Engineering of Chondroitin Sulfate Lyase AC to Improve the Thermostability and Catalytic Efficiency with Free Energy Optimization

Chondroitin sulfate lyase is widely used for the degradation of glycosaminoglycans, but its thermostability frequently limits its practical application. In this study, a stability-oriented protein engineering strategy was applied to enhance the performance of a chondroitin sulfate lyase AC. FoldX and DynaMut2 were employed to perform energy optimization and stability prediction for the mutants, and identified a double mutant, A42D/S62P, with notably improved thermostability. The half-life at 37   o C was increased by 4.48-fold relative to the wild type (WT), along with 23.80% enhancement in specific activity. Molecular dynamics simulations indicated that the main causes of stability improvement were reinforced local interactions and reduced conformational flexibility in dynamic regions. Furthermore, an integrated catalytic performance metric demonstrated that the engineered enzyme achieved nearly two-fold improvement in effective catalytic output under operational conditions. These results highlight the effectiveness of free-energy-based computational design in improving enzyme stability and performance, and demonstrate that a chondroitin sulfate lyase mutant exhibits improved performance for biocatalytic applications.