2007 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.
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. The Agricultural Research Service (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 some phytases are 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.
The producers of poultry and hog feed will benefit from 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.
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. This is now being combined with another discovery that increases the heat tolerance of the molecule. By coupling these two achievements together we have made significant progress in our goal of tailoring phytase for specific applications. 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 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.
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.”
5.Significant Activities that Support Special Target Populations
|Number of active CRADAs and MTAs||1|
|Number of non-peer reviewed presentations and proceedings||2|
Zhang, W., Mullaney, E.J., Lei, X. 2007. Adopting selected hydrogen bonding and ionic interactions from Aspergillus fumigatus phytase structure improves the thermostability of Aspergillus niger PhyA phytase. Applied and Environmental Microbiology. 73(9):3069-3076.