Environmental Chemistry Letters

, Volume 4, Issue 2, pp 69–73 | Cite as

Nitrate reductase for nitrate analysis in water

  • Wilbur H. Campbell
  • Pengfei Song
  • Guillaume G. Barbier
Original Paper


Nitrate analysis in water is one of the most frequently applied methods in environmental chemistry. Current methods for nitrate are generally based on toxic substances. Here, we show that a viable alternative method is to use the enzyme nitrate reductase. The key to applying this Green Chemistry solution for nitrate analysis is plentiful, inexpensive, analytical grade enzyme. We demonstrate that recombinant Arabidopsis nitrate reductase, expressed in the methylotrophic yeast Pichia pastoris, is a highly effective catalyst for nitrate analysis at 37°C. Recombinant production of enzyme ensures consistent quality and provides means to meet the needs of environmental chemistry.


Nitrate reductase Recombinant expression Nitrate Green chemistry Water Enzymes 



We thank Prof. N. M. Crawford for supplying the Arabidopsis NaR2 containing plasmid. We also thank Troy Kinnunen-Skidmore and Michael J. B. Campbell for assistance with the experiments. This work was supported, in part, by the Michigan Tech Research Fund.


  1. Barbier GG, Joshi RC, Campbell ER, Campbell WH (2004) Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris. Prot Expr Purif 37:61–71CrossRefGoogle Scholar
  2. Campbell WH (1999) Nitrate reductase structure, function and regulation: Bridging the gap between biochemistry and physiology. Annu Rev Plant Physiol 50:277–303CrossRefGoogle Scholar
  3. Clesceri LS, Greenberg AE, Eaton AD (1998) Standard methods for the examination of water and wastewater, 20th edn. American Water Works Association, Washington DC, pp 4-99–4-122Google Scholar
  4. Crawford NM, Smith M, Bellissimo D, Davis RW (1988) Sequence and nitrate regulation of the Arabidopsis thaliana mRNA encoding nitrate reductase, a metalloflavoprotein with three functional domains. Proc Natl Acad Sci 85:5006–5010PubMedCrossRefGoogle Scholar
  5. Fisher K, Barbier GG, Hecht H-J, Mendel RR, Campbell WH, Schwarz G (2005) Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site. Plant Cell 17:1167–1179CrossRefPubMedGoogle Scholar
  6. Higgins DR, Cregg JM (1998) Pichia protocols. Humana Press, Totowa, NJ, pp 1–15Google Scholar
  7. Mertens JA, Shiraishi N, Campbell WH (2000) Recombinant expression of molybdenum reductase fragments of plant nitrate reductase at high levels in Pichia pastoris. Plant Physiol 123:743–756CrossRefPubMedGoogle Scholar
  8. Patton CJ, Fischer AE, Campbell WH, Campbell ER (2002) Corn leaf nitrate reductase: A non-toxic alternative to cadmium for photometric nitrate determinations in water samples by air-segmented continuous-flow analysis. Environ Sci Technol 36:729–735CrossRefPubMedGoogle Scholar
  9. Skipper L, Campbell WH, Mertens JA, Lowe DJ (2001) Pre-steady-state kinetics analysis of recombinant Arabidopsis NADH: nitrate reductase. J Biol Chem 276:26995–27002CrossRefPubMedGoogle Scholar
  10. Su W, Mertens JA, Kanamaru K, Campbell WH, Crawford NM (1997) Analysis of wild-type and mutant plant nitrate reductase expressed in the methylotrophic yeast Pichia pastoris. Plant Physiol 115:1135–1143CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Wilbur H. Campbell
    • 1
    • 2
  • Pengfei Song
    • 2
  • Guillaume G. Barbier
    • 1
    • 2
  1. 1.The Nitrate Elimination Co., Inc.Lake LindenUSA
  2. 2.Biological Sciences DepartmentMichigan Technological UniversityHoughtonUSA

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