A Cold-Adapted GH1 β-Glucosidase from Paenibacillus cellulosilyticus with high methanol tolerance for efficient biotransformation of ginsenoside Rb1 to minor ginsenoside F2.

This study characterized a novel cold-adapted GH1 β-glucosidase (Bgl1PC) from Paenibacillus cellulosilyticus and elucidated its catalytic mechanism in the biotransformation of ginseng-derived ginsenosides. The recombinant enzyme exhibited optimal activity at 40 °C and pH 7.5, while retaining more than 32% relative activity at 10 °C, indicating strong cold adaptation. Bgl1PC displayed exceptional glucose tolerance (Ki = 763.33 mM) and methanol resistance, retaining 23% residual activity in 30% methanol. Substrate specificity analysis revealed strict preference for aryl β-glucosides (pNPG > pNPGal > oNPG). The kinetic parameters for p-Nitrophenyl-β-D-glucopyranoside (pNPG) were listed as follows: specific activity, 355.60 U·mg-1; Km, 2.99 mM; Vmax, 450.69 μmol·min-1·mg-1 and kcat/Km, 2223.81 s-1·mM-1. Notably, Bgl1PC sequentially hydrolyzed the major ginsenoside Rb1 to the minor ginsenosides Rd and F2 via a two-step pathway, with the conversion of Rd to F2 being the rate-limiting step. Molecular docking identified Asn171 as a key residue facilitating hydrophobic interactions with the C-20 position of Rd, explaining the enzyme's regioselectivity. Thus, these properties make Bgl1PC a promising biocatalyst for the energy-efficient production of pharmaceutically active minor ginsenosides, demonstrating significant potential for the pharmaceutical and cosmetic industries.