Improve thermostability of Bacillus sp. TS chitosanase through structure-based alignment

Chitosanases can catalyze the release of chitooligosaccharides which have a number of medical applications. Therefore, Chitosanases are good candidates for large-scale enzymatic synthesis due to their favorable thermostability properties and high catalytic efficiency. To further improve the thermostability of a chitosanase from Bacillus sp. TS, which has a half-life of 5.32 min, we mutated specific serine residues that we identified as potentially relevant through structure comparison with thermophilic CelA from Clostridium thermocellum. Out of a total of 15 mutants, three, namely S265G, S276A, and S347G, show higher thermostability. Their half-lives at 60 degrees C were calculated as 34.57 min, 36.79 min and 7.2 min. The K-m values of S265G, S276A and S347G mutants show substrate binding ability comparable to that of the wild-type enzyme, while the S265G mutant displays a significant decrease of enzymatic activities. Additionally, we studied the synergistic effects of combined mutations, observing that all double mutants and the triple mutant are more stable than the wild-type enzyme and single mutants. Finally, we investigated the mechanisms which might give a reasonable explanation for the improved thermostability via comparative analysis of the resulting 3D structures.

Automated summary

Chitosanases can catalyze the release of chitooligosaccharides with numerous medical applications. Mutations targeting specific serine residues in a chitosanase from Bacillus sp. TS, guided by structural comparison with CelA from Clostridium thermocellum, resulted in three mutants with improved thermostability. The mutated enzymes exhibited longer half-lives at 60 degrees Celsius compared to the wild-type enzyme.