Recombinant Pichia pastoris overexpressing bioactive phytase

Phytase gene phyA2 , whose signal peptide encoding sequence and intron sequence had been removed, was modified. The Arg encoding codons CGG and CAG in phyA2 were mutated into synonymous codon AGA. The modified phyA2 was fused behind α factor signal sequence under the control of AOX1 promoter in plasmid pPIC9, then introduced into the host Pichia pastoris by electroporation. The results of Southern blotting analysis and Northern blotting analysis demonstrated that the phyA2 gene had integrated into the genome of P. pastoris and transcribed. The result of SDS PAGE of the phytase expressed by P. pastoris showed that the modified phyA2 had been overexpressed and secreted. The concentration of the phytase expressed by P. pastoris with modified phyA2 exceeded 15 000 U/mL, which had a 3 000 fold increase over that of origin Aspergillus niger 963 and was 37 times higher than that of recombinant P. pastoris with non modified phyA2 .