|Rodriguez, Eric - CORNELL UNIVERSITY|
|Lei, Xin - CORNELL UNIVERSITY|
Submitted to: Biochemical and Biophysical Research Communications
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 24, 1999
Publication Date: N/A
Interpretive Summary: Adding microbial phytase (the enzyme that breaks down phytic acid) to diets of pigs and poultry has been shown to be an effective means to lower phosphorus levels in manure from poultry and swine production operations. Lowering production costs of this enzyme would speed its acceptance by the animal feed industry and this in turn would increase its positive impact on nour environment. One phytase from a mold has shown some promise as a candidate for commercialization. In this study a phytase gene was successfully expressed in a strain of yeast. The yeast strain had been previously used to produce other proteins. The recombinant phytase was expressed at a high level and displayed a desirable level of heat tolerance and activity. In conclusion, this system is a potential means to produce elevated levels of phytase and the phytase produced has several desirable features to support its use as an animal feed additive. This will be of benefit to the animal feed industry.
Technical 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 pPICZ(alpha)A 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 deg 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.