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Title: Impact of Assay conditions on activity estimate and kinetics comparison of Aspergillus niger PhyA and Escherichia coli AppA2 phytases

Author
item WEAVER, JEREMY - CORNELL UNIVERSITY
item Ullah, Abul
item Sethumadhavan, Kandan
item Mullaney, Edward
item LEI, XIN - CORNELL UNIVERSITY

Submitted to: Journal of Agriculture and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/28/2009
Publication Date: 6/1/2009
Citation: Weaver, J.D., Ullah, A.H., Sethumadhavan, K., Mullaney, E.J., Lei, X.G. 2009. Comparisons of activity assay methods and kinetics of Aspergillus niger PhyA and Escherichia coli AppA2 phytases. Journal of Agriculture and Food Chemistry. 57:5315-5320.

Interpretive Summary: The enzyme phytases were developed to combat phosphate pollution emanating from increased poultry and swine production allover the world. These simple-stomached animals are fed soybean rich meal that contains an antinutrient called phytic acid, which cannot be metabolized because these animals lack an enzyme call phytase. Microbes that grow in soil and water rich in nutrients on the other hand could degrade this compound because they have the enzyme phytase, which could systematically breakdown the phosphate rich antinutrient, phytic acid. We have developed an enzyme-based technology to degrade phytic acid from soybean meal. Thus far, only two phytases have been commercialized; one from a soil fungus, Aspergillus niger and the other one from a bacterium that lives in the lower intestine of farm animal such as pig. In this communication we have compared the rate of breakdown of phytic acid by this two commercially important phytase. We have also compared three separate methods by which phosphates released by phytase were measured. We have shown that some of these methods have limitations. Overall, both of these phytases have abilities to degrade phytic acid; however, the bacterial phytase have higher turnover number indicating a faster rate of catalysis. The fungal phytase is somewhat sluggish but it has superior affinity for phytic acid. The kinetic efficiency, which is measured by both the rate of catalysis and affinity of phytic acid to bind to the enzyme, is very similar. Our results have confirmed that these two phytases are indeed a better candidate for commercialization.

Technical Abstract: This study was to compare three phytase activity assays and kinetics of Aspergillus niger PhyA and Escherichia coli AppA2 phytases expressed in Pichia pastoris at the observed stomach pH of 3.5. In Experiment 1, equivalent phytase activities in the crude preparations of PhyA and AppA2 were tested by the commonly-used molybdenum blue method, the molybdovanadate method, and the acetone phosphomolybdate method. Values of PhyA activity varied by 22%, and the assay buffer, the buffer pH, and the unknown factors accounted for 15, 15, and 70% of the total differences, respectively. Values of AppA2 activity varied by 179%, and the assay buffer, the buffer pH, and Triton X-100 and bovine serum albumen accounted for 39, 30, and 32% of the total differences, respectively. In Experiment 2, we compared the Michaelis-Menten constant (Km), maximum velocity (Vmax), turnover number (kcat) and catalytic efficiency (kcat/Km) of purified (>95% homogeneity) PhyA and AppA2. While AppA2 had a higher Km (74 vs. 34 µM, P < 0.01) than PhyA for phytate, this bacterial enzyme displayed a greater (P < 0.01) Vmax (1,070 vs. 120 U mg-1), kcat (840 vs. 170 sec-1) and kcat/Km (1.1x107 vs. 0.5x107 M-1 sec-1) than that of PhyA. PhyA was nearly 20 times more resistant to competitive inhibition by myo-inositol hexasulfate than AppA2 (Ki = 3.9 vs. 0.2 µM). Likewise, 4 times more (1.4 vs. 0.3 M) guanidine hydrochloride was required for 50% inhibition of PhyA activity than for that of AppA2. In conclusion, the activity values of AppA2 were affected by the assay method more greatly than those of PhyA. Overall, AppA2 displayed superior kinetic properties to PhyA at pH 3.5, which helps in catalyzing phytate hydrolysis at gastric conditions of simple-stomached animals. Phytase activity assays produce different results for the same sample.