KAIST Institute for Biocentury, Korea Advanced Institute of Science and Technology, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
J Biotechnol. 2012 Oct 31;161(3):294-301. doi: 10.1016/j.jbiotec.2012.06.021. Epub 2012 Jul 2.
This study focuses on the cloning, expression, and characterization of recombinant ginsenoside hydrolyzing glycosidase from Sanguibacter keddieii in order to biotransform ginsenosides efficiently. The gene, termed bglSk, consists of 1857 bp and revealed significant homology to that of glycoside hydrolase family 3. The enzyme was over-expressed in Escherichia coli BL21 (DE3) using a GST-fused pGEX 4T-1 vector system. The over-expressed recombinant enzymes could convert six major ginsenosides Rb(1), Rb(2), Rc, Rd, Re and Rg(1) into more pharmacologically active rare ginsenosides such as C-Y, C-Mc, C-K, Rg(2)(S), and F(1). Especially, BglSk could completely convert the Rg(1) into F(1). The GST-fused BglSk was purified with GST·bind agarose resin and then characterized. The kinetic parameters for β-glucosidase had apparent K(m) values of 0.456±0.009 and 0.167±0.003 mM and V(max) values of 30.2±0.7 and 4.1±0.1 μmol min(-1) mg of protein(-1) against p-nitrophenyl-β-d-glucopyranoside and Rb(1), respectively.
本研究致力于从血杆菌中克隆、表达和鉴定重组人参皂苷水解糖苷酶，以便有效地进行生物转化。该基因名为 bglSk，由 1857bp 组成，与糖苷水解酶家族 3 具有显著的同源性。该酶在大肠杆菌 BL21(DE3)中使用 GST 融合的 pGEX 4T-1 载体系统过表达。过表达的重组酶可以将六种主要的人参皂苷 Rb(1)、Rb(2)、Rc、Rd、Re 和 Rg(1)转化为更具药理活性的稀有人参皂苷，如 C-Y、C-Mc、C-K、Rg(2)(S)和 F(1)。特别是，BglSk 可以将 Rg(1)完全转化为 F(1)。GST 融合的 BglSk 用 GST·bind 琼脂糖树脂纯化，然后进行特性分析。β-葡萄糖苷酶的动力学参数对 p-硝基苯-β-d-吡喃葡萄糖苷和 Rb(1)的表观 K(m)值分别为 0.456±0.009 和 0.167±0.003 mM，V(max)值分别为 30.2±0.7 和 4.1±0.1 μmol min(-1) mg 蛋白(-1)。
