Hyper production of β-glucosidase by an Aspergillus sp.

Summary An Aspergillus sp. was isolated which secreted high levels of -glucosidase in growth medium. The maximum activity(10 IU/ml of -glucosidase and 22.6 IU/ml of cellobiase) was obtained in cellulose medium supplemented with wheat bran. The pH and temperature optima for this enzyme were 4.5 and 65°C respectively.NCL Communication No. 3616     

The effect of pH on the production of cellulases in Aspergillus terreus

Summary The optimal pH for the production of extracellular cellulolytic enzymes in the wild strain of Aspergillus terreus was shown to be pH 5.0. After 160 h of cultivation, carboxymethyl cellulase reached 9.0 IU/ml, filterpaper, cellulase 0.5 IU/ml and -glucosidase 0.9 IU/ml. The rate of synthesis of CM- and FP-cellulases decreased after 90 h of cultivation but -glucosidase was produced linearly for 160 h. Some of the enzymes produced were released into the medium during the fungal growth while others remained bound. The binding of enzymes to cells and residual crystalline cellulose was strongly affected by the pH of the medium. FP-cellulase and particularly -glucosidase were bound more effectively, at lower pHs. Cold shock treatment of the cell suspension increased the activities of FP- and CM-cellulases but -glucosidase activity was not affected.     

The effect of β-glucosidase on some assays for cellulolytic enzymes

The effect of -glucosidase on three assays for cellulolytic enzymes, i. e. the activities against dyed Avicel, hydroxyethylcellulose (HEC) and filter paper (FPU), was studied using cellulase enzyme derived from Trichoderma reesei VTT-D-80133. The dyed Avicel and HEC assays were only slightly affected by -glucosidase, whereas the FPU assay was linearly dependent on the level of -glucosidase over a wide range of activity of this enzyme.     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

Production of β-glucosidase withAspergillus phoenicis QM329 in aqueous two-phase systems

Summary The production of -glucosidase withAspergillus phoenicis QM 329 was studied in two different aqueous two-phase systems: polyethylene glycol (PEG) 1550 7.5%/Dextran T2000 9.5% and PEG 8000 4%/polyvinyl alcohol (PVA) 14000 8%. The enzyme concentrations in the top phase of the phase systems were 3.4 IU/ml and 3.2 IU/ml, respectively, compared with 2.0 IU/ml obtained in a regular medium. The total amount of -glucosidase obtained in the phase systems was 265 IU and 176 IU, respectively, compared with 200 IU in a regular medium.     

Over-production of β-glucosidase by Aspergillus niger mutant from lignocellulosic biomass

The maximum yield of -glucosidase by A. niger KK2 mutant, grown on the basal medium for 7 days, was 514 I U g^–1 ground rice straw, and was about twice those obtained from wheat straw or bran by previous researchers. Optimal activity of -glucosidase was at 60–70 °C and pH 4.8.     

Solid state fermentation of straw withNeurospora crassa for CMCase and β-glucosidase production

Summary CMCase and -glucosidase were produced by the mutantNeurospora crassa 40b cultivated on untreated wheat straw in a solid state fermentation. Best enzyme activities were observed when the growth medium was composed of wheat straw mixed with certain mineral solutions at a ratio 1:2 (w/v). A partially purified enzyme preparation showed optimum enzyme activities of CMCase and -glucosidase at pH 4.0 and 5.0 and temperature 50 and 60°C respectively. The apparent Km values for the same enzymes were 16.8 g/l and 1.03x10^–4 M respectively. At optimum growth and enzyme assay conditions yields as high as 586.2 U CMCase and 58.4 U -glucosidase per gram of straw were obtained.     

Simultaneous overproduction of extracellular endoglucanase and intracellular β-glucosidase by genetically engineered Bacillus subtilis

Summary Simultaneous overproduction of intracellular -glucosidase and extracellular endoglucanase was attempted by constructing two artificial operon systems comprising the -glucosidase-endoglucanase gene(E) or the endoglucanase--glucosidase gene(E) under the control of a strong engineered promoter, BJ27U88 and expressing them in Bacillus subtilis DB104. Two artificial operon systems contained 30 bp or 5 bp gap between the termination codon of the upstream gene and the SD sequence of the downstream gene, respectively. These operon systems were expressed well in B. subtilis and overproduced the -glucosidase cell extract as well as the endoglucanase supernatant. The level of expression in the operon system was almost the same as that in a single expression system.     

Über den histochemischen Nachweis der β-Glueosidase mit 1-Naphthyl-β-glucopyranosid

Zusammenfassung Es wird eine einfache simultane Azokupplungsmethode zur Darstellung der -Glucosidase im Dünndarm verschiedener Säuger beschrieben und mit anderen histochemischen Verfahren zum Nachweis dieses Enzyms verglichen. Eine intrazelluläre Lokalisation der -Glucosidase ermöglichen nur die Indigogen-und die hier angegebene Technik, nicht dagegen die bisherigen Azofarbstoffmethoden mit 6-Br-2-Naphthyl--glucopyranosid als Substrat und p-Rosanilin zur Simultanoder Fast Blue B zur Postkupplung.Das Inkubationsmedium des neuen Verfahrens enthält 4,5–9,0 mg 1-Naphthyl--glucopyranosid (gelöst in 0,4 ml Dimethylformamid) und 0,4–0.8 ml 2% hexazotiertes p-Rosanilin in 9,0 ml 0,1 M Citronensäure-Phosphat-Puffer, pH 5,5. —Mikrochemische Messungen mit dem gleichen Substrat zeigen, daß die -Glucosidase durch p-Rosanilin in ähnlichem Ausmaß wie durch Ferricyanid im Indigogen-Medium gehemmt wird.Wegen der fraglichen Verwandtschaft von -Glucosidase und neutraler -Galactosidase wurde dieses Enzym mit obigem Ansatz und 1-Naphthyl--galactopyranosid als Substrat untersucht.

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On the histochemical demonstration of -glucosidase with 1-naphthyl--glucopyranoside

Summary A simple simultaneous azo coupling method for the demonstration of -glucosidase in the small intestine of various mammals is described and compared with other histochemical techniques for this enzyme. Strong evidence occurs that a correct intracellular localization of -glucosidase can only be obtained by means of the indigogenic and the assay presented here: the azo dye methods published so far with 6-Br-2-naphthyl--glucopyranoside as substrate and p-rosaniline for simultaneous or Fast Blue B for postcoupling are not able to reflect the true binding sites of intestinal--glucosidase.The recommended incubation medium consists of 4.5–9.0 mg 1-naphthyl--glucopyranoside (dissolved in 0.4 ml NN-dimethyl formamide) and 0.6–0.8 ml 2% hexazonium-p-rosaniline in 9.0 ml 0.1 M citric acid-phosphate buffer, pH 5.5.— Microchemical measurements using the same substrate show that p-rosaniline inhibits -glucosidase to a similar extent as ferricyanide in the indigogenic medium.Because of the presumed relationship between -glucosidase and neutral -galactosidase the latter enzyme has been demonstrated with the above mentioned assay replacing the 1-naphthyl--glucoside by the corresponding -galactopyranoside.The strongest -glucosidase and -galactosidase activity can regularly be observed in the jejunum of rats, mice and guinea-pigs where both enzymes are localized in the brush border region of the enterocytes. In comparison with -galactosidase the -glucosidase reaction is always more intensive and the azo dye production in the microvillous zone of suckling rats and guinea-pigs is far higher than in the intestine of adult animals. Furthermore both enzymes react in a similar way to inhibitors, experiments (thirst, hunger) and pregnancy and do not split naphthol AS BI -glucopyranoside respectively -galactopyranoside.Bloc fixation in formol-calcium and especially in glutaraldehyde improves the localization of the azo dye considerably; but microchemistry reveals that aldehyde fixation supresses the -glucosidase to ca. 50%. The basis activity of the enzyme following pretreatment with formol is reached within the first minute of fixation.

     

Hydrolytic properties of extracellular cellulases fromPleurotus ostreatus

Pleurotus ostreatus mycelium produced extracellular cellulases when grown on ground wheat straw. No cellulase activity was detectable when glucose or aqueous extracts of vegetable material were used as substitutes for straw in culture media. The specific activity of excreted cellulases did not vary significantly by increasing the straw concentration from 1 to 6%. Chromatographic fractionation of extracellular proteins gave rise to five fractions with cellulolytic activity. The hydrolytic properties of these partially purified fractions were analysed by using several substrates (carboxymethylcellulose, cellobiose,p-nitrophenyl--d-cellobioside,p-nitrophenyl--d-lactoside). The results indicate that the cellulase system ofP. ostreatus includes at least a -glucosidase, two endocellulases, an exoglucohydrolase and an exocellobiohydrolase.     

A simple and inexpensive method for cellulase and β-glucosidase production by Neurospora crassa

Summary Elevated levels of cellobiohydrolase, carboxymethylcellulase (CMCase) and -glucosidase were produced by strain STA of Neurospora crassa, grown in solid-state fermentation on untreated wheat straw supplemented with simple mineral salts. Yields as high as 6.1 units of cellobiohydrolase, 969.2 units of CMCase and 169.4 units of -glucosidase per gram of straw were obtained at optimum growth and enzyme assay conditions.     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

Production of cellulases and hemicellulases by an anaerobic mixed culture from lignocellulosic biomass

A comparison of different habitats, biogas plant, rumen fluid and sewage sludge, for cellulolytic organisms indicated sewage studge was the best source. Enrichment cultura gave a mixed culture which exhibited CMCase activity as well as extracellular Avicelase, xylanase, -glucosidase, -xylosidase activities and cell-bound -glucosidase, and -xylosidase production in a synthetic medium with eleven different cellulosic and lignocellulosic substrates. The activity of extracellular -glucosidase and -xylosidase production was significantly higher than endogenous activities. Hemicellulases were induced better than cellulases. The anzyme system was stable under aerobic conditions. Of the different lignocellulosic substrates, kallar grass was the best inducer of extracellular enzymes.

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Résumé La comparaison de différents habitats: digesteur méthanique, fluide du rumen ou boue de station d'épuration, pour leur contenu en organismes cellulolytiques, indiquent que la boue de station d'épuration est la meilleure source. Une culture par enrichissement a produit une culture mixte qui a exhibé aussi bien une activité CMCase que des activitiés extracellulaires avicelasique, xylanasique, -glucosidasique et -xylosidasique et qu'une production de -glucosidase et de -xylosidase liées à la cellule, dans un milieu synthétique et pour onze substrats cellulosiques et lignocellulosiques différents. L'activité de la -glucosidase extracellulaire et la production de -xylosidase sont significativement plus élevées que les activitiés endogènes. Les hemicellulases sont mieux induites que les cellulases. Le système enzymatique est stable dans des conditions aérobies. Parmi les divers substrats lignocellulosiques, l'herbe Kallar est le meilleur inducteur d'enzymes extracellufaires.

     

Cloning of Candida pelliculosa beta-glucosidase gene and its expression in Saccharomyces cerevisiae

Summary Candida pelliculosa var. acetaetherius is a strain of yeast which can utilize cellobiose as the carbon source. From a gene library prepared from this yeast, the -glucosidase gene has been cloned in a S. cerevisiae host using a chromogenic substrate, 5-bromo-4-chloro-3-indolyl--glucoside as an indicator. It was proved by Southern analysis that the DNA fragment carrying the -glucosidase gene originated from C. pelliculosa. -Glucosidase produced by S. cerevisiae transformants was secreted into the periplasmic space. In Candida, -glucosidase was not induced by cellobiose but was derepressed by lowering the concentration of glucose. The regulation of -glucosidase synthesis in S. cerevisiae carrying the cloned -glucosidase was not clear compared with that in Candida, however, the enzyme activity in low glucose medium (0.05%) was reproducibly higher than in high glucose medium (2%). We have found the sequence that controls the expression of the -glucosidase gene negatively in S. cerevisiae.     

Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production

To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed -glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of -agglutinin was used as surface-display motif for the expression of -glucosidase in the cell wall. The surface-displayed -glucosidase had a half-life time (t _1/2) of 100 h in acidic culture broth conditions, while secreted -glucosidase had a t _1/2 of 60 h. With such stabilization of -glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l^–1 over 60 h, while S. cerevisiae secreting -glucosidase into culture broth used 5.8 g cellobiose l^–1 over the same period.     

Synthesis of alkyl β-glucosides from cellobiose with Aspergillus niger β-glucosidase II

An extracellular -glucosidase II of Aspergillus niger catalyzed the synthesis of methyl -glucoside and ethyl -glucoside with 5.0% (v/v) cellobiose as glucosyl donor in a biphasic media containing 20% (v/v) methanol and 30% (v/v) ethanol, respectively. The maximum yield of methyl -glucoside and ethyl -glucoside was 83% (mol/mol; 12 mg/ ml) and 53% (mol/mol; 5.5 mg/ml), based on cellobiose consumed. © Rapid Science Ltd. 1998     

β-Glucosidase excretion by Trichoderma pseudokoningii: Correlation with cell wall bound β-1.3-glucanase activities

The formation and excretion of -glucosidase from Trichoderma pseudokoningii was studied during growth on different carbon sources. The enzyme was present under all conditions examined, but increased activity was found during growth on carbon sources favouring slow growth.Two different patterns of -glucosidase excretion were observed: on carbon sources allowing fast growth a relatively high percentage of total activity was found in the culture fluid, which decreases as the culture grows older, but which increases again during the phase of cell lysis; on carbon sources favouring slow growth, excretion is initially low, but commences at later culture stages.Changes in cell wall composition and cell wall lytic enzyme activities associated with the cell walls were examined during phases of high and low ratios of extracellular to cell-wall bound -glucosidase activities. With no component of the cell wall (chitin, -glucan, -glucan, galactosamine) could correlation with -glucosidase excretion be identified. Among a number of cell-wall lytic, cell-wall associated enzymes (-glucanases, -glucanases, glucosaminidase, galactosaminidase), -1.3-glucanase activity correlated well with the excretion of -glucosidase.The results suggest a possible role of -1.3-glucanases in the mechanism of release of -glucosidase from cell walls of T. pseudokoningii; this is discussed.     

Nucleotide and derived amino acid sequence of the cyanogenic β-glucosidase (linamarase) from white clover (Trifolium repens L.)

The nucleotide sequence and derived amino acid sequence of two different -glucosidase cDNA clones were determined. One clone (TRE104) was identified as the cyanogenic -glucosidase by homology with the N-terminal and internal peptide amino acid sequence of the purified enzyme. The biological function of the other -glycosidase (TRE361) is not known. Co-segregation of genomic restriction fragments uniquely identified by each cDNA clone shows that these two genes are linked in the white clover genome. Both TRE104 and TRE361 fragments co-segregate with cyanogenic -glucosidase activity. Extensive homology was found between the white clover -glucosidase sequences and a group of prokaryote and mammalian -glycosidases. This group of sequences has no homology with a separate set of -glucosidase genes isolated from fungi and the thermophilic bacterium Clostridium thermocellum.     

Cell wall localization of dihydroflavonol-glucoside β-glucosidase in flowers of Petunia hybrida

Limbs of flower buds from Petunia hybrida were investigated for -glucosidase activity with dihydroflavonol-glucosides and 4-methyl-umbelliferyl--D-glucoside as substrates. Dihydroflavonol-glucoside -glucosidase is localized in the cell wall. This activity has an acid pH optimum and is also active toward 4-methyl-umbelliferyl--glucoside. Besides this activity a neutral -glucosidase is present. This activity is soluble and is not active toward dihydroflavonol-glucosides. Using starch gel electrophoresis it was shown that no difference in -glucosidase activity is present between mutants able to convert dihydroflavonols into anthocyanins and mutants accumulating dihydroflavonol-glucosides. It is concluded that -glucosidase activity is not involved in anthocyanin synthesis.Abbreviations 4MU--glc 4-methylumbelliferyl--D-glucopyranoside - dHQ-7-g dihydroquercetin-7-glucoside - dHQ-4-g dihydroquercetin-4-glucoside - dHM-4-g dihydromyricetin-4-glucoside Deceased     

Kinetic properties of β-glucosidase from Aspergillus ornatus

Summary Kinetic properties of extracellular -glucosidase from Aspergillus ornatus were determined. The pH and temperature optima for the enzyme were found to be 4.6 and 60°C, respectively. Under these conditions, the enzyme exhibited a K _m (p-nitrophenyl--glucoside) value of 0.76±0.11 mM. The activation energy for the enzyme was 11.8 kcal/mol. Several divalent metal ions inhibited -glucosidase activity, some of which showed inhibition of enzyme activity only at higher concentrations. Ag²⁺ was the most potent inhibitor. A metal chelating agent, EDTA, also inhibited -glucosidase activity. Except for trehalose, glucose, glucono--lactone, cellobiose, gentiobiose, laminaribiose, maltose and isomaltose inhibited -glucosidase activity. Glucose was found to be a competitive inhibitor, whereas glucono--lactone and other -linked disaccharides were noncompetitive (mixed) inhibitors of the enzyme.