The Purification And Characterization Of Phytases From A.niger A3214,A.niger And Recombinant Pichia Pastoris E22

Phytase can catalyze phytate into myo-inositol and phosphate, relieves phosphorus, metal iron and protein from phytate, thus grids of phytate's nutrition restriction when it's be mixed in the food and feed for one-stomach animal.The trace of phytase is discovered in the animal, plant and microbe. But only the microbe becomes the main source of selling phytase because it provides many kinds of easy-extracted and large-yield phytase. There isn't a perfect product of phytase while tens kind of phytase product came out. It's the direction of phytase research in this time to improve the quality and to raise the yield of phytase by mutation and gene engineering.We gained A3214, a strain ofAspergillus niger from soil, N14, a mutated strain of A3214, and E22, a recombinant Pichia pastoris with the phytase gene of N14 before.Now we purify the phytase of A3214, the phytase of N14, and the phytase of E22 and study the characters of them. The results of our research are followed.1. We purified the phytase of A3214, the phytase of N14, and the phytase of E22 by precipitation with acetone, molecular sieve chromatography and ion exchange chromatography. The recovery ratio of A3214, of N14, and t of E22 is 5.6%, 7.1%, 1.4% accordingly.2. The pH optimum of A3214 and of N14 is 2.5. The phytase of E22 has tow pH optima at 2.5-3.0 and 5.5. The temperature optimum of A3214 and of N14 is 50℃. The phytase of E22 has a temperature optimum of 45℃ at pH2.5 and has another temperature optimum of 55℃ at pH 5.5.3. The phytase of A3214 and the phytase of N14 have 80% activity after 10 minutes incubation at 60℃. The phytase of E22 has 70% activity after 10 minutes incubation at 85℃.The phytase of A3214 and the phytase of N14 have 90% activity after 6 hours incubation with pepsin. The phytase of E22 has almost 100% activity after 6 hours incubation with pepsin and has 80% activity after 6 hours incubation with trypsin. The activity of the A3214 phytase and the N14 phytase is reduced by K⁺, Na⁺, Ca²⁺, Mg²⁺, Fe²⁺ and is not reduced by Zn²⁺, Cu²⁺, Mn²⁺. The activity of the E22 phytase...