Improvement of the fibrinolytic activity, acid resistance and thermostability of nattokinase by surface charge engineering.

Nattokinase is a promising thrombolytic drug due to its powerful fibrinolytic effect and few side effects. However, the low fibrinolytic activity and stability of nattokinase have limited its industrial production and oral application. In this study, the basic and neutral amino acid residues on the surface of recombinant nattokinase AprY from Bacillus mojavensis LY-06 (rAprY) were mutated to acidic amino acid residues by surface charge engineering strategy, and two variants K12D and N109D with 92.6 % and 8.4 % increased fibrinolytic activity were obtained. The R45E variant with enhanced acid stability and thermostability was also screened, its acid stability at pH 4 and t1/2 at 55 °C were 3.7-fold and 1.8-fold higher than that of wild type rAprY, respectively. Bioinformatics analysis showed that the increased activities of K12D and N109D variants were related to the increased flexibility of the region around their active centers. The increased rigidity of 97-103 amino acid residues around the active center of R45E may be the reason for its enhanced stability and reduced catalytic activity. The multipoint mutation K12D-N109D (M2)'s catalytic activity did not increase cumulatively, but its pH stability did. The nattokinase variants generated in this study have potential for industrial production and application.