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PubMed1981

Alpha-glucosidase synthesis in yeast cells depleted of intramitochondrial ATP.

We have studied the dependence on mitochondrial ATP of expression of MAL genes specifying maltose utilization in yeast. It was found that bongkrekic acid does not prevent the maltose induced synthesis of alpha-glucosidase in derepressed cells of the wild-type and corresponding respiratory-deficient mutant of Saacharomyces cerevisiae. The results suggest that expression of nuclear genes specifying alpha-glucosidase and maltose catabolism in yeast is apparently not dependent on the proper function of mitochondrial adenine nucleotide translocase and does not even require the presence of normal levels of ATP in mitochondria.

Adenosine Triphosphate↗
PubMed1982

Purification and characterization of glucosidase II, an endoplasmic reticulum hydrolase involved in glycoprotein biosynthesis.

Rat liver glucosidase II, an endoplasmic reticulum hydrolase involved in the biosynthesis of the N-linked class of glycoproteins, has been purified in good yield to a state approaching homogeneity. The purified enzyme hydrolyzes p-nitrophenyl-alpha-D-glucopyranoside, 4-methylumbelliferyl-alpha-D-glucopyranoside, maltose, and the precursor oligosaccharides glucose1-2mannose9N-acetylglucosamine, but it does not act on glucose3mannose9N-acetylglucosamine or p-nitrophenyl-beta-D-glucopyranoside. The ratio of the rate at which glucose is released from p-nitrophenyl-alpha-D-glucopyranoside to that from glucose2mannose9N-acetylglucosamine or glucose1mannose9N-acetylglucosamine remains constant throughout the 8-step purification procedure; thus it appears that a single enzyme is responsible for the activities toward both the artificial and oligosaccharide substrates. The fact that the enzyme cleaves both of the inner 1,3-linked glucosyl residues from the precursor oligosaccharides supports the view that they are linked in the alpha-configuration. The pH dependence of enzymatic activity is quite similar for different substrates, showing a broad optimum between pH 6 and 7.5. Activity toward p-nitrophenyl-alpha-D-glucopyranoside is enhanced by 12 mM 2-deoxy-D-glucose (260-300% activation) and 25 mM mannose (150% activation), but these two compounds inhibit the action of the enzyme toward the precursor oligosaccharides. By isoelectrofocusing the purified enzyme exhibits one form, which has a pI of 3.5-3.8. Reductive polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicates that glucosidase II has a subunit molecular weight of 65,000. Ferguson plot analysis of the behavior of native enzyme in polyacrylamide gels indicates that it is a 262,000-dalton tetramer. Gel filtration gives a molecular weight of 288,000. Several lines of evidence indicate that the enzyme is a glycoprotein.

Animals↗
PubMed1995

[Glycogenosis type II; acid alpha-glucosidase deficiency].

Knowledge of the general enzymology of lysosomal acid alpha-glucosidase (acid maltase) including that of the intracellular processing mechanism and of the cDNA sequence has advanced recently. Genetic heterogeneity of the mRNA of acid alpha-glucosidase deficient patients has also been reported. Referring to these recent advances, and based on our previously reported results, the pathologic aspects of the disease are reviewed. Heterogeneity of the enzyme molecules themselves and their characteristics in various tissues are also demonstrated.

Glucan 1,4-alpha-Glucosidase↗
PubMed1999

Expression of soluble and catalytically active plant (monocot) beta-glucosidases in E. coli.

Complementary DNAs encoding mature beta-glucosidase proteins Glu1 and Glu2 of maize were amplified by the polymerase chain reaction (PCR) and cloned into the expression vector pET21a. Both Glu1 and Glu2 isozymes were expressed in high yield ( approximately 3.8% of the total soluble protein and 32% of the total expressed protein) in E. coli. Recombinant enzymes were active on a variety of artificial and natural substrates at levels similar to those of their native counterparts isolated from maize seedlings. Western blot analysis confirmed that both recombinant isozymes were immunoreactive with maize anti-beta-glucosidase sera and their molecular sizes were identical to those of the native maize Glu1 and Glu2 isozymes. Zymogram assays in native gels revealed that recombinant enzymes had the same electrophoretic mobility and substrate specificity as their native counterparts.

Amino Acid Sequence↗
PubMed2003

Identification of four novel mutations in the alpha glucosidase gene in five Italian patients with infantile onset glycogen storage disease type II.

Glycogen storage disease type II (GSDII) is an autosomal recessive disorder due to the deficiency of the lysosomal enzyme acid alpha glucosidase. Four novel mutations (C670T, G989A, G2188T, and Delta 23 nt 828-850) were identified in five Italian patients with the infantile form of the disease. The C670T mutation was present in two unrelated patients in heterozygosity; the effect on enzyme activity was assessed by in vitro expression. COS-1 cells expressing the C670T allele had a twofold higher activity than the negative control cells. The G989A and G2188T point mutations lead to the introduction of premature stop signals that results in truncated forms of alpha glucosidase. The in vitro expression of G2188T allele demonstrated no increment in activity compared to negative control. The frame shifting deletion of nucleotides 828-850 was identified in one patient in heterozygosity. The shift in the reading frame introduces a stop codon 135 nucleotides downstream the deletion junction that results in a truncated protein without catalytic activity. Nested PCR screening showed that the mutation was carried by the mother and was absent in the other members of the family. The four novel severe mutations herein described concerned only infantile onset GSDII patients; the loss of enzyme activity is correlated with the severity of the disease.

Age of Onset↗
PubMed2001

Improved oligosaccharide synthesis by protein engineering of beta-glucosidase CelB from hyperthermophilic Pyrococcus furiosus.

Enzymatic transglycosylation of lactose into oligosaccharides was studied using wild-type beta-glucosidase (CelB) and active site mutants thereof (M424K, F426Y, M424K/F426Y) and wild-type beta-mannosidase (BmnA) of the hyperthermophilic Pyrococcus furiosus. The effects of the mutations on kinetics, enzyme activity, and substrate specificity were determined. The oligosaccharide synthesis was carried out in aqueous solution at 95 degrees C at different lactose concentrations and pH values. The results showed enhanced synthetic properties of the CelB mutant enzymes. An exchange of one phenylalanine to tyrosine (F426Y) increased the oligosaccharide yield (45%) compared with the wild-type CelB (40%). Incorporation of a positively charged group in the active site (M424K) increased the pH optimum of transglycosylation reaction of CelB. The double mutant, M424K/F426Y, showed much better transglycosylation properties at low (10-20%) lactose concentrations compared to the wild-type. At a lactose concentration of 10%, the oligosaccharide yield for the mutant was 40% compared to 18% for the wild-type. At optimal reaction conditions, a higher ratio of tetrasaccharides to trisaccharides was obtained with the double mutant (0.42, 10% lactose) compared to the wild-type (0.19, 70% lactose). At a lactose concentration as low as 10%, only trisaccharides were synthesized by CelB wild-type. The beta-mannosidase BmnA from P. furiosus showed both beta-glucosidase and beta-galactosidase activity and in the transglycosylation of lactose the maximal oligosaccharide yield of BmnA was 44%. The oligosaccharide yields obtained in this study are high compared to those reported with other transglycosylating beta-glycosidases in oligosaccharide synthesis from lactose.

Amino Acid Sequence↗
PubMed1997

Analysis of mandelonitrile lyase and beta-glucosidase from sweet almonds by combined electrophoretic techniques.

Almonds are a rich source of mandelonitrile lyase (oxynitrilase) and beta-glucosidase. The isolation of these two enzymes from sweet almonds requires fractional ammonium sulfate precipitation followed by ion-exchange chromatography on diethylaminoethyl-(DEAE) and carboxymethylcellulose (CMC) columns. In the present investigation different electrophoretic techniques such as sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing in immobilized pH gradients (IEF-IPG), and capillary electrophoresis were used to characterize these two enzymes. For the first time, beta-glucosidase and oxynitrilase were separated in an immobilized pH gradient of one pH unit. Capillary zone electrophoresis (CZE) was an excellent tool for analysis of the purity of enzyme preparations, achieving complete separation of various protein constituents in only 15 min. CZE showed a resolving capacity for the separation of enzyme forms comparable to that of isoelectric focusing in an immobilized pH gradient.

Aldehyde-Lyases↗
PubMed1990

Qualitative and quantitative changes in beta-1,4-glucosidase accompanying growth of Aspergillus nidulans.

A comparative study of the wild type A. nidulans and a mutant strain aco-T69 (lacking conidia and cleistothecia) revealed the better production of beta-1,4-glucosidase in the former. The relative distribution of the enzyme levels in various morphological structures viz. somatic hyphae, spores and cleistothecia also showed a variation. Highest specific activity was found in the cleistothecial extracts. The electrophoretic analysis of the wild type strain demonstrated the presence of three isoenzymes of beta-1,4-glucosidase i.e. beta-GLU I, beta-GLU II and beta-GLU III, the number of which varies from one to three during the growth of the organism. The parallel study of the mutant strain showed complete absence of beta-GLU II. All three forms were present in the somatic hyphae and cleistothecial extracts while spore extracts depicted the absence of beta-GLU III in the wild type.

Aspergillus↗
PubMed1994

Investigation of the active site of the cyanogenic beta-D-glucosidase (linamarase) from Manihot esculenta Crantz (cassava). II. Identification of Glu-198 as an active site carboxylate group with acid catalytic function.

The broad-specificity cyanogenic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) (linamarase) from Manihot esculenta Crantz (cassava) was irreversibly inactivated by N-bromoacetyl-beta-D-glucopyranosylamine according to pseudo-first-order kinetics with a second-order efficiency constant (ki/Ki = 0.1 min-1 M-1) identical for p-nitrophenyl-beta-D-glucopyranosidase, p-nitrophenyl-beta-D-galactopyranosidase, and linamarase activities of the enzyme. The competitive inhibitor p-nitrothiophenyl-beta-D-glucopyranoside protected the enzyme from inactivation. pH dependence of the pseudo-first-order rate constant of inactivation revealed the involvement of an amino acid side chain in the inactivation process with pKa 7.0, which is very similar to that of the acid catalyst group of the enzyme (pKE2 = 7.2). The involved amino acid, which has to be ionized for the inactivation, was identified as Glu-198 using 14C-labeled inactivator to label the enzyme, cleaving the labeled protein into peptides and then purifying and sequencing the labeled peptide. This residue is highly conserved in the homologous family A beta-glucosidases and family A1-A5 cellulases and lies in a consensus Asn-Glu-Pro motif occurring in all of these enzymes.

Acetylglucosamine↗
PubMed1994

A spectrophotometric assay for glucosidase I.

A spectrophotometric assay for glucosidase I using the synthetic trisaccharide alpha-D-Glc 1-->2 alpha-D-Glc 1-->3 alpha-D-Glc-O(CH2)8COOCH3 is reported. The terminal glucose is released from the substrate by the enzyme and quantitated using glucose oxidase, peroxidase, and o-dianisidine. The trisaccharide is specific for glucosidase I and provides all the necessary structural features for correct interaction in the enzyme active site. The utility of the assay for monitoring enzyme activity during isolation and for use in kinetic and inhibition studies (i.e., with 1-deoxynorjirimycin) is demonstrated.

1-Deoxynojirimycin↗
PubMed1996

Molecular cloning of cDNA and analysis of expression of the gene for alpha-glucosidase from the hypopharyngeal gland of the honeybee Apis mellifera L.

Previously, we identified and purified an alpha-glucosidase with a molecular mass of 70 kDa from a homogenate of the hypopharyngeal gland of the older worker bee (forager bee) Apis mellifera L. (T. Kubo et al, J. Biochem., 1996, 119, 291-295). Here, we isolated and sequenced a cDNA for the alpha-glucosidase. The cDNA encoded a protein consisting of 650 amino acids, which had high sequence identity with fruit fly and mosquito possible maltase gene products. RT-PCR showed that the gene was expressed specifically in the hypopharyngeal gland of the forager bee.

Amino Acid Sequence↗
PubMed1999

Tyrosinase folding and copper loading in vivo: a crucial role for calnexin and alpha-glucosidase II.

Tyrosinase is the key enzyme of melanin biosynthesis. It is a multiply glycosylated metalloenzyme, which has a long maturation time making it an ideal in vivo model system to probe protein folding and metal loading events. The use of NB-DNJ, an alpha-glucosidase I and II inhibitor has allowed us to dissect these processes. Here we show that tyrosinase folds through several inactive intermediates, at least two of which are recognised by the ER chaperone, calnexin. If the association with calnexin is prevented, more rapid folding occurs, the resulting protein fails to bind copper and is inactive. If dissociation from calnexin is inhibited, folding is prevented; the protein does not go through the normal secretory pathway and is targeted for degradation. Thus, tyrosinase folds off calnexin, giving alpha-glucosidase II a critical role, but the association with calnexin is essential to promote the correct folding which enables it to acquire copper.

Animals↗
PubMed2000

Formation of fibrillar multimers of oat beta-glucosidase isoenzymes is mediated by the As-Glu1 monomer.

Oat beta-glucosidase (EC 3.2.1.21) exists in two isomeric forms of homomultimer (type I) and heteromultimer (type II), which are comprised of two 60 kDa monomers of As-Glu1 and As-Glu2. The cDNA of As-Glu2 was cloned in this study, whereas As-Glu1 was previously cloned as As-P60. The As-Glu2 cDNA encodes a plastid-directing transit peptide of 57 amino acid residues and a mature protein of 521 amino acid residues. The amino acid sequence of As-Glu2 is highly homologous to that of As-Glu1, except for their C-terminal portions. When the two cDNAs of the mature proteins were expressed as T7.Tag-fused proteins in Escherichia coli, they produced soluble and enzymatically active T7.Tag-As-Glu1 and T7.Tag-As-Glu2 proteins. The T7.Tag-As-Glu1 was assembled into a donut-shaped hexamer ring which was in turn stacked in integer numbers to form long fibrillar homomultimers of different lengths with a molecular mass of up to several million daltons. On the other hand, the T7.Tag-As-Glu2 primarily formed a dimer rather than a multimer. When both cDNAs of As-Glu1 and As-Glu2 were co-expressed as T7.Tag-fused mature proteins, they were also assembled into a hexamer ring comprised of the two monomers in a 1:1 stoichiometry. The heteromeric hexamer was stacked in smaller numbers to form the heteromultimer of T7. Tag-As-Glu1 and -As-Glu2. The results indicate that the As-Glu1 monomer plays a crucial role in the formation of both the As-Glu1 homomultimer and the As-Glu1 and As-Glu2 heteromultimer. We describe here a unique structure for the oat beta-glucosidase fibrillar multimer that is formed by stacking the hexamer rings composed of As-Glu1 and/or As-Glu2.

Amino Acid Sequence↗
PubMed1996

Sequential precipitation with reversibly soluble insoluble polymers as a bioseparation strategy: purification of beta-glucosidase from Trichoderma longibrachiatum.

Precipitation of proteins/enzymes with reversibly soluble-insoluble polymers is a simple approach which can be easily scaled up. It is shown here that considerable purification may be achieved if more than one polymer (chosen judiciously) is used in sequence for purification. The principle is illustrated with purification of beta-glucosidase from a commercial preparation of Trichoderma longibrachiatum. Precipitation with chitosan removed cellulase activity from the preparation with 4-fold purification and 99% recovery of beta-glucosidase activity in the supernatant. The treatment of the supernatant with Eudragit S-100 precipitated 98% of the enzyme activity. The two different elution procedures gave 82% recovery (with 14-fold purification) and 86% recovery (with 12-fold purification) of the enzyme activity from the precipitate. A low molecular weight protein still present in these preparations could be removed by gel filtration. This finally yielded an electrophoretically homogeneous enzyme with 74% recovery and 29-fold purification.

Cellulase↗
PubMed1992

Cloning and expression of a thermostable exo-alpha-1,4-glucosidase gene from Bacillus stearothermophilus ATCC12016 in Escherichia coli.

The gene coding for a thermostable exo-alpha-1,4-glucosidase (alpha-glucoside glucohydrolase: EC 3.2.1.20) of Bacillus stearothermophilus ATCC 12016 was cloned within a 2.8-kb AvaI fragment of DNA using the plasmid pUC19 as a vector and Escherichia coli JM109 as a host. E. coli with the hybrid plasmid accumulated exo-alpha-1,4-glucosidase mainly in the cytoplasm. The level of enzyme production was about sevenfold higher than that observed for B. stearothermophilus. The cloned enzyme coincided absolutely with the B. stearothermophilus enzyme in its relative molecular mass (62,000), isoelectric point (5.0), amino-terminal sequence of 15 residues (Met-Lys-Lys-Thr-Trp-Trp-Lys-Glu-Gly-Val-Ala-Tyr-Gln-Ile-Tyr-), the temperature dependency of its activity and stability, and its antigenic determinants.

Cloning, Molecular↗
PubMed1995

Cloning and characterization of the bgxA gene from Erwinia chrysanthemi D1 which encodes a beta-glucosidase/xylosidase enzyme.

A beta-glucosidase/xylosidase gene from Erwinia chrysanthemi strain D1 was cloned and sequenced. This gene, named bgxA, encodes a ca. 71 kDa protein product which, following removal of the leader peptide, resulted in a ca. 69 kDa mature protein that accumulated in the periplasmic space of E. chrysanthemi strain D1 and Escherichia coli cells expressing the cloned gene. The protein exhibited both beta-glucosidase and beta-xylosidase activities but gave no detectable activity on xylan or carboxymethyl cellulose. The enzyme was classified as a type 3 glycosyl hydrolase, but was unusual in having a truncated B region at the carboxyl-terminus. Several E. chrysanthemi strains isolated from corn produced the glucosidase/xylosidase activity but not those isolated from dicot plants. However, bgxA marker exchange mutants of strain D1 were not detectably altered in virulence on corn leaves.

Amino Acid Sequence↗
PubMed1995

Production of beta-glucosidase by Aspergillus terreus.

The production of beta-glucosidase by Aspergillus terreus was investigated in liquid shake cultures. Enzyme production was maximum on the 7th day of growth (2.18 U/ml) with the initial pH of the medium in the range of 4.0-5.5. Cellulose (Sigmacell Type 100) at 1.0% (wt/vol) gave maximum beta-glucosidase activity among the various soluble and insoluble carbon sources tested. Potassium nitrate was a suitable nitrogen source for enzyme production. Triton X-100 at 0.15% (vol/vol) increased the enzyme levels of A. terreus. The test fungal strain showed an ability to ferment glucose to ethanol.

Aspergillus↗
PubMedDate not supplied

Effect of environmental toxicants on enzyme biosynthesis: a comparison of beta-galactosidase, alpha-glucosidase and tryptophanase.

Except for beta-galactosidase, little is known about the effect of environmental toxicants on enzyme induction. The information could be potentially useful for the development of low-cost and rapid ecotoxicity assays. The effect of toxicants on the de novo biosynthesis of three inducible enzymes, beta-galactosidase and tryptophanase in E. coli and alpha-glucosidase in B. subtilis was investigated. Biosynthesis of alpha-glucosidase was the most sensitive to environmental toxicants, particularly pentachlorophenol and sodium dodecyl sulfate. The sensitivity of B. subtilis to toxicants was further increased when Tween 80 was incorporated in the growth medium.

Bacteria↗