A highly selective ginsenoside Rb1-hydrolyzing β-d-glucosidase from Cladosporium fulvum

G-I, a highly selective β-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-β-d-glucopyranoside (pNPG) occurred at pH 6.0 and 45 °C at which the Km against pNPG was 0.18 mM and Vmax was 46.7 μmol nitrophenol/min/mg. G-I was highly stable within pH 4.0–11.0 and below 40 °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 β-linked disaccharide cellobiose, gentiobiose and sophorose, but exhibited very low activities against other aryl-glycosides, methyl-α-glycosides and disaccharides trehalose and sucrose. Moreover, G-I specifically hydrolyzed β-(1 → 6)-glucosidic linkage at the C-20 site of ginsenoside Rb1 to produce ginsenoside Rd, without attack on other β-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.