RESUMEN

Measuring total nitrogen, nitrate, and nitrite is critical for compliance with water safety standards. Previous methods for measuring total nitrogen were hazardous, time consuming, and expensive. Here we report a method for measuring total nitrogen in water and soil using alkaline persulfate digestion combined with a Nitrate Reductase assay. In this method the alkaline persulfate reaction oxidizes all nitrogen present in the sample to nitrate, Nitrate Reductase then is used to catalyze the reduction of nitrate to nitrite in the presence of NADH. The nitrite is then treated with Griess reagents to produce a pink color. The absorbance of this color is measured at 540 nm using a spectrophotometer and when compared to a standard curve of nitrate, treated with both the reduction and colorizing steps, can be used to determine the total nitrogen content of measured samples. This method customizes the measurement of total nitrogen by combining alkaline persulfate digestion with a Nitrate Reductase assay using enzyme based green chemistry. •Customization of total nitrogen analysis by combining alkaline persulfate digestion, driving all nitrogen to nitrate, with a colorimetric nitrate reductase assay•Nitrate reductase catalyzes all nitrate, produced by alkaline persulfate digestion and present in the original sample, to nitrite•Nitrite is measured by the addition of sulfanilamide and N-(1-napthyl)ethylenediamine dihydrochloride, resulting in a pink color.

RESUMEN

Despite the availability of numerous electroanalytical methods for phosphate quantification, practical implementation in point-of-use sensing remains virtually nonexistent because of interferences from sample matrices or from atmospheric O2. In this work, phosphate determination is achieved by the purine nucleoside phosphorylase (PNP) catalyzed reaction of inosine and phosphate to produce hypoxanthine which is subsequently oxidized by xanthine oxidase (XOx), first to xanthine and then to uric acid. Both PNP and XOx are integrated in a redox active Os-complex modified polymer, which not only acts as supporting matrix for the bienzymatic system but also shuttles electrons from the hypoxanthine oxidation reaction to the electrode. The bienzymatic cascade in this second generation phosphate biosensor selectively delivers four electrons for each phosphate molecule present. We introduced an additional electrochemical process involving uric acid oxidation at the underlying electrode. This further enhances the anodic current (signal amplification) by two additional electrons per analyte molecule which mitigates the influence of electrochemical interferences from the sample matrix. Moreover, while the XOx catalyzed reaction is sensitive to O2, the uric acid production and therefore the delivery of electrons through the subsequent electrochemical process are independent of the presence of O2. Consequently, the electrochemical process counterbalances the O2 interferences, especially at low phosphate concentrations. Importantly, the electrochemical uric acid oxidation specifically reports on phosphate concentration since it originates from the product of the bienzymatic reactions. These advantageous properties make this bioelectrochemical-electrochemical cascade particularly promising for point-of-use phosphate measurements.

Asunto(s)

Técnicas Biosensibles/métodos , Técnicas Electroquímicas , Fosfatos/análisis

RESUMEN

Measurement of ortho-phosphate in soil extracts usually involves sending dried samples of soil to a laboratory for analysis and waiting several weeks for the results. Phosphate determination methods often involve use of strong acids, heavy metals, and organic dyes. To overcome limitations of this approach, we have developed a phosphate determination method which can be carried out in the field to obtain results on the spot. This new method uses: •Small volumes.•An enzymatic reaction.•Green chemistry. First, the soil sample is extracted with deionized water and filtered. Next, an aliquot of the soil extract (0.5 mL) is transferred to a disposable cuvette, containing 0.5 mL of reaction mixture [200 mM HEPES, pH 7.6, 20 mM MgCl2, with 80 nmol 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) and 1 unit of recombinant purine nucleoside phosphorylase (PNP; EC 2.4.2.1)], mixed, and incubated for 10 min at field temperature. Absorbance of the completed reaction is measured at 360 nm in open-source, portable photometer linked by bluetooth to a smartphone. The phosphate and phosphorus content of the soil is determined by comparison of its absorbance at 360 nm to a previously prepared standard phosphate curve, which is stored in the smartphone app.

RESUMEN

Monolayers of Cu(II)-complexes on electrode surfaces are frequently applied for the immobilization and controlled orientation of His-tagged redox proteins. However, affinity binding is limited to applications that require potentials less negative than the reduction potential of the metal complexes. In order to overcome this limitation, we used Zn(2+) cations on nitrilotriacetic acid (NTA) modified carbon electrodes for the coordination of His-tagged nitrate reductase (NaR). The NTA modified electrodes were prepared upon diazotation and electrochemical reduction of an aniline functionalized NTA ligand. After coordination of Zn(2+) to the bound NTA ligand, self-assembly of NaR is achieved via coordination of the imidazole groups from the His-tag to the NTA-Zn(II) complex. The electrochemical investigations of the NaR monolayer on NTA-Zn(II) films demonstrate the catalytic activity for reduction of nitrate to nitrite in the presence of methyl viologen. The catalytic current density correlates with the one expected for a fully active enzyme monolayer. Moreover, the reduction of Zn(2+) is not observed at the potential necessary for the reduction of methyl viologen. Therefore, affinity binding based on Zn(2+) may be used for the immobilization and electrochemical applications of His-tagged NaR.

Asunto(s)

Enzimas Inmovilizadas/química , Histidina/química , Nitrato-Reductasa/química , Zinc/química , Animales , Aspergillus niger/enzimología , Carbono/química , Bovinos , Cobre/química , Electroquímica , Electrodos , Enzimas Inmovilizadas/metabolismo , Vidrio/química , Nitrato-Reductasa/metabolismo , Ácido Nitrilotriacético/química , Paraquat/química

RESUMEN

Electroanalytical procedures are often subjected to oxygen interferences. However, achieving anaerobic conditions in field analytical chemistry is difficult. In this work, novel enzymatic systems were designed to maintain oxygen-free solutions in open, small volume electrochemical cells and implemented under field conditions. The oxygen removal system consists of an oxidase enzyme, an oxidase-specific substrate, and catalase for dismutation of hydrogen peroxide generated in the enzyme catalyzed oxygen removal reaction. Using cyclic voltammetry, three oxidase enzyme/substrate combinations with catalase were analyzed: glucose oxidase with glucose, galactose oxidase with galactose, and pyranose 2-oxidase with glucose. Each system completely removed oxygen for 1 h or more in unstirred open vessels. Reagents, catalysts, reaction intermediates, and products involved in the oxygen reduction reaction were not detected electrochemically. To evaluate the oxygen removal systems in a field sensing device, a model nitrate biosensor based on recombinant eukaryotic nitrate reductase was implemented in commercial screen-printed electrochemical cells with 200 µL volumes. The products of the aldohexose oxidation catalyzed by glucose oxidase and galactose oxidase deactivate nitrate reductase and must be quenched for biosensor applications. For general application, the optimum catalyst is pyranose 2-oxidase since the oxidation product does not interfere with the biorecognition element.

Asunto(s)

Técnicas Biosensibles , Técnicas Electroquímicas , Enzimas/metabolismo , Nitratos/análisis , Oxígeno/metabolismo , Aire , Biocatálisis , Deshidrogenasas de Carbohidratos/metabolismo , Galactosa Oxidasa/metabolismo , Glucosa Oxidasa/metabolismo , Concentración de Iones de Hidrógeno , Nitrato-Reductasa/genética , Nitrato-Reductasa/metabolismo , Oxidación-Reducción , Plantas/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo

RESUMEN

Rate-limiting processes of catalysis by eukaryotic molybdenum-containing nitrate reductase (NaR, EC 1.7.1.1-3) were investigated using two viscosogens (glycerol and sucrose) and observing their impact on NAD(P)H:NaR activity of corn leaf NaR and recombinant Arabidopsis and yeast NaR. Holo-NaR has two "hinge" sequences between stably folded regions housing its internal electron carriers: 1) Hinge 1 between the molybdenum-containing nitrate reducing module and cytochrome b domain containing heme and 2) Hinge 2 between cytochrome b and cytochrome b reductase (CbR) module containing FAD. Solution viscosity negatively impacted the activity of these holo-NaR forms, which suggests that the rate-limiting events in catalysis were likely to involve large conformational changes that restrict or "gate" internal electron-proton transfers (IET). Little effect of viscosity was observed on recombinant CbR module and methyl viologen nitrate reduction by holo-NaR, suggesting that these activities involved no large conformational changes. To determine whether Hinge 2 is involved in gating the first step in IET, the effects of viscosogen on cytochrome c and ferricyanide reductase activities of holo-NaR and ferricyanide reductase activity of the recombinant molybdenum reductase module (CbR, Hinge 2, and cytochrome b) were analyzed. Solution viscosity negatively impacted these partial activities, as if Hinge 2 were involved in gating IET in both enzyme forms. We concluded that both Hinges 1 and 2 appear to be involved in gating IET steps by restricting the movement of the cytochrome b domain relative to the larger nitrate-reducing and electron-donating modules of NaR.

Asunto(s)

Nitrato Reductasas/química , Secuencia de Aminoácidos , Arabidopsis/metabolismo , Sitios de Unión , Tampones (Química) , Catálisis , Reductasas del Citocromo/química , Citocromos b/química , Relación Dosis-Respuesta a Droga , Electrones , Ferricianuros/química , Proteínas Fúngicas/metabolismo , Glicerol/farmacología , Cinética , Modelos Biológicos , Modelos Químicos , Datos de Secuencia Molecular , Nitrato-Reductasa , Nitrato Reductasas/metabolismo , Oxidación-Reducción , Paraquat/química , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Protones , Proteínas Recombinantes/química , Homología de Secuencia de Aminoácido , Sacarosa/farmacología , Viscosidad , Zea mays/metabolismo

RESUMEN

Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 angstroms. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis.

Asunto(s)

Células Eucariotas/enzimología , Nitrato Reductasas/química , Nitrato Reductasas/metabolismo , Nitratos/metabolismo , Pichia/enzimología , Animales , Arginina/química , Sitios de Unión/fisiología , Agua Corporal/química , Pollos , Cristalografía por Rayos X , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Molibdeno/química , NADP/química , NADP/metabolismo , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Estructura Terciaria de Proteína/fisiología , Homología de Secuencia de Aminoácido , Triptófano/química

RESUMEN

NAD(P)H:nitrate reductase (NaR, EC 1.7.1.1-3) is a useful enzyme in biotechnological applications, but it is very complex in structure and contains three cofactors-flavin adenine dinucleotide, heme-Fe, and molybdenum-molybdopterin (Mo-MPT). A simplified nitrate reductase (S-NaR1) consisting of Mo-MPT-binding site and nitrate-reducing active site was engineered from yeast Pichia angusta NaR cDNA (YNaR1). S-NaR1 was cytosolically expressed in high-density fermenter culture of methylotrophic yeast Pichia pastoris. Total amount of S-NaR1 protein produced was approximately 0.5 g per 10 L fermenter run, and methanol phase productivity was 5 microg protein/g wet cell weight/h. Gene copy number in genomic DNA of different clones showed direct correlation with the expression level. S-NaR1 was purified to homogeneity in one step by immobilized metal affinity chromatography (IMAC) and total amount of purified protein per run of fermentation was approximately 180 mg. Polypeptide size was approximately 55 kDa from electrophoretic analysis, and S-NaR1 was mainly homo-tetrameric in its active form, as shown by gel filtration. S-NaR1 accepted electrons efficiently from reduced bromphenol blue (kcat = 2081 s(-1)) and less so from reduced methyl viologen (kcat = 159 s(-1)). The nitrate KM for S-NaR1 was 30 +/- 3 microM, which is very similar to YNaR1. S-NaR1 is capable of specific nitrate reduction, and direct electric current, as shown by catalytic nitrate reduction using protein film cyclic voltammetry, can drive this reaction. Thus, S-NaR1 is an ideal form of this enzyme for commercial applications, such as an enzymatic nitrate biosensor formulated with S-NaR1 interfaced to an electrode system.

Asunto(s)

Células Eucariotas/enzimología , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Nitrato Reductasas/aislamiento & purificación , Nitrato Reductasas/metabolismo , Pichia , Sitios de Unión , Reactores Biológicos , Coenzimas/química , Electroquímica , Fermentación , Flavina-Adenina Dinucleótido/química , Proteínas Fúngicas/genética , Hemo/química , Metaloproteínas/química , Metanol/metabolismo , Estructura Molecular , Peso Molecular , Cofactores de Molibdeno , Nitrato-Reductasa , Nitrato Reductasas/genética , Pichia/genética , Pichia/metabolismo , Pteridinas/química

RESUMEN

Development, characterization, and operational details of an enzymatic, air-segmented continuous-flow analytical method for colorimetric determination of nitrate + nitrite in natural-water samples is described. This method is similar to U.S. Environmental Protection Agency method 353.2 and U.S. Geological Survey method 1-2545-90 except that nitrate is reduced to nitrite by soluble nitrate reductase (NaR, EC 1.6.6.1) purified from corn leaves rather than a packed-bed cadmium reactor. A three-channel, air-segmented continuous-flow analyzer-configured for simultaneous determination of nitrite (0.020-1.000 mg-N/L) and nitrate + nitrite (0.05-5.00 mg-N/L) by the nitrate reductase and cadmium reduction methods-was used to characterize analytical performance of the enzymatic reduction method. At a sampling rate of 90 h(-1), sample interaction was less than 1% for all three methods. Method detection limits were 0.001 mg of NO2- -N/L for nitrite, 0.003 mg of NO3-+ NO2- -N/L for nitrate + nitrite by the cadmium-reduction method, and 0.006 mg of NO3- + NO2- -N/L for nitrate + nitrite bythe enzymatic-reduction method. Reduction of nitrate to nitrite by both methods was greater than 95% complete overthe entire calibration range. The difference between the means of nitrate + nitrite concentrations in 124 natural-water samples determined simultaneously bythe two methods was not significantly different from zero at the p = 0.05 level.

Asunto(s)

Monitoreo del Ambiente/métodos , Nitrato Reductasas/farmacología , Nitratos/análisis , Contaminantes del Agua/análisis , Cadmio/química , Colorimetría/métodos , Nitrato-Reductasa , Hojas de la Planta/enzimología , Zea mays

RESUMEN

Nitrate reductase (NR; EC 1.6.6.1-3) catalyzes NAD(P)H reduction of nitrate to nitrite. NR serves plants, algae, and fungi as a central point for integration of metabolism by governing flux of reduced nitrogen by several regulatory mechanisms. The NR monomer is composed of a ~100-kD polypeptide and one each of FAD, heme-iron, and molybdenum-molybdopterin (Mo-MPT). NR has eight sequence segments: (a) N-terminal "acidic" region; (b) Mo-MPT domain with nitrate-reducing active site; (c) interface domain; (d) Hinge 1 containing serine phosphorylated in reversible activity regulation with inhibition by 14-3-3 binding protein; (e) cytochrome b domain; (f) Hinge 2; (g) FAD domain; and (h) NAD(P)H domain. The cytochrome b reductase fragment contains the active site where NAD(P)H transfers electrons to FAD. A complete three-dimensional dimeric NR structure model was built from structures of sulfite oxidase and cytochrome b reductase. Key active site residues have been investigated. NR structure, function, and regulation are now becoming understood.