耐热植酸酶突变体的筛选及性质研究

目的:对大肠杆菌Escherichia coli植酸酶基因进行定向进化,获得热稳定性提高的植酸酶突变体.方法:利用易错PCR技术和96微孔板高通量筛选方法获得突变体基因,并对突变酶进行异源表达、纯化及性质研究.结果:通过筛选获得3株热稳定性明显提高的植酸酶突变体APPA1、APPA2、APPA3.酶学性质分析结果表明,...

Screening and Characterization of Thermostable Phytase Mutants

Objective:Directed evolution of Escherichia coli phytase gene to obtain phytase mutants with improved thermostability.Methods:The mutant gene was obtained by error-prone PCR and the high-throughput screening methods of 96-well plate.After heterologous expression and purification,the mutant enzymes were characterized.Results:Three phytase mutants APPA1,APPA2 and APPA3 with significantly improved thermal stability were obtained through screening.The results of enzymatic properties analysis showed that the molecular weight of the three mutants was about 55 kDa,and the optimum p H was 4.5,which was not significantly different from the wild-type.The thermal stability of mutants was significantly improved compared with the wild-type.Among them,the suitable temperature of APPA3 is 65℃,which is 5℃higher than wild enzyme,and 50%of the enzyme activity is retained after treatment at 90℃for 10 minutes.The three-dimensional structure simulation of the enzyme showed that the five mutation sites introduced new hydrogen bonds in the overall structure of the phytase.Conclusion:Obtaining Escherichia coli phytase mutants with improved thermal stability through directed evolution is of great significance to the industrial application of phytase and the study of the relationship between phytase structure and function.