A novel beta-glucosidase (G-II) was purified to homogeneity from a culture filtrate of the phytopathogenic fungus Cladosporium fulvum (syn. Fulvia fulva). G-II specifically cleaved the beta-(1 -> 6)-glucosidic linkage at the C-20 site of ginsenoside Rb-1 to produce ginsenoside Rd, but did not hydrolyze the other beta-D-glucosidic linkages in protopanaxadiol-type ginsenosides. In specificity tests, G-II was active against pNPG and disaccharides such as cellobiose and gentiobiose, but exhibited very low activities against other aryl-glycosides and methyl-alpha-glycosides. G-II consisted of two identical subunits with a native molecular mass of 180 kDa and a pl of 4.4. The optimal pH of G-II was pH 5.5, and the enzyme was highly stable over a range of pH 5.0-11.0. The optimal temperature was 45 degrees C, and the enzyme became unstable at temperatures above 40 degrees C. The K-m and V-max values against pNPG were 0.19 mM and 57.7 mu mol/ (min mg), respectively. The enzyme was inhibited by Zn2+, Cu2+ (over 50 mM) and SDS (250 mM). However, the inhibition by SDS was partially reversed by 10 mM dithiothreitol. Three oligopeptide fragments obtained after enzymatic digestion of G-II were sequenced by nanoESI-MS/MS. The amino acid sequence homology analysis showed that G-II possessed significant homology with the family 3 beta-glucosidases. (C) 2009 Elsevier Ltd. All rights reserved.