Mutation Of Phytase Gene(phyA) Active Center And Culture Conditions Of Recombinant Yeast

By using long-distance inverse PCR,Arg(cgt) and Arg(cgg) in the expression fragment of phytase phyA gene were mutated synonymously to Arg(aga),which is a bias code of Yeast.The mutated gene phyAm was cloned to puc18 vector,and the two mutated sites were confirmed by sequencing.The expression plasmid pPIC9k-phyAm was constructed and transformed into GS115 strain. By riddling of phenotypes and phytase activities,we selected two high activity strains of mutated and unmutated transformants. Southern blotting analysis to the four yeast transformants showed phyA gene was intergrated into the chromosome genome through single-crossover events with one copy of phyA gene.SDS-PAGE analysis suggust that the expression product could be secreted and over-expressed, the size of enzyme protein is 70.15KD .the test of transformants phytase activity indicated the transformant of PP-NPm-8 which codons was optimized can reach 47600U/ml in malt wort culture medium after being induced with 36h,compared to PP-NP-2 (23667 u/ml) ,1 times was improved. And 1.1 times than that of PP-NP-5 (22598 u/ml) .The genetic stability of transformant is quite good.the culture conditions of PP-NPm-8 strain were studied , the optimal conditions are as follows: seed age 16h, quantity of inoculation3%, initial pH of cell growth 4.5, initial pH of methanol inducing 5.0, methanol inducing concentration 4%, methanol inducing time 42h. culturing PP-NPm-8 strain on the optimal conditions,the phytase expression can get to 85067U/ml which was cultured in malt wort culture medium after being induced with 36h. compared to the unoptimized, about 1 times was improved, (about 4.4 times compared to pPIC9A-7 (15656 u/ml) reported by Yao Bin,which was cultured in BMGY/BMMY culture medium).Compare the substrate cost of mutated strain PP-NPm-8 with unmutated strain PP-NP-5 which were all cultured in malt wort culture medium. To the unmutated strain PP-NP-5 ,the price relations of substrate cost and enzyme yield is 0.0303 /100000 U,and to the mutated strain PP-NPm-8 is 0.009 yuan/100000U,so mutated strain PP-NPm-8 could decreased substrate cost 3.35 times than that of unmutated strain PP-NP-5,besides,compared to BMGY/BMMY culture medium(the price relations of substrate cost and enzyme yield is 1.97104 yuan/100000U), the substrate cost could decreased 218 times, so the cost of production can be greatly reduced.