Thermostability Improvement Of Pseudoalteromonas Carrageenovoraarylsulfatase By Direct Evolution

In this study,in order to improve the thermal stability of arylsulfatase from Pseudoalteromonas carrageenovora,error-prone PCR was used for the direct evolution of this enzyme.The mutants with higher thermal stability were successfully obtained.The secondary and tertiary structure of P.carrageenovora arylsulfatase were predicted.Site-directed mutagenesis and saturation mutagenesis methods were applied to study the relationship between the enzyme's microstructure changes and thermal stability.Finally,the enzymatic proterties of the mutant enzymes were characterized and the mutant enzymes'abilities to remove the sulfate groups from Gracilaria lemaneiformis agar were also investigated.In the previous study,the recombinant plasmid?pET-28a-ars?containing the arylsulfatase gene of P.carrageenovora was constructed.A random mutant library of this arylsulfatase was generated by error-prone PCR with the recombinant plasmid as the template.The mutant frequency of the library was 0.22%,and the library had approximately 2.0×10⁵ random mutants.After the screening,two mutants named 4-138 and 4-153 were observed to display higher thermostability.According to the sequence analysis,the mutant arylsulfatase 4-138 had one alteration of H259L in amino acid residue,and the mutant arylsulfatase 4-153 had two alterations?including D83A and H259L?.Two mutants had the same alteration at the position of 259,the amino acid at this position was predicted to be important for the thermostability of P.carrageenovora arylsulfatse.Mutant 4-138,mutant 4-153 and wild-type arylsulfatases were named H259L,4-153 and WT,respectively.H259L and 4-153 retained 62%and 48%of residual activities after incubation at 55°C for 30 min,while WT could only keep 21%of residual activity at the same conditions.In order to study the microstructure changes of P.carrageenovora arylsulfatase,the secondary and tertiary structures of this enzyme should be simulated according to the amino acid sequence.Online server and circular dichroism spectroscopy were used to predict secondary structure of this arylsulfatase.According to the results,there were 19%-21%of?-helix,29-30%of?-strand and 13.4%of?-turn in the enzyme's secondary structure.Modeller 9.15 was used to simulate the three-dimensional model of the enzyme with the method of multi-templates.Three-dimensional model of the arylsulfatase was optimized by loop refine and energy minimization.This model also was adopted by PSVS to assess the responsibility of the model.Using site-directed mutagenesis method,the single point mutant of D83A was constructed.The thermal stability of two single point mutants?D83A and H259L?and one double points mutant 4-153 were analysed.At 55°C,the half-times of H259L?42 min?and 4-153?23 min?were10.5-fold and 5.7-fold of the one of WT?4 min?.A saturation mutagenesis library of 259 amino acid residue was constructed.And four mutants including H259F,H259Q,H259I and H259R were also observed to have higer thermostability than wild-type arylsulfatase,remaining more than 35%of residual activity after incubation at 55°C for 30 min.The microstructure analysis showed that the numbers of hydrogen bond of the five mutants?H259L,H259F,H259Q,H259I and H259R?increased.In addition,decreasing the hydrophobicity of the surface molecules,increasing enzyme's rigidity,and optimization of surface charge-charge interactions,might be important for the thermostability improvement of the mutants.Finally,some enzymatic properties of 4-153,H259L and WT were investigated.Except the minor difference on the optimum pH,there were no changes on pH stability,the effects of metal ions,inhibitors and detergents on the mutant and wide-type enzymes.The V_maxax and K_m of H259L were lower the ones of the WT,but the kinetic parameters of 4-153 were almost the same as WT.Mutant and wide-type enzymes to applied to remove the sulfate groups on Gracilaria lemaneiformis agar.H259L and 4-153 could remove 82.1%and 79.3%of the sulfate groups,respectively.Under the same conditions,the wild-type arylsulfatase could only remove 76.1%of the sulfate groups.