Bioconversion of ginsenosides Rb₁, Rb₂, Rc and Rd by novel ¦Â-glucosidase hydrolyzing outer 3-O glycoside from Sphingomonas sp. 2F2: Cloning, expression, and enzyme characterization

详细信息    查看全文

• 作者：Liang  ; Wang^a ; Qing-Mei  ; Liu^a ; Bong-Hyun  ; Sung^b ; Dong-Shan  ; An^c ; Hyung-Gwan  ; Lee^a ; Song-Gun  ; Kim^c ; Sun-Chang  ; Kim^a ;   ; ^d ; Sung-Taik  ; Lee^a ; Wan-Taek  ; Im^a ;   ; ^d ;   ; ^{wandra@kaist.ac.kr}
• 关键词：Ginsenoside ; Bioconversion ; ¦Â-Glucosidase ; Sphingomonas
• 刊名：Journal of Biotechnology
• 出版年：2011
• 出版时间：10 November 2011
• 年：2011
• 卷：156
• 期：2
• 页码：125-133
• 全文大小：875 K

文摘

A new ¦Â-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 Rb₁, Rb₂, Rc and Rd to the more pharmacological active rare ginsenosides gypenoside XVII, ginsenoside C-O, ginsenoside C-Mc₁ and ginsenoside F₂, respectively, were purified by two steps with Amylose-affinity and DEAE-Cellulose chromatography and characterized. The kinetic parameters for ¦Â-glucosidase showed the apparent K_m and V_max values of 2.9 ¡À 0.3 mM and 515.4 ¡À 38.3 ¦Ìmol min^? mg of protein^? against p-nitrophenyl-¦Â-d-glucopyranoside. The enzyme could hydrolyze the outer C3 glucose moieties of ginsenosides Rb₁, Rb₂, Rc and Rd into the rare ginsenosides Gyp XVII, C-O, C-Mc₁ and F₂ quickly at optimal conditions of pH 5.0 and 37 ¡ãC. A little ginsenoside F₂ production from ginsenosides Gyp XVII, C-O, and C-Mc₁ was observed for the lengthy enzyme reaction caused by the side ability of the enzyme.