Improving the Thermostability of Serine Protease PB92 from Bacillus alcalophilus via Site-Directed Mutagenesis Based on Semi-Rational Design

Proteases have been widely employed in many industrial processes. In this work, we aimed to improve the thermostability of the serine protease PB92 from Bacillus alcalophilus to meet the high-temperature requirements of biotechnological treatments. Eight mutation sites (N18, S97-S101, E110, and R143) were identified, and 21 mutants were constructed from B-factor comparison and multiple sequence alignment and expressed via Bacillus subtilis. Among them, fifteen mutants exhibited increased half-life (t(1/2)) values at 65 degrees C (1.13-31.61 times greater than that of the wild type). Based on the composite score of enzyme activity and thermostability, six complex mutants were implemented. The t1/2 values of these six complex mutants were 2.12-10.05 times greater than that of the wild type at 65 degrees C. In addition, structural analysis revealed that the increased thermal stability of complex mutants may be related to the formation of additional hydrophobic interactions due to increased hydrophobicity and the decreased flexibility of the structure. In brief, the thermal stability of the complex mutants N18L/R143L/S97A, N18L/R143L/S99L, and N18L/R143L/G100A was increased 4-fold, which reveals application potential in industry.

Automated summary

This study aimed to improve the thermostability of the serine protease PB92 from Bacillus alcalophilus to meet the high-temperature requirements of biotechnological treatments. Mutations were introduced at eight sites, and 21 mutants were constructed, among which 15 mutants showed increased half-life values at 65 degrees C. Six complex mutants with improved thermal stability were identified based on enzyme activity and thermostability scores, and structural analysis suggested that the increased stability might be due to additional hydrophobic interactions and reduced flexibility. Overall, the N18L/R143L/S97A, N18L/R143L/S99L, and N18L/R143L/G100A mutants showed 4-fold increase in thermal stability, indicating potential industrial applications.