Highly regioselective biotransformation of ginsenoside Rb2 into compound Y and compound K by ß-glycosidase purified from Armillaria mellea mycelia.

BACKGROUND: The biological activities of ginseng saponins (ginsenosides) are associated with type, number, and position of sugar moieties linked to aglycone skeletons. Deglycosylated minor ginsenosides are known to be more biologically active than major ginsenosides. Accordingly, the deglycosylation of major ginsenosides can provide the multibioactive effects of ginsenosides. The purpose of this study was to transform ginsenoside Rb2, one of the protopanaxadiol-type major ginsenosides, into minor ginsenosides using ß-glycosidase (BG-1) purified from Armillaria mellea mycelium. METHODS: Ginsenoside Rb2 was hydrolyzed by using BG-1; the hydrolytic properties of Rb2 by BG-1 were also characterized. In addition, the influence of reaction conditions such as reaction time, pH, and temperature, and transformation pathways of Rb2, Rd, F2, compound O (C-O), and C-Y by treatment with BG-1 were investigated. RESULTS: BG-1 first hydrolyzes 3-O-outer ß-d-glucoside of Rb2, then 3-O-ß-d-glucoside of C-O into C-Y. C-Y was gradually converted into C-K with a prolonged reaction time, but the pathway of Rb2 → Rd → F2 → C-K was not observed. The optimum reaction conditions for C-Y and C-K formation from Rb2 by BG-1 were pH 4.0-4.5, temperature 45-60°C, and reaction time 72-96 h. CONCLUSION: ß-Glycosidase purified from A. mellea mycelium can be efficiently used to transform Rb2 into C-Y and C-K. To our best knowledge, this is the first result of transformation from Rb2 into C-Y and C-K by basidiomycete mushroom enzyme.

Purification and characterization of a novel ginsenoside Rc-hydrolyzing ß-glucosidase from Armillaria mellea mycelia.

Ginsenosides are the principal compounds responsible for the pharmacological effects and health benefits of Panax ginseng root. Among protopanaxadiol (PPD)-type ginsenosides, minor ginsenosides such as ginsenoside (G)-F2, G-Rh2, compound (C)-Mc1, C-Mc, C-O, C-Y, and C-K are known to be more pharmacologically active constituents than major ginsenosides such as G-Rb1, G-Rb2, G-Rc, and G-Rd. A novel ginsenoside Rc-hydrolyzing ß-glucosidase (BG-1) from Armillaria mellea mycelia was purified as a single protein band with molecular weight of 121.5 kDa on SDS-PAGE and a specific activity of 17.9 U mg-1 protein. BG-1 concurrently hydrolyzed α-(1 â†’ 6)-arabinofuranosidic linkage at the C-20 site or outer ß-(1 â†’ 2)-glucosidic linkage at the C-3 site of G-Rc to produce G-Rd and C-Mc1, respectively. The enzyme also hydrolyzed outer and inner glucosidic linkages at the C-3 site of G-Rd to produce C-K via G-F2, and inner glucosidic linkage at the C-3 site of C-Mc1 to produce C-Mc. C-Mc was also slowly hydrolyzed α-(1 â†’ 6)-arabinofuranosidic linkage at the C-20 site to produce C-K with reaction time prolongation. Finally, the pathways for formation of C-Mc and C-K from G-Rc by BG-1 were G-Rc â†’ C-Mc1 â†’ C-Mc and G-Rc â†’ G-Rd â†’ G-F2 â†’ C-K, respectively. The optimum reaction conditions for C-Mc and C-K formation from G-Rc by BG-1 were pH 4.0-4.5, temperature 45-60 °C, and reaction time 72-96 h. This is the first report of efficient production of minor ginsenosides, C-Mc and C-K from G-Rc by ß-glucosidase purified from A. mellea mycelia.