Source: THE NITRATE ELIMINATION COMPANY, INC. submitted to
ENZYME-BASED TEST KITS FOR PHOSPHATE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0221712
Grant No.
2010-33610-20885
Project No.
MICK-2010-00130
Proposal No.
2010-00130
Multistate No.
(N/A)
Program Code
8.4
Project Start Date
Aug 1, 2010
Project End Date
Oct 31, 2012
Grant Year
2010
Project Director
Campbell, W. H.
Recipient Organization
THE NITRATE ELIMINATION COMPANY, INC.
334 HECLA STREET
LAKE LINDEN,MI 49945
Performing Department
(N/A)
Non Technical Summary
The goal of this proposal is to develop a recombinant form of the enzyme purine nucleoside phosphorylase (PNP) as the basis for development of simple, reliable, field-usable test kits for available phosphate (also called reactive, ortho, or soluble P). Enzyme-based methods for analytical chemistry have a number of advantages over wet chemistry and instrumental methods, including reduction of sample preparation time and decreased use of hazardous or toxic reagents. NECi, The Nitrate Elimination Co., Inc., has developed a series of enzyme-based nitrate test kits for agriculture. NECi's Ag-NTKs (nitrate test kits) and lab reagents are now in wide usage by farmers, Ag consultants, and major Ag feed/seed companies. These test kits combine ease of use with reliability of results. Here, we propose to apply the knowledge gained in development and implementation of Field Nitrate Test Kits to bring a green, sustainable field-usable Phosphate Test Kit to the market. Test kits for available P complement NECi's existing product line of enzyme-based test kits and reagents for nitrate, or available N. Application and management of N and P are key elements to successful production of agricultural products. Phosphate is an essential nutrient for plant productivity, but unlike nitrogenous fertilizers which can be manufactured, the supply of phosphate is finite and rapidly dwindling. Inappropriately applied phosphate nutrients leach from soils over time and enter the ground water. Excess phosphate is a major water quality problem, leading to eutrophication of ponds, lakes and coastal areas. Loss of recreational opportunities in rural areas has economic and social impacts: there's no fishing or swimming in impaired or algae-filled waters. NECi's products and R&D are based on clean manufacturing practices to develop recombinant enzymes for detection of compounds of environmental concern. Ag producers need better tools for management of N and P. These critical agricultural nutrients are coming under increased regulatory and cost pressures. Adoption of soil and water testing regimens into farm Best Management Practices (BMPs) will assist producers to improve management of soil and water quality, to save money by purchasing and applying the right amount of nutrients, and to stay in regulatory compliance. A test kit for soluble phosphate that provides reliable information on site, in the field, would be a useful tool to USDA Extension agents, farm managers, and technical service providers (TSPs). NECi's nitrate test kits are used by all of these groups, plus academic researchers in agriculture and environmental quality.
Animal Health Component
40%
Research Effort Categories
Basic
20%
Applied
40%
Developmental
40%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
1020110200010%
1020210200010%
1025210200010%
1027299100030%
1027299104030%
1027299200010%
Goals / Objectives
The goal of this proposal is to develop a recombinant form of the enzyme PNP (purine nucleoside phosphorylase) as the basis for development of simple, reliable, field-usable test kits for available (also called reactive, ortho, or soluble) phosphate. The milestones and outputs of this project are: 1) Clone a bacterial form of purine nucleoside phosphorylase (PNP), and express the active enzyme in Pichia pastoris. This recombinant protein expression system is already employed at NECi to produce Nitrate Reductase for NECi Nitrate Test Kits. NECi has a commercial license for production of recombinant proteins in the Pichia system and more than ten years of experience with manufacturing enzymes with it. 2) Develop an effective production protocol for recombinant PNP using the fermenter system already in place at NECi, which will include development of downstream processing methods to manufacture a commercial, analytical grade of PNP. 3) Demonstrate that the NECi recombinant PNP is an effective replacement for commercially available PNP in the Green/Sustainable Field Assay for Phosphate being developed at NECi.
Project Methods
The project involves development, expression, and purification of a recombinant enzyme, purine nucleoside phosphorylase (PNP), and adapting the new enzyme for field usable test kits for phosphate measurement of soil and water samples. In Objective 1, we will clone and express a bacterial form of the enzyme using standard molecular biology techniques. The gene will be synthesized by a commercial service based on our direction. The gene will be transformed into a vector used for expression of proteins in the Pichia pastoris system. Cell lines expressing the active enzyme will be selected and confirmed. Objective 2 involves development of efficient PNP production protocols in fermentation, and downstream processes for enzyme purification and storage. In Objective 3, we will evaluate the recombinant PNP in phosphate test kit formats, using lab and real world samples.

Progress 08/01/10 to 10/31/12

Outputs
OUTPUTS: Phase I CRIS Report Extension Oct 2012 OUTPUTS: This Report is on work performed during the project Extension period that was granted for administrative reasons. The objectives for Phase I were straightforward: To clone the enzyme Purine Nucleoside Phosphorylase (PNP) and express it in active form, and to demonstrate that this enzyme can be used for determination of phosphate. We chose the PNP gene from E.coli, and designed the construct, including modifications to facilitate insertion of the new gene into a cloning vector and a purification "tag" to allow one-step purification. Cloning and transformation into the pET protein expression system and selection processes went smoothly. Commercial scale production of active enzyme was accomplished early in Phase I. Protein was freeze-dried and stored at various controlled temperature environments so that enzyme stability assays could be performed throughout Phase I. These studies show that NECi's new PNP is stable enough for use on site, and has an adequate shelf-life for shipping and commercial sales. A simple assay was developed using the compound MESG (2-amino-6-mercapto-7-methylpurine), which absorbs at 360 nm, as the substrate. The enzyme also performs well in phosphate determination in water, with a detection range of zero to ten nanomoles ( 2 - 50 micromolar, or high ppm range). During the Phase I Extension, stability studies on the freeze-dried PNP and on the detection reagent MESG were continued by Lab Assistant/Chemistry major Kyle Scott and other NECi technicians. PARTICIPANTS: For marketing input, NECi continues to work with John Whittaker and Brent Fitzsimmons of Northern Star Integrated Services, a subsidiary of Au Sable LLC. They assist NECi with access to target customers such as TPAs, Extension agents, farm managers, and grain operators for product evaluations and focus groups. TARGET AUDIENCES: In Phase II, the target audiences will be TPAs, Extension agents, farm managers, and grain operators and others concerned with nutrient management on the farm. The goal of the project is to provide tools to these users to make nutrient management easier. NECi's products are designed to alleviate the burdens of regulatory compliance while ensuring maximum yields. The thinking is that if compliance is affordable and easy, resistance to consideration of water quality will decrease. Protection of ground water is critical to long term success in agriculture and to the health of rural communities. An additional target is the analytical chemistry community, which is more open to Green methods in the 21st century. NECi is working with a number of agencies to achieve Standard Method status for its enzyme-based analytical methods. Consideration is underway at the US Geological Survey, ASTM, and the UP EPA. PROJECT MODIFICATIONS: The Phase I proposal detailed expression of the PNP enzyme in the Pichia pastoris expression system. However, the enzyme expresses well in E coli, so this extra step was not useful. Reproducibility of PNP production was demonstrated during this interim period via repeat fermentations. PARTICIPANTS: PARTICIPANTS: For marketing input, NECi continues to work with John Whittaker and Brent Fitzsimmons of Northern Star Integrated Services, a subsidiary of Au Sable LLC. They assist NECi with access to target customers such as TPAs, Extension agents, farm managers, and grain operators for product evaluations and focus groups. During the extension period, NECi engaged with the Chemistry Department of Michigan Technological University to allow Mr Scott to receive credits for some of his work at NECi. TARGET AUDIENCES: TARGET AUDIENCES: In Phase II, the target audiences will be TPAs, Extension agents, farm managers, and grain operators and others concerned with nutrient management on the farm. The goal of the project is to provide tools to these users to make nutrient management easier. NECi's products are designed to alleviate the burdens of regulatory compliance while ensuring maximum yields. The thinking is that if compliance is affordable and easy, resistance to consideration of water quality will decrease. Protection of ground water is critical to long term success in agriculture and to the health of rural communities. An additional target is the analytical chemistry community, which is more open to Green methods in the 21st century. NECi is working with a number of agencies to achieve Standard Method status for its enzyme-based analytical methods. Consideration is underway at the US Geological Survey, ASTM, and the UP EPA. PROJECT MODIFICATIONS: PROJECT MODIFICATIONS: The Phase I proposal detailed expression of the PNP enzyme in the Pichia pastoris expression system. However, the enzyme expresses well in E coli, so this extra step was not useful. Reproducibility of PNP production was demonstrated during this interim period via repeat fermentations.

Impacts
The work performed during the Phase I Extension period added to the base of information carried on to Phase II. he Phase II project will build on these results to develop and validate phosphate test kits and analytical reagents for use with samples of interest to agriculture. Sample types include runoff, manure ponds, livestock feed, and well or ground water. NECi is also developing a photometer with digital readout in units that ag producers use (such as ppm and per cent) for soil and water. The new photometer will work with NECi's nitrate (NTK) and phosphate (PTK) test kits. The overarching goal of this project is to enable the agricultural community to better manage N and P. Reagents for use in autoanalyzers may be market-ready before test kits are finalized. Conventional test kits and methods in use today require concentrated acids as well as toxic metal reagents. Many users are looking for safer, more environmentally friendly analytical tools. NECi works with Green Chemistry early adopters, including methods development chemists at the US Geological Survey and autoanalyzer equipment manufacturers such as OI Analytical. Kyle Scott, a Chemistry major at MI Technological University, received training in analytical chemistry techniques and protein expression and characterization during the Phase I project.

Publications

  • There were no publications during the report period. Mr Scott, who graduated August 2012 with a BSc in Chemistry, will present a poster on the phosphate assay development work at the Michigan Green Chemistry Conference, Oakland, MI, October 2012.


Progress 08/01/10 to 08/31/11

Outputs
OUTPUTS: The objectives for Phase I were straightforward: To clone the enzyme Purine Nucleoside Phosphorylase (PNP) and express it in active form, and to demonstrate that this enzyme can be used for determination of phosphate. We chose the PNP gene from E.coli, and designed the construct, including modifications to facilitate insertion of the new gene into a cloning vector and a purification "tag" to allow one-step purification. The construct was made for us by a contract firm. Transformation into the pET protein expression system and selection processes went smoothly. A high-expressing cell line was chosen and taken from shake flask culture to fermentation. Commercial scale production of active enzyme was accomplished early in Phase I. Protein was freeze-dried and stored at various controlled temperature environments so that enzyme stability assays could be performed throughout Phase I. These studies show that NECi's new PNP is stable enough for use on site, and has an adequate shelf-life for shipping and commercial sales. A simple assay was developed using the compound MESG (2-amino-6-mercapto-7-methylpurine), which absorbs at 360 nm, as the substrate. The enzyme also performs well in phosphate determination in water, with a detection range of zero to ten nanomoles ( 2 - 50 micromolar, or high ppm range). PARTICIPANTS: For marketing input, NECi has worked with John Whittaker and Brent Fitzsimmons of Northern Star Integrated Services, a subsidiary of Au Sable LLC. They assist NECi with access to target customers such as TPAs, Extension agents, farm managers, and grain operators for product evaluations and focus groups. Kyle Scott, a Chemistry major at MI Technological University, received training in analytical chemistry techniques and protein expression and characterization during the Phase I project. TARGET AUDIENCES: In Phase II, the target audiences will be TPAs, Extension agents, farm managers, and grain operators and others concerned with nutrient management on the farm. The goal of the project is to provide tools to these users to make nutrient management easier. NECi's products are designed to alleviate the burdens of regulatory compliance while ensuring maximum yields. The thinking is that if compliance is affordable and easy, resistance to consideration of water quality will decrease. Protection of ground water is critical to long term success in agriculture and to the health of rural communities. An additional target is the analytical chemistry community, which is more open to Green methods in the 21st century. Many methods used for analytical chemistry today were developed one hundred or more years ago and involve the use of toxic or hazardous reagents. Waste disposal and operator exposure were not considered when these methods were adopted and promulgated. NECi personnel will continue to make presentations at EPA and American Chemical Society conferences. NECi is working with a number of agencies to achieve Standard Method status for its enzyme-based analytical methods. Consideration is underway at the US Geological Survey, ASTM, and the UP EPA. PROJECT MODIFICATIONS: The Phase I proposal detailed expression of the PNP enzyme in the Pichia pastoris expression system. However, the enzyme expresses well in E coli, so this extra step was not useful. NECi personnel have expertise in both protein expression systems. NECi is licensed to work with both technologies.

Impacts
The Phase II project will build on these results to develop and validate phosphate test kits and analytical reagents for use with samples of interest to agriculture. Sample types include runoff, manure ponds, livestock feed, and well or ground water. Reagents for use in autoanalyzers may be market-ready before test kits are finalized. Conventional test kits and methods in use today require concentrated acids as well as toxic metal reagents. Many users are looking for safer, more environmentally friendly analytical tools. NECi works with Green Chemistry early adopters. NECi is also developing a photometer with digital readout in units that ag producers use (such as ppm and per cent) for soil and water. The new photometer will work with NECi's nitrate (NTK) and phosphate (PTK) test kits. The overarching goal of this project is to enable the agricultural community to better manage N and P.

Publications

  • No publications reported this period