Improving enzyme stability in the presence of organic solvent is crucial for non-aqueous catalysis. In this study, directed evolution was applied to improve the tolerance of metalloprotease PT121 towards organic solvent. In presence of acetonitrile and acetone, three mutants (T46Y, H224 F, and H224Y) of PT121 showed excellent solvent stability, which increased their half-lives by 1.2-3.5-fold as compared to the wild-type enzyme. Kinetic constants (K and k values) of the caseinolysis reaction presented H224 F and H224Y mutants have higher affinity than the wild-type, but T46Y mutant were similar to those of the wild-type enzyme. Interestingly, combined mutants T46Y/H224 F and T46Y/H224Y mutants presented awesome stability and excellent caseinolytic activity. Molecular dynamic simulation suggest that improved enzyme stability may be attributed to extensive non-covalent bond network resulting in a more compact structure. Disruption of the disulphide bond formation between Cys-30 and Cys-58 residues in the F56 V mutant is possibly the reason behind its low stability among all the selected mutants. Additionally, T46Y/H224 F and T46Y/H224Y showed a higher peptide synthetic activity in the presence of organic solvents than the wild-type, which renders these mutant enzymes as promising biocatalysts for biotechnological applications.
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