Computer-Guided Engineered Endo- and Exocleaving Glycosidase for Significantly Improving Production of Ginsenoside F1

Ginsenoside F1, a particularly rare and valuable compound known for its health benefits, requires precise deglycosylation due to the extensive glycosylation of ginsenosides in <i>Panax notoginseng</i>. Here, we identified that the β-d-glucosidase BglSK exhibits both endo- and exocleaving glycosidase activities with <i>multi-6-O-</i>glycosides, thereby facilitating the specific production of Ginsenoside F1. The variant BglSK_T137A/L508A, obtained through protein engineering, displayed <i>k</i>_cat/<i>K</i>_M values for the reactions of ginsenoside Rg1 and notoginsenoside R1 that were increased by 13.88-fold and 108.56-fold, respectively, compared with the BglSK_WT. The reduced steric hindrance and the overall increase in loop stability show a higher tendency to adopt a closed conformation and facilitate the prereaction state, which may explain the enhanced catalytic efficiency of the engineered enzyme. These beneficial mutants will deepen our understanding of mechanisms for improving glycosidase activity and provide tools for producing high-value <i>P. notoginseng</i> products.