Molecular modifications of nattokinase have been demonstrated to yield enzyme variants with enhanced catalytic performance, offering valuable insights into the structure-activity relationship of this enzyme. In the present study, PoPMuSiC 2.1, a web-based tool designed for predicting protein stability changes induced by mutations, was employed to investigate the catalytic properties of nattokinase. Through PoPMuSiC prediction and site-saturation mutagenesis, the G131A mutant, which exhibited improved catalytic activity, was identified. Comparative analysis of the G131A mutant and the wild type nattokinase revealed a 1.5-fold increase in enzyme activity and a 4.5-fold extension in half-life, effectively addressing the typical trade-off between enzyme activation and stability in protein engineering. Computational simulations further indicated that the G131A mutant adopts a more stable conformation, displays stronger interactions with the substrate, and features a more optimal substrate entry pocket. The fermentation process of G131A mutant was optimized and a yield of 623.43 U/mL was obtained. These findings offer a novel approach for studying the structure-activity relationship of nattokinase and could be extended to other serine proteases.