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ARS Home » Southeast Area » New Orleans, Louisiana » Southern Regional Research Center » Commodity Utilization Research » Research » Publications at this Location » Publication #137806


item Ullah, Abul
item Mullaney, Edward

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 8/2/2002
Publication Date: 8/19/2002
Citation: Ullah, A.H., Mullaney, E.J. 2002. Structural refinement of fungal phytase (phy a)for alteration of ph optimum (Abstract). Protein Structure.

Interpretive Summary:

Technical Abstract: Aspergillus niger phytase has become the 'benchmark' enzyme amoung all the 'Histidine Acid Phosphatases' (HAPs) because of its high activity, stability, and kinetic perfection. The pH of the monogastric animals' stomach in which phytase has to work is not exactly the same as phytase's inherent pH optimum range. Phytase hydrolysis phosphomonoesters present in phytic acid in a stepwise fashion to yield myo-inositol monophosphate and inorganic orthophosphates. Excess phytate present in both poultry and hog feed creates problems such as binding of metals and peptides by phytate, and creating an excess load of phosphate on the environment because of the lack of gastric phytase in mongastric animals. The ensuing phosphate pollution in groundwater is causing harmful algal blooms and fishkill along the east coast of the U. S. Animal feed is now supplemented with phytase to combat groundwater pollution in many developed nations. The kinetic perfection of phytase indicates that catalytic efficiency of the biocatalyst can be improved through knowledge-based re-designing using site-directed mutagenesis. With available three-dimensional structure of phytase (phy A) and sequence comparison data, it is possible to pinpoint some key residues at the active center of the protein. We have identified a few of the residues as possible candidate for structural alteration via site-directed mutagenesis. We have expressed the mutant proteins in yeast and purified the altered phytase by sequential ion-exchange chromatography to near homogeneity. Catalytic characterization of these mutants has led to identification of a single residue that holds the key to the 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 in 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 yielded crucial information about the substrate binding site of the enzyme. These data will be helpful in any redesign exercise to further improve the K cat parameter of phytase.