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

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.