Synergistic engineering of Nattokinase terminal and calcium-binding regions for enhanced catalytic performance and Oral stability.

Nattokinase,a potent thrombolytic enzyme, is hindered by low activity and poor thermal stability, limiting its therapeutic and industrial applications. This study tackles these issues using an AI-driven approach to boost nattokinase's catalytic performance, based on the hypothesis of synergistic regulation between the terminal structure and calcium-binding region, we utilized the DeepDDG and MPEK servers to predict mutations in the Q275G mutant, focusing on improving both stability and catalytic activity. Our approach identified the Q275G-T174C (M2) mutant, which successfully mitigated the typical stability-activity trade-off. Characterization revealed that M2 exhibits significantly enhanced stability across various pH levels and increased thermostability, with a 6.2-fold longer half-life at 55 °C compared to the wild-type. Molecular dynamics simulations and RMSF analysis revealed that revealed elevated flexibility in M2 around residues 212-214 (β-sheet), but reduced fluctuations in segments 40-45 and 99-102 (loop regions) compared to the wild-type. M2 demonstrated superior resistance to degradation in simulated digestive fluids, suggesting enhanced oral bioavailability. The fermentation process for M2 production in Escherichia coli was also optimized, achieving a high enzyme activity of 528.63 ± 3.42 U/mg. This research provides a novel strategy for engineering nattokinase, offering a promising enzyme molecule with balanced activity and stability for improved thrombosis therapy and industrial applications.