利用loop区域理性设计策略提升硫酸软骨素裂解酶ABC的热稳定性

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

摘要:

硫酸软骨素裂解酶ABC (chondroitinase ABC, ChABC)在生物医药及多糖结构解析等领域具有重要应用价值，但其较差的热稳定性严重限制了该酶的工业化生产及应用。本研究针对来源于普通变形杆菌(Proteus vulgaris)的PvChABC的多结构域特征，以该酶N端结构域与核心催化结构域之间的loop区域为靶点，采用计算机辅助的理性设计策略对其进行分子改造，获得稳定性显著提升的优势突变体M3。该突变体在45 ℃下的半衰期达到(48.12±1.85) h，是野生型的13.7倍，且其比酶活为野生型的110.5%，没有表现出酶活性的损失。分子动力学模拟揭示，loop区域的突变通过引入关键疏水相互作用与氢键网络，增强了结构域间的结合能力，从而稳定了整体蛋白构象。此外，PvChABC热稳定性的提升有助于异源表达该酶的工程菌株在37 ℃下发酵产酶，该温度下M3的产量较野生型提高14.84倍，更适合工业化生产应用。本研究不仅为PvChABC的工业生产和应用奠定了基础，也为其他多结构域蛋白的分子改造提供了思路。

Abstract:

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.