The Mutation Of Acid Phytase And Overexpression Of Phytase PhyL And 168phyA Gene In WB800

Phytic acid is the main storage form of phosphorus in plant feed stuff.However, monogastric animals,such as swine and poultry,are unable to utilize phytic acid due to the low levels of phytase activity in their digestive tracts.Phytase,myo-inositol hexaphosphate phosphohydrolase,which can catalyze phytic acid is a new-type important enzyme product as animal feed additives.It is useful to improve the utility of phosphate in plant feedstuffs and the absorption of nutrients,lessening the crisis of pollution caused by phosphorous.Therefor,phytase has wide prospect in the industry.Although the phytase can be found in many plants,animals and microorganisms,the lower production and thermostability limit the application.Therefore,researchers seek many methods to modify phytase,improving their thermostability,pH optima,activity and so on.In this paper,we screen out a novel Aspergillus niger strain(A-828) which expresses high active acid phytase through combination mutation by Co-60 and UV. In addition,two forms phytase gene phyL and 168phyA were inserted into PMA5 vector and cloned,over-expressed in the B.subtilis WB800.The following results were indicated:1.The biological synthesis,purification and characterization of a acid phytase from an Aspergillus niger A-828:Screening out a novel Aspergillus niger strain (A-828) which expresses high active acid phytase through combination mutation by Co-60 and UV.To research its enzymatic characterization,the acid phytase was purified by the steps of ultrafiltration and Bio-gel-P-150 chromatography.The purification ratio and yield was 11.2 and 29%.Result shows that its molecular weight was estimated to be about 66KD by SDS-PAGE.The phytase activity of A828 reached to the 66;000 U/ml,17 fold than origin.Its optimum pH of the enzyme activity was both at 2.5 and 5.5,but the largest activity at 2.5 was 5 fold than that at 5.5.Its optimum temperature was 55℃.The thermal stability of the phytase could be unchanged during 20℃to 60℃for 20 min.The phytase activity was strongly inhibited by 2mmol/L Cu²⁺,Fe²⁺ and Cr²⁺,while could not be influence by Ca²⁺ Mn ²⁺ EDTA,DTT.2.Overexpression and the biochemical characterization of two phytases from B. subtilis 168 and B.licheniformis:Two phytase gene(phyL) and(168phyA) were identified from Bacillus licheniformis and Bacillus subtilis by PCR.They were cloned and over-expressed in B.subtilis WB800.The phytase activities reached to 350000U/ml and 30000U/ml respective.Result shows that their molecular weights were estimated to be about 45KD by SDS-PAGE.The optimum pH of these enzyme activities were both at 7,and the optimum temperature were both at 65℃and 55℃. The thermal stability of the phytase could be unchanged during 60℃tO 90℃for 20 min.