Purification And Characterization Of The Recombinant Heat-Stable Phytase In Aspergillus Niger

Phytase can hydrolyse myo-inositol hexakisphosphate (phytic acid) to phosphate and myo-inositol. Adding phytase to food and feedstock can improve the utilization of nutrients, reducing the phosphate content in excrete of monogastric animals, thus showing great importance in raising the profit of cattery and reducing the pollution of phytate-phosphorous on environment. Meanwhile the hydrolysed products of phytase in the human body has a very important physiological functions, which brought brighter prospects of phytase to the application of the enzyme. Although phytase has been used in industrialized production, there still exists some problems. Amongs them, the most prominent problem is the thermal in stability of phytase. Thus we need a phytase of higher heat resistance. We cloned the encoding sequence for phytase From the laboratory of domestic acquired Aspergillus fumigatus and got the achievement of its expression of secretory protein in aspergillus niger.We added six consecutive histidine (His-tag) to the C end of the heat-stable phytase. anti-His antibody and anti-phytase antibody's western detection, further confirmed that phytase from Aspergillus fumigatus got the achievement of its expression of secretory protein in aspergillus niger. By changing the composition of medium to explore the best medium, the ultimate use of the 1% soluble starch as a carbon source. 0.5% ammonium sulfate as a nitrogen source, adding 0.5% surfactant Triton-x-100. In optimum conditions, the activity reached the highest 212.4IU/ml than before optimization activity increased 1.66 times. The crude phytase produced by the recombinant Aspergillus niger in the pH 6.0 was purified with G25 sepharose and Q sepharose ion exchange chromatography. Detection by western proved to be derived from Aspergillus fumigatus heat-stable phytase.Part of the recombinant phytase characterization study found : in the condition of pH4.0-6.8 and 37-90℃, it is stable, pH6.2 with a peak activity. The optimum temperature is 62℃and 80℃for 20 min treatment, activity remained at 78%. 90℃ and 100℃for 10 min treatment, the activity respectively remained at 55.8% and 43.9%. It has good thermal stability. Mg²⁺ and K⁺ is on the role of promoting activity, while Mn²⁺, Cu²⁺, Fe²⁺, Zn²⁺ is on the role of inhibiting phytase activity.Experiment by changing the recombinant heat-stable phytase medium components, increased the phytase activity, and through desalination, ion exchange chromatography purificated recombinant heat-stable phytase, and studied part of it's characterization. As the result this experiment provided a group of very meaningful data to the industrialization of phytase.