The enzymatic transformation of PPD and PPT type ginsenoside into 20(s)-Ginsenoside Rg3 and Rg2 using two distinct recombinant β-glucosidases from Mucilaginibacter sp. nov. $GH3-E5^{T}$

Abstract

Ginsenosides are biologically active compounds produced by ginseng plants that are reported to have their own individual health benefits. The interaction of the plant-microbe rhizosphere is potentially of great importance in that these two sustain each other in the environment. Although Ginsenoside possesses antifungal and antibacterial properties in the rhizosphere, some bacteria could keep growing to assimilate ginsenoside using glycosidase produced by themselves. To obtain glycosidase in this way, we screened novel bacterial strains by metabolizing ginsenosides from the rhizosphere soil of a wild-simulated ginseng. The microbial community was evaluated using a 16S rRNA multiplex 454 pyrosequencing approach. This analysis revealed patterns of bacterial diversity similar to those reported for many other soils, and there were high levels of nitrogen-fixing bacteria and Methylotrophs. While members of the phyla Proteobacteria and Acidobacteria were the most abundant bacteria in comparison within phlyla level, the class Sphingobacteria in Bacteroidetes phylum was also included the most abundant group in comparison within class level.The predominant genera found were Methylotenera (phylum Proteobacteria), Bradyrhizobium (phylum Proteobacteria), Gemmatimonas (phylum Gemmatimonadetes), Rhodoplanes (phylum Proteobacteria) and Terrimonas (Phylum Bacteroidetes). Through four rounds of screening, 20 strains were found to be able to transform major ginsenosides into minor ginsenosides. Some bacteria belonging to the Mucilaginibacter genus were able to convert ginsenoside Rb1, Rb2, Rc and Rd to the active metabolites C-K via F2, C-Y and C-Mc, respectively. One of these, the strain Gh3-E5, was determined to have a ginsenoside conversion pathway through microbial transformation: Rb1 → Rd → F2 → C-K; Rb2 → C-O → C-Y → C-K; Rc → C-Mc1 → C-Mc → C-K. These results show that the strain Gh3-E5 contained several enzymes that hydrolyze, consecutively, the terminal glucopyranosyl, ...