Overexpression of Escherichia coli Phytase in Pichia pastoris and Its Biochemical Properties

To obtain a Pichia pastoris mutant with an Escherichia coli phytase gene, which was synthesized according to P. pastoris codon preference, a mature phytase cDNA of E. coli being altered according to the codons usage preference of P. pastoris was artificially synthesized and cloned into an expression vector of pGAPZ alpha C. The final extracellular phytase activity was 112.5 U/mL after 72 h of cultivation. The phytase, with a molecular mass of 46 kDa, was purified to electrophoretical homogeneity after Ni Sepharose 6 Fast Flow chromatography. The yield, purification fold, and specific activity were 63.97%, 26.17, and 1.57 kU/mg, respectively. It had an optimal pH and temperature of 4.0-6.0 and 50 degrees C, respectively, and was stable at pH 3.0-8.0 and 25-40 degrees C. The purified recombinant phytase was resistant to trypsin, highly inhibited by Cu2+, Zn2+, Hg2+, Fe2+, Fe3+, phenylmethylsulfonyl fluoride, and N-tosyl-L-lysine chloromethyl ketone, but activated by Mg2+, Ca2+, Sr2+, Ba2+, glutathione, ethylenediaminetetraacetic acid, and N-ethylmaleimide. It revealed higher affinity to calcium phytate than to other phosphate conjugates.