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

Abstract

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 β-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 → 6)-arabinofuranosidic linkage at the C-20 site or outer β-(1 → 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 → 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 → C-Mc1 → C-Mc and G-Rc → G-Rd → G-F2 → 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 β-glucosidase purified from A. mellea mycelia.

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

“…C-Y was also very slowly transformed into C-K over a prolonged reaction time. However, the use of a large quantity of enzyme (BG-1) may be necessary to rapidly transform C-Y into C-K, because BG-1 enzyme has a weak activity in hydrolyzing the outer 20-α- l -arabinopyranosidic linkage of the aglycone [18] . Meanwhile, when Rd was used as a substrate, BG-1 transformed it into C-K via F2.…”

“…Our attention has been focused on the transformation of major ginsenosides into minor ginsenosides by glycoside hydrolases secreted from basidiomycetes mycelia. In previous studies [17] , [18] , we found that minor ginsenosides, C-K and C-Mc, could be efficiently produced from Rb1 and Rc by a crude enzyme extract or purified β-glycosidase from Armillaria mellea mycelium. Rb2 has α- l -arabinopyranosidic linkage different from Rb1 (β- d -glucosidic) and Rc (α- l -arabinofuranosidic) at the C-20 outer position of the aglycone.…”

“…Unless otherwise stated, all procedures for mushroom mycelia production, preparations of crude enzymes (30–80% salt precipitates), and purification of β-glycosidase from A . mellea mycelium were performed according to the methods described in our previous studies [17] , [18] .…”

“… [20] . TLC, HPLC, and ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF-MS) analysis were performed as described in our previous studies [17] , [18] . The relative ratio of the transformation products in the reaction mixtures was calculated from the peak area percentages in HPLC analysis without considering the detector response factor.…”

Production of β‐glucosidases by European Armillaria species

“…While the smallest enzymes are usually excreted and monomeric, oligomeric β‐glucosidases have also been characterized. Three peaks corresponding to β‐glucosidase eluted separately from DEAE‐cellulose columns when intracellular mycelium of A. mellea was assayed (Upadhyaya et al., 2016). These authors purified one peak to homogeneity, which had a molecular weight of 121.5 kDa.…”