Study On The Main Factors Affecting The Production Of Phytase

Pichia pastoris as an excellent heterologous gene expression system has been widely used in the production of phytase. The processes of recombinant pichia pastoris in baffled flask and in formentor are also explored to ascertain the optimal condition. The optimum conditions of this genetically engineered strain in shake flask stage are: seed age 20 h; inoculation 5%; the pH of medium in growth and induction phases are 5.5 and 6.0, respectively. The mixture of sorbitol - methanol is used in inducing stage. 2.4mL of the solution is added per day constantly. Fermentation period is reduced to 60h.The optimum conditions of this genetically engineered strain in formentor stage are: the pH of medium in growth and induction phases are 6.0 and 6.5, respectively. Fermentation period is reduced to 54 h and the final activity reaches 31985U/mL.At present, the expensive medium in industrial production and the long fermentation cycle increase the production cost of enterprises significantly. Two cheap media----wort - wheat bran growth medium and wort - wheat bran extraction medium were used to cultivate recombinant pichia pastoris in this study, the composition of the media are optimized. 15g wheat bran plus 100g malt powder is used as basic media materials. In the yeast growth stage 4% of the corn syrup is added. The final enzyme activity increased about one-fold after optimizing.The most commonly used determinations of phytase activity are vanadium– ammonium molybdate method and molybdate-blue method. The accuracy and stability of the two methods were studied in this research. The results show that vanadium– ammonium molybdate method which is recommended in the National Standard (GB/T 18634-2002) has the advantages of simple procedure, rapid testing and satisfied accuracy.The culture medium and fermentation processes of recombinant pichia pastoris have been studied and optimized in this research. The yield of phytase has increased and the production cycle has shortened. Provide scientific base for substrate cost decreasing, improving the enzyme yieldand its fermentational production.