Structure-Guided Remodeling of β-Glucosidase Catalytic Performance toward Reverse Hydrolysis for Enhanced Kinsenoside...

Kinsenoside, a bioactive C10 glucoside, is limited by the scarcity of plant sources and the inefficiency of chemical synthesis. β-Glucosidase-catalyzed reverse hydrolysis is a greener alternative but is hindered by an intrinsic hydrolytic bias, resulting in low yields and product degradation. Here, a β-glucosidase from Dictyoglomus thermophilum (DtBGL) was engineered via structure-guided semirational design to enhance kinsenoside production. The optimal mutant M6 increased kinsenoside yield from 17.90% (wild type) to 42.07% while reducing hydrolytic specific activity from 395 to 182 U/mg, indicating a shift in catalytic performance. Structural and substrate-tunnel analysis combined with molecular dynamics simulations attributed this improvement to the remodeled hydrophobic pocket, reduced steric hindrance, and optimized catalytic tunnel, which collectively promoted glycosylation relative to hydrolysis. Moreover, immobilization of M6 on UiO-66-NH₂ improved storage stability at 4 °C and further increased the yield to 54.51%. This work enables sustainable kinsenoside biomanufacturing and offers design principles for reverse-hydrolysis-driven glycoside synthesis.