Ginsenoside F1, a particularly rare and valuable compound known for its health benefits, requires precise deglycosylation due to the extensive glycosylation of ginsenosides in Panax notoginseng. Here, we identified that the β-d-glucosidase BglSK exhibits both endo- and exocleaving glycosidase activities with multi-6-O-glycosides, thereby facilitating the specific production of Ginsenoside F1. The variant BglSK_T137A/L508A, obtained through protein engineering, displayed k_cat/K_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 P. notoginseng products.

中文翻译：

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计算机引导的工程内切和外切糖苷酶可显着提高人参皂甙 F1 的产生


人参皂甙 F1 是一种特别稀有且有价值的化合物，以其健康益处而闻名，由于三七中人参皂甙的广泛糖基化，因此需要精确的糖基释放。在这里，我们确定 β-d-葡萄糖苷酶 BglSK 与多-6-O-糖苷一起表现出内切和外切糖苷酶活性，从而促进人参皂甙 F1 的特异性产生。通过蛋白质工程获得的变体 BglSK_T137A/L508A 在人参皂苷 Rg1 和三参皂甙 R1 反应中显示 k_cat/K_M 值，与 BglSK_WT 相比，分别增加了 13.88 倍和 108.56 倍。空间位阻的降低和环稳定性的整体增加表明，采用封闭构象并促进预反应状态的趋势更高，这可能解释了工程酶催化效率的增强。这些有益的突变体将加深我们对提高糖苷酶活性机制的理解，并为生产高价值的三七产品提供工具。