SBIR-STTR Award

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. 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.

Nutrient management is a touchy subject: there has to be a balance between regulatory concerns and adequate crop nutrition. Phosphate is an essential nutrient for plant productivity but also a major pollutant when applied in excess. On-the-spot testing for phosphate as orthophosphate (available, soluble, or reactive P) is an appropriate solution to give producers and their technical service providers (Extension agents, crop consultants, and farm managers) better control of the use of phosphate fertilizer in the field. NECi, The Nitrate Elimination Company, Inc., has developed a series of enzyme-based, environmentally benign, and reliable nitrate test kits with the assistance of USDA SBIR funding. NECi Ag-NTKs (Nitrate Test Kits for Agriculture) and lab reagents are being adopted by producers, consultants, and agribusiness. We now propose to develop a similar line of test kits for detection of Phosphate. Compliance with nutrient management regulations will be less burdensome when practical, affordable tools are readily available. The photometer (color reader) being perfected in Phase II is designed to work with both nitrate and phosphate test kits. The photometer will provide data on the N and P status of soils, runoff, feed, and crops. This information can be used for making timely, knowledge-based nutrient application decisions with an eye to maximizing profit and yield while staying in compliance. NECi's develops products that enable and encourage testing as an aid to conservation of rural quality of life. In a different context, NECi is deeply committed to Green Chemistry. Many of the methods routinely used by analytical chemists in the lab or out in the field require the use of hazardous or toxic reagents. Simplified methods designed for non-skilled operators tend to lack the accuracy, sensitivity, and selectivity required in order for the information gathered to be relevant and useful. Enzyme-based methods can satisfy both sets of users as the variability and instability characteristics of enzymes are solved though the use of recombinant protein expression and state of the art stabilization techniques. OBJECTIVES: Nutrient management is a touchy subject: there has to be a balance between regulatory concerns and adequate crop nutrition. Phosphate is an essential nutrient for plant productivity but also a major pollutant when applied in excess. On-the-spot testing for phosphate as orthophosphate (available, soluble, or reactive P) is an appropriate solution to give producers and their technical service providers (Extension agents, crop consultants, and farm managers) better control of the use of phosphate fertilizer in the field. NECi, The Nitrate Elimination Company, Inc., has developed a series of enzyme-based, environmentally benign, and reliable nitrate test kits with the assistance of USDA SBIR funding. NECi Ag-NTKs (Nitrate Test Kits for Agriculture) and lab reagents are being adopted by producers, consultants, and agribusiness. We now propose to develop a similar line of test kits for detection of Phosphate. Compliance with nutrient management regulations will be less burdensome when practical, affordable tools are readily available. Field-useable test kits for available P complement NECi's kits for available N. The photometer (color reader) being perfected in Phase II is designed to work with both test kits. The photometer will provide data on the N and P status of soils, runoff, feed, and crops. This information can be used for making timely, knowledge-based nutrient application decisions with an eye to maximizing profit and yield while staying in compliance. Reagentless sensors are the long term goal. NECi's develops products that enable and encourage testing as aids in conservation of rural quality of life. In a different context, NECi is deeply committed to Green Chemistry. Many of the methods routinely used by analytical chemists in the lab or out in the field require the use of hazardous or toxic reagents. Simplified methods designed for non-skilled operators tend to lack the accuracy, sensitivity, and selectivity required in order for the information gathered to be relevant and useful. Enzyme-based methods can satisfy both sets of users as the variability and instability characteristics of enzymes are solved though the use of recombinant protein expression and state of the art stabilization techniques. NECi is working to develop and validate enzyme-based analytical chemistry through methods approval agencies such as the US EPA, the US Geological Survey, and ASTM. APPROACH: The goal of this Phase II Project is to develop a simple, reliable field test for available phosphate. When combined with a dual-wavelength hand-held portable photometer and the previously developed and implemented NECi Nitrate Test Kit, the new NECi Phosphate Test Kit will provide the end-user with easy-to-use and accurate methods for both nitrate and phosphate. NECi will develop manufacturing systems for recombinant purine nucleoside phosphorylase (PNP; EC 2.4.2.1) to deploy in the NECi Field Phosphate Test Kit (PTK), which was shown to be feasible in Phase I. First, we will optimize production of recombinant Ecoli-PNP-1 cell line from the Phase I Project, in Escherichia coli utilizing a fermenter system. Next we optimize the cell disruption/enzyme extraction method and perfect the one-step enzyme purification. The optimum storage conditions for stability of PNP will be determined and long-term stability analyzed. The reagents for the field PTK will be optimized for maximum ease of use. The project will develop a handheld, portable NECi Dual Wavelength Photometer for NECi Field Nitrate and Phosphate Test Kits. In cooperation with Dr. David Coates, we will develop a dual wavelength LED handheld portable photometer for qualifying results of either NECi Nitrate Test Kit (NTK) or NECi Phosphate Test Kit (PTK) with the same instrument. Algorithms will be developed for conversion of raw data to concentrations of nitrate and phosphate in Ag samples. The analyte units reported will be familiar to Ag users. Data will be transferable via a USB port to laptop PCs or other devices such as "Smart Phones". Test kits and photometers will be evaluated in the field to demonstrate that the NECi Phosphate Test Kit (PTK) when employed with the NECi Dual Wavelength Photometer will produce accurate and precise results with real world Ag samples. Field testing of the PTK and photometer will be carried out in cooperation with technical service providers and other collaborators.