Abstract
Site-directed mutagenesis was employed to investigate the structure-function relationship of nattokinase (NK) and its effect on the enzymatic activity, thermostability, pH tolerance, and fibrinolytic properties of NK. Specific mutations (T270S, V271I, E262D, and A259T) were introduced within the nk gene, targeting regions predicted to be involved in substrate binding. The NK(E262D) mutant exhibited a significant increase in enzymatic activity (2-fold) and catalytic efficiency (2.2-fold) as assessed by N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (Suc-AAPF-pNA) hydrolysis, compared to the wild type. In silico analysis supported these findings, demonstrating lower binding energy for the NK(E262D) mutant, suggesting stronger fibrin affinity. Thermostability assays revealed that NK(E262D) and NK(A259T) displayed exceptional stability, retaining enzyme activity at 60°C. All mutants exhibited a broader pH tolerance range (pH 5.0-10.0) compared to the wild-type NK. The fibrinolytic activity assay revealed that the E262D mutant possessed the highest fibrinolytic activity (2414 U/mg), surpassing the wild-type. This study reported an NK variant with improved enzymatic activity, thermostability, and fibrinolytic properties.
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