title: Overexpression of Escherichia coli phytase in Pichia pastoris and its biochemical properties.
abstract: 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 pGAPZαC. 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 °C, respectively, and was stable at pH 3.0-8.0 and 25-40 °C. The purified recombinant phytase was resistant to trypsin, highly inhibited by Cu(2+), Zn(2+), Hg(2+), Fe(2+), Fe(3+), phenylmethylsulfonyl fluoride, and N-tosyl-l-lysine chloromethyl ketone, but activated by Mg(2+), Ca(2+), Sr(2+), Ba(2+), glutathione, ethylenediaminetetraacetic acid, and N-ethylmaleimide. It revealed higher affinity to calcium phytate than to other phosphate conjugates.
authors.affiliation: Department of Food and Nutrition, Providence University , Number 200, Section 7, Taiwan Boulevard, Salu, Taichung 43301, Taiwan.
authors.forename: Hsueh-Ming
authors.initials: HM
authors.lastname: Tai
authors.forename: Li-Jung
authors.initials: LJ
authors.lastname: Yin
authors.forename: Wei-Chuan
authors.initials: WC
authors.lastname: Chen
authors.forename: Shann-Tzong
authors.initials: ST
authors.lastname: Jiang
mesh_terms: D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D015202:Protein Engineering
chemical_list: D029968:Escherichia coli Proteins; D010832:6-Phytase