[A rational loop region engineering strategy for enhancing the thermostability of chondroitinase ABC]

Chondroitinase ABC (ChABC) holds significant value in biomedical applications and polysaccharide structure analysis. However, the poor thermostability of this enzyme severely limits its industrial production and utilization. Focusing on the multidomain characteristics of PvChABC derived from Proteus vulgaris and targeting the loop region connecting the N-terminal domain and the catalytic domain, we obtained an optimized mutant M3 with significantly improved thermostability through a computer-aided rational design strategy. M3 exhibited a half-life of (48.12±1.85) h at 45 ℃, which represented a 13.7-fold increase over that of the wild type. It showed the specific activity 110.5% of that in the wild type, which indicated no loss of enzymatic activity. Molecular dynamics simulations revealed that the mutation in the loop region introduced hydrophobic interaction and hydrogen bond network, thereby enhancing the inter-domain binding and stabilizing the overall protein conformation. Notably, the enhanced thermostability enabled efficient heterologous expression at 37 ℃, with M3 showing a 14.84-fold higher yield than the wild type under industrial fermentation conditions. This work not only establishes a foundation for the industrial production of PvChABC but also provides a generalizable strategy for engineering other multidomain proteins.