G-I, a highly selective beta-glucosidase, was purified from phytopathogenic fungus Cladosporium fulvum (syn. Fulvia fulva). G-I was a monomer with native molecular weight of 85 kDa and pI value of 4.2. The maximal activity to p-nitrophenyl-beta-D-glucopyranoside (pNPG) occurred at pH 6.0 and 45 degrees C at which the K, against pNPG was 0.18 mM and V-max was 46.7 mu mol nitrophenol/min/mg. G-I was highly stable within pH 4.0-11.0 and below 40 degrees C. It was inhibited by Co2+, Cu2+ and Zn2+ (50 mM), but showed resistance to sodium dodecyl sulfonate (SDS, 250 mM). G-I was highly active against beta-linked disaccharide cellobiose, gentiobiose and sophorose, but exhibited very low activities against other aryl-glycosides, methyl-alpha-glycosides and clisaccharides trehalose and sucrose. Moreover. G-I specifically hydrolyzed beta-(1 -> 6)-glucosidilc linkage at the C-20 site of ginsenoside Rbi to produce ginsenosidle Rd, without attack on other beta-D-glucosidic linkages. The oligopeptide fragments of G-I were sequenced by nanoESI-MS/MS and showed similarity to the sequences from the glycoside hydrolase family 3. G-I is different to G-II (a glycosidase previously purified from the same fungus) in composition and molecular weight. It shows more stable and higher selectivity than G-II. (C) 2010 Elsevier Ltd. All rights reserved.