Over-expression Of Phytase Gene PhyA In Pichia Pastoris And Optimization Of Media Recipes For Engineered Yeast

Phytases are a class of phosphatases that can catalyze the hydrolysis of phytate into myo-inositol and phosphate. It can relieve anti-nutrition of phytate and improve the nutritional value of animal feed from plant.The study on phytase, especially acidic phytase produced by micro-organism, has drawn much attention in home and abroard. However, its wide application in manufacture has always been restricted by such a question as low phytase-producing level of wild strains, so before the wild strains which can produce acidic phytase are widely used, their phytase activities must be improved by various means, in which using an efficient expression system to express heterogenous phytase is a main consideration. The objective of this research is to transform the cloned phytase gene into Pichia pastoris in order to obtain high-yield phytase-producing strain and to optimize the engineered yeast media recipes for the scale-up production of phytase. Main results of this research are as follow:1 ,Xba I-linized recombinant plasmid pPIC9K-phyA was transformed into Pichia pastoris by Gene Pulser .98 positive transformants showing measurable phytase activities were screened on MD plates and YPD plates containing G418. They all grew quickly on both MD plates and MM plates, which proved their phenotypes of methanol utilization were Mut+. Specific Pichia clony PCR product showed that foreign phytase gene was integrated into the host cell. The experimental results from flask fermentation and phytase activity assay indicated that phytase gene was effectively expressed by the recombinant Pichia. One highly productive and genetically stable recombinant strain named E-22, which produced phytase with 143958.3U/mL under the condition of flask cultivation, was selected through further screening. The phytase activity of E-22 was 341.13 times as high as that of the original strain (422U/mL).2,During the inducing cultivation, the phytase expressed by recombined yeast E-22 keepsimproving in 168h, approaches maximum at 144h and then stays stable. Its cell wet weight increases in 72h and then keeps constant. Studies on the partial enzyme characters of recombinant phytase showed that the recombinant phytase had two pH optima (2.5-3.0 and 5.0-5.5) and a temperature optimum 55℃, and with higher activity under the pH ranged from 4.5 to 6.0. The result of SDS-PAGE indicated that the molecular weight of the recombinant phytase is between 70kDa-97kDa. Specific activity of the rude recombinant phytase is 461575.8 U/mg.3, A five-factor Uniform Design experiment was conducted to optimize the engineered yeast media recipes for phytase production.Phytase activity of 108758.3 U/mL was observed under the condition of flask cultivation in which the contents of yeast exract, methnol, peptone, YNB, biotin in the media were 2%, 4%, 1%, 0, 0.0001% respectively. The phytase activity in fermentation broth was 1.114 times as high as that obtained from native BMMY (97666.7U/mL) under the same condition (inducing time 120h, phytase activity measured at 37℃).