Engineering the terminal regions of Chondroitinase AC to improve the thermostability and activity

High catalytic activity and thermostability are essential for enzymes, especially industrial enzymes. A Chondroitinase AC derived from Pedobacter xixiisoli (PxChSaseAC) shows high catalytic activity, while its poor thermostability might be a bottleneck that seriously hinders its industrial application. In this study, the structure of PxChSaseAC was analyzed, and six amino acid residues with high flexibility located at the N-terminal and C-terminal domains were selected as candidate mutation sites. Subsequently, a mutation library containing 27 mutants was constructed by combining the mutation prediction module of DynaMut and consensus design. The thermostability of 22 out of the 24 mutants that could be expressed improved to varying degrees, and the circular dichroism (CD) spectroscopy revealed that the spatial structures of these mutants were not appreciably altered. In particular, the half-time of K43G and S673V at 37°C increased by 95.0% and 193.8%, respectively. Surprisingly, the catalytic activity of these two mutants was also 39.7% and 27.13% higher than that of WT. Molecular dynamics (MD) simulations and molecular docking indicated that it may be due to the enhanced local rigidity of the mutated enzyme, as well as the enlargement of the catalytic cleft and the affinity for the substrate.