Modification Of Phytase Gene From Selenomonas Ruminantium And Its Overexpression In Pichia Pastoris

According to bias in codon choice of yeast and the factors in eukaryote which affect the gene transcription, translation and the stability of mRNA , The gene encoding a phytase PHYS of high specific activity from ruminal microorganism Selenomonas ruminantium was modified without changing its amino acid sequence. The phyS-M gene which be fit for expression in Pichia pastoris was designed and synthesized. The pbyS-M gene was cloned into vector of pPIC9 and spliced with the signal piptide encoding sequence of Pichia pastoris a-factor in reading frame correctly, and constituted the recombinant vector of pPIC9-phyS-M. The phyS-M was under the control of the alcohol oxidase gene promoter. A high-expression strain was constructed by transforming pPlC9-phyS-M into Pichia pastoris GS115. The assay result revealed that the recombinant phytase gene was expressed highly and secrete effectually in Pichia pastoris, and the expressed product had the normal bioactivity. In 5L fermentation, the biomass of expressed phytase in recombinant P. pastoris with modified phytase gene was high as 4mg/mL, and the enzyme activity in the medium was reached 1.6xl06U/mL, which is 10 fold of that in recombinant P. pastoris with unmodified phytase gene. SDS-PAGE revealed that the molecular weight of the recombinant expressed phytase is about 38kD. The specific activity of expressed phytase was up to 4xl05U/mg protein.