Computer-Guided\nEngineered Endo- and Exocleaving Glycosidase\nfor Significantly Improving Production of Ginsenoside F1

Ginsenoside\nF1, a particularly rare and valuable compound\nknown\nfor its health benefits, requires precise deglycosylation due to the\nextensive glycosylation of ginsenosides in Panax notoginseng. Here, we identified that the β-d-glucosidase BglSK\nexhibits both endo- and exocleaving glycosidase activities with <i>multi-6-O-</i>glycosides, thereby facilitating the specific\nproduction of Ginsenoside F1. The variant BglSK<sub>T137A/L508A</sub>, obtained through protein engineering, displayed <i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub> values for the reactions\nof ginsenoside Rg1 and notoginsenoside R1 that were increased by 13.88-fold\nand 108.56-fold, respectively, compared with the BglSK<sub>WT</sub>. The reduced steric hindrance and the overall increase in loop stability\nshow a higher tendency to adopt a closed conformation and facilitate\nthe prereaction state, which may explain the enhanced catalytic efficiency\nof the engineered enzyme. These beneficial mutants will deepen our\nunderstanding of mechanisms for improving glycosidase activity and\nprovide tools for producing high-value P. notoginseng products.