beta-Glucosidases from cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum as typical glycosidases.

By ethanol precipitation (v/v) and chromatography on Sephadex SP, DEAE (or DEAE-cellulose), and G-200 beta-glucosidases (EC 3.2.1.21) from the culture filtrates of cellulolytic fungi Aspergillus terreus, Geotrichum candidum, and Trichoderma longibrachiatum grown on the medium with cellulose containing materials were isolated. The enzymes were homogenous as shown by different techniques. The substrate specificities of the obtained enzymes were studied. beta-Glucosidases had higher affinity for p-nitrophenyl-beta-D-glucopyranoside than for cellobiose (Km 1.25, 0.34, 0.20 and 5.4, 2.0, 1.2 mM, respectively) and were able to hydrolyze both laminaribiose and gentiobiose; but they were unable to cleave cotton fiber, carboxymethylcellulose, and other glycans to reducing sugars. They showed transglycosylase activity. Ki values for arylglucosidase activity of beta-glucosidases from A. terreus, G. candidum, and T. longibrachiatum in the presence of either glucose or glucono-1,5-lactone were 12.2, 6.0, 2.1 and 0.20, 0.19, 0.07 mM, respectively. The Mr's were estimated by gel filtration and by sedimentation equilibrium centrifugation to 200,000, 200,000, 350,000, respectively. The isoelectric points of beta-glucosidases were 4.8, 5.9, and 4.2, respectively. The optimum temperatures and pH's were 60, 50, and 50 degrees C and at pH 4.5, 4.5, and 4.8-5.7, respectively. These properties appear to relate beta-glucosidases obtained in the present study to typical glycosidases.

[Transglycosylation reactions catalyzed by Aspergillus niger 15 beta-xylosidase]. / Izuchenie reaktsii transglikozilirovaniia, kataliziruemykh beta-ksilozidazoi Aspergillus niger 15.

The products of transglycosylation formed as a result of action of beta-xylosidase from Aspergillus niger 15 on p-nitrophenyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-xylopyranoside were studied by means of chromatography on sephadexes. They are formed at the substrate concentration of 10--100 mM with the amount 7--10 times less than that of hydrolysis products. Peculiarities of chromatography of substrates, products of transglycosylation and p-nitrophenol on Sephadexes G-15 and G-25 were analysed.

beta-Xylosidase from Aspergillus niger 15: purification and properties.

Homogeneous (as judged by data from gel filtration, ultracentrifugation, polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate (SDS), and isoelectric focusing) beta-xylosidase showing beta-D-xylosidase, beta-D-glucosidase, beta-D-galactosidase, and alpha-L-arabinosidase activities has been isolated from the hemicellulase preparation of the microscopic fungus Aspergillus niger 15 by ethanol fractionation and chromatography on Sephadex G-50, cellulose DE-52, and Sephadexes SP C-50 and G-200. The specific activity of the enzyme toward p-nitrophenyl-beta-D-xylopyranoside (p-NPX) increased 199-fold and was equal to 35.2 units/mg of protein; the activity yield was 43%. The sedimentation coefficient was equal to 10.6 S, and the molecular weight was 253,000 according to the gel filtration data and 122,000 according to the data from SDS electrophoresis. The isoelectric point was at pH 4.9. An amino acid analysis has shown that dicarboxylic and hydrophobic amino acids prevail in the enzyme. beta-Xylosidase had no carbohydrate component, and p-chloromercuribenzoate inhibited its activity. The temperature optimum of beta-xylosidase activity toward p-NPX was at 70 degrees C, and the pH optimum was 3.8-4.0. The enzyme was stable at pH 3 to 8 and did not lose its activity for 1 h at temperatures up to 50 degrees C. D-Xylose was found to be a competitive inhibitor of the beta-D-xylosidase activity of the enzyme with Ki = 2.9 mM. beta-Xylosidase showed transglycosylase activity.

[Properties of the exo-1,4-beta-xylosidase of Aspergillus niger 15]. / Svoistva ékzo-1,4-beta-ksilozidazy Aspergillus niger 15.

Properties of exo-1,4-beta-xylosidase from the fungus Aspergillus niger 15 were investigated. The enzyme was homogeneous during gel filtration, electrophoresis in polyacrylamide gel in the presence and absence of Na dodecyl sulfate, ultracentrifugation and isoelectric focusing. The enzyme had a temperature optimum at 70 degrees, pH optimum 3.8-4.0 for p-nitrophenyl-beta-D-xylopyranoside (p-NPX), was stable at pH 3-8, retained its 100% activity for 1 hour at 50 degrees and 42% activity at 60 degrees. Km was 0.23 mM for p-NPX and 0.67 mM for xylobiose. Xylose was a competitive inhibitor of exo-1,4-beta-xylodidase with Ki = 2.9 mM. The enzyme showed a transglycosilase activity. The aminoacid analysis of exo-1,4-beta-xylosidase showed that the enzyme molecule contained predominantly dicarboxylic and hydrophobic amino acids as well as serine. The enzyme contained no carbohydrates. Its activity was inhibited by p-chloromercury benzoate.

[Purification of exo-1,4-beta-xylosidase from Aspergillus niger 15]. / Ochistka ékzo-1,4-beta-ksilozidazy Aspergillus niger 15.

From the preparation of xylanase of the fungus Aspergillus niger 15 exo-1,4-beta-xylosidase was isolated by means of ethanol fractionation and chromatography on columns of Sephadex G-50, cellulose DE-52, Sephadex SP C-50, Sephadex G-200. The isolated enzyme (with the purification degree as calculated per protein 199, and yield with respect to activity 42.5%) was homogeneous during gel filtration, polyacrylamide gel electrophoresis in the presence and absence of Na dodecyl sulfate, ultracentrifugation and isoelectric focusing. Exo-1,4-beta-xylosidase had a molecular mass of 253,000 as shown by gel filtration and 122,000 as shown by Na dodecyl sulfate polyacrylamide gel electrophoresis; its sedimentation coefficient was 10.6 S and isoelectric point was pI 4.9. The specific activity of the homogeneous enzyme was 35.2 u./mg protein with respect to p-nitrophenyl-beta-D-xylopyranoside and 30.2 u./mg protein with respect to xylobiose.