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 a nd CAG in phyA2 were mutated into synonymous codon AGA. The modified p hyA2 was fused behind alpha-factor signal sequence under the control o f AOX1 promoter in plasmid pPIC9, then introduced into the host Pichia pastoris by electroporation. The results of Southern blotting analysi s 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 mo dified 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 Aspergi llus niger 963 and was 37 times higher than that of recombinant P. pas toris with non-modified phyA2.