Regulation of the NADH supply by nuoE deletion and pncB overexpression to enhance hydrogen production in Enterobacter aerogenes

In this study, seven mutants from E. aerogenes IAM1183 wildtype were constructed via different strategies including deletion of lactate dehydrogenase, disruption of NADH dehydrogenase gene nuoE, overexpression of pncB and a combination of both to regulate of the NADH supply to enhance hydrogen production. Compared with the parental strain, the hydrogen yields of the strains IAM1183-E, IAM1183-L and IAM1183-EL increased by 23.3, 81.7 and 97.9%, respectively. When the pncB gene was overexpressed, the hydrogen yield of IAM1183/P, IAM1183-E/P, IAM1183-L/P and IAM1183-EL/P increased by 39.0, 6.5, 5.9, and 5.1% compared with the respective original knockout strains. Among them, the total hydrogen yield of strain IAM1183-EL/P with highest production efficiency was 58% higher than IAM1183. Further metabolite analysis indicated that the knockout of nuoE and ldhA, combined with the overexpression of pncB, resulted in a redistribution of the metabolic fluxes in E. aerogenes, which led to an improvement of the hydrogen yield.     

Analysis of culture fluorescence by a fiber-optic sensor inNicotiana tabacum plant cell culture

The on-line sensing of viable cell weight in plant cell culture process is applied to analysis and control of process. The fiber-optic fluorescence sensor was constructed to measure the NADH-dependent fluorescence inNicotiana tabacum plant cell culture and the analysis of fluorescence signal was done to be correlated with the viable cell weight. The structured kinetic model for cell growth was proposed to estimate the theoretical viable cell weight. The dimensional analysis was proposed for the interpretation of fluorescence signal, in which the path length, the inner filter effect and the hydrodynamic conditions were considered as the key factors on fluorescence signal. The dimensional analysis and empirical correlation of fluorescence signal to viable cell weight was applied to the interpretation of the detected fluorescence signal during cultivation. The proposed interpretation of fluorescence signal using dimensional analysis was well correlated with the viable cell weight estimated by the structured kinetic model as well as by empirical correlation.     

Nile blue adsorbed onto silica gel modified with niobium oxide for electrocatalytic oxidation of NADH

A study of modified carbon paste electrode employing Nile blue (NB) adsorbed on silica gel modified with niobium oxide (SN) for electrocatalytic oxidation of reduced nicotinamide adenine dinucleotide (NADH) is described. The adsorbed organic dye on SN was used to prepare a modified carbon paste electrode to investigate its electrochemical properties. The formal potential (E°′) of the adsorbed NB (−230 mV vs. saturated calomel electrode, SCE) showed a shift of 70 mV towards a more positive potential value, compared to NB dissolved in aqueous solution. In solutions with pH between 6.0 and 8.0 did stability and E°′ remained almost constant. However, for a solution pH lower than 6.0 the E°′ was affected by the acidity of the contacting solution, shifting the E°′ towards more positive values. For the solution pHs between 6.0 and 8.0 the electrocatalytic activity remained almost constant. A linear response range for NADH between 1.0×10⁻⁵ and 5.2×10⁻⁴ mol l⁻¹, at pH 7.0, was observed for the electrode, with an applied potential of −200 mV versus SCE. The formation of an intermediate charge transfer (CT) complex was proposed to the CT reaction between NADH and adsorbed NB. The heterogeneous electron transfer rate, k_obs, was 1400 M⁻¹ s⁻¹ and the apparent Michaelis-Menten constant, was 0.21 mM at pH 7.0 evaluated from rotating disk electrode (RDE) experiments with an electrode coverage of about 5.2×10⁻⁹ mol cm⁻². The increase in the reaction rate between NADH and the immobilized NB compared to those obtained with dissolved NB was assigned to the shift of the E°′ towards more positive values.     

Electrocatalytic properties of NDGA and NDGA/FAD hybrid film modified electrodes for NADH/NAD redox reaction

The fabrication of monolayers composed of nordihydroguaiaretic acid (NDGA), and hybrid films composed of NDGA-flavin adenine dinucleotide (FAD) adsorbed films was performed in neutral aqueous solutions to produce electrochemically active thin films exhibiting one and two redox couples, respectively. An electrochemical quartz crystal microbalance and cyclic voltammetry were used to study the in situ growth of the NDGA and hybrid NDGA/FAD film monolayers. The NDGA modified film electrocatalytically oxidized NADH, ascorbic acid, dopamine, and N₂H₄ in neutral aqueous solutions. Well-separated voltammetric peaks were observed for dopamine and uric acid mixtures, and also for ascorbic acid and uric acid mixtures using the NDGA/GC modified electrode. When transferred to various aqueous buffered solutions, the two redox couples of the NDGA/FAD hybrid film and their formal potentials were observed to be pH-dependent. The electrocatalytic oxidation and reduction of NADH and NAD⁺ by a NDGA/FAD hybrid film in neutral aqueous solutions was carried out, and the electrocatalytic oxidation of NADH was performed using a NDGA/FAD hybrid film.     

Electrocatalytic activity of oxidation products of guanine and 5′-GMP towards the oxidation of NADH

We have studied the potential electrocatalytic activity towards the oxidation of NADH of several oxidation products of guanine and its derivative guanosine-5′-monophosphate (5′-GMP) on pyrolytic graphite electrodes (PGE). The distribution of products generated strongly depends on the experimental conditions. Our investigations focused on the oxidation products that are adsorbed on the electrode surface, are redox active and, exhibited electrocatalytic activity toward the oxidation of NADH. These compounds were electrochemically and kinetically characterized in terms of dependence of the formal potential on pH and electron transfer rate constant (k_s). The voltammetric and catalytic behavior of both guanine and 5′-GMP oxidation products was compared with that of other guanine derivatives we have previously studied. Some mechanistic aspects concerning the generation of the catalysts are also discussed.     

碳纳米管修饰电极的制备及其对NADH的电催化氧化

采用丝网印刷技术,制备出羧基化多壁碳纳米管修饰的丝网印刷碳电极,并采用循环伏安法研究了该电极对还原型烟酰胺腺嘌呤二核苷酸(NADH)的电催化氧化性能.结果表明,与未修饰丝网印刷碳电极相比,多壁碳纳米管修饰丝网印刷碳电极显著降低了NADH的氧化峰电位,消除了反应产物对电极的污染及其它电化学反应对测量的干扰.将修饰电极与流...     

Poly-(3-methylthiophene)/carbon nanotubes hybrid composite-modified electrodes

The preparation of poly-(3-methylthiophene)—multi-walled carbon nanotubes hybrid composite electrodes is reported. The hybrid electrode shows a synergic effect of the electrocatalytic properties, and high active surface area of both the conducting polymer and carbon nanotubes, which gives rise to a remarkable improvement of oxidation of NADH with respect to polymer-modified electrodes, and CNTs-modified electrodes. SEM showed that carbon nanotubes served as nanosized backbone for P3MT electropolymerization. The amperometric NADH detection at +300 mV provided fast responses, a range of linearity between 5.0 × 10⁻⁷ and 2.0 × 10⁻⁵ mol l⁻¹, and a detection limit of 1.7 × 10⁻⁷ mol l⁻¹, which compares advantageously with those reported for other NADH CNT-based amperometric sensors. Furthermore, the direct electrochemistry of cytochrome c and FAD at the hybrid electrode is also checked.     

Redox-flexible NADH oxidase biosensor: A platform for various dehydrogenase bioassays and biosensors

A generic amperometric bioassay based on the enzymatic oxidation catalysed by the stable NADH oxidase (NAox) from Thermus thermophilus has been developed for NADH measurements. The NAox uses O₂ as its natural electron acceptor and produces H₂O₂ in a two-electron process. Electrochemical and spectrophotometric experiments showed that the NAox used in this work, presents a very good activity towards its substrate and, in contrary to previously mentioned NADH oxidases, does not require the addition of any exogenous flavin cofactor neither to promote nor to maintain its activity. In addition, the NAox used also works with artificial electron acceptors like ferrocene derivatives. O₂ was successfully replaced by redox mediators such as hydroxymethyl ferrocene (FcCH₂OH) for the regeneration of the active enzyme. Combining the NAox with the mediator and the horseradish peroxidase we developed an original, high sensitive “redox-flexible” NADH amperometric bioassay working in a large window of applied potentials in both oxidation and reduction modes. The biosensor has a continuous and complementary linearity range permitting to measure NADH concentrations starting from 5 × 10⁻⁶ M in reduction until 2 × 10³ M in oxidation. This redox-flexibility allows choosing the applied potential in order to avoid electrochemical interferences. The association of the “redox-flexible” concept with NADH dependent enzymes opens a novel strategy for dehydrogenases based bioassays and biosensors. The great number of dehydrogenases available makes the concept applicable for numerous substrates to analyse. Moreover it allows the development of a wide range of biosensors on the basis of a generic platform. This gives several advantages over the previous manufacturing techniques and offers a general and flexible scheme for the fabrication of biosensors presenting high sensitivities, wide calibration ranges and less affected by electrochemical interferences.