[Overexpression of Citrobacter braakii phytase with high specific activity in Pichia pastoris]

Utilization of the phytase with high specific activity is an effective way to improve the fermentation potency of phytase in recombinant host and decrease the production cost. Up to now, the phytase APPA from Citrobacter braakii exhibits the highest specific activity in the all phytases recorded previously. The gene AppA encoding phytase was modified according to the bias in codon choice of the high expression gene in Pichia pastoris without changing the amino acid sequence and artificially synthesized. The modified gene, AppA ( m) , was inserted into the Pichia pastoris expression vector pPIC9 under the control of AOX1 promoter, and the resulted expression vector pPIC9-AppA ( m) was introduced into the host Pichia pastoris by electroporation. PCR analysis of the recombinant yeast indicated that AppA (m) gene was integrated into the chromosome of Pichia pastoris. The Pichia pastoris recombinants for phytase overexpression were screened by enzyme activity analysis and SDS-PAGE. The recombinant phytase APPA was purified by simple methods, such as dialysis, ultrafiltration and chromatography. After the simple purification, the purity of the recombinant phytase reached to electrophoresis purity, and the recombinant phytase was shown to be glycosylated by Endo-H treatment. The specific activity of the purified recombinant APPA was 3.5 x 10(6) IU/mg of protein. Recombinant phytase APPA showed activity at pH values from 2.0 through 7.0 with the optimum at 4.5. The temperature optimum was 55 degrees C at pH 4.5.The Km value for sodium phytate was 0.165mmol/L with a Vmax of 3.3 x 10(6)IU/mg min. In 5-liter fermentor in fed-batch fermentation, the expression level of phytase in recombinant Pichia pastoris was 3.2mg/mL and the fermentation potency exceeded 1.4 x 10(7) IU/mL, which is the highest level among all of the reported phytase recombinant strains at present.