Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea.
J Biotechnol. 2011 Nov 10;156(2):125-33. doi: 10.1016/j.jbiotec.2011.07.024. Epub 2011 Aug 27.
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.
从人参种植地分离得到的人参转化鞘氨醇单胞菌菌株 2F2 中克隆出一种新的β-葡萄糖苷酶基因（bglSp）。bglSp 由 1344bp（447 个氨基酸残基）组成，预测分子量为 49399Da。使用 bglSp 序列进行 BLAST 搜索显示与糖苷水解酶超家族 1 具有显著同源性。该酶在大肠杆菌 BL21（DE3）中使用 pET21-MBP（TEV）载体系统过表达。通过两步亲和层析和 DEAE-纤维素层析，可将过表达的重组酶分别转化为人参皂苷 Rb(1)、Rb(2)、Rc 和 Rd 为更具药理活性的稀有皂苷 gypenoside XVII、ginsenoside C-O、ginsenoside C-Mc(1) 和 ginsenoside F(2)。β-葡萄糖苷酶的动力学参数表明，该酶对 p-硝基苯基-β-d-葡萄糖苷的表观 K(m)和 V(max)值分别为 2.9±0.3mM 和 515.4±38.3μmol min(-1)mg 蛋白(-1)。该酶在最佳条件 pH5.0 和 37°C 下，能够快速将人参皂苷 Rb(1)、Rb(2)、Rc 和 Rd 的外 C3 葡萄糖基水解成稀有皂苷 Gyp XVII、C-O、C-Mc(1)和 F(2)。由于酶的侧活性，在较长的酶反应中，观察到从 gypenoside XVII、C-O 和 C-Mc(1)产生少量的 ginsenoside F(2)。
