Phytase Expressed By PIAβ8 And PGAPZαA Vectors And Analysis Of Its Biochemical Characters

SummeryOur objectives were to determine the biochemical characters of phytase from A. ficuum,to compare the ability of phytase gene expression in E.coli by pIAβ8 and pGAPZαA vectors,and to choose the optimum vector for fresher researches.A.ficuum was incubated with solid medium.Phytase was extracted and purified by salting-out process,dialysis,laminar analysis.The purified phytase was used for thermostability and pH determination.The optimum pH points of phytase were 2.5 and 5.5,and optimum temperature was 50℃.It is effective to hydrolyze phytate when this kind of phytase was used in animal feed.The genomic DNA from A.ficuum has been extracted successfully.A 1.4 Kb phytase gene was amplified by PCR.Then the PUCm-T vector and 1.4-Kb DNA were ligated.PCR detection and digestion with restriction endonucleases of XbaⅠand KpnⅠwere used to check the inserted DNA.The nucleotide sequence of phytase gene was analyzed,and it has been deposited in the GenBank nucleotide sequence database (Ref.EF206311).The homologies of the gene nucleotide sequences and deduced amino sequences between the amplified gene products and the target sequence of phytase were 99.5%and 99.6%,respectively(Ref.AY013315).The deduced amino acid sequences have conserved the sequence in the active site of phytase.The recombinant PUCm-T vectors were partially digested with XbaⅠand KpnⅠto isolate phytase gene and then purified with the kits as above.The plasmids(pIAβ8 and pGAPZαA) were also partially digested with XbaⅠand KpnⅠand purified with the kits, respectively.Ligase was used to construct the recombinant of pIAβ8-phyA and pGAPZαA-phyA vectors.The recombinant shuffle vectors were inserted into E.coli by chemical method.By the comparison of the expressed phytase activity between pGAPZαA-phyA and pIAβ8-phyA in the supernatant of culture and the cells,it could be concluded that the recombinant of pIAβ8-phyA could secrete intracellular phytase(9.04 U/ml,P＜0.05), while pGAPZαA-phyA could secrete extracellular phytase(8.04U/ml,P＜0.05).The molecular weight of the expressed phytase in this study was 54.61 KDa screted in E.coli. It had the same molecular mass as that of the commercial phytase as determined by SDS-PAGE.