Progress 10/01/08 to 09/30/09
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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.
Impacts
(N/A)
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.
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Progress 06/17/04 to 06/16/09
Outputs
Progress Report 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. 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. Progress concluded with the 2009 Annual Report for project 6435-13410-003- 00D. See 2009 Annual Report for the last reported progress. Related work on phytase (enzyme that can break down the undigestible phytic acid (phytate) part found in grains oil seeds and thus release digestible phosphorus, calcium and other nutrients) is reported in the bridging project 6435-13410-004-00D. See 2010 Annual Report for 6435-13410-004- 00D for the progress in FY-2010.
Impacts
(N/A)
Publications
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Progress 10/01/07 to 09/30/08
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations A patent was granted entitled �Using mutations to improve Aspergillus.� This technology was employed to create a phytase (an enzyme that helps animals digest food)for animal feed with higher activity. This resulted in faster growth and lower phosphorus levels in their manure. A study was published that details the different effects that common salt has on the activity of fungal and bacterial phytase. Calcium chloride extended the pH range of fungal phytase to 8.0 and enhanced its activity. In E. coli phytase, it shifted the optimum pH from 5.5 to 2.0 with no enhancement of activity. Since the two phytases share the same catalytic mechanism, other structural components can account for the observed catalytic and salt effect differences. We broadened the pH range of a second fungal phytase, PhyB to a more characteristic pH range in the stomach of animals through site-directed mutagenesis. Amino acids of the substrate specificity site of PhyA were changed and the resulting mutants characterized. A shift in the optimum pH of mutant E272K from 2.5 to 3.2 meant it more closely matched the gastric environment. This change in pH range of PhyB makes it better suited for development as an animal feed additive. NP 306, Component: 2, Problem Area: c. Technology Transfer Number of New/Active MTAs(providing only): 1 Number of Invention Disclosures submitted: 2 Number of New Patent Applications filed: 1
Impacts
(N/A)
Publications
- Kadan, R.S., Phillippy, B.Q. 2007. Effects of yeast and bran on phytate degradation and minerals in rice bread. Journal of Food Science and Technology. 72(4):C208-C211.
- Weaver, J.D., Mullaney, E.J., Lei, X.G. 2007. Altering the substrate specificity site of aspergillus niger PhyB shifts the pH optimum to pH 3.2. Applied Microbiology and Biotechnology. (2007)76:117-122.
- Ullah, A.H., Sethumadhavan, K., Mullaney, E.J. 2008. Salt effect on the pH profile and kinetic parameters of microbial phytases. Journal of Agricultural and Food Chemistry. 56:3398-3402.
- Lei, X. G., Porres, J. M., Mullaney, E. J. and Brinch-Pedersen, H. 2007. Phytase: source, structure and application. In: Industrial Enzymes, structure, function and applications. Editied by Polina, J. and MacCabe, A. P. Springer, Dordrecht, The Netherlands, p. 505-529.
- Weaver, J.D., Ullah, A.H., Sethumadhavan, K., Mullaney, E.J., Lei, X.G. 2007. Enzymatic comparisons of Aspergillus Niger PhyA and Escherichia Coli AppA2 Phytases (abstract). Journal of Animal Science. 85(Supplement 1):647.
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Progress 10/01/06 to 09/30/07
Outputs
Progress Report 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. 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. Significant Activities that Support Special Target Populations 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. Accomplishments 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.� Technology Transfer Number of Active CRADAS and MTAS: 1 Number of Non-Peer Reviewed Presentations and Proceedings: 2
Impacts
(N/A)
Publications
- 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.
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Progress 10/01/05 to 09/30/06
Outputs
Progress 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. 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)
Impacts
(N/A)
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 environment enhances its effectiveness in animal feeding. Applied and Environmental Microbiology. 72:4397-403.
- Mullaney, E.J., Ullah, A.H. 2005. Phytases: attributes, catalytic mechanisms and applications. Inositol Phosphates in the Plant and Soil System. In: Bouyoucos Conference on Inositol Phosphates in the Soil-Plant- Animal System: Linking Agriculture and Environment, August 21-25, 2005, Sun Valley, ID. 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.
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Progress 10/01/04 to 09/30/05
Outputs
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? What does it matter? Most of the natural element phosphorous 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 animals 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 an enzyme, called phytase, that has been demonstrated to effectively reduce phosphorus levels in manure. However, no phytase specifically engineered for animal feed is commercially available today. Now that the molecular structure of phytase is available, research is possible to optimize reactivity 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 raw material 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 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 the milestones (indicators of progress) from your Project Plan. (FY 2005) Produce a structurally altered 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 the natural elements chromium and molybdenum. (FY 2006) Report on a study or pursue a patent
application on our method of making phytase enzymes that are stable at high temperatures (thermostable). Modify the pH profile of the phytase enzyme, named 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 (an organic acid) on phytase activity. (FY 2007) Determine the degree of success achieved for specific applications by structurally changing the enzyme. A goal of increasing the specific activity of a phytase enyzyme, named phyA, three times over the activity level of phyB, which is a better enzyme at pH 2.5, but not at pH 3.5 to 5. 0. (FY 2008) Assess the needs for new enzyme combinations and start animal feed trials on selected enzymes. Determine if there is a correlation between the substitution of specific amino acids in the reactive center of the enzyme and increasing the raw material utilization ability of the phytase enzyme phyA. (FY 2009) Combine selected
thermostable enzymes 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 methods to introduce additional changes in part of the DNA that has the instructions on how to make the phytase phyA enzyme to combine high specific activity for phytase and heat tolerance. 3a List the milestones that were scheduled to be addressed in FY 2005. For each milestone, indicate the status: fully met, substantially met, or not met. If not met, why. 1. Produce an altered phytase that will display enhanced activity over the wild type that is currently being marketed. Milestone Substantially Met 2. Complete a study that will detail the effect of phytase on solubility of chromium and molybdenum. Milestone Not Met Other 3b List the milestones that you expect to address over the next 3 years (FY 2006, 2007, and 2008). What do you expect to
accomplish, year by year, over the next 3 years under each milestone? (FY 2006) Milestone: Publish a study or pursue a patent application on our method of making phytase enzymes that are stable at high temperatures (thermostable). Progress: We have started generating single changes to enzymes and will start combining changes to enzymes to determine if heat tolerance is additive. Milestone: Modify the pH profile of the phytase enzyme, named phyB, to determine if this can increase its commercial potential and acceptance by the animal feed industry. Progress: We have generated enzymes that have a different pH profile and they will be tested shortly. Milestone: Publish a study that will detail the effect of oxalate (an organic acid) on phytase activity. Progress: Because of budgetary constraints and the permanent loss of an SY, this milestone may not be completed. The completion of this milestone is not critical to the overall success of the research project. (FY 2007) Milestone:
Determine the degree of success achieved for specific applications by structurally changing the enzyme. A goal of increasing the specific activity of a phytase enyzyme, named phyA, three times over the activity level of phyB,which is a better enzyme at pH 2.5, but not at pH 3.5 to 5.0. Progress: Some specific activity for phyA has already been achieved. The work with phyB if successful will make it more commercially viable and provide another enzyme for market. Milestone: Combine selected thermostable mutants and measure the additive effects. Progress: Because of budgetary constraints and the permanent loss of an SY, this milestone may not be completed. The completion of this milestone is not critical to the overall success of the research project. (FY 2008) Milestone: Assess the needs for new enzyme combinations and start animal feed trials on selected enzymes. Progress: The initial animal feed trials were successful and showed significant weight gains in animals on the modified
phytase. Milestone: Determine if there is a correlation between the substitution of specific amino acids in the reactive center of the enzyme and increasing the raw material utilization ability of the phytase enzyme. Progress: Initial test of enzymes with altered reative center indicates this is feasible. The anticipated impact is an improved phytase enzyme, stable at high temperatures and active over the acid-base spectrum. Its benefits will have been demonstrated in animal feed trials. The potential market for phytases is $500 million. 4a What was the single most significant accomplishment this past year? Weight gain demonstrated in swine. The results of feed trials with swine demonstrated significant weight gain in swine whose diet contained our engineered phytase over animals fed a diet containing a commercially available phytase. This represents the work of several years and validates the potential of this research to produce a superior product. The impact of the findings
suggest that significant saving may be realized if the feed can be utilized more efficiently in swine. Positive environmental impacts may also be realized as less run-off is anticipated. 4b List other significant accomplishments, if any. The folding pathway of the second phytase, phyB, was determined. The protein remains in dimer state throughout unfolding and refolding state. Lack for the enzyme stabilizing bridges play an important role in phytase folding. Any disturbance of bridge formation leads to the failure in refolding the protein. The engineering of new bridge may help attain further stability in phytase. The impact of this finding, is that enzymes may be altered in the future to make them more stable at high temperatures or at target pH. 4c List any significant activities that support special target populations. Our long term goal is to make changes to traditional crops so that phytase may be produced in the plant leaves. This would result in small farmers having a
value-added product that could be commercialized for animal feed. 4d Progress report. Research under a Specific Cooperative Agreement between ARS and the Florida Institute of Oceanography was completed and the agreement is terminated. Additional details of research can be found in the report for the subordinate project, 6435-13410-003-01S, Relationship of Water/Sediment Quality to Occurrence and Abundance of Harmful Algal Species. 5. Describe the major accomplishments over the life of the project, including their predicted or actual impact. The amino acid residues that compose the reaction center of the enzyme and acid-base profile were identified. Specific changes 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. The protein folding mechanism in phytase was determined. 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 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. The commercial impact of phytases has exceeded $500 million (Science, 283, 2015). The financial benefits are thus considerable and the ability to further conserve the worlds limited phosphorus reserves are priceless. Production of phytase in traditional crops such as tobacco, alfalfa, and potato looks very promising since phytases biochemical properties were unchanged when the protein was expressed in the leaves of the crop plants. These findings can be directly linked to the National
Program's Action Plan as described in question 1. 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. 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). Scientific Publications: Mullaney, E. J., Ullah, A. H.
J. 2005. Conservation of cysteine residues in fungal histidine acip phytases. Biochemical and Biophysical Research Communications. v. 328. p. 404-408. Ullah, A.H.J., Sethumadhavan, K., Mullaney, E.J. 2005. Monitoring of unfolding and refolding in fungal phytase (phyA) by dynamic light scattering. Biochemical and Biophysical Research Communications. v. 327. p. 993-998.
Impacts
(N/A)
Publications
- Mullaney, E.J., Ullah, A.H. Conservation of an eight-cysteine motif in fungal histidine acid phosphatases. Mycological International Conference Proceedings, July 30-August 4, 2005, Hilo, Hawaii. p. 169.
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