KAIST Institute for Biocentury, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Korea ; College of Biotechnology, Dalian Polytechnic University, Dalian 116034, China.
J Ginseng Res. 2012 Oct;36(4):418-24. doi: 10.5142/jgr.2012.36.4.418.
This study focused on the enzymatic biotransformation of the major ginsenoside Rb1 into Rd for the mass production of minor ginsenosides using a novel recombinant β-glucosidase from Flavobacterium johnsoniae. The gene (bglF3) consisting of 2,235 bp (744 amino acid residues) was cloned and the recombinant enzyme overexpressed in Escherichia coli BL21(DE3) was characterized. This enzyme could transform ginsenoside Rb1 and gypenoside XVII to the ginsenosides Rd and F2, respectively. The glutathione S-transferase (GST) fused BglF3 was purified with GST-bind agarose resin and characterized. The kinetic parameters for β-glucosidase had apparent Km values of 0.91±0.02 and 2.84±0.05 mM and Vmax values of 5.75±0.12 and 0.71±0.01 μmol·min(-1)·mg of protein(-1) against p-nitrophenyl-β-D-glucopyranoside and Rb1, respectively. At optimal conditions of pH 6.0 and 37℃, BglF3 could only hydrolyze the outer glucose moiety of ginsenoside Rb1 and gypenoside XVII at the C-20 position of aglycon into ginsenosides Rd and F2, respectively. These results indicate that the recombinant BglF3 could be useful for the mass production of ginsenosides Rd and F2 in the pharmaceutical or cosmetic industry.
本研究利用来自黄杆菌属的新型重组β-葡萄糖苷酶，专注于将主要人参皂苷 Rb1 酶促生物转化为 Rd，以大规模生产低含量人参皂苷。该基因（bglF3）由 2235bp（744 个氨基酸残基）组成，在大肠杆菌 BL21（DE3）中进行了克隆和重组酶的过表达，并对其进行了表征。该酶可以将人参皂苷 Rb1 和绞股蓝皂苷 XVII 分别转化为人参皂苷 Rd 和 F2。谷胱甘肽 S-转移酶（GST）融合的 BglF3 用 GST 结合琼脂糖树脂进行纯化并进行了表征。β-葡萄糖苷酶的动力学参数具有明显的 Km 值，分别为 0.91±0.02 和 2.84±0.05mM，Vmax 值分别为 5.75±0.12 和 0.71±0.01μmol·min(-1)·mg of protein(-1)，对 p-硝基苯-β-D-吡喃葡萄糖苷和 Rb1。在最佳条件 pH 6.0 和 37℃下，BglF3 只能在糖苷配基的 C-20 位置水解人参皂苷 Rb1 和绞股蓝皂苷 XVII 的外源性葡萄糖单元，分别生成人参皂苷 Rd 和 F2。这些结果表明，重组 BglF3 可用于在制药或化妆品行业中大规模生产人参皂苷 Rd 和 F2。
