Nitrate reductase for nitrate analysis in water

Abstract

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.

Effects of seasonal variation and land cover on riparian denitrification along a mid-sized river

“…NH 4 C ÀN was measured colorimetrically using a microplate reader and a method that replaces phenol with the sodium salt of 2-phenylphenol (PPS) as the substrate for the Berthelot reaction (Sims et al 1995;Rhine et al 1998;Sims 2006). NO 3 ¡ ÀN was also measured colorimetrically on a microplate reader using the methods of Campbell et al (2006) and Ringuet et al (2011). Briefly, AtNaR2 (acquired from NECi, Lake Linden, MI) quantitatively reduced nitrate to nitrite in a phosphate buffer.…”

Nitrate assimilatory enzymes of Spirulina (Arthospira) platensis are more thermotolerant than those of rice

“…For example, at 60 °C and 80 °C, the specific activity of rice NR declined to negligible levels within 5 min indicating total inactivation, whereas in Spirulina, even after 30 min, NR retained full activity at 40 °C, over half the activity at 60 °C, and 28 % of the initial activity at 80 °C. In view of the fact that earlier attempts to enhance the stability of higher plant NR by genetic manipulation met with limited success (Campbell et al, 2006), our finding that Spirulina could be a natural source for stable and thermotolerant NR bears significance.…”

“…The detrimental environmental effects of excess nitrate/ nitrite and other forms of reactive nitrogen generated from unutilized fertilizers and industrial effluents have been highlighted recently (Abrol et al, 2007;Galloway et al, 2008). Purified nitrate assimilatory enzymes are used in environmental testing (Patton et al, 2002;Campbell et al, 2006), nitrate decontamination (Mellor et al, 1992), meat processing (Gotterup et al, 2007), biosensors (Chen et al, 2008) and other applications (Angeby et al, 2002), while whole organisms are useful for bioremediation (Hu et al, 2000). However, the poor stability and thermosensitivity of these enzymes is a major limitation in their widespread use.…”

Modelling combined effect of chloramine and copper on ammonia-oxidizing microbial activity using a biostability approach

“…The reaction between NH 2 Cl and salicylate ions at around pH 12.6 in the presence of sodium nitroprusside produces a blue compound that is measured spectrophotometrically at a wavelength of 660 nm (EPA, 1981). NO 2 -N was measured by the sulphanilamide method (4500-NO 2 B) (APHA, 1998), and NO x -N was measured by catalytically reducing NO 3 -N to NO 2 -N by nitrate reductive enzyme in the presence of reduced nicotinamide dinucleotide (Campbell et al, 2006, Patton et al, 2002. The chloramine residual was reduced to 0 mgCl 2 L À1 using 0.5% sodium thiosulfate stock/ascorbic acid solution before measuring NOx-N.…”