Development Of NAD Research Articles

The development of NAD+-dependent dehydrogenase screen-printed biosensor based on enzyme and nanoporous gold co-catalytic strategy

Given the significance of dihydronicotinamide adenine dinucleotide (NADH) and nicotinamide adenine dinucleotide (NAD+) in numerous biochemical fields such as clinical diagnostics and fermentation monitoring, a synergistic strategy was proposed based on the co-catalysis of NAD+-dependent dehydrogenases and nanoporous gold (NPG) towards the oxidation of substrates and NADH, respectively. An NAD+-dependent dehydrogenase/NPG/SPE biosensing platform was developed by modifying screen-printed electrode (SPE) with NPG and NAD+-dependent dehydrogenase for the electrochemical detections of NADH, ethanol, and glucose. Owing to the exceptional oxidation activity of NPG towards NADH, the amperometric detection of NADH exhibited good linearity from 50 μM to 2.0 mM with a low detection limit (LOD) of 15.18–16.39 μM and a satisfactory sensitivity of 1.58–1.72 μA mM−1 within a wide pH range. With alcohol dehydrogenase (ADH) and glucose dehydrogenase (GDH) as model enzymes, ADH/NPG/SPE and GDH/NPG/SPE exhibited excellent analytic characteristics (sensitivity of 0.66 μA mM−1 and 2.04 μA mM−1, LOD of 40.72 μM and 14.83 μM) of ethanol and glucose detection in buffer solution as well as in human serum and fermentation broth. The sensitive micro-sample detections of ethanol and glucose in both real samples were achieved using the proposed biosensors with comparable accuracy (deviation rates of 0.85–7.92%) as automatic analyzers. The proposed biosensing platform elicited many advantageous properties in practical applications, including micro-sample analysis, cost-efficient, easy fabrication, and flexible adaptability, which made it a promising candidate for the clinical blood test, urinalysis, and fermentation monitoring.

Age-Dependent Decline of NAD+-Universal Truth or Confounded Consensus?

Nicotinamide adenine dinucleotide (NAD+) is an essential molecule involved in various metabolic reactions, acting as an electron donor in the electron transport chain and as a co-factor for NAD+-dependent enzymes. In the early 2000s, reports that NAD+ declines with aging introduced the notion that NAD+ metabolism is globally and progressively impaired with age. Since then, NAD+ became an attractive target for potential pharmacological therapies aiming to increase NAD+ levels to promote vitality and protect against age-related diseases. This review summarizes and discusses a collection of studies that report the levels of NAD+ with aging in different species (i.e., yeast, C. elegans, rat, mouse, monkey, and human), to determine whether the notion that overall NAD+ levels decrease with aging stands true. We find that, despite systematic claims of overall changes in NAD+ levels with aging, the evidence to support such claims is very limited and often restricted to a single tissue or cell type. This is particularly true in humans, where the development of NAD+ levels during aging is still poorly characterized. There is a need for much larger, preferably longitudinal, studies to assess how NAD+ levels develop with aging in various tissues. This will strengthen our conclusions on NAD metabolism during aging and should provide a foundation for better pharmacological targeting of relevant tissues.

NAD+ : A key metabolic regulator with great therapeutic potential.

Nicotinamide adenine dinucleotide (NAD+ ) is a ubiquitous metabolite that serves an essential role in the catabolism of nutrients. Recently, there has been a surge of interest in NAD+ biology, with the recognition that NAD+ influences many biological processes beyond metabolism, including transcription, signalling and cell survival. There are a multitude of pathways involved in the synthesis and breakdown of NAD+ , and alterations in NAD+ homeostasis have emerged as a common feature of a range of disease states. Here, we provide an overview of NAD+ metabolism and summarise progress on the development of NAD+ -related therapeutics.

NAD(P)H-Dependent Dehydrogenases for the Asymmetric Reductive Amination of Ketones: Structure, Mechanism, Evolution and Application.

Asymmetric reductive aminations are some of the most important reactions in the preparation of active pharmaceuticals, as chiral amines feature in many of the world's most important drugs. Although many enzymes have been applied to the synthesis of chiral amines, the development of reductive amination reactions that use enzymes is attractive, as it would permit the one‐step transformation of readily available prochiral ketones into chiral amines of high optical purity. However, as most natural “reductive aminase” activities operate on keto acids, and many are able to use only ammonia as the amine donor, there is considerable scope for the engineering of natural enzymes for the reductive amination of ketones, and also for the preparation of secondary amines using alkylamines as donors. This review summarises research into the development of NAD(P)H‐dependent dehydrogenases for the reductive amination of ketones, including amino acid dehydrogenases (AADHs), natural amine dehydrogenases (AmDHs), opine dehydrogenases (OpDHs) and imine reductases (IREDs). In each case knowledge of the structure and mechanism of the enzyme class is addressed, with a further description of the engineering of those enzymes for the reductive amination of ketones towards primary and also secondary amine products.

Discovery and Biochemical Characterization of UDP-Glucose Dehydrogenase from Granulibacter bethesdensis.

UDP-glucose dehydrogenases (EC 1.1.1.22) are responsible for the conversion of UDP-glucose to UDP-glucuronic acid, a key precursor in the biosynthesis of glycoconjugates. Herein we report the discovery and characterization of a UDPglucose dehydrogenase (GbUGD) from Granulibacter bethesdensis, a bacterium originally isolated from the lymph nodes of patients with chronic granulomatous disease (CGD). The recombinant form of the protein was expressed in high yield and the purified enzyme showed highest activity at 37°C/pH 9.0 and was strongly inhibited by Zn(2+) ions, sodium dodecyl sulfate (SDS) and urea. UDP-xylose, an allosteric feedback inhibitor, reduced significantly the activity of the enzyme. High activities were observed using the co-substrates UDP-glucose and NAD+, whereas no activity could be detected using other nucleotide sugars or NADP(+) as potential alternative substrates. The high activity combined with the simple purification procedure used make GbUGD a valuable new alternative biocatalyst for the synthesis of UDP-glucuronic acid or the development of NAD+ regeneration systems.

Development of Nad+ Regeneration Process in Microreactors of Different Materials

Coenzyme NAD+ (nicotinamide adenine dinucleotide) is used in numerous biocatalytic oxidation reactions where NAD+ is reduced to NADH (reduced form of nicotinamide adenine dinucleotide). As the price of coenzyme NAD+ is extremely high, it is essential to regenerate the reduced form of coenzyme back into the oxidized form. In this study regeneration of coenzyme NAD+ was carried out by reversible oxidation of ethanol to acetaldehyde using different biocatalysts and microreactors of different materials. Based on experimental results, by measuring dependence of the reaction rate on the concentration of all components of the reaction system, kinetics of coenzyme regeneration was estimated and the mathematical model of the process was developed. The enzyme kinetics was modeled as a pseudo-homogeneous process with the double substrate Michaelis–Menten rate expression. Validation of developed mathematical model of the process was carried out on a series of independent experiments. Based on results and good agreement between model predictions and experimental results, obtained model simulation results could be used for further process optimization and development of new microreactors. Additionally, comparison of NAD+ coenzyme regeneration within a microreactor and in a batch reactor using immobilized enzyme and enzyme suspension was made. The best results were noticed when glass microreactor with suspended ADH was used (X = 95.89 %; t = 2 s).

On the development of NAD(P)H-sensitive fluorescent probes

In contrast to current, multi-reagent assay systems, the development of a single reagent that can be used to assay NAD(P)H is described. The reagent eliminates the fluorophore 4-methylumbelliferone from a quinoxalinium adduct upon reduction and the chemistry of this process is described.

The development of NAD(P)H-dependent and ferredoxin-dependent glutamate synthase in greening barley and pea leaves

The activity of NAD(P)H-dependent glutamate synthase (E.C. 1.4.1.14) has been demonstrated in extracts from etiolated shoots of pea (Pisum sativum L.) and barley (Hordeum vulgare L.). This activity does not significantly alter upon greening of the etiolated shoots, and is at a similar level in light-grown material. Ferredoxin-dependent glutamate synthase (E.C. 1.4.7.1) has low activity in etiolated shoots but increases rapidly on greening. In light grown leaves ferredoxin-dependent activity is 30-40-fold higher than NAD(P)H-dependent activity. It is not considered that the NAD(P)H-dependent glutamate synthase plays an important role in ammonia assimilation in the photosynthetic tissue of higher plants.

Development of NAD(P)H: and NADH:Nitrate Reductase Activities in Soybean Cotyledons

The cotyledons of soybean begin to develop photosynthetic capacity shortly after emergence. The cotyledons develop nitrate reductase (NR) activity in parallel with an increase in chlorophyll and a decrease in protein. In crude extracts of 5- to 8-day-old cotyledons, NR activity is greatest with NADH as electron donor. In extracts of older cotyledons, NR activity is greatest with NADPH. Blue-Sepharose was used to purify and separate the NR activities into two fractions. When the blue-Sepharose was eluted with NADPH, NR activity was obtained which was most active with NADPH as electron donor. Assays of the NADPH-eluted NR with different concentrations of nitrate revealed that the highest activity was obtained in 80 millimolar KNO(3). Thus, this fraction has properties similar to the low nitrate affinity NAD(P)H:NR of soybean leaves. When 5- to 8-day-old cotyledons were extracted and purified, further elution of the blue-Sepharose with KNO(3), subsequent to the NADPH elution, yielded an NR fraction most active with NADH. Assays of this fraction with different nitrate concentrations revealed that this NR had a higher nitrate affinity and was similar to the NADH:NR of soybean leaves. The KNO(3)-eluted NR fraction which was purified from the extracts of 9- to 14-day-old cotyledons, was most active with NADPH. The analysis of these fractions prepared from the extracts of older cotyledons indicated that residual NAD(P)H:NR contaminated the NADH:NR. Despite this complication, the pattern of development of the purified NR fractions was consistent with the changes observed in the crude extract NR activities. It was concluded that NADH:NR was most active in young cotyledons and that as the cotyledons aged the NAD(P)H:NR became more active.