The Cloning, Site-directed Mutation And High Expression Of Phytase Gene From Aspergillus Niger

Phytase has a bright and broad application prospects as an important feed additive and potential food additive. However, the low activity of microbe secretion expression phytase is the main barrier of its application. Therefore, the research and development of high-activity-phytase-producing technology has an very important significance. This paper studied the fermentation of phytase by Aspergillus niger NL-1, meanwhile cloned, site-directed mutated and optimized the inducible expression condition of the phytase gene.(1) The phytase gene, about 1.5 kb, was successfully cloned by PCR method from Aspergillus niger NL-1. And then we aligned the sequence with AY426977 registered on the Genbank. The coding-sequence homology was 98.75%.(2) The Pichia painstoris fusion expression plasmid pPICZαA-PhyA was successfully constructed. Then we screened the wanted transformant after eletro-transforming the plasmid into Pichia pastoris GS115. The inducible expression results suggested that the phytase gene was highly expressed in Pichia pastoris. The phytase activity could be 223 times to the original strain in proper conditions. And it's the first time to clong the neutral phytase from Aspergillus niger.(3) During the inducible expression of phytase, the enzyme activity could be as high as 142.82 U/mL when appropriate amount of 1% methanol was added every 36h in 75% BMMY +25% malt meal medium after 96h culture.(4) The proper phytase reaction temperature is 50℃and the proper pH is 5.0-7.0. Phytase has a good stability below 70℃when pH was 3.0-7.0. Also, the metal ion has some effects on the activity of phytase.(5) Some of the Arg coding sequence were synonymous mutated, on the condition of amino acid sequence was intact. And we got the 3-sites and 6-sites mutated recombined plasmids and then successfully transformed them into Pichia pastoris GS115. Then, after 5 days of recombined-strain inducible expression, the phytase activity produced by 6-sites mutated recombined yeast reached to the peak as 149.31U/mL and was 1.61 times to pre-mutated recombined Pichia pastoris strain.