Submitted to: European Symposium of the Protein Society
Publication Type: Abstract Only
Publication Acceptance Date: October 21, 2002
Publication Date: March 29, 2003
Citation: Ullah, A.H., Mullaney, E.J. 2003. Alteration of the ph versus activity profile of fungal phytase by site-directed mutagenesis (abstract). European Symposium of the Protein Society. Technical Abstract: Aspergillus niger phytase has become the benchmark enzyme among HAPs due to its high activity, stability, and kinetic perfection. Monogastric animals lack phytase; thus, excess phytates present in animal feed bind metals and peptides, and creates excess load of phosphate on environment. The kinetic perfection of the biocatalyst could be improved through structural modification. In addition, the pH of the animals' stomach is slightly different from phytase's inherent pH optimum range. By analyzing the three-dimensional structure of phytase and sequence comparison data, it was possible to pinpoint some key residues at the active center of the protein that may play a role in determining the pH versus activity profile in phytase. Mutant phytases were expressed in yeast and purified. Catalytic characterization of these mutants has led to identification of a single residue that holds the key to pH optimum profile in A. niger phytase. We were able to abolish the dip in activity at about pH 3-3.5. The rise in activity at this pH range would improve the catalytic function of phytase because the gastric pH of poultry falls into this range. Further structural refinement in fungal phytase will aid in improving catalytic function of the biocatalyst not at the expense of stability because only one crucial amino acid will be mutated to effect the change. Mutations at the same site that did not result in significant change as far as pH effect is concerned gave crucial information about the substrate binding site of the enzyme. These data will be helpful in any redesign exercise to further improve the Kcat parameter of phytase.