Abstract: This study aimed to improve the thermostability of κ-carrageenase from Pseudoalteromonas sp. JMUZ2 by rational design. A total of 10 single-site mutants were chosen using the PoPMuSiC program to analyze the sequence of the κ-carrageenase gene. A mutant gene was obtained by site-directed mutagenesis. The mutant enzyme was expressed under induced conditions, purified and identified. The mutant K155A could maintain the specific enzyme activity and had improved thermostability. After treatment at 50, 55, and 60 ℃ for 40 min, the thermal stability of the mutant K155A was 1.8, 2.7, and 4.5 times higher than that of the wild-type enzyme (WT), respectively. The structure and molecular dynamic simulation analysis showed that the increase in hydrophobic interactions and structural rigidity may be the cause of the improved thermal stability of K155A. This study is of great significance for improving the properties of κ-carrageenase, expanding its application scope, and studying its structural-functional relationship.

Key words: κ-carrageenase; thermostability; rational design