Purification and characterization of piceid-β-d-glucosidase from Aspergillus oryzae

The piceid-β-d-glucosidase that hydrolyzes the β-d-glucopyranoside bond of piceid to release resveratrol was isolated from Aspergillus oryzae sp.100 strain, and the enzyme was purified and characterized. The enzyme was purified to one spot in SDS polyacrylamide gel electrophoresis, and its molecular weight was about 77 kDa. The optimum temperature of the piceid-β-d-glucosidase was 60 °C, and the optimum pH was 5.0. The piceid-β-d-glucosidase was stable at less than 60 °C, and pH 4.0–5.0. Ca²⁺, Mg²⁺ and Zn²⁺ ions have no significant effect on enzyme activity, but Cu²⁺ ion inhibits enzyme activity strongly. The K_m value was 0.74 mM and the V_max value was 323 nkat mg⁻¹ for piceid.     

Cloning,expression, and characterization of the β-glucosidase hydrolyzing secoisolariciresinol diglucoside to secoisolariciresinol from Bacteroides uniformis ZL1

Previously, from the human intestinal flora we isolated the bacterial strain Bacteroides uniformis ZL1, which could convert secoisolariciresinol diglucoside (SDG) to its aglycone secoisolariciresinol (SECO) in vivo. In this study, 24 putative β-glucosidase genes were screened from the genome of B. uniformis ATCC 8492, which were used as templates to design PCR primers for the target genes in B. uniformis ZL1. Fifteen genes (bgl1–bgl15) were amplified from strain ZL1, and among them we identified bgl8 as the gene encoding the SDG-hydrolyzing β-glucosidase. We sequenced the bgl8 gene, cloned it into the expression vector and then transformed Escherichia coli to construct the recombinant bacteria that could synthesize the target β-glucosidase (BuBGL8). We purified and characterized BuBGL8, which showed maximal activity and stability under the culture conditions of pH 6.0 and 30 °C. SDG (2.0 mg/ml) was converted to SECO by both the purified BuBGL8 (0.035 mg/ml) and crude enzyme extract (0.23 mg crude protein/ml) with the efficiency of more than 90 % after 90 min at the reaction conditions. This is, to our knowledge, the first report of using recombinant bacteria to synthesize the SDG-hydrolyzing β-glucosidase, which could be used to produce SECO from SDG conveniently and highly efficiently.     

Purification and characterization of α-glucosidase from Aspergillus carbonarious

Summary An -glucosidase was purified from Aspergillus carbonarious CCRC 30414 over 20 fold with 37 % recovery. Its molecular mass was estimated to be 328 kDa by gel filtration with an optimum pH from 4.2 to 5.0, and pI=5.0. The optimum temperature is at 60°C over 40 min. The enzyme was partially inhibited by 5 mM Ag⁺, Hg²⁺, Ba²⁺, Pb²⁺, and Aso₄ ⁺.     

Purification and Properties of Leucine Aminopeptidase I–III from Aspergillus oryzae

An aminopeptidase from Aspergillus oryzae 460 was purified from the rivanol precipitable fraction. The partially purified enzyme was not homogeneous in disc electrophoresis, although symmetric profiles were obtained for enzyme protein and activity in Sephadex gel filtration. Its optimum pH is at pH 8.5 for l-leucyl-β-naphthylamide. The enzyme activity was inhibited by metal chelating agents and S-S dissociating agents, but not inhibited by SH reagents. The molecular weight of the enzyme was estimated to be about 26,500 by gel filtration. The enzyme was named leucine aminopeptidase I of Asp. oryzae 460, since it preferentially hydrolyzed oligopeptides that possess leucine as the amino terminal amino acid.     

Purification and properties ofβ-fructofuranosidase from Aspergillus japonicus

-Fructofuranosidase fromAspergillus japonicus, which produces 1-kestose (O--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) and nystose (O--d-fructofuranosyl-(21)--d-fructofuranosyl-(21)--d-fructofuranosyl -d-glucopyranoside) from sucrose, was purified to homogeneity by fractionation with calcium acetate and ammonium sulphate and chromatography with DEAE-Cellulofine and Sephadex G-200. Its molecular size was estimated to be about 304,000 Da by gel filtration. The enzyme was a glycoprotein which contained about 20% (w/w) carbohydrate. Optimum pH for the enzymatic reaction was 5.5 to 6. The enzyme was stable over a wide pH range, from pH 4 to 9. Optimum reaction temperature for the enzyme was 60 to 65°C and it was stable below 60°C. The K_m value for sucrose was 0.21m. The enzyme was inhibited by metal ions, such as those of silver, lead and iron, and also byp-chloromercuribenzoate.     

Purification and characterization of an extracellular β-D-xylosidase from Aspergillus carbonarius

The production of an extracellular -D-xylosidase (-D-xyloside xylohydrolase, EC 3.2.1.37) by four Aspergillus strains (A. carbonarius, A. nidulans, A. niger and A. oryzae) grown on wheat bran medium was compared. The highest amount of the enzyme was found in the culture of A. carbonarius. The -D-xylosidase from A. carbonarius was purified to homogeneity by a rapid procedure, using hydrophobic interaction chromatography, chromatofocusing and affinity chromatography. The purified enzyme possessed not only -D-xylosidase activity, but also -L-arabinosidase activity. Mixed substrate experiments revealed that a single active centre was responsible for the splitting of the corresponding synthetic substrates. The molecular weight of the purified enzyme proved to be 100,000 Da, as estimated by SDS–PAGE. The isoelectric point was at pH 4.4. The pH and temperature optima were 4.0 and 60 °C, respectively. The enzyme remained stable over a pH range of 3.5–6.5 and up to 50 °C for 30 min. The Michaelis constant for p-nitrophenyl -D-xyloside was 0.198 mM. Kinetic studies demonstrated that the lack of the C-5 hydroxylmethyl group and the configuration of the C-4 hydroxyl group on the pyranoside ring play an important role in both substrate binding and splitting.     

Purification and Characterization of Chitosanase and Exo-β-D-Glucosaminidase from a Koji Mold,Aspergillus oryzae IAM2660

Chitosan-degrading activity was detected in the culture fluid of Aspergillus oryzae, A. sojae, and A. flavus among various fungal strains belonging to the genus Aspergillus. One of the strong producers, A. oryzae IAM2660 had a higher level of chitosanolytic activity when N-acetylglucosamine (GlcNAc) was used as a carbon source. Two chitosanolytic enzymes, 40 kDa and 135 kDa in molecular masses, were purified from the culture fluid of A. oryzae IAM2660. Viscosimetric assay and an analysis of reaction products by thin-layer chromatography clearly indicated the endo- and exo-type cleavage manner for the 40-kDa and 135-kDa enzymes, respectively. The 40-kDa enzyme, designated chitosanase, catalyzed a hydrolysis of glucosamine (GlcN) oligomers larger than pentamer, glycol chitosan, and chitosan with a low degree of acetylation (0-30%). The 135-kDa enzyme, named exo-β-D-glucosaminidase, released a single GlcN residue from the GlcN oligomers and chitosan, but did not release GlcNAc residues from either GlcNAc oligomer or colloidal chitin.     

Purification and characterization of a (1,4)-β-d-arabinoxylan arabinofuranohydrolase from Aspergillus awamori

Summary An enzyme able to split off arabinose sidechains from cereal arabinoxylans was isolated from a cell-free culture filtrate of Aspergillus awamori CMI 142717 containing milled oat straw as the carbon source. The enzyme was highly specific for arabinoxylans and, unlike other -l-arabinofuranosidases reported in the literature, did not show any activity towards p-nitrophenyl -l-arabinofuranoside, arabinans and arabinogalactans. This novel enzyme, which can be described as a (1,4)--d-arabinofuranohydrolase, had a molecular mass of 32 000 Da when determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and a specific activity of 22 units/mg on wheat arabinoxylan. Offprint requests to: A. G. J. Voragen     

Purification and biochemical characterization of a novel α-glucosidase from Aspergillus niveus

An extracellular α-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS–PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical α-glucosidase activity, hydrolyzing p-nitrophenyl α-d-glucopyranoside and presented an optimum temperature and pH of 65°C and 6.0, respectively. In the absence of substrate the purified α-glucosidase was stable for 60 min at 60°C, presenting t ₅₀ of 90 min at 65°C. Hydrolysis of polysaccharide substrates by α-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and β-ciclodextrin were poor substrates, and sucrose and α-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an α-helical content of 31% and a β-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.     

Purification of α-galactosidase and invertase by three-phase partitioning from crude extract of Aspergillus oryzae

alpha-Galactosidase and invertase were accumulated in a coherent middle phase in a three-phase partitioning system under different conditions (ammonium sulphate, ratio of tert-butanol to crude extract, temperature and pH). alpha-Galactosidase and invertase were purified 15- and 12-fold with 50 and 54% activity recovery, respectively. The fractions of interfacial precipitate arising from the three-phase partitioning were analyzed by SDS-PAGE. Both purified preparations showed electrophoretic homogeneity on SDS-PAGE.     

Kinetic characterization of a crude β-d-glucosidase from Aspergillus wentii Pt 2804

The kinetic characteristics of β-d-glucosidase (cellobiase, β-d-glucosidase glucohydrolase, EC 3.2.1.21) from the filtered broth of a well grown culture of Aspergillus wentii have been studied. Both cellobiose and 4-nitrophenyl-β-d-glucoside (4NPG) were used as substrates and values of K_m, V_max for both the substrates were determined. Activity was maximum over a pH range of 4.5–5.5 but declined sharply beyond 5.5 for both substrates. The optimum temperature was between 60 and 65°C. Half-life of the cellobiase was ～38.0 h at 60°C and ～6.3 h at 65°C. However, the enzyme was found to be quite stable at 50°C. The activation and deactivation energies for 4NPG hydrolysis were 33.2 and 111.3 kJ mol^?1 K^?1, and 43.6 and 63.7 kJ mol K^?1 for cellobiose hydrolysis. Product inhibition was found to be of the competitive type. Preliminary experiments showed that marked synergistic activity exists between Trichoderma reesei and A. wentii cellulases [see 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] for cellulose hydrolysis.     

Molecular cloning,purification and characterization of two endo-1,4-β-glucanases from Aspergillus oryzae KBN616

Two endo-1,4-β-glucanase genes, designated celA and celB, from a shoyu koji mold Aspergillus oryzae KBN616, were cloned and characterized. The celA gene comprised 877 bp with two introns. The CelA protein consisted of 239 amino acids and was assigned to the cellulase family H. The celB gene comprised 1248 bp with no introns. The CelB protein consisted of 416 amino acids and was assigned to the cellulase family C. Both genes were overexpressed under the promoter of the A. oryzae taka-amylase A gene for purification and enzymatic characterization of CelA and CelB. CelA had a molecular mass of 31 kDa, a pH optimum of 5.0 and temperature optimum of 55 °C, whereas CelB had a molecular mass of 53 kDa, a pH optimum of 4.0 and temperature optimum of 45 °C. Received: 3 July 1996 / Accepted: 15 July 1996     

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.     

Synthesis of propyl-β-d-galactoside with free and immobilized β-galactosidase from Aspergillus oryzae

Synthesis of propyl-β-galactoside catalyzed by Aspergillus oryzae β-galactosidase in soluble form was optimized using response surface methodology (RSM). Temperature and 1-propanol concentration were selected as explanatory variables; yield and productivity were chosen as response variables. Optimal reaction conditions were determined by weighing the responses through a desirability function. Then, synthesis of propyl-β-galactoside was evaluated at the optimal condition previously determined, with immobilized β-galactosidase in glyoxyl-agarose and amino-glyoxyl-agarose, and with cross-linked aggregates (CLAGs). Yields of propyl-β-galactoside obtained with CLAGs, amino-glyoxyl-agarose and glyoxyl-agarose enzyme derivatives were 0.75, 0.81 and 0.87 mol/mol and volumetric productivities were 5.2, 5.6 and 5.9 mM/h, respectively, being significantly higher than the corresponding values obtained with the soluble enzyme: 0.47 mol/mol and 4.4 mM/h. As reaction yield was increased twofold with the glyoxyl-agarose derivative, this catalyst was chosen for evaluating the synthesis of propyl-β-galactoside in repeated batch operations. Then, after ten sequential batches, the efficiency of catalyst use was 115% higher than obtained with the free enzyme. Enzyme immobilization also favored product recovery, allowing catalyst reuse, and avoiding browning reactions. Propyl-β-galactoside was recovery by extraction in 90%v/v acetone with a purity higher than 99% and its synthesis was confirmed by mass spectrometry.     

Protodioscin-glycosidase-1 hydrolyzing 26-O-β-d-glucoside and 3-O-(1 → 4)-α-l-rhamnoside of steroidal saponins from Aspergillus oryzae

A novel protodioscin-(steroidal saponin)-glycoside hydrolase, named protodioscin-glycosidase-1 (PGase-1), was purified and characterized from the Aspergillus oryzae strain. The molecular mass of this enzyme was determined to be about 55 kDa based on SDS-polyacrylamide gel electrophoresis. PGase-1 was able to hydrolyze the terminal 26-O-β-d-glucopyranoside of protodioscin (furostanoside) to produce dioscin (spirostanoside), and then further hydrolyze the terminal 3-O-(1?→?4)-α-l-rhamnopyranoside of dioscin to form progenin III. However, PGase-1 could hardly hydrolyze the 3-O-(1?→?2)-α-l-rhamnopyranoside of progenin III, 3-O-β-d-glucoside of trillin, and the 1-O-glycosides of ophiopogonin D (steroidal saponin). In addition, PGase-1 also could hydrolyze the α-d-galactopyranoside, β-d-glucopyranoside, and β-d-galactopyranoside of p-nitrophenyl-glycosides, but the enzyme could not hydrolyze the α-d-mannopyranoside, α-l-arabinopyranoside, α-d-glucopyranoside, β-d-xylopyranoside, and α-l-rhamnopyranoside of p-nitrophenyl-glycosides. These new properties of PGase-1 were significantly different from those of previously described steroidal saponin-glycosidases and the glycosidases currently described in Enzyme Nomenclature by the NC-IUBMB. The gene (termed as pgase-1) encoding PGase-1 was cloned, sequenced, and expressed in Pichia pastoris GS115. The complete nucleotide sequence of pgase-1 consists of 1,725 bp. The recombinant PGase-1 from recombinant P. pastoris GS115 strain also showed the activity hydrolyzing glycosides of steroidal saponins which was similar to that of the wild-type PGase-1 from A. oryzae. The PGase-1 gene is highly similar to Aspergilli α-amylase (EC 3.2.1.1), and PGase-1 should be classified as glycoside hydrolase family 13 by the method of gene sequence-based classification. But the enzyme properties of PGase-1 are different from those of α-amylase in this family.     

Purification and characterization of β-xylosidase from Aureobasidium pullulans

Summary -Xylosidase was obtained from Aureobasidium pullulans CBS 58475 with an activity of 0.35 units/ml culture filtrate. The production of the enzyme was strongly inducible. -Xylosidase was purified in two steps by anion exchange and gel-permeation chromatography to high purity. The enzyme is a glycoprotein with an apparent molecular mass of 224 kDa in sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and separates into two subunits of equal molecular mass. After SDS-PAGE -xylosidase could be renatured and stained with methylumbelliferyl--xylopyranoside. The enzyme was able to split substrates of other glycosidases. The maximum activity was reached at pH 4.5 and 80° C. -Xylosidase showed high stability over a broad pH range from pH 2.0 to 9.5 and up to 70° C. Analysis of cleavage patterns revealed that the enzyme was a typical glycosidase. Larger oligosaccharides consisting of xylose were degraded by an exomechanism together with a transxylosylation reaction.     

Purification and characterization of glycyrrhizin-β-d-glucuronidase and baicalin-β-d-glucuronidase from a commercial enzyme preparation

Abstract

Five commercial enzyme preparations were screened for hydrolysis of the glucuronic acid units of glycyrrhizin (GL) and baicalin. Two preparations hydrolyzing GL to glycyrrhetic acid (GA) and four enzyme preparations hydrolyzing baicalin to baicalein were obtained. One enzyme preparation with the ability to hydrolyze both GL and baicalin, namely Rapidase Pineapple, was purified by anion exchange, cation exchange and molecular sieve chromatography. The results of purification indicated that the enzymes containing the glycyrrhizin-β-d-glucuronidase (GBDG) and baicalin-β-d-glucuronidase (BBDG) activities were distinct, with different substrate specificities, molecular weights and enzymatic characteristics. GBDB hydrolyzed GL to GA, but had no detectable activity on baicalin, and BBDG hydrolyzed baicalin to baicalein, but could not hydrolyze GL. However, both GBDG and BBDG could hydrolyze the artificial substrate p-nitrophenyl- β-d-glucuronide (pNPGA).     

Purification and characterization of β‐methylaspartase from Fusobacterium varium

Methylaspartase (EC 4.3.1.2) was purified 20fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAESepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg²⁺ (K_m = 0.27 ± 0.01 mM) and K⁺ (K_m = 3.3 ± 0.8 mM) for maximum activity. Methylaspartasecatalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)3methylaspartate (major product) and (2S,3R)3methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)3methylaspartate (K_m = 0.51 ± 0.04 mM) was observed at pH 9.7. The Nterminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.     

Purification and characterization of three β-glycosidases exhibiting high glucose tolerance from Aspergillus niger ASKU28

Production and utilization of cellulosic ethanol has been limited, partly due to the difficulty in degradation of cellulosic feedstock. β-Glucosidases convert cellobiose to glucose in the final step of cellulose degradation, but they are inhibited by high concentrations of glucose. Thus, in this study, we have screened, isolated, and characterized three β-glycosidases exhibiting highly glucose-tolerant property from Aspergillus niger ASKU28, namely β-xylosidase (P1.1), β-glucosidase (P1.2), and glucan 1,3-β-glucosidase (P2). Results from kinetic analysis, inhibition study, and hydrolysis of oligosaccharide substrates supported the identification of these enzymes by both LC/MS/MS analysis and nucleotide sequences. Moreover, the highly efficient P1.2 performed better than the commercial β-glucosidase preparation in cellulose saccharification, suggesting its potential applications in the cellulosic ethanol industry. These results shed light on the nature of highly glucose-tolerant β-glucosidase activities in A. niger, whose kinetic properties and identities have not been completely determined in any prior investigations.     

Purification and characterization of endo-1,4-β-xylanase from Paecilomyces varioti Bainier

An endo-xylanase (1,4-β-d-xylanxylanohydrolase EC 3.2.1.8) was isolated from the culture filtrate of Paecilomyces varioti Bainier. The enzyme was purified 3.2 fold with a 60% yield by gel filtration and ion exchange chromatography. The purified enzyme had a molecular weight of 25,000 with a sedimentation coefficient of 2.2 S. The isoelectric point of the enzyme was 3.9. The enzyme was obtained in crystalline form. The optimum pH range was 5.5–7.0 and the temperature, 65°C. The Michaelis constant was 2.5 mg larchwood xylan/ml. The enzyme was found to degrade xylan by an endo mechanism producing arabinose, xylobiose, xylo- and arabinosylxylo-oligosaccharides, during the initial stages of hydrolysis. On prolonged incubation, xylotriose, arabinosylxylotriose and xylobiose were the major products with traces of xylotetraose, xylose and arabinose.