Mechanism Exploration of Targeted Mutagenesis Strategy To Enhance the Catalytic Performance of Oenococcus oeni β-Glucosidase

β-Glucosidase from Oenococcus oeni can hydrolyze glycoside substrates to release aromatic ligands, significantly improving the food flavor. However, its catalytic activity and stability still need to be improved. In this study, three double mutants with significantly enhanced catalytic performance were obtained by site-directed mutagenesis on the key amino acids of Oenococcus oeni β-glycosidase. The enzymatic activities of mutants I/II/III were 4.24, 3.76, and 2.82 times that of the wild type, respectively. The thermal stability was also significantly improved. The Km of mutants I/II/III for catalyzing substrate p-NPG were decreased by 15.2, 30.3, and 24.2%, respectively. Molecular simulation indicated that the increase in surface charge and the decrease of B-factor in loop region improved the stability of the mutants. The increase of hydrogen bonds and π–π aromatic interaction were main driving forces for enhanced activity of mutants. This study is of great significance for the industrial application of Oenococcus oeni β-glucosidase.