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United States Department of Agriculture

Agricultural Research Service

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Research Project: DEVELOP ENHANCED PHYTASES FOR ANIMAL FEED AND FOR INCORPORATION INTO NEW PLANT CULTIVARS REQUIRING LESS PHOSPHORUS FERTILIZERS

Location: Commodity Utilization Research

2006 Annual Report


1.What major problem or issue is being resolved and how are you resolving it (summarize project aims and objectives)? How serious is the problem? Why does it matter?
Most of the phosphorus in seed and grains is in the form of phytic acid. When these plant products are used as feed for swine and poultry, nearly all this phosphorus is nutritionally unavailable and ends up in the animal's manure. Modern agricultural operations have concentrated the output of this manure in certain areas creating serious environmental problems. ARS research has already made possible the production of a fungal phytase that has been demonstrated to effectively reduce phosphorus levels in manure. However, today no phytase specifically engineered for animal feed is commercially available. Now that the molecular structure of phytase is available, research is possible to optimize catalytic and other features of this enzyme to further its utilization and reduce the antinutritional effects of phytic acid. Today, there is a need for such an enhanced phytase to protect our watersheds and coastal environments. This study also has the potential to expand the use of phytase to aquaculture and to increase the capacity of plants to better acquire soil phosphorus. All these are important components as we search for effective means to conserve our limited phosphate reserves for future generations.

The project has these three goals:.
1)to develop phytases with significantly higher specific activity and broad substrate utilization;.
2)to engineer higher heat stability in phytases and combine this with increased activity to produce a more cost effective enzyme for the animal feed industry; and.
3)to optimize the enzymatic and nutritional properties of phytases for specific applications.

This research supports National Program 306 - Quality and Utilization of Agricultural Products. It directly addresses the NP 306 Action Plan, Component 2, New Processes, New Uses, and Value-Added Foods and Biobased Products, Problem Area 2c "to reduce the negative impact of excess phosphate from animal manure caused by phytic acid in plants meals, enzyme technologies will be developed to increase the bioavailability of this nutrient."

The producers of poultry and hog feed will benefit from an improved phytases. Federal, State and local government will benefit from reduced incidents of fish kills from algal blooms in waterways. Farmers subject to clean water legislation will also benefit by having improved tools to maintain production levels and comply with these regulations.


2.List by year the currently approved milestones (indicators of research progress)
Produce a mutant phytase that will display enhanced activity over the wild type that is currently being marketed.

Complete a study that will detail the effect of phytase on solubility of chromium and molybdenum.

(FY 2006)

Report on a study or pursue a patent application on our method of developing thermostable mutants and decide on the most effective method.

Modify the pH profile of A. niger phyB to determine if this can increase its commercial potential and acceptance by the animal feed industry.

Report on a study that will detail the effect of oxalate on phytase activity.

(FY 2007)

Determine the degree of success achieved for specific applications by mutagenesis.

A goal of increasing the specific activity of phyA 3 fold is targeted based on the activity level in A. niger NRRL 3135 phyB, which is a better catalyst at pH 2.5, but not at pH 3.5 to 5.0.

(FY 2008)

Assess the needs for new mutant combinations and start animal feed trials on selected mutants.

Determine if there is a correlation between the substitution of specific amino acids in the SSS and increasing the substrate utilization ability of NRRL 3135 phyA.

(FY 2009)

Combine selected thermostable mutants and measure the additive effects. Combine the successful modifications of the project to engineer a superior phytase for use by the animal feed industry and other phytases for other applications.

Utilize selected oligonucleotides to introduce additional changes in mutant A. niger PhyA gene to combine high specific activity for phytase and heat tolerance.


4a.List the single most significant research accomplishment during FY 2006.
By modification of the enzyme's pH profile to match the pH level of the stomach, we found the means to increase the specific activity of phytase in the stomach of animals.


4b.List other significant research accomplishment(s), if any.
Successfully modified the pH profile of A. niger phyB phytase using the same technique that we developed for A. niger phyA phytase.

Performed a comparison of A. niger phyA phytase and E. coli phytase to measure their catalytic parameters.


4c.List significant activities that support special target populations.
None.


4d.Progress report.
None.


5.Describe the major accomplishments to date and their predicted or actual impact.
The amino acid residues that compose the substrate specificity site and pH profile were identified. Specific mutations of the key amino acid residues in this domain had resulted in altering the pH optima that is more suitable for degrading phytic acid in the stomach of poultry and swine. The tailor making of phytase to suit a specific application is now possible. This research also serves as an excellent example of how ARS research uses basic science to solve practical problems in agriculture, environment and human health. The commercial value of phytases has exceeded $500 million (Science, 283, 2015). The financial benefits are thus considerable and the ability to further conserve the world's limited phosphorus reserves are priceless. Biofarming of phytase [Producing phytase in crop plants instead of using traditional fermentation technology] in traditional crops such as tobacco, alfalfa, and potato looks very promising since phytase's biochemical properties were unchanged when the protein was expressed in the leaves of the crop plants. Now small farmers may produce phytase in the leaves of traditional crops to enhance their income. The protein folding mechanism in phytase was elucidated. Protein engineering, by site-directed mutations can now change the structure of the phytases molecule to make the enzyme more stable. Technology has also been developed that allows for precise measurement of the effects of phosphorus from agricultural operation has on the growth and development of microorganisms causing harmful algal bloom in our waterways. This will provide information on how to develop strategies to better prevent fish kills and other environmental harmful effects from these operations.


6.What science and/or technologies have been transferred and to whom? When is the science and/or technology likely to become available to the end-user (industry, farmer, other scientists)? What are the constraints, if known, to the adoption and durability of the technology products?
Both scientific knowledge and several cloned phytases genes were made available to scientists all around the world. This has provided the foundation for the development of enhanced phytases for utilization as an animal feed additive, the discovery of its potential to reduce the need for phosphorus fertilizers in crop plants, and the development of a farm-based method to produce phytases for the commercial market. During this period the proceedings of a major conference on the importance of inositol phosphates [breakdown products of phytic acid] in the soil-plant-animal system was compiled and edited. When published later this year this will provide a major references source for both science and industry.


7.List your most important publications in the popular press and presentations to organizations and articles written about your work. (NOTE: List your peer reviewed publications below).
"Phytase Reinvented" Agricultural Research magazine, ARS/USDA July 2006 p. 14.

"Feed Additive Helps Agriculture and the Environment" Western Agri-Radio Network, Inc. July 2006 (www.westernagri-radio.net)


Review Publications
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.

Mullaney, E.J., Ullah, A.H. 2005. Phytases: attributes, catalytic mechanisms and applications (abstract). Inositol Phosphates in the Plant and Soil System. p.17-18.

Fitzmorris, K.B., Lima, I.M., Marshall, W.E., Reimers, R.S. 2006. Anion and cation removal from solution using activated carbons from municipal sludge and poultry manure. Journal of Residuals Science & Technology. 3(3):161-167.

Last Modified: 4/17/2014
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