Modification of the active centre of nattokinase to enhance its thermostability using a strategy based on molecular dynamics simulation, steered dynamics simulation, and conservative prediction.

Introduction: The poor thermostability of nattokinase represents a significant limitation in its potential applications. Additionally, there is a notable absence of studies focused on modifying residues within the active site region of nattokinase with the aim of enhancing its catalytic properties. Furthermore, the direct utilisation of directed evolution often yields unfavourable outcomes, with a considerable workload being a common consequence. Methods: In order to solve the above problems, a new method based on molecular dynamics simulation, steered dynamics simulation and conservative analysis with site-directed mutagenesis was proposed to screen nattokinase mutants with improved thermal stability. Molecular dynamics simulation was used to explain the mechanism of catalytic performance improvement of positive mutants. Finally, the fermentation process of the positive mutant was optimized. Results and discussion: Based on these findings, the mutant A216K was selected for a 5.7-fold increase in half-life at 55°C with a small increase in activity, which further enhanced the mutation library of the thermal stability enhancement site in the enzyme's active centre. The results of the molecular dynamics simulation indicated that the enhancement of the number of hydrogen bonds within the protein and between the protein and the solvent, as well as the augmentation of the rigidity around the calcium ion binding site and the mutation site, were the primary factors contributing to the improvement of the thermal stability of A216K. It is anticipated that this strategy will provide novel insights into enzyme engineering research.