title: Expression of the Aspergillus fumigatus phytase gene in Pichia pastoris and characterization of the recombinant enzyme.
abstract: Aspergillus fumigatus phytase is a heat-stable enzyme of great potential. Our objective was to determine if a high level of functional expression of the A. fumigatus phytase gene could be produced in Pichia pastoris and how the recombinant phytase reacted to different substrates, heating conditions, and proteases. A 1.4-kb DNA fragment containing the coding region of the gene was inserted into the expression vector pPICZalphaA and expressed in P. pastoris as an active, extracellular phytase (r-Afp). The yield was 729 mg of purified protein per liter of culture, with a specific activity of 43 units/mg of protein. The enzyme r-Afp shared similar pH and temperature optima, molecular size, glycosylation extent, and specificity for p-nitrophenyl phosphate and sodium phytate to those of the same enzyme expressed in A. niger. Given 20 min of exposure to 65 to 90 degrees C, the enzyme retained 20 to 39% higher residual activity in 10 and 200 mM sodium acetate than that in sodium citrate. The enzyme seemed to be resistant to pepsin digestion, but was degraded by high levels of trypsin. In conclusion, P. pastoris is a potential host to express high levels of A. fumigatus phytase and the thermostability of the recombinant enzyme is modulated by the specificity of buffer used in the heat treatment.
authors.affiliation: Department of Animal Science, Cornell University, Ithaca, New York 14853, USA.
authors.forename: E
authors.initials: E
authors.lastname: Rodriguez
authors.forename: E J
authors.initials: EJ
authors.lastname: Mullaney
authors.forename: X G
authors.initials: XG
authors.lastname: Lei
mesh_terms: D010832:6-Phytase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D001232:Aspergillus fumigatus / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006031:Glycosylation; D010447:Peptide Hydrolases / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D010832:6-Phytase; D010447:Peptide Hydrolases