High expression of codon optimized phytase-encoding gene appA-P in Pichia Pastoris

According to the bias in codon choice of the high expression gene in Pichia pastoris,the gene encoding the mature portion of Escherchia coli phytase(AppA) with high specific activity was optimized and artificially synthesized without changing the amino acid sequence,and the modified gene was inserted into the Pichia pastoris expression vector pHBM905A,then the linearized recombinant plasmid,pHBM905A-appA-P,was introduced into Pichia pastoris GS115 by the PEG1000 transformation method.A high expression recombinant Pichia pastoris GS115-appA-P was obtained.After induced by 1%(V/V) methanol for 168 h at 25℃,the activity of phytase achieved 422 U/mL in the supernatant of the 100 mL shaking flask culture,which was among the highest of those recombinant strains reported now.The phytase expressed by GS115-appA-P has a temperature optimum of 60℃ and a pH optimum of 4.5,and are active at temperature ranged from 20℃ to 50℃ and pH ranged from 1.0 to 6.0.