Construction of highly active and stable recombinant nattokinase by engineered bacteria and computational design

Nattokinase (NK) is an enzyme that has been recognized as a new potential thrombolytic drug due to its strong thrombolytic activity. However, it is difficult to maintain the enzyme activity of NK during high temperature environment of industrial production. In this study, we constructed six NK mutants with potential for higher thermostability using a rational protein engineering strategy integrating free energy-based methods and molecular dynamics (MD) simulation. Then, wild-type NK and NK mutants were expressed in Escherichia coli (E. coli), and their thermostability and thrombolytic activity were tested. The results showed that, compared with wild-type NK, the mutants Y256P, Q206L and E156F all had improved thermostability. The optimal mutant Y256P showed a higher melting temperature (Tm) of 77.4 °C, an increase of 4 °C in maximum heat-resistant temperature and an increase of 51.8 % in activity at 37 °C compared with wild-type NK. Moreover, we also explored the mechanism of the increased thermostability of these mutants by analysing the MD trajectories under different simulation temperatures. [Display omitted] •The mutants in this study improved thermostability compared to wild-type NK.•The maximum heat-resistant temperature of mutant Y256P was increased by 4 °C.•The activity of the mutant Y256P was increased by 51.83 % at 37 °C.•The melting temperature of mutant Y256P was higher than wild-type NK.•This study explored the mechanism of increased thermostability of NK mutants.