Rational Engineering of a GH18 Chitinase for Enhanced Thermostability and Catalytic Efficiency via Dynamic Allosteric Modulation

Engineering chitinases with simultaneous improvements in catalytic efficiency and thermostability remains challenging. Here, we present a computational strategy targeting the insertion domain (CID), a noncatalytic auxiliary region. Using CaChi18A_ΔChBDs as the starting scaffold, folding free energy calculations identified a stabilizing mutation, A347P, within the CID hydrophobic core, which significantly enhanced thermostability by extending the half-life at 45 °C by 56 min. Based on this stabilized scaffold, evolutionary coupling analysis and stability predictions guided the design of secondary mutations, yielding several double mutants with improved catalytic performance. The best variants (A347P/A137S, A347P/A158G, and A347P/E219A) exhibited increases in specific activity of up to 99% compared with the wild-type and substantially prolonged half-lives at 50 °C. Notably, A347P/A137S produced 4.9-fold more GlcNAc during colloidal chitin hydrolysis. These results demonstrate that CID-targeted engineering provides an effective strategy for optimizing multidomain chitinases.