Improving the Thermostability and Catalytic Performance of the Bacillus subtilis Chitosanase BsCsn46A via Computational Design

Chitosanase plays a pivotal role in the production of chitooligosaccharide. Nevertheless, there is untapped potential for enhancing both its catalytic efficiency and thermostability, which could significantly bolster its therapeutic and biotechnological applications. In this study, two computer-aided protein design methods, namely Fireprot and PROSS, were utilized to pinpoint 6 single Bacillus subtilis chitosanase BsCsn46A mutants (S126A, D191A, K70A, L159I, K104P, and A129L) as well as 4 multiple mutants (K70A/S126A, K70A/S126A/K104P, K70A/S126A/L159I, and K70A/S126A/K104P/L159). The wild-type (WT) and all 10 BsCsn46A mutants displayed robust adaptability across a broad pH range, exhibiting peak activity within the pH spectrum of 5.5 to 9.5. The results demonstrated that, compared to the WT, 9 out of 10 mutants exhibited significantly heightened chitosanase activity, with the sole exception being the D191A mutant, which displayed activity levels nearly identical to the WT. Notably, the A129L displayed an impressive 20% increase in the enzyme activity at elevated temperatures, specifically in the range of 55-80 degrees C. Assessing protein stability, results indicated that all samples maintained stability when incubated at 30 degrees C for 1 h. However, when subjected to a higher temperature of 40 degrees C for 1 h, only the A129L mutant retained stability, which persisted even after an extended incubation period of 3 h at 40 degrees C. Furthermore, a thermal stability analysis revealed noteworthy differences between the WT and the mutants. The WT chitosanase activity diminished by 50% after brief 30-min incubation at 50 degrees C, whereas the K70A/S126A, K70A/S126A/K104P, and A129L mutants maintained 50% of their activity for approximately 2 h under the same conditions. In summary, the study provides valuable insights into the thermostability and catalytic activity of chitosanase, highlighting promising candidates for industrial chitosanase applications.