| In this study,the thermal stability of Pseudoalteromonas carrageenovora arylsulfatase was improved based on the rational design.Single point mutation,saturation mutation,and complex mutation were performed,and mutants with significantly improved thermal stability were obtained.The structures of the mutant and wild-type arylsulfatase were compared by circular dichroism,fluorescence spectroscopy,protein surface hydrophobicity and microstructure,and then the reasons for the increase in thermal stability were analysed.The mutants with improved thermostability were predicted by PoPMuSiC online server,and10 single point mutants were selected.Using the wild-type arylsulfatase recombinant plasmid as the template,and the selected sites were mutated by the site directed mutagenesis kit,and the enzymes were purified by affinity chromatography.The results of thermal stability analysis showed that two mutants of K253 N and P314 F with improved thermostability were screened.After treatment at 55oC for 30 min,K253 N and P314 F maintained 27.1% and 25.8% residual activity,respectively,while wild type arylsulfatase(WT)only retained 21% of the residual activity.It is speculated that amino acid residues 253 and 314 are the key sites that affect the thermal stability of P.carrageenovora arylsulfatase.In order to further study the effects of amino acid residues on the thermostability of P.carrageenovora arylsulfatase,saturation mutation was performed at 253 and 314 sites.The thermostability analysis of the mutant enzymes showed that the mutants with the most enhanced thermal stability at positions 253 and 314 were K253 H and P314 T,respectively.The optimal temperature of 55oC for K253 H and P314 T is the same as that of WT.The optimal pH of the two mutants shifted from 7.5 to 8.0 compared with WT.Kinetic results showed that K253 H had a higher affinity for the substrate than WT.Based on the synergistic effect,the single mutants of K253 H,H260L and P314 T with improved thermal stability were selected for the compound mutation.The results of thermal stability analysis showed that the best mutant was K253H/H260 L.Its half-life at 55oC was 70.3 min,while that of WT was only 9.1 min.Moreover,the enzyme activity of K253H/H260 L was also increased by 7% compared with that of WT.In order to analyze the reasons for the improvement of thermal stability,the structural changes of K253H/H260 L and wide-type enzyme were explored.The secondary and tertiary structures of K253H/H260 L and WT were analyzed by circular dichroism(CD)and fluorescence spectroscopy,respectively.The results showed that the secondary and tertiary structures of P.carrageenovora arylsulfatase did not change significantly before and after mutation.Using ANS as a probe,the surface hydrophobicity of the enzyme was measured.The results showed that the surface hydrophobicity of K253H/H260 L was lower than that of WT.The decrease of surface hydrophobicity might be the reason for the increase of thermal stability.The microstructure analysis of P.carrageenovora arylsulfatase showed that the number of hydrogen bond in the mutant K253H/H260 L increased.In addition,the optimization of the surface charge-charge interaction,the enhancement of rigidity,and the enhancement of ionic interaction might also be the influence factors that improve the thermal stability of K253H/H260 L. |
