Enzyme-based nitrate determination receives USGS Standard Method designation


Source: NECi Superior Enzymes

Based on NECi's reagent grade nitrate reductase enzyme

12 Feb 2012, Lake Linden, MI US -- RECOMBINANT PROTEIN TECHNOLOGY for GREEN ANALYTICAL CHEMISTRY:  USGS Standard Methods I-2457-11 and I-2458-11

 Analytical chemistry for environmental monitoring often requires hazardous reagents.  NECi’s enzyme-based nitrate reagents replace cadmium reduction with reagent-grade, environmentally benign enzymes.  The resulting nitrite is determined by standard Griess reagent chemistry.  The reaction approaches 100% reduction, is irreversible, and goes to completion.  The sensitive method works for most aqueous samples, including soil extracts and low levels in seawater.

Environmental protection requires chemistry.  In particular, it requires analytical chemistry.  And analytical chemistry involves the use of many reagents and solvents that are inimical to environmental quality and human health.  It seems ironic and unfair that the people most closely associated with environmental monitoring and health need to compromise the health of their own environment in order to understand the processes going on around them. 


Biotechnology has a role in the future of environmental protection.  Chemistry requires harsh environments because energy is required to affect most reactions.  Catalysts reduce this activation energy to conditions more in tune to the standard environment (STP).  Enzymes are protein catalysts that biological systems use to accomplish the complex chemical reactions that metabolism requires.  For most chemical reactions, there is an enzyme that can make it happen efficiently at low energy.  These reactions can be adapted for detection of each enzyme’s target substrate.


NECi is dedicated to the application of enzyme systems to the solution of environmental challenges, especially for wet chemistry analytical methods.  Current focus is on analysis of nitrate and phosphate, the two critical agricultural nutrients important in water quality monitoring.  NECi’s test kits and reagents for Nitrate determination have been on the market since 1993.    


Enzymatic reactions have great specificity and selectivity.  They are also efficient and rapid.   NaR reduces nitrate to nitrite in any aqueous solution, from tissue culture media to agricultural runoff to seawater.  The resulting nitrite is determined by standard Griess reagent chemistry.  The reaction approaches 100% reduction, is irreversible, and runs to completion.  Results are detected by spectroscopy at 540nm (+/- 10nm).


NECi has developed two forms of recombinant nitrate reductase (NaR), an enzyme in plants and some microbes that reduces nitrate to nitrite.  The enzymes are produced in the Pichia pastoris protein expression system by fermentation under tightly controlled conditions, which means that quality is as stable as for any reagent grade chemical.  NECi has formulated nitrate test kits and reagents for manual, automated, and on-site applications.  Enzyme-based phosphate enzymes and methods will be market-ready in 2012.  Development of the reagents and methods has been funded by the Small Business Innovation Research (SBIR) programs of NIH and the USDA.


 Prepared by Ellen R Campbell, Vice President, NECi




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