Mining and Modification of Key Functional Regions of the Nattokinase Propeptide

The nattokinase propeptide acts as an intramolecular chaperone that is essential for the correct folding of the mature peptide. Understanding key catalytic regions and sites in this propeptide is crucial for enhancing the nattokinase folding efficiency. Through bioinformatics and enzyme activity screening, we pinpointed critical sites affecting folding efficiency and identified mutants Y106V and A103T. In fermentation, their crude enzyme activities reached 277.83 and 205.63% of those of WT, respectively. Mutant Y106V exhibited a 19.2% increase in the specific enzyme activity, improving both the folding efficiency and peptide conformation. Molecular dynamics simulations elucidated the catalytic changes in Y106V and A103T. Optimization of fermentation processes for these mutants yielded nattokinase levels of 317.042 and 336.65 U/mL. This study lays a foundation for further research on nattokinase propeptide function and modification.