title: Bioconversion of ginsenosides Rb(1), Rb(2), Rc and Rd by novel β-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: cloning, expression, and enzyme characterization.
abstract: 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 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 β-glucosidase showed the apparent K(m) and V(max) values of 2.9±0.3 mM and 515.4±38.3 μmol min(-1)mg of protein(-1) against p-nitrophenyl-β-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°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.
authors: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea.
authors: Liang
authors: L
authors: Wang
authors: Qing-Mei
authors: QM
authors: Liu
authors: Bong-Hyun
authors: BH
authors: Sung
authors: Dong-Shan
authors: DS
authors: An
authors: Hyung-Gwan
authors: HG
authors: Lee
authors: Song-Gun
authors: SG
authors: Kim
authors: Sun-Chang
authors: SC
authors: Kim
authors: Sung-Taik
authors: ST
authors: Lee
authors: Wan-Taek
authors: WT
authors: Im
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D036145:Ginsenosides / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D020578:Sphingomonas / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D036145:Ginsenosides; D006027:Glycosides; D001617:beta-Glucosidase