NECi now produces two forms of recombinant
enzyme
Nitrate Reductase (NaR) :
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Yeast NAD(P)H: NaR - called YNaR1
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Plant NADH: NaR - called AtNaR2
Both forms
are "Superior Stock NaR" since they are more stable and more robust forms of the
enzyme than found from natural sources (1, 2). Both Superior Stock NaRs
are recombinantly manufactured in Pichia pastoris. These isoenzymes
are made in large quantities, with consistent properties, and low production
cost. Thus, both YNaR1 and AtNaR2 are analytical grade enzymes ready to be used
in analytical chemical analysis of water and other aqueous solutions.
NECi has been championing
enzyme-based nitrate analysis for a number of years, since 1993. Our original
product was the nitrate reductase (NaR) naturally present in corn (Zea mays)
seedlings. Building on years of academic research, we made purified and
stabilized NADH-nitrate reductase commercially available for nitrate analysis.
It was clear from early on that anticipated demand could not be met using the
native enzyme. Efforts to develop more reliable recombinant sources of this
enzyme were initiated in earnest in 1999, with funding from the Small Business
Innovation Research (SBIR) program of the National Institutes of Health.
Recombinant enzymes can be produced under the stringently controlled conditions
required to meet today’s QA/QC standards, and production is scalable to meet
demand. In addition, their underlying genes can be modified to facilitate
purification and, sometimes, to improve functionality.
Today NECi produces two
different nitrate reductase enzymes: YNaR1, introduced in 2002, and AtNaR2,
introduced in 2005. Both forms of NaR have shelf-life and working solution
stability characteristics suited to virtually any lab or field environment.
They differ mainly in operation temperature range. For applications where the
analysis is run between 5 – 30C, YNaR1 is better. For applications at
physiological temperatures or in the range of 20 – 45 C, AtNaR2 is best. AtNaR2
is slightly more suited to high salt (sodium chloride) environments.
A major advantage of
enzyme-based nitrate analysis over standard chemical methods is sensitivity,
which allows a small sample size in relation to the total assay volume. In most
cases, potential interferences such as pH extremes and turbidity are diluted
out. The selectivity of enzymatic reactions means that fewer compounds will
interfere, permitting analysis in complex matrices with little sample
preparation required. Their high specificity reduces false positives.
Superior Stock NaRs
have been shown to effective as catalysts for the
NECi NaR Nitrate Analysis
Method (1, 2). Superior Stock
NaRs work well in automated analyzers as shown by Patton and coworkers at USGS
(3) and by Lachat Instruments (4). Recently, the NECi NaR Nitrate Analysis
Method was "qualified" on discrete analyzers using YNaR1 at room temperature (4)
and AtNaR2 at 37°C (5).
Superior Stock NaRs, YNaR1 and AtNaR2,
are the highest specific activity NaRs (ie. most highly purified) commercially
available, anywhere in the world. Superior Stock NaR forms are highly
stable and can be shipped at room temperature. Since NECi YNaR1 and AtNaR2
utilize NADH as electron donor to drive nitrate reduction, they are less
expensive to use than NADPH-dependent fungal NaR forms.
For more information on the
properties of this unique enzyme, see the detailed review of the biochemistry
of NaR published by W. H. Campbell (6).
References:
Barbier, Guillaume G., and Wilbur H. Campbell (2000)
Expression of Pichia angusta nitrate reductase (YNaR1) in Pichia
pastoris. Current Topics in Gene Expression Systems: Program and
Abstracts, Abstract # PY4, p. 64.
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Campbell,
Wilbur H., P Song, GG Barbier (2006) Nitrate Reductase for Nitrate Analysis in
Water. Environmental Chemistry Letters, 4: 69-73.
Download PDF.
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Campbell, Wilbur H, T Kinnunen-Skidmore, MJ Brodeur-Campbell & ER Campbell (2004)
New and improved nitrate reductase for enzymatic nitrate analysis. American
Laboratory 22(10): 12. read it
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Campbell, Wilbur H., Ellen R.
Campbell, Lynn Egan (2006) Green Chemistry Nitrate Determination: An
Alternative Nitrate Analysis Method. American Laboratory, February, 2006.
See:
http://www.americanlaboratory.com/ OR
Download
PDF
-
Patton, C. J.,
et al. (2006) Nitrate Analysis with Nitrate Reductase on the Discrete
Analyzer. In preparation. See:
http://pubs.acs.org/subscribe/journals/esthag-a/40/i03/html/020106news4.html
Campbell, Wilbur H. (1999) Nitrate Reductase
Structure, Function and Regulation: Bridging the Gap between Biochemistry and
Physiology, Annual Review of Plant Physiology and Plant Molecular Biology
50:277-303.
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1-888-NITRATE (648-7283)
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