Studies On Optimal Conditions For Phytase Producing And The Expression Of Phytase Gene

Phytase is a group of enzyme which can catalyze phytate into myoinositol and phosphate, and release phosphorus, mineral element and protein from phytate. Phytase shows broad application prospects in the animal feed industry, food industry, pharmacy and other fields. The purpose of this study was to screen phytase-producing strain, and to obtain the optimum conditions for phytase producing. A phytase-producing strain was isolated and identified as A. niger FZ56.Liquid fermentation was studied and the optimal fermentation medium was obtained as followed, starch 4%, NH₄NO₃ 0.08%, KCl 0.05%, MgSO₄·7H₂O 0.05 %, FeSO₄·7H₂O 0.003%, MnSO₄·7H₂O 0.003%, and intial pH 6.0. Extracellular phytase production was 2221 U/mL when fermentation was carried out at 30℃and 220 r/min for 84 h.The phytase was extracted from liquid state leavening of A. niger FZ56. The crude product was purified by (NH₄)₂SO₄ precipitation, gel filtration and ion exchange chromatography. The purified phyatse was 10.3 times of the raw products, and the extraction ration was 25.2%. The study on the enzymology of phyatse showed that the optimal temperature and pH was 55@@@ and 6.0, respectively. The Km value of the phytase was 0.14 mmol/L.The phy A gene was produced by PCR from the genome of Aspergillus niger FZ56,and then inserted into the chromosome DNA of Pichia pastoris GS115 and was expressed under the induction.,while the same time the condition of induction was optimized. The recombinant enzyme was purified using protein purification methods and characterized by SDS/PAGE electrophoresis. And the result indicated a molecular mass of 61 kD for this phyA.But the native phy A was 50 kD,from which we can infer that the protein has a degree of glycosylation. Finally,the enzymatic propertieswas determined.The optimum pH was 6.0 and the optimum temperature was 55@@@,which confirmed that phyA was correctly expressed.