Bioconversion of ginsenosides Rb-1, Rb-2, Rc and Rd by novel beta-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp 2F2: Cloning, expression, and enzyme characterization

摘要

A new beta-glucosidase gene (bglSp) was cloned from the ginsenoside converting Sphingomonas sp. strain 2F2 isolated from the ginseng cultivating filed. The bglSp consisted of 1344 bp (447 amino acid residues) with a predicted molecular mass of 49,399 Da. 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-1, Rb-2, Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc(1) and ginsenoside F-2, respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for beta-glucosidase showed the apparent K-m and V-max values of 2.9 +/- 0.3 mM and 515.4 +/- 38.3 mu mol min(-1) mg of protein(-1) against p-nitrophenyl-beta-D-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb-1, Rb-2, Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc(1) and F-2 quickly at optimal conditions of pH 5.0 and 37 degrees C. A little ginsenoside F-2 production from ginsenosides Gyp XVII, C-O, and C-Mc(1) was observed for the lengthy enzyme reaction caused by the side ability of the enzyme