Study On The High-Density Fermentation Conditions Of Recombinant Producing Phytase Pichia Pastoris PP-MS-NP～(m-4)-16

Abstract: Synthesizing new signal peptide MS which was based on signal peptide of Pichia pastoris and codes of yeast, and new Pichia pastoris vector pPIC9k-MS and the expression plasmid pPICgk-MS-phyA^m-4 were constructed and were transformed into GS115 strain, one high activity strains of transformant PP-MS-Np^m-4-16 was selected. In this research, the media of PP-MS-NP^m-4-16 was studied, the result indicated that the 40% chrysalis culture, 190mg/ml nutrition, salt added, phytase activity was increased 36.8% on the base ofcomparision.In baffled flask the single element experiment indicated that the relevance rotation speeds was 220r/min, initial pH of cell growth 5.0, initial pH of methanol inducing 5.5. by using orthogonal experiment, the result indicated that inoculate quantity 4%, methanol inducing concentration 4%,bean oil concentration during growth phase 1.3%, bean oil concentration during inducement phase 3.5%. when it was finished inducing the hignest phytase activity reached 192.4 ku/ml, which was 1.6 times as high as that of before optimizing.In the 5L automatic fermenter, the fermentation condition was optimized as the follows: the temperature 28℃, stirring speed 750r/min, inoculate quantity 4%, methanol velocity 8ml/L/h, glycerol velocity during inducing phase lml/L/h, induce time 84 h, when fermentation end, the thalli density attain 175.2, the paytase activity reached to 472.933KU/ml, which was 2.458 times as high as that of flask and increased 0.8 times than the activity of PP-Np^m-8.The preparation of phytase product and its thermostability were studied, the coarse enzyme was obtained by freeze centrifugal of fermentation liquid, then the 10mg/ml mannitol was added, coat of rice was used as carrier, mixed the coat of rice and fermentation according to the proportion 1: 1 , Na₂SO₄ was 20% w/w to coat of rice, sodium alginate was 0.5% w/w to coat of rice, the result indicated that after coating, the relative retaining activites of coated phytase under the temperature of 80℃reached 92.6%.The ratio of substrate cost of PP-MS-Np^m-4-16 strain and enzyme yield was 0.005461yuan/10⁶u, the price of product of 5000u/g was about 18yuan/kg.In this research we brought out phytase fermentation and product procession, which increased phytase activity,increased thermostability of phytase and provided scientific base for its fermentational production and cost decreasing as well.