Thermostability Improvement of the Chitinase from Bacillus circulans for Efficient Chitin Oligosaccharide Production...

The chitinase A1 from Bacillus circulans WL-12 (BcChiA1) exhibits promising potential for producing chitin oligosaccharides (CHOs), while its application is limited by its poor thermal stability. In this study, a set of thermostable variants were obtained by modifying BcChiA1 using a comprehensive strategy based on a computer-aided design. A combination of five beneficial single-point mutations (S67G/K177R/A220V/N257Y/N271E) to BcChiA1 generated a markedly improved variant, Mu5. Mu5 exhibited a half-life of 295 min at 60 °C, which was 59 times higher than that of BcChiA1. Furthermore, Mu5 was reused for chitin conversion, releasing 86.14 ± 3.73 mM of CHOs after five reaction cycles. Molecular dynamics simulation and structural analysis revealed that these enhancements were driven by increased structural rigidity and compactness, resulting in a protein conformation that was less prone to thermal denaturation. This combined approach through computational design yielded a thermostable BcChiA1 variant, potentially facilitating CHOs production in economical way.