Cloning,expression,characterization And Application Of A β-propeller Phytase Bap2062

Background and purpose:Phytase catalyze the hydrolysis of phytate to lower inositol phosphates and inorganic phosphate at the mild condition.Hence,this class of enzyme have been applied to plant-based feeds as feed additive to improve the negative interactions of phytate with nutrients,reduce phosphorus concentrations in animal excreta and protect the environment against P pollution.Compared to the histidine acid phytase,which widely used in animal feed,β-propeller phytase displays greater thermostability,a more basic pH profile and play an important role in small intestine.This research aims to clone,express and characterize aβ-propeller phytase Bap2062 from Bacillus amyloliquefaciens DSM7 and provide a new choice for commercial application of phytase.Experimental method:The gene of Bap2062 was achieved via PCR from Bacillus amyloliquefaciens DSM7.The recombinant plasmid pET22b-bap2062 was constructed using pET22b as vector and expressed in E.coli transetta（DE3）.Then,the Bap2062 was purified and characterized.Results:The sequencing result shows that the gene got from Bacillus amyloliquefaciens DSM7 has no mutation.The optimum temperature of Bap2062 is75℃.The optimum pH of Bap2062 is 6.5.The K_m and k_cat are 7.02 mM and 156.51s^-1,respectively.And it showed significant effects on hydrolysis of the soybean meal and corn meal.Conclusions:This research constructed the recombinant plasmid pET22b-bap2062 and expressed it in E.coli transetta（DE3）successfully.Protein was purified by Ammonium sulfate precipitation and Anion exchange column chromatography.Bap2062 showed great thermostability and catalytic properties.