Purification and some properties of .BETA.-xylosidase from Trichoderma viride.

Abstract

β-Xylosidase was purified 25 fold from a culture filtrate by ammoniumsulfate fractionation, DEAE-Sephadex chromatography, column electrophoresis, gel filtration on Biogel P-100, and isoelectric focusing. The purified β-xylosidase was found to be homogeneous on SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis and on disc electrophoresis. A molecular weight of 101, 000 was estimated by chromatography on Sephadex G-200, and 102, 000 was obtained by SDS polyacrylamide gel electrophoresis. The purified β-xylosidase had an isoelectric point at pH 4.45, and contained 4.5% carbohydrate residue. The optimum activity for the enzyme was found to be at pH 4.5 and 55°C. The enzyme activity was inhibited by Hg2+, and JV-bromosuccinimide at a concentration of 1 x10-3 m. The purified enzyme hydrolyzed phenyl β-D-xyloside (Ko= 13.0 sec-1), p-nitrophenyl β-D-xyloside (KO= 21.3 sec-1), o-nitrophenyl β-D-xyloside (KO=22.2 sec-1), o- chlorophenyl β-D-xyloside (ko=20.0 sec-1), p-methylphenyl β-D-xyloside (ko=9.0 sec-1), omethylphenyl β-D-xyloside (ko= 10.7 sec-1), β-methoxyphenyl β-D-xyloside (ko= l0.3 secβ-1), omethoxyphenylβ β-D-xyloside (ko= 10.9 sec-1), xylobiose (ko=36A sec-1), xylotriose (ko=34.5 sec-1), xylotetraose (ko= 17.4 sec-1), and xylopentaose (ko= 13.0 sec-1). On enzymic hydrolysis of phenyl β-D-xyloside, the reaction product was found to be β-D-xylose with retention of configuration. The purified β-xylosidase was practically free of α-xylosidase and β-glucosidase activities.