The Genetic Modify Of Phytase Trichoderma Strain

Phytases (myo-inositol hexakisphosphate phosphohydrolases) catalyze the hydrolysis of phytates to myoinositol and phosphates. Phytases are varied and widespread in nature, which occurr in plants, microorganisms, and animal tissues. Phytases, in general, are known to enhance phosphate and mineral uptake in monogastric animals such as human beings, poultry, swine, and fish, which cannot metabolize phytate besides reducing environmental pollution significantly.In this paper, phytase of Trichoderma was studied, and Trichoderma strains of high phytase yield were selected from those strains collected from multiple areas.NO.11 strain of high phytase yield was isolated from soil samples and, which was selected by transparent circle method and the phytase activity method of liquid fermentation. The research is aimed to improve phytase activity by genome shuffling, and the results are as follows. NO.11 strain was treated with ultraviolet radiation and NTG, respectively.Then good mutants were obtained.The protoplasts of goog mutants were heat-killed and UV-inactivated, and combined randomly.At last fusants which were high phytase yield were selected.Wiedtype strain was treated with ultraviolet radiation and NTG, respectively. Then the optimal dose for mutant were selected. Lots of mutants were selected with phytase activity method. The high phytase yield strain of UV radiation and NTG mutant was UV-Ⅱand NTG-4, the phytase activity of which were 1.21±0.013 U / L (p <0.05) and 0.94±0.014 U / L (p <0.05), respectively.The conditions for protoplast preparation and regeneration were studied. The effects of strain age, the lysozyme concentration, the lysozyme treating time and temperature on preparation and regeneration of protoplast were studied. The chief constituent of fungal cell wall is polysaccharide, which is made chiefly of chitin and few cellulose. It was prepared for Trichoderma lysozyme protoplasts with UV-Ⅱstrain and snail enzyme. The optimum conditions of Trichoderma protoplast are as follow: snail enzyme concentration 2.0mg/mL, strain age 20h, lysozyme time 2.0 h, lysozyme temperature 30℃.UV-Ⅱand NTG-4 protoplasts were heat-killed and UV-inactivated, and combined randomly with PEG as the integration agents of protoplast fusion. Fu-②was obtained from genome reorganization, phytase activity of which was 2.4±0.016 U / L and higher 320% compared with the original strains . The fusant is genetically stable.The result was found thatⅡliquid medium was the best medium. The mediumⅡ: Glucose 3.0 g, soluble starch 3.0 g, wheat bran 0.05 g, NH4NO30.5 g, MgSO4 ? 7H2O0.05 g, MnSO4 ? 7H2O 0.003 g, FeSO4 ? 7H2O 0.003 g, CaCO30.5 g, pH value of 5.5 -6.0,115℃30min sterilization. The conditions of Fu-②optimization: shaking speed 115r/min, medium volume 100 ml (500 ml volumetric flask), inoculation number 3%, temperature 30℃, fermentation time 120h, phytase activity 3.68 U / L and the enzyme activity was increased 33.3%.