Site-Directed Mutagenesis Increased the Catalytic Activity and Stability of β-Glucosidase: Characterization of Enzymatic Properties and Exploration of Mechanisms.

β-glucosidase can significantly improve food flavor, but its catalytic activity and stability performance need to be enhanced. In this study, the two dominant mutants III and IV were obtained by site-directed mutation of key amino acid residues in the catalytic pocket of β-glucosidase. Compared with the wild-type enzyme, the activities of mutants III and IV were increased by 2.81 and 3.18 times, respectively. Their thermal stability was also significantly improved. Mutants III and IV showed a significant increase in affinity for the standard substrate -NPG, with the value decreasing by 18.2% and 33.3%, respectively. Molecular docking analysis indicated that hydrogen bonding and π-π aromatic interactions were the primary factors influencing the changes in enzyme properties, with F133 and N181 identified as key amino acids affecting catalytic activity and stability. This research is of great significance for enhancing food flavor and expands the potential industrial applications of β-glucosidase.