A novel ginsenoside Rb1-hydrolyzing β-d-glucosidase from Cladosporium fulvum

Abstract: A novel β-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 β-(1→6)-glucosidic linkage at the C-20 site of ginsenoside Rb1 to produce ginsenoside Rd, but did not hydrolyze the other β-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-α-glycosides. G-II consisted of two identical subunits with a native molecular mass of 180kDa and a pI 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°C, and the enzyme became unstable at temperatures above 40°C. The K m and V max values against pNPG were 0.19mM and 57.7μmol/(minmg), respectively. The enzyme was inhibited by Zn2+, Cu2+ (over 50mM) and SDS (250mM). However, the inhibition by SDS was partially reversed by 10mM 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 β-glucosidases. [Copyright &y& Elsevier]