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Developmental lignification and seasonal variation in beta-glucosidase and peroxidase activities in xylem of Scots pine, Norway spruce and silver birch.

We examined the relationship between beta-glucosidase and peroxidase activities and xylem lignification in the stems of Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies (L.) Karst.) and silver birch (Betula pendula Roth) during the 1999 growing season. Examination of stem cross sections stained with safranin and Alcian blue for lignin and cellulose, respectively, indicated that radial growth of pine and spruce xylem began in late May, whereas the growth of birch xylem was initiated 2 weeks later. Lignification began soon after thickening of the newly formed cell walls, i.e., upon deposition of cellulose. Hydrolysis of the synthetic beta-glucosidase substrate p-nitrophenyl-beta-O-D-glucopyranoside was correlated with radial growth and lignification in the xylem of both conifers, but the relationship between lignification and the hydrolysis of coniferin by beta-glucosidase was not obvious. Beta-glucosidase activities in the xylem of silver birch were low and did not correlate with growth or lignification with either substrate. An increase in peroxidase activity was detected at the initiation of growth and lignification in the conifers and during growth and lignification in silver birch, but high peroxidase activities were also measured outside the growth period during late autumn, winter and early spring.

Betula↗

Histochemical localization and quantification of alpha-glucosidase in the epididymis of men and laboratory animals.

The seminal marker of epididymal function alpha-1, 4-glucosidase was localized histochemically in the cytoplasm of the efferent duct epithelium and the brush border of the entire length of the human epididymis. Quantification using the specific inhibitor castanospermine revealed strongest activity in the corpus and cauda regions. Selective inhibition of the brush border enzyme activities by maltotriose identified these as the neutral isoenzymes. Despite detection of alpha-glucosidase in the renal tubules of all the animals studied, the enzyme was not detectable in epididymides of hamsters or mice. In rabbits and monkeys, it was absent from the entire brush border but present weakly in the cytoplasm of the proximal epididymides. An enzyme distribution pattern similar to that in the human epididymis was found in rats, except for the absence of histochemical staining at pH 6.5 from the initial segment and distal cauda epididymidis. Experiments in which endogenous testosterone was depleted in rats demonstrated the dependence of epididymal alpha-glucosidase on androgen, albeit with a low sensitivity. This study suggests the rat to be a suitable model for the investigation of the role of epididymal alpha-glucosidase in fertility.

Alkaloids↗

Structure and expression of a dhurrinase (beta-glucosidase) from sorghum.

Sorghum (Sorghum bicolor L. Moench) has two isozymes of the cyanogenic beta-glucosidase dhurrinase: dhurrinase-1 (Dhr1) and dhurrinase-2 (Dhr2). A nearly full-length cDNA encoding dhurrinase was isolated from 4-d-old etiolated seedlings and sequenced. The cDNA has a 1695-nucleotide-long open reading frame, which codes for a 565-amino acid-long precursor and a 514-amino acid-long mature protein, respectively. Deduced amino acid sequence of the sorghum Dhr showed 70% identity with two maize (Zea mays) beta-glucosidase isozymes. Southern-blot data suggested that beta-glucosidase is encoded by a small multigene family in sorghum. Northern-blot data indicated that the mRNA corresponding to the cloned Dhr cDNA is present at high levels in the node and upper half of the mesocotyl in etiolated seedlings but at low levels in the root-only in the zone of elongation and the tip region. Light-grown seedling parts had lower levels of Dhr mRNA than those of etiolated seedlings. Immunoblot analysis performed using maize-anti-beta-glucosidase sera detected two distinct dhurrinases (57 and 62 kD) in sorghum. The distribution of Dhr activity in different plant parts supports the mRNA and immunoreactive protein data, suggesting that the cloned cDNA corresponds to the Dhr1 (57 kD) isozyme and that the dhr1 gene shows organ-specific expression.

Amino Acid Sequence↗

Characterization of beta-glucosidase activity in yeasts of oenological origin.

Three hundred and seventeen strains representing 20 species of yeasts were screened for the presence of beta-glucosidase activity. All of the strains of the species Debaryomyces castellii, Deb. hansenii, Deb. polymorphus, Kloeckera apiculata and Hansenula anomala showed beta-glucosidase activity, but only one of 153 strains of Saccharomyces cerevisiae. The other species behaved differently, depending upon the strain. The strains that hydrolysed arbutin were checked to localize the beta-glucosidase activity. A strain of Deb. hansenii exhibited the highest exocellular activity and some wall-bound and intracellular activity. The beta-glucosidase synthesis from this yeast was enhanced by aerobic conditions of growth, was repressed by high glucose concentration (9%) and occurred during exponential growth. The optimum conditions for enzymatic preparations of Deb. hansenii were between pH 4.0 and 5.0 and 40 degrees C. A high concentration of ethanol and glucose did not reduce the enzymatic activity. The enzymatic preparations of Deb. hansenii released monoterpenols and other alcohols from a grape glycoside extract.

Arbutin↗

Optimization of a rapid method for studying the cellular location of beta-glucosidase activity in wine yeasts.

AIMS: To improve a method for determining beta-glucosidase activity and to apply it in yeasts isolated from wine ecosystems from "La Mancha" region and to know its cellular location. METHODS AND RESULTS: A total of 82 wine yeasts were identified (PCR/RFLP) and evaluated for their beta-glucosidase activity. First, they were qualitatively evaluated by growth on YNB cellobiose, the activity was quantified using different culture media, under aerobic and anaerobic conditions and cells after 24-72 h of growth. To study the location activity, five fractions were obtained (supernatant, whole cell, cell wall, cytosol and cell membrane). The enzymatic assays were optimized, being: growth in YP cellobiose for 72 h in aeration conditions and, after cell removing, enzyme analysis with 128 g l(-1) of cellobiose as substrate, for 30 min at 30 degrees C. The genus that displayed the greatest activity were Pichia, Hanseniaspora and Rhodotorula, and the activity was intracellular. CONCLUSIONS: The study showed that beta-glucosidase activity was induced by the carbon source and was aerobic dependent. The non-Saccharomyces species displayed the greatest activity, which was intracellular and strain-dependent. SIGNIFICANCE AND IMPACT OF THE STUDY: This study developed a reliable method for screening beta-glucosidase activity in yeasts isolated from wine ecosystems. This activity is very important in the release of monoterpenols from glycoside precursors for the enhancement of wine aromas.

Cell Membrane↗

Km mutant of acid alpha-glucosidase in a case of cardiomyopathy without signs of skeletal muscle involvement.

A male patient is reported with a mutation of acid alpha-glucosidase causing an altered Km toward natural substrates. Cardiac arrhythmia was found at 12 years of age, and he died of heart failure at 15 years. No skeletal muscle involvement was observed either clinically or histologically. Acid alpha-glucosidase activity in fibroblasts was moderately low (43% of the control mean) with normal Km for 4-methylumbelliferyl alpha-D-glucoside. The hydrolysis of glycogen was markedly decreased (14% of the control mean), and the Km for maltose was increased 4-fold and for glycogen 5-fold. The biosynthesis and the posttranslational processing of the mutant enzyme appeared normal, but the total amount of the enzyme was lower than normal. This mutant enzyme comigrated with normal acid alpha-glucosidase on starch gel electrophoresis, and not with the rare isozyme, acid alpha-glucosidase 2. A possible role of this mutant enzyme in the pathogenesis of this disease and the relationship to glycogenesis II are discussed.

Adolescent↗

Assembly of asparagine-linked oligosaccharides in baby hamster kidney cells treated with castanospermine, an inhibitor of processing glucosidases.

We have shown previously that the processing of asparagine-linked oligosaccharides in baby hamster kidney (BHK) cells is blocked only partially by the glucosidase inhibitors, 1-deoxynojirimycin and N-methyl-1-deoxynojirimycin [Hughes, R. C., Foddy, L. & Bause, E. (1987) Biochem. J. 247, 537-544]. Similar results are now reported for castanospermine, another inhibitor of processing glucosidases, and a detailed study of oligosaccharide processing in the inhibited cells is reported. In steady-state conditions the major endo-H-released oligosaccharides contained glucose residues but non-glycosylated oligosaccharides, including Man9GlcNAc to Man5GlcNAc, were also present. To determine the processing sequences occurring in the presence of castanospermine, BHK cells were pulse-labelled for various times with [3H]mannose and the oligosaccharide intermediates, isolated by gel filtration and paper chromatography, characterized by acetolysis and sensitivity to jack bean alpha-mannosidase. The data show that Glc3Man9GlcNAc2 is transferred to protein and undergoes processing to produce Glc3Man8GlcNAc2 and Glc3Man7GlcNAc2 as major species as well as a smaller amount of Man9GlcNAc2. Glucosidase-processed intermediates, Glc1Man8GlcNAc2 and Glc1Man7GlcNAc2, were also obtained as well as a Man7GlcNAc2 species derived from Glc1Man7GlcNAc2 and different from the Man7GlcNAc2 isomer formed in the usual processing pathway. No evidence for the direct transfer of non-glucosylated oligosaccharides to proteins was obtained and we conclude that the continued assembly of complex-type glycans in castanospermine-inhibited BHK cells results from residual activity of processing glucosidases.

Alkaloids↗

Alpha-glucosidase in the human epididymis: topographic distribution and clinical application.

Alpha-glucosidase activity (EC.3.2.1.20) is present in human seminal plasma, and the neutral form of the enzyme originates almost exclusively from the epididymis. In this study, the specific immunocytochemical location of alpha-glucosidase in the human epididymis was evaluated using a polyclonal antibody. Furthermore, a spectrophotometric assay was employed to assess epididymal obstruction in infertile patients. The enzymatic activity of alpha-glucosidase free of prostate isoform (AGFPI) was determined spectrophotometrically at 405 nm. According to AGFPI activity, patients with leucocytospermia, oligozoospermia and azoospermia were recorded as having normal values or low values indicating epididymal obstruction. Specific immunochemistry staining was demonstrated in the cytoplasmic cells at the epithelial level, in the transition area and in the efferent ducts. The values of the three groups and the control were as follows (mean +/- SEM): normozoospermia (control): 20.2 +/- 1.4 mU ml(-1); azoospermia: normal value: 17.6 +/- 2.2 mU ml(-1), low value: 7.4 +/- 1.8 mU ml(-1); oligozoospermia: normal value: 22.3 +/- 2.5 mU ml(-1), low value: 7.3 +/- 0.7 mU ml(-1); leucocytospermia: increase value: 38.9 +/- 3.7 mU ml(-1), low value: 11.1 +/-1.3 mU ml(-1). This study suggests that determination of alpha-glucosidase might be helpful to evaluate functions of the epididymis and particularly to exclude epididymal obstruction.

Adult↗

A carbohydrate-degrading enzyme from Candida albicans: correlation between alpha-glucosidase activity and fungal growth.

Attempts to detect inducible carbohydrate-degrading enzymes from C. albicans were performed using carbohydrate-restricted media. Although alpha-amylase (polysaccharide hydrolase) was not induced from media containing starch or dextrin, alpha-glucosidase (disaccharide hydrolase) was induced from maltose-containing medium. alpha-Glucosidase activity was detected from the intact cells in suspension and from the supernatant of both spheroplasted and mechanically broken cells, but not from the media. Enzyme activity in intact cells was pH independent and increased during logarithmic growth phase and in media containing lower concentrations of maltose. The addition of 5 mM 1-deoxynojirimycin, a competitive inhibitor of alpha-glucosidase, suppressed fungal growth by more than 50%. These results suggest that alpha-glucosidase activity is crucial for fungal growth in medium containing maltose as a sole carbon source.

Candida albicans↗

An investigation of the possible influence of neutral alpha-glucosidases on the clinical heterogeneity of glycogenosis type II.

The lysosomal storage disorder glycogenosis type II, caused by a deficiency of lysosomal alpha-glucosidase, is very heterogeneous in its clinical presentation. It has been suggested that this heterogeneity may be due to differential expression of neutral alpha-glucosidases. We have therefore analysed the activity of the major neutral alpha-glucosidases in cultured fibroblasts or muscle cells from 26 patients with glycogenosis type II. The results indicate that there is no correlation between the expression of neutral alpha-glucosidase isoenzymes and the clinical phenotype of this disease.

Cells, Cultured↗

Molecular cloning of a gene encoding acid alpha-glucosidase from Tetrahymena pyriformis.

Lysosomal acid alpha-glucosidase is essential for the degradation of glycogen to glucose in lysosomes. The ciliated protozoan Tetrahymena pyriformis secretes acid alpha-glucosidase into its culture medium. We have earlier reported the purification and characterization of acid alpha-glucosidase from T. pyriformis. The exact molecular mechanism of secretion of this enzyme has not yet been clarified. In the present study we have isolated a full length cDNA clone encoding acid alpha-glucosidase from T. pyriformis. The isolated clone (3019 bp) contained an open reading frame encoding 923 amino acids, and has an estimated molecular mass of 104 kDa. Northern blot analysis revealed that the isolated cDNA hybridized to a 2.8-kb mRNA transcript. N-terminal amino acids after the first methionine fulfilled the requirement of a signal peptide. The deduced amino acid sequence contains the amino acid sequences determined of several peptides derived from the purified enzyme, and was found to have 34% identity and 45% similarity with that of human lysosomal enzyme, with 75% identity in the 16 amino acids at the proposed active site.

Amino Acid Sequence↗

Induction and preliminary characterization of intracellular beta-glucosidases from a cellulolytic Streptomyces strain.

The cellulolytic actinomycete Streptomyces sp. QM-B814 possess an intracellular beta-glucosidase system which is induced by cellobiose and carboxymethylcellulose. Maximal beta-glucosidase activity was attained 8-10 h after inducer addition to exponential phase growing cultures. The induction is depressed in the presence of glucose. The system is composed of two electrophoretically different beta-glucosidases forms showing relative molecular masses of about 60 and 35 kDa, and pI values in the range 4.2-4.5. Both beta-glucosidases are synthesized de novo. The enzymes share substrate preference and are both inhibited by delta-gluconolactone and p-chloromercuribenzoate. The induction pattern and glucose inhibition are similar for both enzymes.

Cellobiose↗

Regulation of beta-glucosidase biosynthesis in Aspergillus nidulans.

beta-Glucosidase in Aspergillus nidulans was found to be both intracellular and extracellular. The intracellular beta-glucosidase was synthesized after the exhaustion of carbon source in the medium. The extracellular enzyme appeared with autolysis of the mycelium. Biosynthesis of beta-glucosidase was not induced by various carbohydrates but repressed to varying extents in the presence of glucose, glycerol, and 2-deoxyglucose. This repression was not relieved by addition of cAMP. The repression was relieved much more by mutations in the creA gene than by one in the creC gene. Thus, beta-glucosidase synthesis in A. nidulans is subject to carbon catabolite repression.

Aspergillus nidulans↗

Overproduction of beta-glucosidase in active form by an Escherichia coli system coexpressing the chaperonin GroEL/ES.

beta-Glucosidase from Cellvibrio gilvus was successfully overproduced in soluble form in Escherichia coli, with the coexpression of GroEL/ES. Without the GroEL/ES protein, the beta-glucosidase overexpressed in E. coli constituted a huge amount (80%) of the total cellular protein, but was localized in the insoluble fraction, and little activity was detected in the soluble fraction. Coexpression of the E. coli GroEL/ES had a drastic impact on the proper folding of the beta-glucosidase; 20% of the overexpressed enzyme was recovered in the soluble fraction in active form. In addition, the synergistic effect of GroEL/ES and the low induction temperature led to 70% solubilization of the total expressed target protein and more than a 20-fold increase in activity. Similar effects of GroEL/ES were also observed on the overexpressed beta-glucosidase from Agrobacterium tumefaciens.

Chaperonin 60↗

Cloning of a beta-glucosidase gene from Ruminococcus albus and its expression in Escherichia coli.

A HindIII fragment of R. albus DNA encoding beta-glucosidase was cloned into E. coli. The DNA sequence (3158 bp) was determined, and the longest potential encoding sequence consisted of 2,841 bp (947 amino acids with the calculated molecular weight of 104,276. The deduced NH2-terminal amino acid sequence from the first (methionine) to the twentieth (glycine) was identical to that of the purified enzyme, suggesting that the gene for beta-glucosidase does not encode a signal peptide. The enzyme purified from the culture supernatant of the transformant had a molecular weight of 120,000 and its maximum activity was revealed at pH 6.5 and 30 degrees C. Reducing reagents activated the enzyme, whereas the sulfhydryl group-blocking reagents and reaction products (glucose) inhibited the activity. Hydrolyzates of celloorigomers contained glucose as a major product, indicating that the enzyme acts as beta-glucosidase. The enzyme from the transformant revealed similar properties to that from R. albus, and both enzyme proteins were immunologically the same to each other, indicating that the cloned gene encodes beta-glucosidase from R. albus.

Amino Acid Sequence↗

Beta-glucosidase of Trichoderma: its biosynthesis and role in saccharification of cellulose.

The extracellular beta-glucosidase of Trichoderma viride generally is present in low levels when the organism is cultured on cellulose because it is inactivated under the acid conditions which develop in the medium while the other enzymes of the cellulase complex are more stable. With the appropriate pH control, inactivation of beta-glucosidase is prevented and the activity of this enzyme increases during growth. In the saccharification of crystalline cellulose, or of cellulose at low concentrations, much of the glucose produced is the result of the cleavage of cellobiose by beta-glucosidase. However when high concentrations (10%) of pretreated cellulose are saccharified, significant quantities of glucose are produced by action of enzymes other than beta-glucosidase.

Cell-Free System↗

Purification, characterization, gene cloning, and sequencing of a new beta-glucosidase from Bacillus circulans subsp. alkalophilus.

An intracellular beta-glucosidase was purified from cell extracts of Bacillus circulans subsp. alkalophilus by NAD affinity and high-performance anion-exchange chromatographies. The enzyme was active against a wide range of aryl-beta-glucosides and beta-linked disaccharides. The structural gene for beta-glucosidase was cloned in Escherichia coli. The beta-glucosidase gene consisted of an open reading frame of 1,350 bp encoding a protein of 450 amino acids with a calculated M(r) of 51,303. The enzyme exhibited from 45 to 66% identity with five bacterial beta-glucosidases.

Amino Acid Sequence↗

Genes encoding two different beta-glucosidases of Thermoanaerobacter brockii are clustered in a common operon.

A 5.9-kb fragment of chromosomal DNA coding for beta-glucosidase activity of the thermophilic anaerobe Thermoanaerobacter brockii was sequenced. Two genes, cglT and xglS, encoding a cellodextrin-cleaving beta-glucosidase and a xylodextrin-degrading xylo-beta-glucosidase, respectively, were located directly adjacent to each other. The 5' region contained two additional genes, cglF and cglG, whose products exhibited similarity to integral membrane proteins of metabolite transport systems. The two beta-glucosidases, CglT and XglS, with deduced molecular masses of 52 and 81 kDa, belong to different families of glycosyl hydrolases. Both enzymes were overexpressed in Escherichia coli and could be detected after protein gel electrophoresis and activity staining. The enzyme CglT was purified by fast protein liquid chromatography and identified by N-terminal sequencing. The enzyme was thermostable at 60 degrees C for at least 24 h, and the temperature optimum was 75 degrees C. The ki for glucose inhibition was calculated to 200 mM. The enzyme released glucose from the nonreducing end of beta-1,4-cello oligomers as well as from various disaccharides. CglT was active on glucosides, galactosides and on fucosides, while XglS cleaved beta-glucosides and beta-xylosides as well. The cglT gene was also expressed in Bacillus subtilis, and the enzyme was mainly intracellular during exponential growth but was efficiently released into the supernatant after cultures entered the stationary phase.

Amino Acid Sequence↗