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PubMed1993

Purification and characterization of an extremely thermostable beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus.

Cell-free extracts of cellobiose-grown cells of the hyperthermophile Pyrococcus furiosus contain very high activities (19.8 U/mg) of a beta-glucosidase. The cytoplasmic enzyme was purified 22-fold to apparent homogeneity, indicating that the enzyme comprises nearly 5% of the total cell protein. The native beta-glucosidase has a molecular mass of 230 +/- 20 kDa, composed of 58 +/- 2-kDa subunits. The enzyme has a pI of 4.40. Thiol groups are not essential for activity, nor is the enzyme dependent on divalent cations or a high ionic strength. The enzyme shows optimum activity at pH 5.0 and 102-105 degrees C. From Lineweaver-Burk plots, Vmax values of 470 U/mg and 700 U/mg were found for cellobiose (Km = 20 mM) and p-nitrophenyl-beta-D-glucopyranoside (Km = 0.15 mM), respectively. The purified enzyme also exhibits high beta-galactosidase activity and beta-xylosidase activity, but shows no activity towards alpha-linked disaccharides or beta-linked polymers, like cellulose. The purified beta-glucosidase shows a remarkable thermostability with a half life of 85 h at 100 degrees C and 13 h at 110 degrees C.

Archaea↗
PubMed1993

Effect of bromoconduritol on glucosidase II from rat liver. A new kinetic model for the binding and hydrolysis of the substrate.

Bromoconduritol inhibits the p-nitrophenyl-glucosidase and maltase activities of glucosidase II purified from rat liver, an enzyme that removes the two alpha-1,3-linked glucose residues of the protein-bound oligosaccharide Glc2Man9GlcNAc2 in the processing of N-glycoproteins. The inactivation process exhibits pseudo-first-order kinetics. Previously, we have demonstrated the occurrence of two binding (active) sites in the glucosidase II for the substrates p-nitrophenyl alpha-D-glucopyranoside (pNphGlc) and maltose (high- and low-affinity sites). The inhibition kinetic studies with bromoconduritol indicate that the high- and low-affinity sites for pNphGlc correspond to high- and low-affinity sites for maltose, respectively. Bromoconduritol has no effect on the binding of the substrates (pNphGlc and maltose) to the high-affinity site, although it does modify the low-affinity site and hinders the binding of the two indicated substrates to this site. These results, together with previous reports, have prompted us to propose a new kinetic model of binding and hydrolysis of the physiological substrate of the enzyme, in which the outermost glucose residue would bind and be released at the high-affinity site, whereas the innermost glucose residue would do so at the low-affinity site.

Animals↗
PubMed1994

A beta-glucosidase gene (bgl3) from Streptomyces sp. strain QM-B814. Molecular cloning, nucleotide sequence, purification and characterization of the encoded enzyme, a new member of family 1 glycosyl hydrolases.

A beta-glucosidase gene (bgl3) from Streptomyces sp. QM-B814 (American Type Culture Collection 11238) has been cloned by functional complementation of a beta-glucosidase-negative mutant of Streptomyces lividans. An open-reading frame of 1440 nucleotides encoding a polypeptide of 479 amino acids was found by sequencing. The encoded protein (Bgl3) shows extensive similarity (over 45% identity) with beta-glycosidases from family-1 glycosyl hydrolases. The cloned enzyme, purified following ammonium sulphate precipitation and two chromatographic steps, is monomeric with molecular mass 52.6 kDa, as determined by mass spectrometry, and an isoelectric point of pI 4.4. The enzyme appears to be a beta-glucosidase with broad substrate specificity, is active on cellooligomers, and performs transglycosylation reactions. The estimated apparent Km values for p-nitrophenyl-beta-D-glucopyranoside and cellobiose are 0.27 mM and 7.9 mM, respectively. The Ki values for glucose and delta-gluconolactone, using p-nitrophenyl-beta-D-glucopyranoside as a substrate, are 65 mM and 0.08 mM, respectively. The purified enzyme has a pH optimum of pH 6.5 and the temperature optimum for activity is 50 degrees C.

Amino Acid Sequence↗
PubMedDate not supplied

Alpha-glucosidase activity and sperm motility.

We correlated the activity of alpha-glucosidase in seminal plasma with the motility and differential motility of sperm. Significant positive correlations were found between the alpha-glucosidase activity and both motility and the percentage sperm with good forward progression. This supports the use of alpha-glucosidase in semen as a marker of epididymal function and specifically of the development of motility.

Humans↗
PubMedDate not supplied

A comparative study of the EpiScreen kit and a conventional method for the determination of seminal alpha-glucosidase activity.

Low alpha-glucosidase activity in seminal plasma is a marker of epididymal obstruction. The criterion standard for determining activity is the epididymal specific method, whereby the neutral iso-enzyme, specifically produced by the epididymis, is measured. A kit that determines total alpha-glucosidase activity (neutral iso-enzyme and the acid iso-enzyme originating from the prostate) has become available. The objective of the laboratory-based study was to compare alpha-glucosidase activity values measured by both the EpiScreen and the epididymal specific method, to determine if the kit may provide reliable results to substitute the neutral iso-enzyme specific method in the routine clinical setting. The neutral iso-enzyme activity according to both methods was measured in seminal plasma of 23 post-vasectomy and 24 normozoospermic patients. Significant differences (P < 0.05) were found between the activities measured according to both methods, but these differences pertained mostly to high values (> 40 mU ejaculate-1), which was not clinically significant. In conclusion, the epididymal specific method is best suited for research purposes, but the EpiScreen kit is convenient for routine use in infertility clinics.

Analysis of Variance↗
PubMed1995

Cloning and nucleotide sequence of the bglA gene from Erwinia herbicola and expression of beta-glucosidase activity in Escherichia coli.

Genomic DNA fragments encoding beta-glucosidase activity from the wild-type strain WD4 of Erwinia herbicola were cloned into Escherichia coli. Two clones containing a common fragment encoded a polypeptide of 58,000 Da. Cloned beta-glucosidase, expressed in E. coli, showed activity against natural beta-glucoside sugars except for cellobiose. An open reading frame of 1442 bp termed bglA was identified by nucleotide sequencing and it coded for a protein of 480 amino acids (M(r) 53,896) which showed significant homology with beta-glucosidases from glycosyl hydrolase family 1.

Cloning, Molecular↗
PubMed1976

Production of extracellular alpha-glucosidase by a thermophilic Bacillus species.

Production of extracellular alpha-glucosidase was studied with strain KP 1006 of a new species of thermophilic Bacillus, which was isolated from soil samples by enrichment at 65 C. alpha-Glucosidase production was maximum at 60 C and at an initial pH of 6.5. The final enzyme yield was increased by starch, maltose, glycerol, peptone, and yeast extract but reduced by acetate and gluconate, alpha-Glucosidase was formed in the cytoplasm and accumulated as a large pool during the logarithmic growth phase. At a midpoint of this period, the enzyme appeared in the culture broth, and its level increased until the end of the stationary phase.

Bacillus↗
PubMed2001

Assessing the diversity of marine bacterial beta-glucosidases by capillary electrophoresis zymography.

We propose a new method for the fast separation and detection of beta-glucosidases in environmental samples. With this approach, beta-glucosidases extracted from bacteria are evidenced by substrate-incorporated capillary electrophoresis (CE zymography) and their kinetic parameters can be determined by repeated injections using different substrate concentrations. Preliminary results obtained with natural bacterial communities from the coastal North Sea suggest that the diversity of beta-glucosidases in the marine environment might be much higher than previously observed.

Electrophoresis, Capillary↗
PubMed2001

The celA gene, encoding a glycosyl hydrolase family 3 beta-glucosidase in Azospirillum irakense, is required for optimal growth on cellobiosides.

The CelA beta-glucosidase of Azospirillum irakense, belonging to glycosyl hydrolase family 3 (GHF3), preferentially hydrolyzes cellobiose and releases glucose units from the C(3), C(4), and C(5) oligosaccharides. The growth of a DeltacelA mutant on these cellobiosides was affected. In A. irakense, the GHF3 beta-glucosidases appear to be functional alternatives for the GHF1 beta-glucosidases in the assimilation of beta-glucosides by other bacteria.

Amino Acid Sequence↗
PubMed2002

Enzymatic properties and intracellular localization of the novel Trichoderma reesei beta-glucosidase BGLII (cel1A).

This paper describes the characterization of an intracellular beta-glucosidase enzyme BGLII (Cel1a) and its gene (bgl2) from the cellulolytic fungus Trichoderma reesei (Hypocrea jecorina). The expression pattern of bgl2 is similar to that of other cellulase genes known from this fungus, and the gene would appear to be under the control of carbon catabolite repression mediated by the cre1 gene. The BGLII protein was produced in Escherichia coli, and its enzymatic properties were analyzed. It was shown to be a specific beta-glucosidase, having no beta-galactosidase side activity. It hydrolyzed both cellotriose and cellotetraose. BGLII exhibited transglycosylation activity, producing mainly cellotriose from cellobiose and sophorose and cellobiose from glucose. Antibodies raised against BGLII showed the presence of the enzyme in T. reesei cell lysates but not in the culture supernatant. Activity measurements and Western blot analysis of T. reesei strains expressing bgl2 from a constitutive promoter further confirmed the intracellular localization of this beta-glucosidase.

Gene Expression↗
PubMed2005

Cloning and characterization of the glucosidase II alpha subunit gene of Trichoderma reesei: a frameshift mutation results in the aberrant glycosylation profile of the hypercellulolytic strain Rut-C30.

We describe isolation and characterization of the gene encoding the glucosidase II alpha subunit (GIIalpha) of the industrially important fungus Trichoderma reesei. This subunit is the catalytic part of the glucosidase II heterodimeric enzyme involved in the structural modification within the endoplasmic reticulum (ER) of N-linked oligosaccharides present on glycoproteins. The gene encoding GIIalpha (gls2alpha) in the hypercellulolytic strain Rut-C30 contains a frameshift mutation resulting in a truncated gene product. Based on the peculiar monoglucosylated N-glycan pattern on proteins produced by the strain, we concluded that the truncated protein can still hydrolyze the first alpha-1,3-linked glucose residue but not the innermost alpha-1,3-linked glucose residue from the Glc2Man9GlcNAc2 N-glycan ER structure. Transformation of the Rut-C30 strain with a repaired T. reesei gls2alpha gene changed the glycosylation profile significantly, decreasing the amount of monoglucosylated structures and increasing the amount of high-mannose N-glycans. Full conversion to high-mannose carbohydrates was not obtained, and this was probably due to competition between the endogenous mutant subunit and the introduced wild-type GIIalpha protein. Since glucosidase II is also involved in the ER quality control of nascent polypeptide chains, its transcriptional regulation was studied in a strain producing recombinant tissue plasminogen activator (tPA) and in cultures treated with the stress agents dithiothreitol (DTT) and brefeldin A (BFA), which are known to block protein transport and to induce the unfolded protein response. While the mRNA levels were clearly upregulated upon tPA production or BFA treatment, no such enhancement was observed after DTT addition.

Amino Acid Sequence↗
PubMed1969

Isozymes of beta-glucosidase in Dictyostelium discoideum.

The activity of beta-glucosidase (EC 3.2.1.21) in extracts of Dictyostelium discoideum was investigated. The specific activity increased early in development, declined during pseudoplasmodium formation, and increased again during sorocarp formation. The beta-glucosidase which was present in growing amoebae and during the first stages of multicellular development was electrophoretically distinct from the enzyme which accumulated during the final stages of morphogenesis. Ribonucleic acid synthesis and protein synthesis during development were required for the accumulation of the later isozyme. Analysis of beta-glucosidase activity in a number of morphological mutants suggests that the enzyme which accumulates late in morphogenesis is developmentally controlled.

Centrifugation, Density Gradient↗
PubMed1972

Biochemical and genetic characterization of -glucosidase mutants in Saccharomyces lactis.

Mutants with reduced activity for beta-glucosidase (beta-d-glucoside glucohydrolase EC 3.2.1.21) were isolated from the haploid yeast Saccharomyces lactis. Tetrad analysis indicated that in each mutant a single genetic factor, closely linked or allelic to the structural gene for beta-glucosidase (B locus), is responsible for the decreased activity. beta-Glucosidases produced by wild-type and mutant strains are similar in molecular size and charge but differ in catalytic properties, thermal stability, and serological specificity, indicating that mutants are in the structural gene. All mutants retained their capacity to be induced by either methyl-beta-d-glucoside or glucose. In all cases, the mutant phenotype was dominant in heterozygous diploids.

Animals↗
PubMed1990

Purification and characterization of an alpha-glucosidase from a hyperthermophilic archaebacterium, Pyrococcus furiosus, exhibiting a temperature optimum of 105 to 115 degrees C.

Pyrococcus furiosus is a strictly anaerobic hyperthermophilic archaebacterium with an optimal growth temperature of about 100 degrees C. When this organism was grown in the presence of certain complex carbohydrates, the production of several amylolytic enzymes was noted. These enzymes included an alpha-glucosidase that was located in the cell cytoplasm. This alpha-glucosidase has been purified 310-fold and corresponded to a protein band of 125 kilodaltons as resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited optimum activity at pH 5.0 to 6.0 and over a temperature range of 105 to 115 degrees C. Kinetic analysis conducted at 108 degrees C revealed hydrolysis of the substrates p-nitrophenyl-alpha-D-glucopyranoside (PNPG), methyl-alpha-D-glucopyranoside, maltose, and isomaltose. Trace activity was detected towards p-nitrophenyl-beta-D-glucopyranoside, and no activity could be detected towards starch or sucrose. Inhibition studies conducted at 108 degrees C with PNPG as the substrate and maltose as the inhibitor yielded a Ki for maltose of 14.3 mM. Preincubation for 30 min at 98 degrees C in 100 mM dithiothreitol and 1.0 M urea had little effect on enzyme activity, whereas preincubation in 1.0% sodium dodecyl sulfate and 1.0 M guanidine hydrochloride resulted in significant loss of enzyme activity. Purified alpha-glucosidase from P. furiosus exhibited remarkable thermostability; incubation of the enzyme at 98 degrees C resulted in a half life of nearly 48 h.

Archaea↗
PubMed1998

Identification and enzymatic characterization of the maltose-inducible alpha-glucosidase MalL (sucrase-isomaltase-maltase) of Bacillus subtilis.

A gene coding for a putative alpha-glucosidase has been identified in the open reading frame yvdL (now termed malL), which was sequenced as part of the Bacillus subtilis genome project. The enzyme was overproduced in Escherichia coli and purified. Further analyses indicate that MalL is a specific oligo-1,4-1,6-alpha-glucosidase (sucrase-maltase-isomaltase). MalL expression in B. subtilis requires maltose induction and is subject to carbon catabolite repression by glucose and fructose. Insertional mutagenesis of malL resulted in a complete inactivation of the maltose-inducible alpha-glucosidase activity in crude protein extracts and a Mal- phenotype.

Bacillus subtilis↗
PubMed2000

Alpha-glucosidase inhibitors reduce dengue virus production by affecting the initial steps of virion morphogenesis in the endoplasmic reticulum.

We report that endoplasmic reticulum alpha-glucosidase inhibitors have antiviral effects on dengue (DEN) virus. We found that glucosidase inhibition strongly affects productive folding pathways of the envelope glycoproteins prM (the intracellular glycosylated precursor of M [membrane protein]) and E (envelope protein): the proper folding of prM bearing unprocessed N-linked oligosaccharide is inefficient, and this causes delayed formation of prME heterodimer. The complexes formed between incompletely folded prM and E appear to be unstable, leading to a nonproductive pathway. Inhibition of alpha-glucosidase-mediated N-linked oligosaccharide trimming may thus prevent the assembly of DEN virus by affecting the early stages of envelope glycoprotein processing.

1-Deoxynojirimycin↗
PubMed1996

Purification and characterization of a beta-glucosidase from solid-state cultures of Humicola grisea var. thermoidea.

The thermophilic fungus Humicola grisea var. thermoidea produced beta-glucosidase activity when grown in a solid-state culture on wheat bran as carbon source. A beta-glucosidase was purified to apparent homogeneity by ultrafiltration, gel filtration chromatography on Sephacryl S-100, and ion-exchange chromatography on S-Sepharose, as judged by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE) on a 12.5% (w/v) slab gel. The enzyme had a molecular mass of 82 and 156 kDa, as estimated by SDS-PAGE and gel filtration on a high performance liquid chromatographic column, respectively, suggesting that the native enzyme may consist of two identical subunits. The purified enzyme was thermostable at 60 degrees C for 1 h with a half-life of 15 min at 65 degrees C, and displayed optimum activity at 60 degrees C and a pH range. of 4.0-4.5. The Km and Vmax values for p-nitrophenyl beta-D-glucopyranoside were determined to be 0.316 mM and 0.459 IU.mL-1, respectively. D-Glucose, D-gluconic acid lactone, Hg2+, Cu2+, and Mn2+ inhibited beta-glucosidase activity. The enzyme activity was competitively inhibited by D-glucose (ki = 0.6 mM). The purified enzyme was very active against cellobiose and p-nitrophenyl beta-D-glucopyranoside.

Cations, Divalent↗
PubMed1977

The beta-glucosidases of porcine kidney.

Some of the properties of a partially purified particle bound and soluble beta-glucosidase (EC 3.2.1.21) from pig kidney were compared. The soluble beta-glucosidase (1) hydrolyzed 4-methylumbelliferyl-beta-D-glucoside (4-MU-beta-D-glucoside) 17 alpha-estradiol 3beta-glucoside. 17 alpha-estradiol 17beta-glucoside, and salicin, but not glucosylceramide, (2) possessed a broad pH optimum (5.5-7.0), (3) had an isoelectric point of 4.9, and (4) was inhibited by Triton X-100. Several compounds were found to be competitive inhibitors of its hydrolytic activity, gluconolactam and estrone beta-glucoside being the most effective. In contrast, a particulate beta-glucodidase purified from the same tissue (1) had an acidic pH optimum (5.0), (2) was stimulated by sodium taurocholate and 'Gaucher's factor' for the hydrolysis of both 4-MU-beta-glucosidase and glucosylceramide, and (3) was capable of catalyzing a transglucosylation reaction employing 4-MU-beta-D-glucoside or glucosylceramide as the glucosyl donor, and [14C]ceramide as acceptor.

Animals↗