Enhancing thermostability of alkaline κ-carrageenase from Pseudoalteromonas tetraodonis by rational design of disulfide bonds

Abstract

The industrial applications of the κ-carrageenases have been restricted by their poor thermostability. In this study, based on the structural information of alkaline κ-carrageenase from Pseudoalteromonas tetraodonis (WT) and with the aid of Disulfide by Design2 computational tool, two disulfide bond mutants of K44C-A119C and T120C-Q250C were identified with improved thermostability and enzymatic activity. K44C-A119C and T120C-Q250C increased the enzyme activity by 19.0% and 7.3%, respectively. After treatment at 50 °C for 30 min, the residual activities of K44C-A119C, T120C-Q250C, and WT were 80.1%, 78.6%, and 45.4%, respectively. The Tm values of K44C-A119C, T120C-Q250C, and WT determined by differential scanning calorimetry were 56.0 °C, 54.8 °C, and 51.2 °C, respectively. The increasement of disulfide bond and its adjacent non-covalent interactions would account for the increased thermostability of the mutants. Molecular dynamics simulation analysis revealed that the improved thermostability and catalytic activities of K44C-A119C and T120C-Q250C could be attributed to the increased flexibility of finger F6 and the less structural deviation in some loops and β-sheets. In addition, the κ-carrageenan hydrolysates by K44C-A119C exhibited higher tyrosinase inhibitory activity than the untreated κ-carrageenan. These findings provide a useful strategy to improve κ-carrageenases’ thermostability and enzyme activity for their better industrial applications.