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United States Department of Agriculture

Agricultural Research Service

Related Topics


Location: Commodity Utilization Research

2009 Annual Report

1a.Objectives (from AD-416)
First, develop phytases with significantly higher specific activity and broad substrate utilization by employing molecular biology techniques. Second, engineer higher heat stability in phytase and combine this with increased specific activity to produce a more cost effective enzyme for the animal feed industry. This will be followed by optimizing the enzymatic and nutritional properties of phytase for specific applications.

1b.Approach (from AD-416)
Analyze the sequence and molecular structural data on phytase molecules to achieve higher specific activity for phytic acid and then mutate the substrate specificity site and neighboring amino acid residues in A. niger NRRL 3135 phyA to effect these changes. These same techniques will also be employed to widen the variety of substrates that can be utilized. Increase heat tolerance in phytase will be effected by replacement of specific amino acid residues in the phyA molecule with amino acids occurring at higher frequency in stable proteins; this will result in a mutant phyA with increased heat tolerance. Once this goal is achieved, a combination of increased thermostability with the mutations conferring the higher specific activity will be undertaken. Determination of whether phytate binds to and affects the solubility of chromium and molybdenum, and whether phytate and oxalate interact via cross-linking by calcium and/or magnesium are also planned.

3.Progress Report
This project is expiring and this report constitutes the final report. This project will be terminated and the resources directed to new research activities.

During the last five years, considerable progress has been made on the fundamental understanding of the enzyme called phytase, and what improvements can be made when the structure is changed. The research group obtained a patent for the changes that could be made to the enzyme structure, thus making it a better additive to swine feed for increasing the degradation of phytic acid and availability of phosphorous in the animal diet. The improved version worked better in the sour (acidic) stomach of the animal.

The enzyme phytase exists as form A and form B. By doing a study, we proved that the two forms are different in structure. We proved this by unfolding the enzyme in a special solution of the chemical guanidium chloride and then refolding the enzyme. The refolded enzymes were different, and thus, it was proven the two forms were different in structure. A difference in structure may be important as the structure is an important part of the enzyme's function and its ability to carry out those functions.

It was discovered that phytase’s (form A) normal chemical reactions could be stopped by adding chemicals containing the metal ion vanadium. When the normal reactions were stopped, other reactions took place. Thus, the vanadium compounds could be used as a switch to turn on or off certain chemical reactions.

Our group was the first group to show that the enzyme phytase, form B, could carry out a reaction that breaks up a chemical called phosphotyrosine. We showed that this was true with form B phytases that we isolated from two different molds. This reaction is important for cell growth and division.

Our group conducted work in changing the structure of the phytase, form A, by systematically changing one component (at the gene level) at a time in the structure of the gene and resultant enzyme derived from that modified gene. In this fashion, we could measure how the enzyme reacted to different changes and what effect it had on its function. By doing this, we were able to change the effectiveness of the enzyme at different temperatures.

1. Adapting phytase for higher activity in a gastric environment. By altering the optimum pH of fungal phytase to match the pH of the stomach of an animal, more of the phytin phosphorus is made available to the animal. This promotes faster animal growth and reduces phosphorus levels in the manure. We have successfully engineered our phytase to perform more effectively in the digestive tract of animals. This makes more of the phytin phosphate available to the animal and also lowers the amount lost in manure. A low phosphorus level in manure is important because it protects our environment and helps conserve the world’s phosphorus reserves. Application of this technology to a second phytase demonstrates it has the potential for a wide application.

2. Increasing thermostability of phytase. Animal feed is sometimes heated briefly during processing. In order to retain the high level of activity, a heat tolerant phytase is desirable. Earlier studies have focused on finding a phytase with thermostability and attempting to incorporate several desirable features to make it commercially viable. We successfully took a different approach to this problem by starting with a widely marketed phytase and increased its thermostability. This technique provides the animal feed producers with an improved phytase that has an already proven track record.

6.Technology Transfer


Review Publications
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.

Ullah, A.H., Sethumadhavan, K., Mullaney, E.J. 2008. Kinetic characterization of o-phospho-L-tyrosine phosphohydrolase activity of two fungal phytases. Journal of Agriculture and Food Chemistry. 56(16):7467-7471.

Ullah, A.H., Sethumadhavan, K., Mullaney, E.J. 2008. Unfolding and Refolding of Aspergillus Niger PhyB Phytase: Role of Disulfide Bridges. Journal of Agriculture and Food Chemistry. 56(17):8179-8183.

Last Modified: 4/19/2014
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