Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/4/2006
Publication Date: 6/1/2006
Citation: Kim, T., Mullaney, E.J., Porres, J.M., Roneker, K.R., Crowe, S., Rice, S., Ko, T., Ullah, A.H., Daly, C.B., Welch, R.M., Lei, X.G. 2006. Shifting the ph profile of Aspergillus niger PhyA phytase to match the stomach ph enhances its effectiveness as an animal feed additive. Applied and Environmental Microbiology. 72(6):4397-4403. Interpretive Summary: When used as an animal feed additive fungal phytase has been shown to be a very successful means to lower phosphorus levels in manure. This special enzyme allows the animals with simple digestive systems (poultry and swine) to utilize the phosphorus found in phytic acid, which is abundant in seeds and grains. Without this enzyme the animal absorbs very little of this phosphorus and instead it ends up in the manure were it can potentially harm the environment by spurring the growth of harmful algae in waterways. These algal blooms have been known to cause large fish kills. The enzyme that is current marketed was found in nature and adapted to this use. ARS scientists have now improved on this native phytase by changing its chemical structure to make it more active in the stomach of pigs. By changing the pH optima of this phytase to perform better in the actual conditions found in the stomach of animals, a superior phytase has been produce. A minor change in the chemical structure of the enzyme has resulted in a phytase that now performs significantly better under these conditions in laboratory test. Animal feeding trial then showed the superiority of this new phytase and validated the potential value of this new product to agriculture and to protecting the environment.
Technical Abstract: Environmental pollution of phosphorus (P) from animal waste is a major problem in agriculture because simple-stomached animals such as swine, poultry, and fish cannot digest phosphorus (as phytate) present in plant feeds. To alleviate this problem, a phytase from Aspergillus niger PhyA is widely used as a feed additive to hydrolyze phytate-phosphorus. However, it has the lowest relative activity at the pH of stomach (3.5) where the hydrolysis occurs. Our objective was to shift the pH optima of PhyA to match the stomach condition by substituting amino acids in the substrate-binding site with different charges and polarities. Based on the crystal structure of PhyA, we prepared 21 single or multiple mutants at Q50, K91, K94, E228, D262, K300, and K301 and expressed them in Pichia pastoris yeast. The WT PhyA showed the unique bi-hump, two pH optima profile, whereas 17 mutants lost one pH optimum or shifted the pH optimum from pH 5.5 to more acidic side. The mutant E228K exhibited the best overall changes, with a shift of pH optimum to 3.8 and 266% greater (P < 0.05) hydrolysis of soy phytate at pH 3.5 than the WT enzyme. The improved efficacy of the enzyme was confirmed in an animal feeding trial and was characterized by biochemical analysis of the purified mutant enzymes. In conclusion, it is feasible to improve the function of PhyA phytase at the stomach pH condition by rational protein engineering.