Improving the catalytic activity and stability of Bacillus alcalophilus serine protease BAPB92 by rational design

The catalytic activity and stability of proteases are essential for their application in the detergent industry. To enhance the catalytic properties of BAPB92, homologous sequence comparison combined with rational design was employed. Six mutants were generated: BAPB92 (A188P), BAPB92 (V262I), BAPB92 (Q239R), BAPB92 (A188P/V262I), BAPB92 (Q239R/V262I), and BAPB92 (Q239R/A188P). Remarkably, the mutant BAPB92 (A188P/V262I) exhibited the most significant improvement, exhibiting a 4.30-fold increase in kcat/Km compared to the wild type, and a 0.75-fold enhancement in thermal stability at 60 °C. The enzymatic activity of BAPB92 (A188P/V262I) reached 6511.81 U/mg, which was 2.95 times higher than that of the wild type BAPB92. Furthermore, the optimal reaction temperature of this mutant increased from 50 °C to 60 °C. The BAPB92 (A188P/V262I) mutant also showed a marked improvement in detergent stability. In sodium tripolyphosphate liquid detergent, its washing efficacy was 17.84 % higher than that of the wild type, and in methyl glycine diacetate liquid detergent, the improvement was 18.51 %. These findings suggested that BAPB92 (A188P/V262I) holds significant potential as a detergent protease in the washing industry. Structural analysis and molecular dynamics simulations further confirmed the enhanced stability of this mutant compared to the wild type. This study provides valuable theoretical insights for the application of the serine protease BAPB92 in detergent formulations. •An efficient rational design strategy for improving the catalytic activity and stability of Bacillus alcalophilus serine protease BAPB92.•Mutant A188P/V262I showed a 4.30-fold increase in catalytic activity compared to the wild type.•Molecular dynamics simulations indicated the mutant had more stable active structure.