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PubMed2002

Microbial beta-glucosidases: cloning, properties, and applications.

Beta-glucosidases constitute a major group among glycosylhydrolase enzymes. Out of the 82 families classified under glycosylhydrolase category, these belong to family 1 and family 3 and catalyze the selective cleavage of glucosidic bonds. This function is pivotal in many crucial biological pathways, such as degradation of structural and storage polysaccharides, cellular signaling, oncogenesis, host-pathogen interactions, as well as in a number of biotechnological applications. In recent years, interest in these enzymes has gained momentum owing to their biosynthetic abilities. The enzymes exhibit utility in syntheses of diverse oligosaccharides, glycoconjugates, alkyl- and aminoglucosides. Attempts are being made to understand the structure-function relationship of these versatile biocatalysts. Earlier reviews described the sources and properties of microbial beta-glucosidases, yeast beta-glucosidases, thermostable fungal beta-glucosidase, and the physiological functions, characteristics, and catalytic action of native beta-glucosidases from various plant, animal, and microbial sources. Recent efforts have been directed towards molecular cloning, sequencing, mutagenesis, and crystallography of the enzymes. The aim of the present article is to describe the sources and properties of recombinant beta-glucosidases, their classification schemes based on similarity at the structural and molecular levels, elucidation of structure-function relationships, directed evolution of existing enzymes toward enhanced thermostability, substrate range, biosynthetic properties, and applications.

Biotechnology↗
PubMed1995

Castanospermine analogues: their inhibition of glycoprotein processing alpha-glucosidases from porcine kidney and B16F10 cells.

We have used a simple and efficient procedure for the synthesis of N-5-carboxypentyl-1-deoxynojirimycin, an affinity ligand for alpha-glucosidase I (Bernotas, R. C. and Ganem, B., Biochem. J., 270, 539-540, 1990). The affinity gel was used to purify alpha-glucosidase I in one step from crude extract. In subsequent steps, partially purified alpha-glucosidase II was obtained. We have synthesized several analogues of castanospermine and studied their inhibition of alpha-glucosidase I in vitro using purified alpha-glucosidase I and in vivo in cultured B16F10 cells. Although the castanospermine analogues were significantly less active against the purified enzyme (IC50 approximately 1-23 micrograms/ml) as compared to castanospermine (IC50 = 0.02 microgram/ml), several compounds had up to 30-fold higher activity than castanospermine against alpha-glucosidase I in B16F10 cells, based on the accumulation of G3M7-9N2 oligosaccharide-containing glycoproteins. These results suggest that these analogues with lipophilic side chains cross the membrane barrier more efficiently than castanospermine. Once inside the cell, they may be converted to their active metabolite, castanospermine, by cellular esterases to give enzyme inhibition.

Animals↗
PubMed2002

(Arg)3 within the N-terminal domain of glucosidase I contains ER targeting information but is not required absolutely for ER localization.

Glucosidase I is an endoplasmic reticulum (ER) type II membrane enzyme that cleaves the distal alpha1,2-glucose of the asparagine-linked GlcNAc2-Man9-Glc3 precursor. To identify sequence motifs responsible for ER localization, we prepared a protein chimera by transferring the cytosolic and transmembrane domain of glucosidase I to the luminal domain of Golgi-Man9-mannosidase. The GIM9 hybrid was overexpressed in COS 1 cells as an ER-resident protein that displayed alpha1,2-mannosidase activity, excluding the possibility that the glucosidase I-specific domains interfere with folding of the Man9-mannosidase catalytic domain. After substitution of the Args in position 7, 8, or 9 relative to the N-terminus by leucine, the GIM9 mutants were transported to the cell surface indicating that the (Arg)3 sequence functions as an ER-targeting motif. Cell surface expression was also observed after substitution of Arg-7 or Arg-8 but not Arg-9 in GIM9 by either lysine or histidine. Thus the side chain structure, including its positive charge, appears to be essential for signal function. Analysis of the N-linked glycans suggests that the (Arg)3 sequence mediates ER localization through Golgi-to-ER retrograde transport. Glucosidase I remained localized in the ER after truncation or mutation of the N-terminal (Arg)3 signal, in contrast to comparable GIM9 mutants. ER localization was also observed with an M9GI chimera consisting of the cytosolic and transmembrane domain of Man9-mannosidase and the glucosidase I catalytic domain. ER-specific targeting information must therefore be provided by sequence motifs contained within the glucosidase I luminal domain. This structural information appears to direct ER localization by retention rather than by retrieval, as concluded from N-linked Man9-GlcNAc2 being the major glycan released from the wild-type enzyme.

Amino Acid Motifs↗
PubMed1999

Molecular cloning and expression of the novel fungal beta-glucosidase genes from Humicola grisea and Trichoderma reesei.

A novel fungal beta-glucosidase gene (bgl4) and its homologue (bgl2) were cloned from the cellulolytic fungi Humicola grisea and Trichoderma reesei, respectively. The deduced amino acid sequences of H. grisea BGL4 and T. reesei BGL2 comprise 476 and 466 amino acids, respectively, and share 73.1% identity. These beta-glucosidases show significant homology to plant beta-glucosidases belonging to the beta-glucosidase A (BGA) family. Both genes were expressed in Aspergillus oryzae, and the recombinant beta-glucosidases were purified. Recombinant H. grisea BGL4 is a thermostable enzyme compared with recombinant T. reesei BGL2. In addition to beta-glucosidase activity, recombinant H. grisea BGL4 showed a significant level of beta-galactosidase activity, while recombinant T. reesei BGL2 showed weak beta-galactosidase activity. Cellulose saccharification by Trichoderma cellulases was improved by the addition of recombinant H. grisea BGL4.

Amino Acid Sequence↗
PubMed2004

Effect of adding and removing N-glycosylation recognition sites on the thermostability of barley alpha-glucosidase.

The thermostability of alpha-glucosidase is important because the conversion of starch to fermentable sugars during the industrial production of beer and fuel ethanol typically occurs at relatively high temperatures (60-75 degrees C). Barley (Hordeum vulgare) alpha-glucosidase is unstable at these elevated temperatures; however, the alpha-glucosidase from sugar beet (Beta vulgaris) is stable at these temperatures. An alignment of the deduced amino acid sequences of barley and sugar beet alpha-glucosidases revealed considerable differences in the number and position of N-glycosylation recognition sites (NGRS). Other researchers have shown that additions or removals of NGRS resulted in either the stabilization or destabilization of the enzymes at elevated temperatures. NGRS present in the barley sequence and absent in the sugar beet sequence were removed via site-directed mutagenesis from the barley protein. Recognition sites absent in the barley sequence and present in the sugar beet sequence were added via mutagenesis into the barley alpha-glucosidase. Two mutations significantly increased thermostability, one mutation significantly decreased thermostability and five mutations had little effect on alpha-glucosidase thermostability.

Alanine↗
PubMed1990

Cloning, sequencing and analysis of expression of a Butyrivibrio fibrisolvens gene encoding a beta-glucosidase.

The cloning, expression and nucleotide sequence of a 3.74 kb DNA segment on pLS215 containing a beta-glucosidase gene (bglA) from Butyrivibrio fibrisolvens H17c was investigated. The B. fibrisolvens bglA open reading frame (ORF) of 2490 bp encoded a beta-glucosidase of 830 amino acid residues with a calculated Mr of 91,800. In Escherichia coli C600(pLS215) cells the beta-glucosidase was localized in the cytoplasm and these cells produced an additional protein with an apparent Mr of approximately 94,000. The bglA gene was expressed from its own regulatory region in E. coli and a single mRNA initiation point was identified upstream of the bglA ORF and adjacent to a promoter consensus sequence. The primary structure of the beta-glucosidase showed greater than 40% similarity with a domain of 237 amino acids present in the beta-glucosidases of Kluyveromyces fragilis and Clostridium thermocellum. The B. fibrisolvens beta-glucosidase hydrolysed cellobiose to a limited extent, cellotriose to cellobiose and glucose, and cellotetraose and cellopentaose to predominantly glucose.

Amino Acid Sequence↗
PubMed2000

The 1-kb-repeat-encoded DNA-binding protein as repressor of an alpha-glucosidase operon flanking the amplifiable sequence AUD1 of Streptomyces lividans.

High-copy-number amplification of the AUD1 element is frequently associated with the large chromosomal deletions responsible for genetic instability in Streptomyces lividans TK64. Five ORFs were found in a 7 kb region directly adjacent to AUD1. The putative products of ORF1, ORF2 and ORF3 showed similarities to ATP-binding cassette (ABC) sugar transporters, the deduced protein sequence of ORF4 displayed similarities to alpha-glucosidases whilst no homology to proteins with known functions was found for ORF5. ORF4 (renamed aglA) was expressed in Escherichia coli and the protein purified and characterized. An alpha-glucosidase activity was detected using the synthetic alpha-glucoside p-nitrophenyl alpha-D-glucopyranoside. Of the many oligosaccharides tested, only sucrose was hydrolysed at a measurable rate [specific activity 32.4 units (mg protein)(-1)] but no growth of S. lividans TK64 on sucrose was observed. A strain in which aglA was disrupted showed the same low alpha-glucosidase activity as strain TK64 and in both strains no stimulation of activity was seen by sucrose, trehalose or maltose; dextrin increased alpha-glucosidase activity about 10-fold. This probably resulted from induction of a second alpha-glucosidase-encoding gene. The AUD1 element contains three 1 kb repeats which encode DNA-binding proteins necessary for high-frequency amplification. In strains with a unique 1 kb repeat, disruption of the repeat led to a significant increase in the alpha-glucosidase activity. These results strongly suggest that the 1-kb-repeat-encoded proteins of AUD1 have a dual function: they are the repressors of the agl genes and they promote amplification of AUD1.

ATP-Binding Cassette Transporters↗
PubMed1995

A beta-glucosidase from lodgepole pine xylem specific for the lignin precursor coniferin.

Coniferin, the glucoside of the monolignol coniferyl alcohol, accumulates to high levels in gymnosperms during spring-cambial reactivation. A cinnamyl alcohol glucoside/beta-glucosidase system is thought to play a key role in lignification by releasing the monolignol aglycones. Investigation of such an enzyme system in the xylem of Pinus contorta var latifolia Engelm. revealed two major beta-glucosidases. One efficiently hydrolyzed the native substrate, coniferin, and the other was more active against synthetic glucosides. The coniferin beta-glucosidase was purified to apparent homogeneity using anion exchange, hydrophobic interaction, and size-exclusion chromatography. The apparent native molecular weight was estimated to be 60,000. A dominant 28-kD protein and a minor 24-kD protein were detected in the purified preparation following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Immunological evidence from polyclonal antibodies directed against the synthetic N-terminal peptide of the 24-kD protein suggested that the native protein is a dimer of 28-kD subunit size. The N-terminal sequence showed that coniferin beta-glucosidase has high homology to known plant beta-glucosidases. Coniferin, syringin, and a synthetic coniferin analog were preferred substrates for the coniferin beta-glucosidase. In situ localization using the chromogenic coniferin analog showed the exclusive presence of beta-glucosidase activity in the differentiating xylem, similar to peroxidase activity.

Amino Acid Sequence↗
PubMed1995

Expression of a zeatin-O-glucoside-degrading beta-glucosidase in Brassica napus.

A beta-glucosidase was purified from seeds of Brassica napus L. (oilseed rape). The 130-kD native enzyme consisted of a disulfide-linked dimer of 64-kD monomers. Internal amino acid sequences were used to construct degenerate primers for polymerase chain reaction-mediated cloning of cDNA for the enzyme. One nearly full-length and one partial beta-glucosidase-encoding cDNA clone were isolated and sequenced. Southern hybridization showed that beta-glucosidase is encoded by a small gene family in B. napus. Northern hybridization showed that the genes are expressed in the seed, with a low degree of expression in other tissues. In the seed, the expression started at 30 days after pollination (DAP), with the highest expression at 40 DAP. The size of the transcript was approximately 1900 nucleotides. In situ hybridization to developing seeds of B. napus showed that the beta-glucosidase expression started at 30 DAP around the provascular tissue in the embryo axis. In the cotyledons, mRNA initially accumulated around the provascular tissues but was detected first at 35 DAP. At 40 DAP, expression occurred in most parts of the seed. In situ hybridization also detected beta-glucosidase mRNA in shoots, young roots, and the basal part of the hypocotyls. Zeatin-O-glucoside was identified as a natural substrate for B. napus beta-glucosidase.

Amino Acid Sequence↗
PubMed1989

Purification and characterization of trimming glucosidase I from pig liver.

Trimming glucosidase I has been purified about 400-fold from pig liver crude microsomes by fractional salt/detergent extraction, affinity chromatography and poly(ethylene glycol) precipitation. The purified enzyme has an apparent molecular mass of 85 kDa, and is an N-glycoprotein as shown by its binding to concanavalin A-Sepharose and its susceptibility to endo-beta-N-acetylglucosaminidase (endo H). The native form of glucosidase I is unusually resistant to non-specific proteolysis. The enzyme can, however, be cleaved at high, that is equimolar, concentrations of trypsin into a defined and enzymatically active mixture of protein fragments with molecular mass of 69 kDa, 45 kDa and 29 kDa, indicating that it is composed of distinct protein domains. The two larger tryptic fragments can be converted by endo H to 66 kDa and 42 kDa polypeptides, suggesting that glucosidase I contains one N-linked high-mannose sugar chain. Purified pig liver glucosidase I hydrolyzes specifically the terminal alpha 1-2-linked glucose residue from natural Glc3-Man9-GlcNAc2, but is inactive towards Glc2-Man9-GlcNAc2 or nitrophenyl-/methyl-umbelliferyl-alpha-glucosides. The enzyme displays a pH optimum close to 6.4, does not require metal ions for activity and is strongly inhibited by 1-deoxynojirimycin (Ki approximately 2.1 microM), N,N-dimethyl-1-deoxynojirimycin (Ki approximately 0.5 microM) and N-(5-carboxypentyl)-1-deoxynojirimycin (Ki approximately 0.45 microM), thus closely resembling calf liver and yeast glucosidase I. Polyclonal antibodies raised against denatured pig liver glucosidase I, were found to recognize specifically the 85 kDa enzyme protein in Western blots of crude pig liver microsomes. This antibody also detected proteins of similar size in crude microsomal preparations from calf and human liver, calf kidney and intestine, indicating that the enzymes from these cells have in common one or more antigenic determinants. The antibody failed to cross-react with the enzyme from chicken liver, yeast and Volvox carteri under similar experimental conditions, pointing to a lack of sufficient similarity to convey cross-reactivity.

Animals↗
PubMed1995

Cloning and expression of glucosidase I from human hippocampus.

Glucosidase I, the first enzyme in the N-linked oligosaccharide processing pathway, cleaves the distal alpha 1,2-linked glucose residue from the Glc3-Man9-GlcNAc2 oligosaccharide precursor highly specifically. A human hippocampus cDNA library was screened against oligonucleotide probes, generated by PCR using primers derived from the amino acid sequences of tryptic peptides of pig liver glucosidase I. Two independent lambda clones were isolated which allowed the construction of a full-length glucosidase I cDNA of 2881 bp. This cDNA construct encodes, in a single open reading frame, a polypeptide of 834 amino acids corresponding to a molecular mass of 92 kDa. The 92-kDa protein contains a single N-glycosylation site of the Asn-Xaa-Thr/Ser type at Asn655, as well as a strongly hydrophobic sequence close to its N-terminus (amino acids 38-58) which, most likely, functions as a transmembrane anchor. The amino acid sequences of all tryptic peptides of the pig liver enzyme were found, with little deviation, within the coding sequence. This demonstrates the authenticity of the cDNA construct and the close evolutionary relationship between the enzymes from human hippocampus and pig liver. In contrast, the nucleotide and amino acid sequence revealed no homology with other processing enzymes cloned so far. Transfection of COS 1 cells with the glucosidase I cDNA construct resulted in overexpression (about fourfold) of enzymic activity, which was inhibited strongly by 1-deoxynojirimycin or N,N-dimethyl-deoxynojirimycin. The expressed enzyme, with a molecular mass of 95 kDa, is degraded by endoglycosidase H to a 93-kDa form, indicating that it carries a high-mannose oligosaccharide chain at Asn655. The presence of this glycan is in line with the localization of glucosidase I in the lumen of the endoplasmic reticulum, shown by immunofluorescence microscopy. The hydrophobicity profile as well as the removal by trypsin of an approximately 4-kDa polypeptide from the membrane-associated glucosidase I in intact microsomal structures, supports the view that the enzyme is a type-II transmembrane glycoprotein, which contains a short cytosolic tail of approximately 37 amino acids, followed by a single transmembrane domain and a large C-terminal catalytic domain located on the luminal side of the endoplasmic reticulum membrane.

Amino Acid Sequence↗
PubMedDate not supplied

Processing and secretion of lysosomal acid alpha-glucosidase in Tetrahymena wild type and secretion-deficient mutant cells.

The proteolytic processing and secretion of a lysosomal enzyme, acid alpha-glucosidase, was studied by pulse-chase labeling with [35S]methionine in Tetrahymena thermophila CU-399 cells treated with ammonium chloride. This cell secreted a large amount of acid alpha-glucosidase into the cultured medium during starvation. The secretion was found to be repressed by addition of ammonium chloride (NH4Cl). Acid alpha-glucosidase was produced as a precursor form (108 kDa) and then processed to a mature polypeptide (105 kDa) within 60 min. This mature enzyme was secreted into the media within 2-3 h after chase, whereas the precursor form was not secreted by either control cells or NH4Cl-treated cells. NH4Cl did not affect the processing of the precursor acid alpha-glucosidase. Processing profile of this enzyme was apparently indistinguishable from that of the mutant MS-1 defective in lysosomal enzyme secretion. Furthermore, the purified extracellular (CU-399) and intracellular (MS-1) acid alpha-glucosidases were the same in molecular mass (105 kDa) and enzymatic properties. They contained no mannose 6-phosphate residues in N-linked oligosaccharides. These results suggested that unlike mammalian cells, Tetrahymena acid alpha-glucosidase may be transferred to lysosomes by a mannose 6-phosphate receptor-independent mechanism, and also that low pH was not essential for the proteolytic processing of precursor polypeptide.

Ammonium Chloride↗
PubMed2006

Molecular cloning and transcriptional expression analysis of an intracellular beta-glucosidase, a family 3 glycosyl hydrolase, from the edible straw mushroom, Volvariella volvacea.

A beta-glucosidase, with a molecular mass of 95 kDa, was isolated from extracts of Volvariella volvacea mycelium grown on crystalline cellulose. Degenerate primers based on the N-terminal sequences of purified beta-glucosidase and two protease-generated peptides were used to generate cDNA fragments encoding a portion of the beta-glucosidase gene (bgl), and rapid amplification of cDNA ends was used to obtain full-length cDNA clones. The cDNA of bgl contained an ORF of 2586 bp coding for 862 amino acids. Alignment of the deduced amino-acid sequence of beta-glucosidase with deduced amino acid sequences of other microbial beta-glucosidases showed the highest overall homology with glycoside hydrolase family 3 beta-glucosidases from fungi. Transcripts of bgl were detected in total RNA extracted from mycelium grown on cellulose and cellobiose, and from mycelium pre-grown for 72 h in basal medium containing 1% (w/v) sorbitol following addition of alpha-lactose, beta-lactose, cellobiose, d- xylose, l-sorbose, beta-gentiobiose, sophorose or d-galactose. Addition of l-sorbose and d-glucosamine to mycelium grown on 1% (w/v) crystalline cellulose greatly increased the level of bgl expression. bgl Was expressed at various stages of the mushroom developmental cycle (substrate colonization to mature fruit body), although the number of bgl transcripts in pinhead and button stages was slightly smaller.

Agaricales↗
PubMed1989

Inhibition of alpha-glucosidase in cattle by Castanospermum australe: an attempted phenocopy of Pompe's disease.

Pompe's disease is characterised by an absence of lysosomal alpha-glucosidase, but this enzyme is also inhibited by Castanospermum australe seeds. Four calves were fed C. australe seeds at the rate of 0.15 g/kg body weight for periods from 1 to 4 days. Lymphocyte alpha-glucosidase activity was reduced by at least 90%, with the majority of inhibition occurring within 8 h of dosing. Several weeks elapsed before activity returned to normal. Significant inhibition of muscle alpha-glucosidase occurred and the ratio of plasma alpha-glucosidase activity measured at pH 5.6 relative to that at pH 3.7 was depressed. In an attempt to induce Pompe's disease, 2 calves were dosed with 1.2 g C. australe seed/kg body weight/day for 13 months. Lymphocyte and muscle alpha-glucosidase activities were markedly reduced over the entire period of feeding, but the animals showed no clinical signs of disease. Tissue cells were not vacuolated nor did they show any apparent accumulation of glycogen. Despite significant inhibition of alpha-glucosidase in skeletal and cardiac muscle, liver, kidney and brain, it is suggested that there was sufficient residual enzyme to prevent induction of a phenocopy of Pompe's disease.

Animals↗
PubMed1996

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).

Candida↗
PubMed1998

Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase from Aspergillus oryzae.

Aspergillus oryzae was found to secrete two distinct beta-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3',4',5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total beta-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant beta-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-beta-D-glucoside as the substrate, we found that the enzyme was optimally active at 50 degreesC and pH 5.0 and had a specific activity of 1,066 micromol min-1 mg of protein-1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1. 36 M) or glucono-delta-lactone (Ki, 12.5 mM), another powerful beta-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal beta-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1-->3)- and (1-->6)-beta-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-beta-D-glucosides in a grape must (pH 2.9, 90 g of glucose liter-1). Other flavor precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.

Aspergillus oryzae↗
PubMed1999

Histoplasma capsulatum strain variation in both H antigen production and beta-glucosidase activity and overexpression of HAG1 from a telomeric linear plasmid.

The H antigen of the dimorphic fungal pathogen Histoplasma capsulatum was first described over 40 years ago. It is a secreted glycoprotein that is immunogenic during infection. Recent cloning of the H antigen gene (HAG1) indicated sequence homology with genes for fungal beta-glucosidases. To understand the biological role of this immunodominant antigen in H. capsulatum, enzymatic assays were performed to determine whether H. capsulatum contained a beta-glucosidase enzyme activity and whether this activity was encoded by the HAG1 gene. Substrate gels with H. capsulatum culture supernatants revealed beta-glucosidase activity near the predicted mobility of the H antigen. Quantitative microtiter plate assays revealed marked differences in secreted beta-glucosidase activities from three H. capsulatum restriction fragment length polymorphism (RFLP) classes, with RFLP class II strains displaying high levels of enzyme activity, in contrast to the low levels of activity exhibited by class I and III strains. Immunoblotting of culture supernatants with an H antigen-specific antiserum demonstrated differences in H protein expression levels between the H. capsulatum classes, with a correlation between secreted enzyme activity and H protein levels. We took advantage of these class differences to demonstrate multicopy plasmid H gene overexpression by transformation of an HAG1 plasmid into H. capsulatum. Both a class II strain (G217Bura5-23) and a class III strain (G184ASura5-11) transformed with the telomeric overexpression plasmid pMAD401 displayed increased levels of beta-glucosidase enzyme activity and H protein expression compared to the levels in control transformants containing only the single genomic copy of HAG1. This is the first demonstration of telomeric plasmid-mediated protein overexpression in this pathogenic fungus, and the findings support the identification of the H antigen as a beta-glucosidase.

Animals↗
PubMed1996

Contribution of a neopullulanase, a pullulanase, and an alpha-glucosidase to growth of Bacteroides thetaiotaomicron on starch.

Bacteroides thetaiotaomicron, a gram-negative colonic anaerobe, can utilize three forms of starch: amylose, amylopectin, and pullulan. Previously, a neopullulanase, a pullulanase, and an alpha-glucosidase from B. thetaiotaomicron had been purified and characterized biochemically. The neopullulanase and alpha-glucosidase appeared to be the main enzymes involved in the breakdown of starch, because they were responsible for most of the starch-degrading activity detected in B. thetaiotaomicron cell extracts. To determine the importance of these enzymes in the starch utilization pathway, we cloned the genes encoding the neopullulanase and alpha-glucosidase. The gene encoding the neopullulanase (susA) was located upstream of the gene encoding the alpha-glucosidase (susB). Both genes were closely linked to another starch utilization gene, susC, which encodes a 115-kDa outer membrane protein that is essential for growth on starch. The gene encoding the pullulanase, pulI, was not located in this region in the chromosome. Disruption of the neopullulanase gene, susA, reduced the rate of growth on starch by about 30%. Elimination of susA in this strain allowed us to detect a low residual level of enzyme activity, which was localized to the membrane fraction. Previously, we had shown that a disruption in the pulI gene did not affect the rate of growth on pullulan. We have now shown that a double mutant, with a disruption in susA and in the pullulanase gene, pulI, was also able to grow on pullulan. Thus, there is at least one other starch-degrading enzyme besides the neopullulanase and the pullulanase. Disruption of the alpha-glucosidase gene, susB, reduced the rate of growth on starch only slightly. No residual alpha-glucosidase activity was detectable in extracts from this strain. Since this strain could still grow on maltose, maltotriose, and starch, there must be at least one other enzyme capable of degrading the small oligomers produced by the starch-degrading enzymes. Our results show that the starch utilization system of B. thetaiotaomicron is quite complex and contains a number of apparently redundant degradative enzymes.

Amino Acid Sequence↗