Site-directed Mutagenesis And Thermal Stability Study Of Phytase From E.Coli

Phytase is one kind of the enzymes acidic phosphorylation, which can catalyze hydrolysis of phytate to inositol and inorganic phosphate. As additives, phytases are currently used in breeding industry, food processing and pharmaceutical production. As an agricultural feed additive, phytase is used to increase animals’utilization of phosphorus in feed, improve the production performance of animal, and reduce phosphorus pollution of environment caused by high phosphorus in animal feces. Pichia pastor is is an eukaryotic expression system developed in the80s, takes the advantages of easy to operate, easy to cultivate, fast growth, and high level of expression but low cost. Pichia pastoris is widely used in the field of medicine production and scientific research, for the unique advantages in the expression of exogenous protein.In this study, sitedirected mutagenesis was performed using the recombinant plasmid pGEM-T-appA~m containing the appAm gene which was sourced from E.coli as a template, in order to screening an industrial production engineering bacterium which can produce phytase with good thermal stability and more conducive to industrial production.By using of the relevant knowledge of biochemistry and molecular biology, six mutations were designed and six complementary primers were designed. The mutated genes were inserted into pPIC9vector, and then transformed into P. pastoris GS115by the method of LiCl. By the way of SDS-PAGE and the phytase activity assay, six positive transformants containing different mutations were obtained successfully (M1,K24E,K43E,C2,M2,M4).The purity of APPA and the6mutants comply with the requirements of the downstream analysis through three steps:ammonium sulfate precipitation, ion exchange chromatography and glucan gel chromatography.Through determination of the phytase activity by the method of molybdate blue, the optimal reaction temperature and pH, heat resistance and pH stability, Km, Vm was determined.The results show that the optimum reaction temperature of mutant M2and M4has been increased5℃, the optimal pH of mutant K24E has been reduced0.5, all the phytase remained activity more than80%after been treatmented in pH2～8for2h except C2and K43E, after been treatmented in70℃for20min, the phytase APPA had almost no residual activity, but mutant M2, the M4, K24E Ml and remained more than50%. Through all of the experiments, we finally obtained the mutant K24E, which showing the best performance. The optimum temperature for60℃, the optimum pH4.0.The residual activity incubation in70℃,80℃and90℃for20min, and more than80%after incubation in the buffer pH2-8for2h. In addition, the kinetic parameters indicated that the mutant K24E53%lower km value and20%higher Vm than that of APPA.