Rational engineering of a metalloprotease to enhance thermostability and activity

The rational design of enzymes with enhanced thermostability is efficient. Solvent-tolerant metalloprotease from Pseudomonas aeruginosa PT121 presents high Z-aspartame (Z-APM) synthesis activity, but insufficient thermostability. In this study, we enhanced enzyme thermostability using a rational strategy. Molecular dynamics (MD) simulation was applied to rapidly identify that the D28 and D116 mutations are likely to exhibit increased thermostability, and experimentation verified that the D28N and D116N mutants were more stable than the wild-type (WT) enzyme. In particular, the T-m of the D28N and D116N mutants increased by 6.1 degrees C and 9.2 degrees C, respectively, compared with that of the WT enzyme. The half-lives of D28N and D116N at 60 degrees C were 1.07- and 1.8-fold higher than that of the WT, respectively. Z-APM synthetic activities of the mutants were also improved. The potential mechanism of thermostability enhancement rationalized using MD simulation indicated that increased hydrogen bond interactions and a regional hydration shell were mostly responsible for the thermostability enhancement. Our strategy could be a reference for enzyme engineering, and our mutants offer considerable value in industrial applications.

Automated summary

The rational design of enzymes with enhanced thermostability is efficient. This study successfully improved the thermostability and synthetic activity of a metalloprotease through a rational strategy, resulting in mutants with increased stability and enhanced Z-aspartame synthesis activity. Molecular dynamics simulation revealed that increased hydrogen bond interactions and a regional hydration shell were key factors in enhancing thermostability. This research provides valuable insights for enzyme engineering and has significant implications in industrial applications.