Research Of Site-directed Mutagenesis Of Phytase Gene

Site-directed mutagenesis (site directed mutagenesis, SDM) technology is abio-engineering research in recent years in an area of rapid development. Through site-directedmutagenesis can transform the purpose of in vitro DNA molecules, thus the study of gene expression and protein structure and function relationship. Site-directed mutagenesis techniques can be divided into PCR-mediated and non-PCR-mediated, as in recent years the rapid development of PCR, the-mediated site-directed mutagenesis method is also more and more widely applied, in particular the large primer site-directed mutagenesis method method has a decisive position. But this method has some inadequacies: excess primers and wild mutation affected the efficiency of the template. In this study, the traditional methods of large mutation primers improved through the use of asymmetric amplification to eliminate the excess primers and adding enzyme DpnI to eliminate the wild template efficiency of the mutant and in the process of eliminating the need for gel purification, simplifying traditional methods of steps to save time. Phytase in the application of this method of site-directed mutagenesis studies, the mutation made the expected results.Phytase is catalyzed hydrolysis of phytic acid and its salts into lower phosphorylated inositol and phosphoric acid (phosphate) is a general term for a class of enzymes. Phytase in the feed of animals will not only enhance the absorption of phosphorus, reducing phosphorus pollution of the environment, but also the lifting of phytic acid on the metal ions, the chelating amino acids greatly enhanced the efficiency of feed utilization, feed industry at home and abroad has a broad application prospects. So this year, more and more people to its transformation, the big test to take to improve primer method, Q278, K281 mutation and two mutations in the gene transferred into the eukaryotic expression hosts. With the yeast of the GA-22 phytase enzyme produced by the comparative analysis of the nature, GA-22 in pH3.0 and pH5.0 have an activity peak, K281E mutant strains (EE-1) enzyme live for 81696U/mL, increased the activity of 20%, the optimum pH value of 5.0, only one activity peak. K281EQ278L the mutant strain (EE-2) enzyme activity for the 62376U/mL, the optimum pH value of 5.5, only one activity peak in the thermal stability of their study found that at 60℃and 80℃processing 30min K281E mutant strain after the relative residual activity of 86.6%, 79.8%, at 60℃and 80℃to deal with K281EQ278L after 30min the residual mutant enzyme activity relative to 91.2%, 57.2%, while the GA-22 through the same processing After the relative residual activity of 79.8%, 60.2% and was informed that the two-point mutation of only a slight increase in the engineered strain GA-22 of the thermal stability.