Loop Dynamics Perturbation Enhances the Catalytic Stability of GH18 Family Chitinases

Enhanced thermostability is crucial for industrial biocatalysts, yet it often compromises the catalytic activity by restricting functional flexibility. Loop regions, with their intrinsic structural flexibility, have become key targets to improve thermostability while maintaining catalytic efficiency. In this study, we revealed that the dynamics of heat-sensitive loop2 and loop4 played a critical role in regulating the catalytic stability of GH18 chitinase Ss Chi18A by molecular dynamics simulations coupled with alanine scanning, providing a basis for function-driven enzyme optimization. The high flexibility of loop2 likely facilitated substrate capture and enhanced catalytic stability at elevated temperatures. Mutations A43T, Q41K, and Q41R increased enzyme half-life by 3, 6, and 7.5 h, respectively, and boosted activity by 23–30%, mainly by stabilizing the loop2 edge. Loop4 enhanced thermal stability by dynamically stabilizing the proton-donor Glu through intramolecular interactions, with hyperthermophilic chitinases featuring a 10-residue extended loop4 that contributes up to a value of −2979 kJ/mol in intramolecular interactions. The A189 V mutation of Ss Chi18A extended the half-life by 35 h and boosted chitinase activity by 34% through enhancing hydrophobic interactions. The function-driven design strategy precisely modulated loop dynamics through intramolecular interactions (e.g., hydrogen bonding and hydrophobic packing), overcoming the activity–stability trade-off and providing a framework for developing thermostable biocatalysts.