyme was also evident in the extracellular region extending approximately 15 microm all around the hyphal tip and trailing back along the length of the hypha. The regions of the hypha located some distance from the apical region appeared to be devoid of intracellular beta-glucosidase, and the enzyme appears to be associated almost exclusively with, or located on the outside surface of, the hyphal wall.

Journal Article↗

PubMedDate not supplied

Troglitazone combination therapy in obese type 2 diabetic patients poorly controlled with alpha-glucosidase inhibitors.

The efficacy and safety of treatment with troglitazone combined with an alpha-glucosidase inhibitor, in obese type 2 diabetic patients who were previously administered alpha-glucosidase inhibitors alone, in improving glycaemic control and reducing insulin resistance were studied. Obese type 2 diabetic patients, poorly controlled with alpha-glucosidase inhibitors, were randomized to receive either oral troglitazone 200 mg twice daily (22 patients: group A) or a placebo (20 patients: group B) in addition to their usual alpha-glucosidase inhibitor. In group A, significant decreases in the mean levels of haemoglobin A1c and basal plasma insulin levels were observed 6 months after the start of combined therapy. Serum triglyceride levels significantly decreased but serum lactic acid dehydrogenase and body weight significantly increased. New systemic oedema was observed in six patients. Combined therapy with troglitazone and alpha-glucosidase inhibitors may be effective for diabetic metabolic abnormalities, although the potential development of adverse effects such as body-weight gain and systemic oedema demands vigilance.

Acarbose↗

PubMed1998

Potential of alpha-glucosidase inhibitors in elderly patients with diabetes mellitus and impaired glucose tolerance.

The prevalence of diabetes mellitus (DM) among the elderly, who constitute > 20% of the population in developed countries, is high (up to 40%). Indeed, elderly people represent the bulk (approximately 50%) of the diabetic population. There is much evidence that better glycaemic control can reduce the morbidity associated with this disease. alpha-Glucosidase inhibitors are well tolerated in the treatment of DM in this population. They reduce postprandial hyperglycaemia and have a moderate effect on fastign plasma glucose levels, resulting in a significant reduction in glycated haemoglobin (HbA1C) levels. alpha-Glucosidase inhibitors can be used either as monotherapy or in combination with other oral hypoglycaemic agents or insulin. The good safety profile of these drugs makes them suitable for use in elderly patients with type 2 (non-insulin-dependent) DM, because they can achieve substantial metabolic improvements without any additional risks. Thus, the use of alpha-glucosidase inhibitors should be considered: (i) as a first-choice treatment in newly diagnosed patients; (ii) in individuals whose DM is not well controlled with any other type of treatment; (iii) as an alternative to sulphonylureas or biguanides in patients at risk from hypoglycaemia or lactic acidosis, respectively. Despite the numerous potential advantages of alpha-glucosidase inhibitors in elderly patients with type 2 DM, there is a lack of studies focusing specifically on that population. However, such studies are under way. In addition, the potential of alpha-glucosidase inhibitors in the prevention of type 2 DM and/or on macrovascular disease is currently under study.

1-Deoxynojirimycin↗

PubMed1996

An importance of carbohydrate ingestion for the expression of the effect of alpha-glucosidase inhibitor in NIDDM.

OBJECTIVE: To examine the usefulness of alpha-glucosidase inhibitors in glycemic control of patients with NIDDM. The involvement of carbohydrate ingestion in manifestation of the effects of alpha-glucosidase inhibitors was also investigated. RESEARCH DESIGN AND METHODS: A total of 41 patients hospitalized with NIDDM (22 patients receiving sulfonylurea and 19 receiving insulin therapy) were given alpha-glucosidase inhibitors during the period when their blood glucose levels were well controlled. They were followed for 3 weeks as inpatients and for an additional 6 months as outpatients. They were retrospectively divided into two groups according to the percentage of carbohydrates in all sources of calories during outpatient management: the < 50% group and the > 50% group. Between these two groups, we compared circadian variation in blood glucose levels, HbA1c, and urine C-peptide. RESULTS: Treatment with alpha-glucosidase inhibitors during the hospital stay markedly improved circadian variation in blood glucose levels and HbA1c and decreased urine C-peptide in both groups. While HbA1c returned to its pretreatment level at 6 months after the treatment in the < 50% group, HbA1c had further improved in the > 50% group at 6 months. CONCLUSIONS: alpha-Glucosidase inhibitors are useful for glycemic control in patients with NIDDM and the percentage of carbohydrate in all calorie sources is an important factor for the expression of their effects.

Biomarkers↗

PubMed2003

[Study on the inhibition of alpha-glucosidase by soyasaponins].

OBJECTIVE: To study the inhibitory effects of soyasaponins on alpha-glucosidase (EC3.2.1.20). METHODS: Soyasaponins were isolated by ODS column chromatography and high-performance liquid chromatography (HPLC) from hypocotyls of soybean. The inhibitory activities of each component of soyasaponins against alpha-glucosidase were tested by colorimetric method. RESULTS: Soyasaponins showed potent inhibitory activities against alpha-glucosidase. Group B, group E and DDMP (2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one) saponins showed stronger potency, which were non-competitive inhibitors of alpha-glucosidase with IC50 values of 10-40 mumol/L. While group A saponins showed a little lower potency with IC50 values of about 2 mmol/L. CONCLUSION: The results suggest soyasaponins, which exhibit inhibitory effects on alpha-glucosidase, seem physiologically useful for suppressing postprandial hyperglycemia in patients with diabetes mellitus.

Chromatography, High Pressure Liquid↗

PubMed1979

Association of beta-glucosidase with intact cells of Thermoactinomyces.

The location of the B-glucosidase activity in a whole culture broth of the thermophilic organism Thermoactinomyces has been studied. Little beta-glucosidase activity was found in the culture filtrate, while the culture solids contained the major part of the activity of the whole culture broth. The activity does not appear to be adsorbed to the culture solids; rather there is evidence that it is an intracellular soluble enzyme(s). The pH and temperature optima for a crude beta-glucosidase preparation were determined to be pH 6.5 and 50--55 degrees C. Enzyme activity studies indicate that the same enzyme(s) accounts for the beta-glucosidase and the cellobiase activities. The validity of using the filter paper activity of culture filtrates from Thermoactinomyces to predict the total saccharification of cellulosic materials to glucose is discussed.

Cellulose↗

PubMedDate not supplied

Alpha-glucosidase as a specific epididymal enzyme marker. Its validity for the etiologic diagnosis of azoospermia.

Azoospermic semen was obtained from 39 vasectomized men and 93 patients consulting for infertility. The latter underwent bioclinical investigations including measurement of plasma FSH and testicular biopsies. Carnitine content and alpha-glucosidase activity in semen samples were measured. The activity of alpha-glucosidase was determined systematically by a semiquantitative microtechnique and was verified by a spectrophotometric assay. A positive correlation was observed between carnitine and alpha-glucosidase activity. Both parameters were severely diminished in semen from the vasectomized men and the patients suffering from a complete obstruction of the genital tract. Enzyme activity was the most discriminant parameter in terms of sensitivity and specificity. The measurement of alpha-glucosidase in semen is a simple and sensitive method for determining the origin of azoospermia when used in conjunction with assays for plasma FSH.

Carnitine↗

PubMed2004

Biopsy-proven alpha-glucosidase deficiency with normal lymphocyte enzyme activity.

Alpha-glucosidase deficiency is a rare cause of muscle disease in adults. The diagnosis relies on recognition of the salient clinical features and determination of significantly reduced alpha-glucosidase (GAA) activity. Lymphocytes are the usual tissue for diagnostic enzymology; discrepant results from analyses of different tissues are unusual. We report a patient with clinical, electromyographic, and biopsy findings indicative of alpha-glucosidase deficiency whose muscle and lymphocyte enzyme results were markedly discrepant on multiple analyses. As a result, we conclude that all patients with suspected alpha-glucosidase deficiency and a normal lymphocyte GAA assay should also have a determination of GAA activity in muscle or skin fibroblasts.

Action Potentials↗

PubMed1989

Mature 98,000-dalton acid alpha-glucosidase is deficient in Japanese quails with acid maltase deficiency.

We compared acid alpha-glucosidase of acid maltase-deficient Japanese quails, an animal model of human late-onset glycogenosis type II, with that of normal controls. Antibody produced in a rabbit against acid alpha-glucosidase purified from chicken pectoral muscle cross-reacted with that of Japanese quails. The presence of a 110K and 98K form of acid alpha-glucosidase was confirmed in normal controls by immunoblotting. However the 98K form was absent in the affected quails. Subcellular distribution studies demonstrated that the 98K form, but not the 110K form, was localized in the lysosomes. This suggests that the 110K form is a precursor of the mature 98K form of acid alpha-glucosidase. In the affected quails, the 110K precursor is synthesized, but maturation to the 98K form does not occur or may be extremely deficient.

Animals↗

PubMed1983

Physical and kinetic properties of beta-glucosidase in Gaucher disease.

Membrane-bound 'acid' beta-glucosidase of human spleen was solubilized with either sodium cholate or 'Cutscum'. The solubilized enzyme in type 1 (adult) Gaucher disease was less heat-stable than the normal enzyme, and when precipitated by ammonium sulphate it had a higher apparent molecular weight than the corresponding normal enzyme. The normal beta-glucosidase was activated by taurocholate, whereas the Gaucher enzyme was inhibited. The decrease in 'acid' beta-glucosidase activity in Gaucher disease was associated with a profound deficiency of that form of the enzyme which bound to Concanavalin A. The results are consistent with faulty processing of newly synthesized 'acid' beta-glucosidase in type 1 Gaucher disease.

Chemical Phenomena↗

PubMed1986

Glucosylceramide and the level of the glucosidase-stimulating proteins.

The concentration of beta-glucosidase-stimulating proteins (called cohydrolase here) was measured in mouse liver and brain by immunoassay. Factors that might influence the levels of cohydrolase were examined. Injecting mice with an inactivator of glucosidase (conduritol B epoxide) rapidly produced elevations in liver glucosylceramide (the enzyme's substrate) and in liver and brain cohydrolase. Injection of glucosylceramide emulsified with Myrj 52 produced the same two effects in liver but not in brain. The increases in cohydrolase level induced by the enzyme inhibitor persisted in both organs for at least seven days, reaching 61-70% above the normal level. Injection of emulsified galactocerebroside, sphingomyelin and mixed glucosphingolipids but not of ceramide also produced rises in cohydrolase level. An increase in cohydrolase level resulted from injection of phenylhydrazine, which produces hemolysis and consequently an increased workload for the glucosidase of liver. When the enzyme inhibitor and/or larger amounts of glucosylceramide emulsion were injected (750 mg/kg body weight), increases in liver weight of 13 to 37% appeared within one day. The increased weight was characterized by increases in the weights of protein, total lipid and DNA and a very high increase in glucosylceramide level. These procedures have produced a rapidly developing model version of Gaucher disease in mice. Injected glucocerebroside also induced an elevated level of glucosidase activity.

Animals↗

PubMed1983

Castanospermine, a tetrahydroxylated alkaloid that inhibits beta-glucosidase and beta-glucocerebrosidase.

Castanospermine (1,6,7,8-tetrahydroxyoctahydroindolizine) was tested against a variety of commercially available glycosidases and found to be a potent inhibitor of almond emulsin beta-glucosidase, and also to inhibit fungal beta-xylosidase. This alkaloid was inactive on yeast alpha-glucosidase, alpha- or beta-galactosidase, alpha-mannosidase, beta-N-acetylhexosaminidase, beta-glucuronidase, alpha-L-fucosidase. Fifty-percent inhibition of beta-glucosidase required about 10 micrograms/ml of castanospermine. The amount of inhibition was uniform throughout the time course, and the inhibition with regard to substrate concentration (p-nitrophenyl-beta-D-glucopyranoside) appeared to be of the mixed type. Castanospermine was also a potent inhibitor of beta-glucocerebrosidase when assayed with fibroblast extracts using either a fluorimetric or a radioactive assay. Interestingly enough, castanospermine also inhibited the lysosomal alpha-glucosidase, and this inhibition required comparable levels of alkaloid to that required for inhibition of beta-glucocerebrosidase. However, a number of other lysosomal glycosidases were not sensitive to castanospermine (i.e., alpha- or beta-galactosidase, alpha- or beta-mannosidase, alpha- or beta-L-fucosidase, beta-N-acetylhexosaminidase, beta-glucuronidase).

Alkaloids↗

PubMed1986

Characterization of calf liver glucosidase I and its inhibition by basic sugar analogs.

Glucosidase I, the enzyme catalyzing the first step of N-linked oligosaccharide processing, has been purified from calf liver crude membranes [H. Hettkamp, G. Legler, and E. Bause, (1984) Eur. J. Biochem. 142, 85-90]. Binding experiments with concanavalin A-Sepharose suggest that glucosidase I is a glycoprotein with high-mannose carbohydrate chain(s). The enzyme has a subunit molecular mass of approximately 83 kDa and specifically hydrolyzes the terminal alpha-1,2-linked glucose residue from the natural Glc3-Man9-GlcNAc2 oligosaccharide. Studies with a variety of substrates modified in the aglycon moiety suggest that the Glc2 branch rather than the more distant domains of the substrate molecule are important for binding and hydrolysis. Glucosidase I does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (dNM) and its N-alkyl derivatives. Ki values range from 0.07 microM for N-methyl-dNM to 1.0 microM for dNM, measured at the pH-optimum of enzyme activity. The pH dependence of inhibition indicates that the cationic form of the inhibitors is the active species. Comparison of the Ki for N-decanoyl-dNM (approximately 70 microM) with that of N-decyl-dNM (approximately 0.4 microM) suggests that electrostatic interactions at the catalytic site of the enzyme are important for inhibitor binding. 1-Deoxymannojirimycin, previously assumed to be a specific mannosidase inhibitor, as well as its N-methyl and N-5-carboxypentyl derivatives, inhibit glucosidase I with Ki values around 190, 17, and 100 microM, respectively. This apparent lack of specificity shows that in vivo experiments on N-glycoprotein processing as well as the interpretation of results with these mannosidase inhibitors may give misleading results when these compounds are used in the millimolar range.

1-Deoxynojirimycin↗

PubMed1986

beta-Glucosidase inhibition in murine peritoneal macrophages by conduritol-B-epoxide: an in vitro model of the Gaucher cell.

Murine peritoneal macrophages were cultured in the presence of conduritol-B-epoxide, a specific covalent inhibitor of beta-glucosidase. The inhibition was found to be dose and time dependent. Upon removal of the inhibitor from the culture medium, beta-glucosidase activity recovered to half maximum by 2.2 days. Treatment of macrophages with this inhibitor for 15 days did not affect cell viability, lysosomal enzyme release to the medium, or levels of intracellular lysosomal enzymes, other than beta-glucosidase activity. This inhibition results in the accumulation of glucocerebroside. In vitro studies on the pathobiology of such macrophages whose beta-glucosidase activity has been reduced may be useful toward understanding the pathogenesis of Gaucher disease.

Animals↗

PubMed1984

Cloning a cDNA for the lysosomal alpha-glucosidase.

Messenger RNA was isolated from monkey testes and size-fractionated on sucrose gradients. In vitro translation of these mRNA fractions resulted in nascent, labeled alpha-glucosidase that could be precipitated with anti human alpha-glucosidase antiserum. A cDNA library was constructed from the most enriched fraction. The library was screened with cDNA made from mRNA obtained from immunoselected polysomes. Five cross-hybridizing clones were isolated and identified by their selection of alpha-glucosidase mRNA, as shown by hybrid released translation and further by their ability to hybridize with DNA from human chromosome 17, on which the gene coding for acid alpha-glucosidase is located.

Animals↗

PubMed1989

Addition of cloned beta-glucosidase enhances the degradation of crystalline cellulose by the Clostridium thermocellum cellulose complex.

A thermostable beta-glucosidase from Clostridium thermocellum which is expressed in Escherichia coli was used to determine the substrate specificity of the enzyme. A restriction map of the beta-glucosidase gene cloned in plasmid pALD7 was determined. Addition of the E. coli cell extract (containing the beta-glucosidase) to the cellulase complex from C. thermocellum increased the conversion of crystalline cellulose (Avicel) to glucose. The increase was specifically due to hydrolysis of the accumulated cellobiose. A cellulose degradation process using beta-glucosidase to assist the potent cellulase complex of C. thermocellum, as shown here can open the way for industrial saccharification of cellulose to glucose.

Cellulase↗