The Extraction/Purification And Characterization Of Recombinant Phytase In Transgenic Corn Seed

Phytase, one of the most widely used feed enzyme additives, can catalysis phytic acid into inorganic phosphorus and inositol by hydrolysis, improve the utilization efficiency of phosphorus phytate in monogatric animals and reduce the pollution of phytate phosphorus to the environment and, therefore, is well worth further studies. High-performance expression of phyA gene from Aspergillus niger (ficuum) NRRL3135 in Pichia pastoris with genetic engineering technology has been successfully industrialized, but it has the disadvantages of high cost and energy consumption. Plant bioreactors used for production of enzyme preparations can effectively reduce the cost and energy consumption. Transgenic maize expressing high phytase was successfully generateded by Fan Yunliu's lab in Chines Academy of Agricultural Sciences.In this paper, phytase maize seeds mentioned above was used as the material, a standard process for extraction and purification of phytase was established by optimization of conditions for extraction, fractional ammonium sulfate precipitation and concentration, and anion-exchange chromatographic purification to provide plenty of samples for further studies on phytase and also referable experience for extraction and purification.of other recombination protein from maize seeds.It was found that phytase extracted by shaking at 720 rpm for 0.5h at room temperature using pH3.0 Gly-HCl buffer at a liquid-solid ratio of 10 had the highest specific activity, so it is considered as the best extraction condition. At an ammonium sulfate saturation of 60-80%, most of the phytase in the crude extract could be precipitated and the specific activity was the highest, so it is considered as the best concentration condition. The concentrated enzyme solution was purified by anion-exchange chromatography; the penetration peaks corresponded to impurities, which were free of phytase protein; the elution peak corresponded to phytase protein, which was SDS-PAGE electrophoretically pure, making it possible to directly determine the enzymological properties.Comparison of enzymological properties of maize/yeast expressed phytase indicated that the optimal pH was 5.8 for both and a second low peak appeared when the pH was 2.0; when the pH was between 2.0 and 8.0, both were stable; the optimal temperature for maize-expressed phytase was 45℃, while that for yeast-expressed phytase was 50℃; after incubation for 1h at 80℃, the residual activity of maize-expressed phytase was over 30%, while that of yeast-expressed phytase was about 11%, indicating the former had better thermal stability than the latter; within 0-30min, the ratio of enzyme activity to reaction time kept stable for both; for maize and yeast-expressed phytase, K_m was 0.202mmol/L and 0.355mmol/L, and V_max was 47mmol/min and 161mmol/min, respectively, suggesting maize-expressed phytase had higher affinity to the substrate while the maximum reaction velocity was relatively lower. Comprehensively considered, maize seeds as a bioreactor for phytase production not only outweigh microbes in production cost and energy consumption, but also facilitate feed processing in enzymological properties, promising an extensive application prospect.