基于计算机辅助的理性设计提高假交替单胞菌κ-卡拉胶酶的热稳定性

Enhancing the thermostability of Pseudoalteromonas sp. κ-carrageenase through computer-aided rational design

摘要:

κ-卡拉胶寡糖因其优异的生物活性在食品与医药领域应用前景广阔，κ-卡拉胶酶是工业化生产高价值卡拉胶寡糖的关键工具酶，但热稳定性不足限制了其应用。本研究旨在通过计算机辅助的理性设计策略，对假交替单胞菌κ-卡拉胶酶进行设计改造，以构建并筛选出热稳定性显著提升的酶突变体。通过分析κ-卡拉胶酶的结构信息，利用Discovery Studio 2019软件对假交替单胞菌κ-卡拉胶酶进行多位点突变筛选。结果表明，突变体G198S的催化效率与热稳定性均得到提升，其比活性和50 ℃下的半衰期较野生型(WT)分别增加了20.5%和27.8 min。分子对接与动力学模拟分析揭示，突变后酶与底物间新增了Pi-Sigma与Pi-Alkyl作用，氢键网络增强，这些是突变体G198S酶活性与热稳定性同时提高的可能原因。分子动力学模拟分析表明，G198S的热稳定性和催化活性的提高可能归因于突变后F5和F6指、关键残基R151的柔性增高，以及一些loop区和β折叠区域刚性的增强。本研究为提高κ-卡拉胶酶热稳定性提供了一种有用的策略，促进了κ-卡拉胶酶结构与功能关系研究以及该酶的工业应用。

Abstract:

κ-carrageenan oligosaccharides hold broad application prospects in the food and pharmaceutical fields due to their excellent bioactivity. κ-carrageenase is a key enzyme for the industrial production of high-value carrageenan oligosaccharides, while the insufficient thermostability limits its application. The objective of this study is to rationally redesign the κ-carrageenase derived from Pseudoalteromonas through a computer-aided strategy, with the aim of constructing and screening enzyme variants exhibiting significantly enhanced thermostability. Discovery Studio 2019 was used for multi-site mutation screening based on the structural information of κ-carrageenase. The results showed that the mutant G198S presented improved catalytic efficiency and thermostability, with its specific activity and half-life at 50 °C increasing by 20.5% and 27.8 min, respectively, compared with those of the wild-type enzyme. Molecular docking and molecular dynamics simulations revealed that new Pi-Sigma and Pi-Alkyl interactions were formed between the enzyme and substrate after mutation, and the hydrogen bond network was strengthened, which may explain the simultaneous enhancement of both enzymatic activity and thermostability of the mutant G198S. Molecular dynamics simulation analysis indicated that the improved thermostability and catalytic activity of G198S may be attributed to increased flexibility of the F5 and F6 fingers and the key residue R151, as well as enhanced rigidity in certain loop regions and β-sheet areas. This study provides a useful strategy for improving the thermostability of κ-carrageenase, contributing to the research on the structure-function relationship and promoting the industrial application of this enzyme.