Improving Process Stability and Activity of B. subtilis GH46 Chitosanase via Directed Evolution: Insights into Active-Site Cleft Dynamics

The enzymatic production of low-molecular-weight chitosan and chitooligosaccharides (COS), with broad application potential in agriculture, food, medicine, and cosmetics, has emerged as an attractive alternative to chemical chitosan depolymerization owing to its substrate specificity and environmentally benign catalytic action. However, the functional properties of available chitosanases need to be enhanced to meet the demands of industrial COS manufacturing under high temperature and substrate concentrations. In this work, we performed directed evolution on a recombinant Bacillus subtilis chitosanase to increase chitosan hydrolysis performance and thermal resistance. Three rounds of directed evolution screening (~9000 clones) yielded variants MT1, MT2, and MT3 with higher specific activity, achieved through Vmax improvement and increased T1/2 at 60 °C. HPLC, DLS, and MALDI-TOF results indicate differences in the hydrolysis kinetics and size distribution of COS products over reaction time, suggesting a narrower distribution and a lower average molecular weight. Molecular dynamics simulations and docking studies revealed potential modulation of chitosanase activity via changes in the opening and closing dynamics of the active-site cleft. These results suggest that future efforts targeting the cleft interface could significantly advance both the catalytic performance and the mechanistic understanding of GH46 family chitosanases.