초록
A new β-glucosidase gene (bglSp) was cloned from the ginsenoside converting Sphingomonas sp. strain 2F2 isolated from the ginseng cultivating filed. The bglSp consisted of 1344bp (447 amino acid residues) with a predicted molecular mass of 49,399Da. A BLAST search using the bglSp sequence revealed significant homology to that of glycoside hydrolase superfamily 1. This enzyme was overexpressed in Escherichia coli BL21 (DE3) using a pET21-MBP (TEV) vector system. Overexpressed recombinant enzymes which could convert the ginsenosides Rb<SUB>1</SUB>, Rb<SUB>2</SUB>, Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc<SUB>1</SUB> and ginsenoside F<SUB>2</SUB>, respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for β-glucosidase showed the apparent K<SUB>m</SUB> and V<SUB>max</SUB> values of 2.9+/-0.3mM and 515.4+/-38.3μmolmin<SUP>-1</SUP>mg of protein<SUP>-1</SUP> against p-nitrophenyl-β-d-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb<SUB>1</SUB>, Rb<SUB>2</SUB>, Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc<SUB>1</SUB> and F<SUB>2</SUB> quickly at optimal conditions of pH 5.0 and 37<SUP>o</SUP>C. A little ginsenoside F<SUB>2</SUB> production from ginsenosides Gyp XVII, C-O, and C-Mc<SUB>1</SUB> was observed for the lengthy enzyme reaction caused by the side ability of the enzyme.