Discovery and Functional Characterization of a β-Glucosidase from <i>Pleurotus ostreatus</i> for the Biocatalytic Conversion of Mogroside V into Mogroside IV and Siamenoside I

Mogroside IV (MG-IV) and Siamenoside I (MG-SI) are key components responsible for the characteristic sweetness and flavor of <i>Siraitia grosvenorii</i>. However, their naturally low abundance limits both flavor enhancement and large-scale industrial application. In this study, A β-glucosidase, PoBgl3B, was identified from <i>Pleurotus ostreatus</i> and exhibited superior catalytic efficiency in converting Mogroside V (MG-V) into MG-IV and MG-SI. Molecular docking, isothermal titration calorimetry, and molecular dynamics (MD) analyses revealed distinct substrate-binding modes and affinity differences. Exploiting the enzyme's moderate glucose sensitivity, a glucose-regulated bioconversion strategy was established to control intermediate accumulation and promote the selective enrichment of desired products. Under optimized conditions, PoBgl3B (0.5 μg/mL) increased the relative contents of MG-IV and MG-SI by 5.54- and 3.52-fold, respectively, within 15 min. Thermal, pH, and metal ion tolerance analyses confirmed the catalytic stability, underscoring its potential as a highly efficient and sustainable biocatalyst for mogroside biotransformation.