Production, Characterization, and Properties of β-Glucosidase and β-Xylosidase from a Strain of Aureobasidium sp.

-Glucosidase and -xylosidase production by a yeastlike Aureobasidium sp. was carried out during solid-state and submerged fermentation using different carbon sources and crude enzymes were characterized. -Glucosidase and -xylosidase exhibited optimum activities at pH 2.0–2.5 and 3.0, respectively. These enzymes had the maximum activities at 65°C and were stable in a wide pH range and at high temperatures.     

Characterization of a Novel β-Glucosidase from a Compost Microbial Metagenome with Strong Transglycosylation Activity

The β-glucosidase encoded by the td2f2 gene was isolated from a compost microbial metagenomic library by functional screening. The protein was identified to be a member of the glycoside hydrolase family 1 and was overexpressed in Escherichia coli, purified, and biochemically characterized. The recombinant β-glucosidase, Td2F2, exhibited enzymatic activity with β-glycosidic substrates, with preferences for glucose, fucose, and galactose. Hydrolysis occurred at the nonreducing end and in an exo manner. The order of catalytic efficiency for glucodisaccharides and cellooligosaccharides was sophorose > cellotetraose > cellotriose > laminaribiose > cellobiose > cellopentaose > gentiobiose, respectively. Intriguingly, the p-nitrophenyl-β-d-glucopyranoside hydrolysis activity of Td2F2 was activated by various monosaccharides and sugar alcohols. At a d-glucose concentration of 1000 mm, enzyme activity was 6.7-fold higher than that observed in the absence of d-glucose. With 31.3 mm d-glucose, Td2F2 catalyzed transglycosylation to generate sophorose, laminaribiose, cellobiose, and gentiobiose. Transglycosylation products were detected under all activated conditions, suggesting that the activity enhancement induced by monosaccharides and sugar alcohols may be due to the transglycosylation activity of the enzyme. These results show that Td2F2 obtained from a compost microbial metagenome may be a potent candidate for industrial applications.     

Purification and Characterization of Extracellular β-Xylosidase and β-Glucosidase from Aspergillus fumigatus

A β-xylosidase (β-d-xyloside xylohydrolase, EC 3.2.1.37) and β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) extracted from a wheat bran culture of Aspergillus fumigatus were purified up to 90-fold and 131-fold, respectively, by ammonium sulfate precipitation, gel filtration, ion exchange chromatography, and hydroxylapatite chromatography. Molecular weights of the β-xylosidase and β-glucosidase were 360,000 and 380,000, respectively, each consisting of four identical subunits. The isoelectric points of β-xylosidase and β-glucosidase were at pH 5.4 and 4.5, respectively. The optimum temperature for the β-xylosidase was 75°C, being stable up to 65°C for 20 min and for the β-glucosidase was 65°C, being stable up to 60°C for 20 min. The optimum pH for both enzymes was about 4.5, being stable between 2 and 8 at 50°C for 20 min. Both enzymes were inhibited by Fe³⁺, Cu²⁺, Hg²⁺, SDS, and p-chloromercuribenzoate. The apparent Michaelis constants of the β-xylosidase were 2.0 and 23.8 mM for p-nitrophenyl-β-xyloside and xylobiose, respectively, and those of the β-glucosidase were 1.4, 11.4, and 24.8 mM for p-nitrophenyl-β-glucoside, gentiobiose, and cellobiose, respectively. To produce xylose from crude xylooligosac-charides prepared by steam-explosion of cotton seed waste (DP ≤10, 53%, total sugars = 150 g/ liter), the crude enzyme from A. fumigatus (β-xylosidase activity = 14.7 units/ml, xylanase activity = 20 units/ml) could hydrolyze the substrate at 55°C and pH 4.5 resulting in almost complete conversion to xylose (160 g/liter).     

Biochemical and Proteomic Characterization of a Novel Extracellular β-Glucosidase from Trichoderma citrinoviride

β-Glucosidases are of pivotal importance in bioconversion of carbonic biomass into fermentable and other useful metabolites, food industry, biotransformation, glyco-trimming of metabolome, etc. Trichoderma citrinoviride when grown on delignified Lantana camara produced a β-glucosidase and secreted it out in the medium. The extracellularly secreted β-glucosidase of T. citrinoviride was homogeneity purified and then characterized for its kinetic properties and proteomic characteristics. The 90 kDa enzyme was monomeric in nature, optimally active at pH 5.5 and the catalytic reaction rate was highest at 55°C. Uniquely, the enzyme was insensitive to inhibition by glucose (up to 5 mM). It also possessed catalytic ability of transglycosylation, as it could catalyze conversion of geraniol into its glucoside. MALDI-TOF assisted proteomic analysis revealed its high degree of sequence similarity with family 3 glycoside hydrolases.     

Expression, Purification and Characterization of a Recombinant β-Glucosidase from Volvariella Volvacea

For the first time, a β-glucosidase gene from the edible straw mushroom, Volvariella volvacea V1-1, has been over-expressed in E. coli. The gene product was purified by chromatography showing a single band on SDS-PAGE. The recombinant enzyme had a molecular mass of 380 kDa with subunits of 97 kDa. The maximum activity was at pH 6.4 and 50 °C over a 5 min assay. The purified enzyme was stable from pH 5.6–8.0, had a half life of 1 h at 45 °C. The β-glucosidase had a K_m of 0.2 mM for p-nitrophenyl-β-D-glucopyranoside.     

Isolation and Characterization of Two β-d-Glucosidases from Bifidobacterium breve 203

Two β-d-glucosidases were purified to homogeneity from Bifidobacterium breve 203: one ( β-d-glucosidase I; molecular weight, 96,000) showed reactivity toward p-nitrophenyl (p-NP) β-d-fucoside, 74% of that to p-NP β-d-glucoside, and the other ( β-dglucosidase II; molecular weight, 450,000) did not. They also differed in their thermal and pH stabilities. Laminaribiose, cellobiose and gentiobiose were hydrolyzed by β-d-glucosidase I, with 53%, 34% and 3% of the reactivity in the case of p-NP β-d-glucoside, and by β-dglucosidase II, with 53%, 6% and 107% of the reactivity. The reaction of β-dglucosidase I with p-NP β-dfucoside was enhanced by the addition of glucose and other monosaccharides to the reaction mixture, whereas that with p-NP β-dglucoside was not affected. The activity of β-dglucosidase II with p-NP β-dglucoside was inhibited by glucose.     

Production and Purification of Thermostable β-Glucosidase from Mucor miehei YH-10

A thermophilic fungus, Mucor miehei YH-10, isolated from manure was selected to produce thermostable β-glucosidase among 207 isolates. When Mucor miehei YH-10 was grown on wheat bran medium, the maximal accumulation of thermostable β-glucosidase was obtained after 4 days at 50°C, The β-glucosidase had an optimal temperature of 60°C and retained 73% of original activity after heating at 95°C for 5 min. The β-glucosidase was fractionated by Sephadex G-100 chromatography into two components during the purification steps. These components were further purified by consecutive column chromatographies until they were homogeneous on disc electrophoresis. One retained 56% of original activity after heating at 95°C for 5 min, whereas the other was completely inactivated after heating at 80°C for 5 min.     

Cloning and Biochemical Characterization of BglC, a β-Glucosidase from the Cellulolytic Actinomycete Thermobifida fusca

An operon, bglABC, that encodes two sugar permeases and a β-glucosidase was cloned from a cellulolytic actinomycete, Thermobifida fusca, into Escherichia coli and sequenced. The bglC gene encoding an intracellular β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) belonging to glycosyl hydrolase family 1 was subcloned and expressed in E. coli. The purified enzyme (MW 53,407 Da; pI 4.69) hydrolyzed substrates containing both β 1 → 4 and β 1 → 2 glycosidic bonds, and was most active against cellobiose (V_max= 29, K _m = 0.34 mm), cellotriose, cellotetraose, and sophorose. The enzyme also showed aryl-β-glucosidase activity on p-nitrophenyl-β-d-glucopyranoside and p-nitrophenyl-β-d-cellobioside. BglC had a pH optimum of 7.0 and a temperature optimum of 50°C. The enzyme was stable at 60°C, but was rapidly inactivated at 65°C. BglC was inhibited by low concentrations of gluconolactone, but was insensitive to end-product inhibition by glucose and was not affected by Ca or Mg ions or EDTA. Its properties are well suited for use in a process to hydrolyze biomass cellulose to glucose. Received: 21 August 2000 / Accepted: 4 October 2000     

Isolation and Characterization of β-1,4- Endoxylanase from Trichoderma pseudokonigi

13-1,4-endoxylanase from Triehoderma pseudokonigi Rifai has been purified by anion-exchange chromatography on DEAE-Sephadex A50, DEAE-Sepharose CL-6B and mono Q. The endoxylanase was shown to be homogeneous by Native-PAGE and SDS-PAGE. This endoxylanase is a single-peptide chain protein with a molecular weight estimated as 66 kD. The endoxylanase was purified by 10-fold with a specific activity of 15.87 U·mg－1 Optimum endoxylanase activity was obtained when the enzyme was incubated at pH 4.5, 55 ℃ with a Km of 20 mg/mL and Vmax of 3.3 μmol·min－1·mg－1. Hg2 + and Cu2 + have a strong inhibition while Fe2 + and Mn2 + have a increasing effect on the enzymatic reaction rate.     

Characterization of a Cold-Active β-Glucosidase from Paenibacillus xylanilyticus KJ-03 Capable of Hydrolyzing Isoflavones Daidzin and Genistin

Paenibacillus xylanilyticus KJ-03 isolated from konjac field, showed β-glucosidase activity on tryptic soy agar plate supplemented with 0.1 % esculin and 0.25 % ferric ammonium citrate. A genome library was constructed to obtain the β-glucosidase gene and a recombinant clone, pGlc2-3 was selected. The 2,247 bp gene encoding KJ-03 β-glucosidase consisted of 749 amino acids. The deduced amino acids of BglA were 61 % homologous with that of the β-glucosidase from Bacillus cereus AH1272, which belongs to the glycoside hydrolase family 3. His-tagged β-glucosidase was purified by using His-Trap column and characterized. KJ-03 β-glucosidase was showed as a single band with about 82 kDa on SDS-PAGE. The purified enzyme has optimal activity at 20 °C and pH 7.0 using p-NPβG and 72 % of the maximal activity was still remaining at 10 °C. The β-glucosidase has optimal activity at low temperatures indicating that it is a cold-active enzyme. The substrate specificity showed that the purified enzyme hydrolyzed aryl β-glucoside substrates and isoflavones such as daidzin and genistin.     

Isolation and Characterization of Novel β-Cyanoalanine Synthase and Cysteine Synthase Genes from Cassava

Cassava is an important staple food crop, feeding 600 million people worldwide, which produce cyanogenic glycosides. Cyanogenic glycosides in cassava are known to act as a deterrent for herbivores as well as serve as a mobile source of reduced nitrogen. Cassava is also equipped with a cyanide detoxification pathway, mediated by β-cyanoalanine synthase (β-CAS) which converts cyanide into asparagine. β-CAS, belonging to the Bsas family of enzymes, is multi functional and shares sequence homology with cysteine synthase (CS). Using rapid amplification of cDNA end-polymerase chain reaction (RACE-PCR), two cDNA sequences were isolated from cassava. The two clones named MANes;BsasA (accession no. EU350583) and MANes;BsasB (accession no. HQ257219), showed high homology to known β-CAS enzymes (80% and 75% amino acid similarity to Arabidopsis and 76% and 82% similarity to spinach, respectively). The kinetic properties of the two clones were characterized in a Escherichia coli NK3 mutant strain which lacks activity for any of the Bsas proteins. Kinetic studies showed that MANes;BsasB is a β-CAS with a CAS/CS activity ratio of 72 while MANes;BsasA is a CS showing bifunctional capabilities and with a CAS/CS activity ratio of 11. The isolation of cassava β-CAS and CS genes reported here paves the way for their utilization in genetically enhancing the cyanide detoxification potential of cassava and/or increase of the essential amino acid cysteine, which has been found to be low in nutritionally compromised individuals.     

Purification and Characterization of a Novel Fungal α-Glucosidase from Mortierella alliacea with High Starch-hydrolytic Activity

The fungal strain Mortierella alliacea YN-15 is an arachidonic acid producer that assimilates soluble starch despite having undetectable α-amylase activity. Here, a α-glucosidase responsible for the starch hydrolysis was purified from the culture broth through four-step column chromatography. Maltose and other oligosaccharides were less preferentially hydrolyzed and were used as a glucosyl donor for transglucosylation by the enzyme, demonstrating distinct substrate specificity as a fungal α-glucosidase. The purified enzyme consisted of two heterosubunits of 61 and 31 kDa that were not linked by a covalent bond but stably aggregated to each other even at a high salt concentration (0.5 M), and behaved like a single 92-kDa component in gel-filtration chromatography. The hydrolytic activity on maltose reached a maximum at 55°C and in a pH range of 5.0-6.0, and in the presence of ethanol, the transglucosylation reaction to form ethyl-α-D-glucoside was optimal at pH 5.0 and a temperature range of 45-50°C.     

Purification,Characterization, and Substrate Specificity of a Novel Highly Glucose-Tolerant β-Glucosidase from Aspergillus oryzae

Aspergillus oryzae was found to secrete two distinct β-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 β-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant β-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-β-d-glucoside as the substrate, we found that the enzyme was optimally active at 50°C and pH 5.0 and had a specific activity of 1,066 μmol min⁻¹ mg of protein⁻¹ and a K_m of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (K_i, 1.36 M) or glucono-δ-lactone (K_i, 12.5 mM), another powerful β-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 β-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1→3)- and (1→6)-β-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-β-d-glucosides in a grape must (pH 2.9, 90 g of glucose liter⁻¹). Other flavor precursors (benzyl- and 2-phenylethyl-β-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 β-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.β-Glucoside glucohydrolases, commonly called β-glucosidases, catalyze the hydrolysis of alkyl- and aryl-β-glucosides, as well as diglucosides and oligosaccharides. These enzymes are widely used in various biotechnological processes, including the production of fuel ethanol from cellulosic agricultural residues (4, 27, 48) and the synthesis of useful β-glucosides (21, 38). In the flavor industry, β-glucosidases are also key enzymes in the enzymatic release of aromatic compounds from glucosidic precursors present in fruits and fermentating products (13, 39). Indeed, many natural flavor compounds, such as monoterpenols, C-13 norisoprenoids, and shikimate-derived compounds, accumulate in fruits as flavorless precursors linked to mono- or diglycosides and require enzymatic or acidic hydrolysis for the liberation of their fragrances (41, 45). Finally, β-glucosidases can also improve the organoleptic properties of citrus fruit juices, in which the bitterness is in part due to a glucosidic compound, naringin (4′,5,7-trihydroxyflavanone-7-rhamnoglucoside), whose hydrolysis requires, in succession, an α-rhamnosidase and a β-glucosidase (33).It is now well-established that certain monoterpenols of grapes (e.g., linalol, geraniol, nerol, citronelol, α-terpineol, and linalol oxide), which are linked to diglycosides, such as 6-O-α-l-rhamnopyranosyl-, 6-O-α-l-arabinofuranosyl-, and 6-O-β-d-apiofuranosyl-β-d-glucosides, contribute significantly to the flavor of wine (15, 44). The enzymatic hydrolysis of these compounds requires a sequential reaction; first, an α-l-rhamnosidase, an α-l-arabinofuranosidase, or a β-d-apiofuranosidase cleaves the (1→6) osidic linkage, and then, the flavor compounds are liberated from the monoglucosides by the action of a β-glucosidase (18, 19). Unlike acidic hydrolysis, enzymatic hydrolysis is highly efficient and does not result in modifications of the aromatic character (16). However, grape and yeast glucosidases exhibit limited activity on monoterpenyl-glucosides during winemaking, and a large fraction of the aromatic precursors remains unprocessed (9, 16, 35). The addition of exogenous β-glucosidase during or following fermentation has been found to be the most effective way to improve the hydrolysis of the glycoconjugated aroma compounds in order to enhance wine flavor (2, 14, 39, 40). The ideal β-glucosidase should function and be stable at a low pH value (pH 2.5 to 3.8) and should be active at a high concentration of glucose (10 to 20%) and in the presence of 10 to 15% ethanol. However, most microbial β-glucosidases are very sensitive to glucose inhibition (4, 12, 47), as well as to inhibition by glucono-δ-lactone, another powerful β-glucosidase inhibitor produced by grape-attacking fungi which can be found in wine must at concentrations up to 2 g/liter (10).The need for more suitable enzymes has led us and other workers to search for novel β-glucosidases with the desired properties. Recently, we showed that an extracellular glucose-tolerant and pH-stable β-glucosidase can be produced by Aspergillus strains (17). However, the enzyme of interest represented only a minor fraction of total β-glucosidase activity, and the major form was highly sensitive to glucose inhibition. Aspergillus oryzae appeared to be the best producer of the minor form when it was grown on quercetin (3,3′,4′,5,7-pentahydroxyflavone), a phenolic flavonoid found in plant cell walls. This paper presents further data on the production and characterization of this novel highly glucose-tolerant β-glucosidase (designated HGT-BG) purified from the extracellular culture filtrate of A. oryzae grown on quercetin.     

Isolation and Characterization of Mutations in the β-Tubulin Gene of SACCHAROMYCES CEREVISIAE

Of 173 mutants of Saccharomyces cerevisiae resistant to the antimitotic drug benomyl (BenR), six also conferred cold-sensitivity for growth and three others conferred temperature-sensitivity for growth in the absence of benomyl. All of the benR mutations tested, including the nine conditional-lethal mutations, were shown to be in the same gene. This gene, TUB2, has previously been molecularly cloned and identified as the yeast structural gene encoding beta-tubulin. Four of the conditional-lethal alleles of TUB2 were mapped to particular restriction fragments within the gene. One of these mutations was cloned and sequenced, revealing a single amino acid change, from arginine to histidine at amino acid position 241, which is responsible for both the BenR and the cold-sensitive lethal phenotypes. The terminal arrest morphology of conditional-lethal alleles of TUB2 at their restrictive temperature showed a characteristic cell-division-cycle defect, suggesting a requirement for tubulin function primarily in mitosis during the vegetative growth cycle. The TUB2 gene was genetically mapped to the distal left arm of chromosome VI, very near the actin gene, ACT1; no CDC (cell-division-cycle) loci have been mapped previously to this location. TUB2 is thus the first cell-division-cycle gene known to encode a cytoskeletal protein that has been identified in S. cerevisiae.