| Phytase are enzymes that catalyze the hydrolysis of phytate into myo-inositol and inorganic phosphate. Monogastric animals like pig and poultry, for the lack of phytase in the alimentary tract, couldn't assimilate phytate that abounded in feed. The problem could be solved by added phytase into livestock food. In addition, phytate can also act as an anti-nutrient factor in animal feed, it would also increase the feed value by removing this anti-nutrient factor. However, due to the high cost and the weak thermostabiliry of the phytase products, only a limited amout of commercial microbial phytases are used in feedstuff. Thus economical and thermostable phytases are badly required in the feedstuff industry.In this study, three pairs of primers had been designed and synthesized . The nucleotide sequence of GAP^ SS andphyA had been amplified respectively from template DNA as follow: total DNA of Pichia pastoris GS115,pPIC9K and pPIC9K-phyAby PCRmethod, then fusing fragment FGSP had been amplificated by fusing PCR to link GAP. SS andphyA together. After digested with AatU and EcoR I , FGSP and pAO815 had been linked together and transformed into E.coli DHScu then some transformants owned recombinant expression plasmid pAOGSP had been selected.To verify the linkage of FGSP and pAO815, pAOGSP was digested by enzymes Aatll and î–©oRI and analysized by electropherosis. The recombinant expression vector pAOGSP was electroporated into the host cell Pichia pastoris GS115, in which the plasmid would integrate into the genome by the crossover event of homological DNA fragments. As long as it occurs, the constitutive expression of phytase gene in Pichia pastoris GS115 is avalible. The result could be detected by PCR. The interst clones would come out with a hydrolytic circle on a selective solid medium contained calcium phytate.Transformants were applied into fermentation tests. After 96h liquid cultivation in flask, the phytase activity was 1397 u/ml.
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