Research On The Mutation-expression Of Phytase Gene And The Thermostability Of Mutated-protein

Phytase can effectively help monogastric animals digest thephytate in the plant seeds of corn and oil crop,imrove availability of phosphorus, decrease the environment pollution especially water resourses pollution caused by undigested phosphate, and also improve the absorption of nutrition material such as inorganic salt, amino acid and so on, promote the animal growth. So the phytase obtains extentive research and utilize.The major purpose of this thesis is to research on the thermostablity of phytase. Through the method of mutation-PCR, the gene (phyA) for Aspergillus niger phytase was mutated. Building two expression-system PTE21a(+)-phyA/BL21(DE3) and PTE35b(+)-phyA/BL21(DE3) based on mutated-gene, the purpose protein was effectively expressed in two systems. Research on the expression of recombinant protein and the biology activity through comparing the PTE21a(+)-phyA/BL21 and PTE35b(+)-phyA/BL21. In PTE21a(+) -phyA/BL21, the recombinant protein was in the form of inclusion body and reached 30% in thallus protein.The biology activity of renature protein reached 1500U/mg;In PTE35b(+)-phyA/BL2l, the recombinant protein in the form of soluble protein and reached 25% in thallus protein, The biology activity of refolded protein was very low. Through comparing, the PTE21a(+)-phyA/BL21 was used to undertake continue experiment.The method for renaturation was improved.Research on the thermostablity of recombinant protein .Finding a mutated-strain named PTE21a(+)-phyA/BL21-2. The recombinant protein (phyA-mut2) expressed by it has better thermostablity, after heat treatment 75C-95C, 30min, still kept biological activity and has the phenomenon of activation.The highest bio-activity reached 5000 U/mg.Through sequencing,find the mutation of primary structure in phyA-mut2.