Improving The Expression Of Phytase Gene(phyA) By Changing The Signal Peptide Of Pichia Pastoris

phytase can improve utilization of P and degrade seed phytic acid, The use of phytases to degrade seed phytic acid has very important signification for reducing the negative environmental impact of livestock production and improving the benefit of enterprise.increasing phytase activity is very important for industrialization of phytase.Synthesizing new signal peptide MS which is based on signal peptide of Pichia pastoris and codes of yeast ,and constructed new Pichia pastoris vector pPIC9k-MS. theninsert mutated gene phyA^m-4 and phyA into pPIC9k-MS vector. The expression plasmid pPIC9k-MS-phyA^m and pPIC9k-MS-phyA^m-4 were constructed and transformed into GS115 strain. By riddling of phenotypes and phytase activities,we selected two high activity strains of transformants which are PP-MS-NP^m-77 and PP-MS-NP^m-4-16.SDS-PAGE analysis suggust that the expression product could be secreted and over-expressed, the size of enzyme protein was 70.15KD.PCR analysis to these yeasttransformants showed the mutated gene phyA and phyA^m-4 were intergrated into thechromosome genome. Additionally, The genetic stability of transformant is quite good.The phytase activity of PP-MS-NP^m-77 reached 179150 u/ml in BMGY/BMMY culture medium after being induced with 36h, which was the 2.81 times higher than PP-NP^m-8(63667u/ml) ,and The phytase activity of PP-MS-NP^m-77 reached 188130u/ml in malt wort culture medium after being induced with 36h, which was the 3.95 times of the original strain PP-NP^m-8(47600 u/ml).The phytase activity PP-MS-NP^m-4-16 reached 201800 u/ml in BMGY/BMMY culture medium after being induced with 36h, which was the 1.51 times higher than...