Production of $Rh_1(S)$ from $Rg_1$ by using the recombinant β-glucosidase from Mucilaginibacter kaistensis QM49T

Abstract

Ginseng saponin, the most important secondary metabolite of ginseng, has various pharmacological activities. To obtain minor ginsenoside from major ginsenoside by using microbial biotransformation, a great number of soil bacteria which have strong β-glucosidase activity were screened. Through four round screenings, 153 strains were found to be able to transform major ginsenosides into minor ginsenosides. One bacterial strain QM49 could convert ginsenosides $Rb_1$, $Rb_2$, Rc, Rd to the active metabolites C-K via F2, C-Y, C-Mc, respectively. Phylogenetically, it was found to belong to genus Mucilaginibacter and was very closely related with Mucilaginibacter kameinonensis $SCK^T$ (97.5% similarity based on 16S rRNA sequence). Enzymatic production of C-K occurred by consecutive hydrolyses of the terminal glucopyranosyl, arabinopyranosyl, arabinofuranosyl moieties at the C-20 carbon and hydrolyses of the terminal and inner C-3 carbon of ginsenosides $Rb_1$, $Rb_2$, Rc showing the biotransformation pathway: $Rb_1$ → Rd → F2 → C-K; $Rb_2$ → C-O → C-Y → C-K; Rc → $C-Mc_1$ → C-Mc → C-K, respectively. The rare minor ginsenoside, C-Mc1, C-Mc, C-O, C-Y were identified by NMR spectrometry and HPLC/MS/MS system. Two new β-glucosidases from a novel strain of Mucilaginibacter kaistensis ($QM49^T$) obtained from the soil of the field of KAIST (Korea Advanced Institute of Science and Technology) were cloned and characterized. One gene was designated as bgl QM49-1 (2346bp, 781 aa) and the other was designated as bgl QM49-2 (2385 bp, 795 aa). Both enzymes share a low level of similarity which was not aligned easily. However, the function of both enzymes converting ginsenoside was not differentiated too much. Both enzymes catalyzed the conversion of ginsenoside $Rb_1$ to the more pharmacologically active ginsenosides $Rg_3(S)$ via Rd and $Rg_1$ to $Rh_1(S)$. A BLAST search of the sequence of bgl QM49-1 and bgl QM49-2 revealed significant homology to family 3 glycoside hydrolases....