Reconstruction of Novel Bacterial Nattokinase Variants through Evolutionary Biological Records

Nattokinase (NK) is a potential therapeutic enzyme obtained from Bacillus subtilis natto. It has fibrinolytic activity and is recognized for breaking down fibrin and preventing blood clot formation. In recent years, NK has been a promising natural alternative for treating thrombotic conditions and offering advantages over chemical synthetic agents, such as being safe, natural, orally active, and cost-effective. In this study, the evolutionary biological records were used to reconstruct the new bacterial NK variant using advanced bioinformatics. Evolutionary sequence conservation and structural-functional information-driven ancestral sequence reconstruction were carried out. The bacterial serine protease (NK) has significant sequence and structural similarities with their homologs. Interestingly, it was observed that active sites and some structural and functionally important residues are highly conserved during the evolution. The reconstructed new microbial NK variants show a similar structure fold to that in reported peptidase family S8. All designed new forms of NKs comprised signal and propeptide regions that play important functional roles. This novel information guides the design of new natural NK variants and enzyme engineering. This approach not only produces NK variants with significantly improved stability, activity, and therapeutic potential for cardiovascular health but also instills hope for the promising future of this enzyme engineering.