A disulfide bond mutant of Pseudoalteromonas porphyrae κ-carrageenase conferred improved thermostability and catalytic activity and facilitated its utilization in κ-carrageenan industrial waste residues recycling

In this study, Discovery Studio was employed to predict the potential disulfide bond mutants of the catalytic domain of Pseudoalteromonas porphyrae kappa-carrageenase to improve the catalytic activity and thermal stability. The mutant N205C-G239C was identified with significantly increased catalytic activity toward kappa-carrageenan substrate, with activity 4.28 times that of WT. The optimal temperature of N205C-G239C was 55 degrees C, 15 degrees C higher than that of WT. For N205C-G239C, the t(1/2) value at 50 degrees C was 52 min, 1.41 times that of WT. The microstructural analysis revealed that the introduced disulfide bond N205C-G239C could create a unique catalytic environment by promoting favorable interactions with kappa-neocarratetraose. This interaction impacted various aspects such as product release, water molecule network, thermodynamic equilibrium, and tunnel size. Molecular dynamics simulations demonstrated that the introduced disulfide bond enhanced the overall structure rigidity of N205C-G239C. The results of substrate tunnel analysis showed that the mutation led to the widening of the substrate tunnel. The above structure changes could be the possible reasons responsible for the simultaneous enhancement of the catalytic activity and thermal stability of mutant N205C-G239C. Finally, N205C-G239C exhibited the effective hydrolysis of the kappa-carrageenan industrial waste residues, contributing to the recycling of the oligosaccharides and perlite.