Dependent Alcohol Dehydrogenase Research Articles

Biotransformation of Deoxynivalenol by a Dual-Member Bacterial Consortium Isolated from Tenebrio molitor Larval Feces.

In this study, a dual-member bacterial consortium with the ability to oxidize deoxynivalenol (DON) to 3-keto-DON, designated SD, was first screened from the feces of Tenebrio molitor larvae. This consortium consisted of Pseudomonas sp. SD17-1 and Devosia sp. SD17-2, as determined by 16S rRNA-based phylogenetic analysis. A temperature of 30 °C, a pH of 8.0-9.0, and an initial inoculum concentration ratio of Devosia to Pseudomonas of 0.1 were optimal single-factor parameters for the DON oxidation activity of the bacterial consortium SD. Genome-based bioinformatics analysis revealed the presence of an intact PQQ biosynthesis operon (pqqFABCDEG) and four putative pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenase (ADH) genes in the genomes of Pseudomonas strain SD17-1 and Devosia strain SD17-2, respectively. Biochemical analyses further confirmed the PQQ-producing phenotype of Pseudomonas and the DON-oxidizing enzymatic activities of two of four PQQ-dependent ADHs in Devosia. The addition of PQQ-containing a cell-free fermentation supernatant from Pseudomonas activated DON-oxidizing activity of Devosia. In summary, as members of the bacterial consortium SD, Pseudomonas and Devosia play indispensable and complementary roles in SD's oxidation of DON. Specifically, Pseudomonas is responsible for producing the necessary PQQ cofactor, whereas Devosia expresses the PQQ-dependent DON dehydrogenase, together facilitating the oxidation of DON.

Enzymatic properties of alcohol dehydrogenase PedE_M.s. derived from Methylopila sp. M107 and its broad metal selectivity.

As an important metabolic enzyme in methylotrophs, pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases play significant roles in the global carbon and nitrogen cycles. In this article, a calcium (Ca2+)-dependent alcohol dehydrogenase PedE_M.s., derived from the methylotroph Methylopila sp. M107 was inserted into the modified vector pCM80 and heterologously expressed in the host Methylorubrum extorquens AM1. Based on sequence analysis, PedE_M.s., a PQQ-dependent dehydrogenase belonging to a methanol/ethanol family, was successfully extracted and purified. Showing by biochemical results, its enzymatic activity was detected as 0.72 U/mg while the Km value was 0.028 mM while employing ethanol as optimal substrate. The activity of PedE_M.s. could be enhanced by the presence of potassium (K+) and calcium (Ca2+), while acetonitrile and certain common detergents have been found to decrease the activity of PedE_M.s.. In addition, its optimum temperature and pH were 30°C and pH 9.0, respectively. Chiefly, as a type of Ca2+-dependent alcohol dehydrogenase, PedE_M.s. maintained 60-80% activity in the presence of 10 mM lanthanides and displayed high affinity for ethanol compared to other PedE-type enzymes. The 3D structure of PedE_M.s. was predicted by AlphaFold, and it had an 8-bladed propeller-like super-barrel. Meanwhile, we could speculate that PedE_M.s. contained the conserved residues Glu213, Asn300, and Asp350 through multiple sequence alignment by Clustal and ESpript. The analysis of enzymatic properties of PedE_M.s. enriches our knowledge of the methanol/ethanol family PQQ-dependent dehydrogenase. This study provides new ideas to broaden the application of alcohol dehydrogenase in alcohol concentration calculation, biosensor preparation, and other industries.

Four PQQ-Dependent Alcohol Dehydrogenases Responsible for the Oxidative Detoxification of Deoxynivalenol in a Novel Bacterium Ketogulonicigenium vulgare D3_3 Originated from the Feces of Tenebrio molitor Larvae.

Deoxynivalenol (DON) is frequently detected in cereals and cereal-based products and has a negative impact on human and animal health. In this study, an unprecedented DON-degrading bacterial isolate D3_3 was isolated from a sample of Tenebrio molitor larva feces. A 16S rRNA-based phylogenetic analysis and genome-based average nucleotide identity comparison clearly revealed that strain D3_3 belonged to the species Ketogulonicigenium vulgare. This isolate D3_3 could efficiently degrade 50 mg/L of DON under a broad range of conditions, such as pHs of 7.0-9.0 and temperatures of 18-30 °C, as well as during aerobic or anaerobic cultivation. 3-keto-DON was identified as the sole and finished DON metabolite using mass spectrometry. In vitro toxicity tests revealed that 3-keto-DON had lower cytotoxicity to human gastric epithelial cells and higher phytotoxicity to Lemna minor than its parent mycotoxin DON. Additionally, four genes encoding pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases in the genome of isolate D3_3 were identified as being responsible for the DON oxidation reaction. Overall, as a highly potent DON-degrading microbe, a member of the genus Ketogulonicigenium is reported for the first time in this study. The discovery of this DON-degrading isolate D3_3 and its four dehydrogenases will allow microbial strains and enzyme resources to become available for the future development of DON-detoxifying agents for food and animal feed.

Cu/nucleotide coordination self-assembling to in situ regenerate NAD(P)+ and co-immobilize dehydrogenase with robust activity and stability

Enzyme is an efficient green catalyst, but its vulnerability and difficulties of separation from products limited the application in industry. Furthermore, a large number of oxidase enzymes rely on dihydronicotinamide adenine dinucleotide (NADH) or dihydronicotinamide adenine dinucleotide phosphate (NADPH) cofactors. These cofactors are too expensive to be used in industrial processes. Thus, cofactor regeneration is an important method to avoid the consumption of large quantities of oxidized cofactor NAD(P)+ in enzyme-catalyzed reactions. In this paper, Cu based metal nucleotides coordination polymers (MNCPs) with NAD(P)H oxidase activity were constructed by self-assembly of copper ions and nucleotides. Cu2+/AMP and Cu2+/GMP showed high catalytic activity and high reusability. Then, Cu2+/AMP with NADH oxidase activity was combined with NAD+ dependent alcohol dehydrogenase (ADH) to construct a bienzyme cascade system with circulating cofactor, which applied to the conversion of benzyl alcohol to benzaldehyde. In this system, ADH exhibited high activity due to the increase of substrate concentration and the inhibition of the diffusion of intermediate products. Besides, the bienzyme cascade system also showed high stability and reusability. After 8 cycles, the immobilized ADH remained more than 57% of the initial activity. The prepared bienzyme cascade system can extensively extended its application in industrial production.

D-Allulose (D-Psicose) Biotransformation From Allitol by a Newly Found NAD(P)-Dependent Alcohol Dehydrogenase From Gluconobacter frateurii NBRC 3264 and the Enzyme Characterization.

The NAD(P)-dependent alcohol dehydrogenase (ADH) gene was cloned from Gluconobacter frateurii NBRC 3264 and expressed in Escherichia coli BL21 star (DE3). The expressed enzyme was purified and the characteristics were investigated. The results showed that this ADH can convert allitol into D-allulose (D-psicose), which is the first reported enzyme with this catalytic ability. The optimum temperature and pH of this enzyme were 50°C and pH 7.0, respectively, and the enzyme showed a maximal activity in the presence of Co2+. At 1 mM Co2+ and allitol concentrations of 50, 150, and 250 mM, the D-allulose yields of 97, 56, and 38%, respectively, were obtained after reaction for 4 h under optimal conditions, which were much higher than that obtained by using the epimerase method of about 30%.

Computationally driven design of an artificial metalloenzyme using supramolecular anchoring strategies of iridium complexes to alcohol dehydrogenase.

Artificial metalloenzymes (ArMs) confer non-biological reactivities to biomolecules, whilst taking advantage of the biomolecular architecture in terms of their selectivity and renewable origin. In particular, the design of ArMs by the supramolecular anchoring of metal catalysts to protein hosts provides flexible and easy to optimise systems. The use of cofactor dependent enzymes as hosts gives the advantage of both a (hydrophobic) binding site for the substrate and a cofactor pocket to accommodate the catalyst. Here, we present a computationally driven design approach of ArMs for the transfer hydrogenation reaction of cyclic imines, starting from the NADP+-dependent alcohol dehydrogenase from Thermoanaerobacter brockii (TbADH). We tested and developed a molecular docking workflow to define and optimize iridium catalysts with high affinity for the cofactor binding site of TbADH. The workflow uses high throughput docking of compound libraries to identify key structural motifs for high affinity, followed by higher accuracy docking methods on smaller, focused ligand and catalyst libraries. Iridium sulfonamide catalysts were selected and synthesised, containing either a triol, a furane, or a carboxylic acid to provide the interaction with the cofactor binding pocket. IC50 values of the resulting complexes during TbADH-catalysed alcohol oxidation were determined by competition experiments and were between 4.410 mM and 0.052 mM, demonstrating the affinity of the iridium complexes for either the substrate or the cofactor binding pocket of TbADH. The catalytic activity of the free iridium complexes in solution showed a maximal turnover number (TON) of 90 for the reduction of salsolidine by the triol-functionalised iridium catalyst, whilst in the presence of TbADH, only the iridium catalyst with the triol anchoring functionality showed activity for the same reaction (TON of 36 after 24 h). The observation that the artificial metalloenzymes developed here lacked stereoselectivity demonstrates the need for the further investigation and optimisation of the ArM. Our results serve as a starting point for the design of robust artificial metalloenzymes, exploiting supramolecular anchoring to natural NAD(P)H binding pockets.

Substituted benzoic acid amides as the modifiers of the ethanol bioelectrooxidation using NAD+-dependent alcohol dehydrogenase

One of the methods for the alcohols determination is based on ethanol bioelectrooxidation with NAD+-dependent alcoholdehydrogenase. This enzyme has been used as a bioselective compound in ethanol electrochemical biosensors. The development issues of this type of biosensors are associated with the need to create special conditions for the NADH electrooxidation, the loss of catalytic activity and the instability of the active complex ADH-NAD+ on the electrode surface. Two substituted benzoic acid amides , 2,4-dichlorbenzamide and 2,4-dichlor-5-methylbenzamide, have been as modulators of alcohol dehydrogenase (ADH) activity. 2,4-dichlorbenzamide inhibited the rate of the forward ADH reaction by 30% and 2,4-dichlor-5-methylbenzamide - by 60%, respectively, in the concentration of 100 µM. This can stabilize the structure of the enzyme and temporarily prevent its inactivation in the paste electrode. The rate of the reverse ADH reaction was suppressed up to 80%. Inhibition of the reverse ADH reaction creates conditions for the accumulation of NADH and its subsequent regeneration. The studies have shown that the introduction of several substituents into the aromatic ring causes a more complex interaction of benzoic acid amides with ADH compared to unsubstituted amides.

Heterologous expression, purification, and characterization of proteins in the lanthanome.

Recent work has revealed that certain lanthanides-in particular, the more earth-abundant, lighter lanthanides-play essential roles in pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenases from methylotrophic and non-methylotrophic bacteria. More recently, efforts of several laboratories have begun to identify the molecular players (the lanthanome) involved in selective uptake, recognition, and utilization of lanthanides within the cell. In this chapter, we present protocols for the heterologous expression in Escherichia coli, purification, and characterization of many of the currently known proteins that comprise the lanthanome of the model facultative methylotroph, Methylorubrum extorquens AM1. In addition to the methanol dehydrogenase XoxF, these proteins include the associated c-type cytochrome, XoxG, and solute binding protein, XoxJ. We also present new, streamlined protocols for purification of the highly selective lanthanide-binding protein, lanmodulin, and a solute binding protein for PQQ, PqqT. Finally, we discuss simple, spectroscopic methods for determining lanthanide- and PQQ-binding stoichiometry of proteins. We envision that these protocols will be useful to investigators identifying and characterizing novel members of the lanthanome in many organisms.

Expression, purification and properties of the enzymes involved in lanthanide-dependent alcohol oxidation: XoxF4, XoxF5, ExaF/PedH, and XoxG4.

In this chapter we describe logistics, protocols and conditions for expression, purification and characterization of Ln3+-dependent alcohol dehydrogenases representing three distinct phylogenetic clades of these enzymes, classified as XoxF4, XoxF5 and ExaF/PedH. We present data on the biochemical properties of a dozen enzymes, all generated by our group, in a comparative fashion. These enzymes display a range of properties in terms of substrate and metal specificities, pH and ammonium requirement, as well as catalytic constants. In addition, we describe a single novel cytochrome, XoxG4, that likely serves as a natural electron acceptor from XoxF5 in methanotrophs of the Gammaproteobacteria class.

Facile Stereoselective Reduction of Prochiral Ketones by using an F420 -dependent Alcohol Dehydrogenase.

Effective procedures for the synthesis of optically pure alcohols are highly valuable. A commonly employed method involves the biocatalytic reduction of prochiral ketones. This is typically achieved by using nicotinamide cofactor‐dependent reductases. In this work, we demonstrate that a rather unexplored class of enzymes can also be used for this. We used an F420‐dependent alcohol dehydrogenase (ADF) from Methanoculleus thermophilicus that was found to reduce various ketones to enantiopure alcohols. The respective (S) alcohols were obtained in excellent enantiopurity (>99 % ee). Furthermore, we discovered that the deazaflavoenzyme can be used as a self‐sufficient system by merely using a sacrificial cosubstrate (isopropanol) and a catalytic amount of cofactor F420 or the unnatural cofactor FOP to achieve full conversion. This study reveals that deazaflavoenzymes complement the biocatalytic toolbox for enantioselective ketone reductions.

Bioelectrocatalytic platforms based on chemically modified nanodiamonds by diazonium salt chemistry

Detonation nanodiamonds immobilized onto screen-printed gold electrodes have been modified with a phenothiazine (Azure A) by electrografting of the corresponding in situ generated diazonium salt in acidic medium in the presence of nitrite. The resulting disposable electrochemical platform has been extensively characterized, confirming that is very stable and highly reactive. It shows an excellent electrocatalytic activity towards the oxidation of substances of interest and can be employed to prepare bioelectrocatalytic platforms. Hence, as proof of concept, nicotinamide adenine dinucleotide (NAD+)-dependent alcohol dehydrogenase has been directly immobilized on the Azure A electroactive film to develop an ethanol biosensor based on the measurement of the enzymatically generated β-nicotinamide adenine dinucleotide (NADH). Considering the excellent results obtained, it can be concluded that the modification of electrodes with detonation nanodiamonds can be a good strategy to generate sensing and biosensing electrochemical devices.

Engineered PQQ-Dependent Alcohol Dehydrogenase for the Oxidation of 5-(Hydroxymethyl)furoic Acid

Furan-2,5-dicarboxylic acid (FDCA) is a bio-based platform chemical with the potential to replace terephthalic acid in the production of polymers. A critical step for enzymatic and whole-cell production of FDCA from 5-(hydroxymethyl)furfural (HMF) is the transformation of 5-(hydroxymethyl)furoic acid (HMFA) into 5-formylfuroic acid (FFA). Here, we establish periplasmic pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases (ADHs) as biocatalytic tools for the oxidation of HMFA, HMF, and 5-formylfurfural (FFF). Further, we identify several amino acid residues including the “lid loop” of the substrate channel as promising targets for future engineering steps toward a fully periplasmic oxidation pathway to FDCA.

Metagenome-assembled genomes reveal unique metabolic adaptations of a basal marine Thaumarchaeota lineage.

Thaumarchaeota constitute an abundant and ubiquitous phylum of Archaea that play critical roles in the global nitrogen and carbon cycles. Most well-characterized members of the phylum are chemolithoautotrophic ammonia-oxidizing archaea (AOA), which comprise up to 5 and 20% of the total single-celled life in soil and marine systems, respectively. Using two high-quality metagenome-assembled genomes (MAGs), here we describe a divergent marine thaumarchaeal clade that is devoid of the ammonia-oxidation machinery and the AOA-specific carbon-fixation pathway. Phylogenomic analyses placed these genomes within the uncultivated and largely understudied marine pSL12-like thaumarchaeal clade. The predominant mode of nutrient acquisition appears to be aerobic heterotrophy, evidenced by the presence of respiratory complexes and various organic carbon degradation pathways. Both genomes encoded several pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases, as well as a form III RuBisCO. Metabolic reconstructions suggest anaplerotic CO2 assimilation mediated by RuBisCO, which may be linked to the central carbon metabolism. We conclude that these genomes represent a hitherto unrecognized evolutionary link between predominantly anaerobic basal thaumarchaeal lineages and mesophilic marine AOA, with important implications for diversification within the phylum Thaumarchaeota.

Real-Time Imaging of Transcutaneous Ethanol Using Biofluorometric Gas-Imaging System (Sniff-cam) with Body Shape Fitting Interface for 2D Enzyme Immobilized Mesh Sheet

Introduction Human derived volatile organic compounds (H-VOCs) had been utilized as one of the methods for disease screening and assessment of metabolism since the era of ancient Greece. A part of blood H-VOCs that reflect internal body conditions is released as breath and transcutaneous H-VOCs to the external body unconsciously; therefore, a measurement of H-VOCs in exhaled breath or transcutaneous gas enables to non-invasive disease screening and assessment of metabolism [1]. In this work, we developed a highly sensitive biofluorometric gas-imaging system (Sniff-cam) utilizing nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase (ADH) for real-time quantitative imaging of ethanol in transcutaneous gas. Also, we investigated a relationship between the spatiotemporal change of emission concentration of transcutaneous ethanol (TrEtOH) and skin conditions by imaging TrEtOH at various body parts of the subject after drinking alcohol. Materials and Methods Fig. 1a shows the detection principle of ethanol (EtOH). Quantitative imaging of gaseous EtOH could be possible by capturing the auto-fluorescence emitted from reduced form of NAD (NADH, lex = 340 nm, lem = 490 nm) that is coproduct of the catalytic reaction of ADH with EtOH and oxidized form of NAD (NAD+). The Sniff-cam was constructed with a consumer-available mirrorless camera (ILCE-7S, Sony), UV-LED ring-light (Custom made, Dowa electronics), two bandpass filters (BPF), and an ADH-immobilized mesh as shown in Fig. 1b. The ADH-immobilized mesh was obtained by the glutaraldehyde crosslinking of ADH onto a cotton mesh. Besides, mesh shape-shifting interface that allows equalizing the distance between the curved shape of the body and the ADH-immobilized mesh according to the irregularities of the skin surface was used.In the characterization of the Sniff-cam, standard VOCs sample that was prepared by standard gas generator (Gastec) was used. Various concentration (0.005–300 ppm) of standard EtOH gas was applied to the ADH-immobilized mesh for 20 sec under a flow rate of 100 mL/min to determine a dynamic range. Also, 1 ppm of various kinds of VOCs contained in transcutaneous gas was applied to the ADH-immobilized mesh to evaluate the selectivity.The experiments of real-time imaging of TrEtOH were conducted under the permission of the ethical review committee of Tokyo Medical and Dental University (code: 2018-M160). Subjects took alcohol at the amount of 0.4 g of EtOH per 1kg of body weight for 15 min. We measured TrEtOH using the Sniff-cam for 45 min while a drinking session. Results and Discussions When applying standard EtOH gas, the distribution of fluorescence intensity that reflects the concentration distribution of EtOH was observed on the ADH-immobilized mesh. As a result of an image differential analysis [2] that computed a reaction rate of ADH-mediated reaction to the fluorescence video, the time course of differential value agreed with an application scenario of standard EtOH gas was obtained. A reaction time that was defined as the time requiring the signal reaching the maximum of differential value was 30 sec. Also, the maximum reaction rate was varied with the concentration of standard EtOH gas. The dynamic range that was calculated based on the maximum reaction rate was 0.02–300 ppm, which encompassed the concentration range of TrEtOH after drinking. Moreover, high selectivity against EtOH that was required to measure TrEtOH was observed, which is originated from the substrate specificity of ADH.Fig. 1c shows typical results at palm and wrist at around 40 min after drinking. The intermittent emission and higher concentration of TrEtOH at palm than at the wrist were observed. Currently, emission pathways of TrEtOH are considered as the result of direct emanation of blood VOCs via the interstitial fluid or of vaporization of sweat that contains blood VOCs. It is known that the palm has a higher amount of sweat glands that reacts not only to thermal stimuli but also to mental stimuli than the wrist [3]. Thus, we considered that a higher concentration of TrEtOH emitted because of increased activity of mental sweating at the palm of the subject affected by alcohol. Conclusions Real-time imaging of TrEtOH was possible by the Sniff-cam utilizing biofluorometry technique. The concentration distribution of gaseous EtOH could be quantified through measuring the distribution of fluorescence intensity of NADH. Response time was 30 sec when the image differential analysis was applied to fluorescence video. Also, the Sniff-cam showed enough dynamic range (0.02–300 ppm) and selectivity against EtOH for imaging of TrEtOH. Finally, we observed the regional difference of spatiotemporal change in TrEtOH from subjects who consumed alcohol. In the future, we would like to expand the usefulness of the Sniff-cam by employing other NAD-dependent enzymes to measure disease or metabolism-related VOCs such as acetaldehyde, acetone, and 2-propanol.

Fabrications of metal organic frameworks derived hierarchical porous carbon on carbon nanotubes as efficient bioanode catalysts of NAD+-dependent alcohol dehydrogenase

Herein, we developed an efficient anode catalyst for alcohol biofuel cell by integrating multi-walled carbon nanotubes (MWCNTs) into an isoreticular metal organic framework derived porous carbon. The derived porous carbon (PC) intercalated by MWCNTs (PC/MWCNTs) serviced as the anode component in catalyzing the electrooxidation of reduced β-Nicotinamide adenine dinucleotide (NADH), enabling catalysis to ethanol electrooxidation by alcohol dehydrogenase with NAD+ as both free coenzyme and electron transfer mediators (onset potential, 0.14 V vs. SCE, pH 8.0). Hierarchy of PC/MWCNTs with micro-, meso-, and macropores provides improved immobilization of electroactive enzymes, aids the facile transportations of electrolyte and increases the conductivity and specific surface areas of anode and results in a much higher catalytic current density for NADH (1.17 mA cm−2 for 10 mM NADH, pH 9.0) and alcohol bioanode (maximum steady-state current density, 0.25 ± 0.03 mA cm−2, pH 8.0) than its singular component analogues (PC and MWCNTs). The high Michaelis-Menten constant (166 ± 16.8 mM) favorites the detection of ethanol at high concentrations. The linear range of ethanol is 10–300 mM, with the sensitivity and detection limit as 24 nA mM−1 and 3 mM. The study applies porous carbon nanomaterials as both electrocatalysts for coenzyme and scaffolds for enzymes, benefiting to feasible constructions of bioelectrodes with notable current densities.

Kinetics aspects of Gamma-hydroxybutyrate dehydrogenase

Two groups of metabolically related enzymes, the Group III family of Fe2+-dependent alcohol dehydrogenases (ADHs) and the separate subfamily of nucleoside diphosphates linked to x (nudix) hydrolases that activate Group III ADHs are under-characterized. Here we report the steady-state initial-velocity forward direction (alcohol → aldehyde) reaction of a Group III ADH, namely gamma-hydroxybutyrate dehydrogenase (GHBDH, UniProt: Q59104), cloned from Cupriavidus necator as a fusion protein. We also report the effects of nudix hydrolases on the GHBDH reaction. At optimal pH 9.0, the GHBDH reaction is activated ~2-fold by two different saturating purified nudix hydrolases, namely Bacillus methanolicus activator (ACT, UniProt: I3EA59) and Escherichia coli NudF (UniProt Q93K97) proteins. At physiological pH values of ~7.0, ACT activates by >3.5-fold. Initial-rate characterization at pH 9.0 of the forward direction un-activated and ACT-activated reactions show for both cases competitive inhibition by the product succinic semialdehyde versus GHB, and noncompetitive inhibitions by the three other substrate-product combinations. This pattern is consistent with NAD+ binding first in Mono-Iso Theorell-Chance kinetics. Mutants of some possibly important residues in GHBDH also were characterized. H265, conserved among all Group III ADHs and previously proposed to be a critical general base, is only ~4-fold helpful for GHBDH activity relevant to H265A. The four previously proposed conserved Fe2+ chelators (D193, H197, H261 and H280) each are essential for GHBDH activity. A 2-step explanation for cross-species stimulation by sub-stoichiometric ACT in the forward direction and confirmed lack of ACT stimulation in the reverse direction reaction is proposed.

Transcutaneous Blood VOC Imaging System (Skin-Gas Cam) with Real-Time Bio-Fluorometric Device on Rounded Skin Surface.

A skin-gas cam that allows continuous imaging of transcutaneous blood volatile organic compounds (VOCs) emanated from human skin was developed. The skin-gas cam is able to reveal the relationship between the local skin conditions and transcutaneous blood VOCs in the field of volatile metabolomics (volatolomics). A ring-type ultraviolet (UV) light-emitting diode was mounted around a camera lens as an excitation light source, which enabled the simultaneous excitation and imaging of fluorescence. A nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase (ADH) was used to detect ethanol as a model sample. When gaseous ethanol was applied to an ADH-immobilized mesh that was wetted with an oxidized NAD solution placed in front of the camera, a reduced form of NAD (NADH) was produced through an ADH-mediated reaction. NADH emits fluorescence by UV excitation, and thus, the concentration distribution of ethanol was visualized by measuring the distribution of the fluorescence light intensity from NADH on the ADH-immobilized mesh surface. In this study, a new gas application method that mimicked the release mechanism of transcutaneous gas for quantification of the transcutaneous gas concentration was evaluated. Also, spatiotemporal changes of transcutaneous ethanol for various body parts were measured. As a result, we revealed a relationship between local skin conditions and VOCs that could not be observed previously. In particular, we demonstrated the facile measurement of transdermal gases from around the ear where capillaries are densely distributed below a thin stratum corneum.

Membraneless ethanol,O2 enzymatic biofuel cell based on laccase and ADH/NAD+ bioelectrodes

This work describes EtOH,O2 membraneless enzymatic biofuel cells (EtOH,O2 MlessEBFCs) that employ laccase-based biocathodes and ADH/NAD+ bioanode. Laccase biocathodes were prepared by immobilizing a polypyrrole film containing different redox mediators (ruthenium and osmium complexes). The bioanode for EtOH,O2 MlessEBFCs was fabricated by immobilizing multiwalled carbon nanotubes, NAD+-dependent alcohol dehydrogenase enzyme (ADH), poly-methylene green, and poly(amidoamine) (PAMAM) dendrimer onto a carbon cloth platform. Maximum power density and current density were 21.0 ± 0.2 mW cm-2 and 0.15 ± 0.07 mA cm-2, respectively, in PBS (pH 6.5). Lifetime tests conducted for EtOH,O2 MlessEBFCs showed promising perspectives for their future application in miniaturized devices.

ІНГІБУВАННЯ АМІДАМИ ЗАМІЩЕНИХ БЕНЗОЙНИХ КИСЛОТ ЦИТОПЛАЗМАТИЧНОЇ АЛКОГОЛЬДЕГІДРОГЕНАЗИ НЕЧІНКИ ЩУРІВ

The main purpose of this work was to investigate the influence of substituted benzoic acid amides on the activity of NAD+ dependent alcohol dehydrogenase in vitro, to study the kinetic nature of the effective inhibitors interaction with the enzyme, to establish the relationship between the chemical structure of compounds and their influence on the rate of the enzymatic reaction

Characterization and Application of a Robust Glucose Dehydrogenase from Paenibacillus pini for Cofactor Regeneration in Biocatalysis.

Glucose dehydrogenases are important auxiliary enzymes in biocatalysis, employed in the regeneration of reduced nicotinamide cofactors for oxidoreductase catalysed reactions. Here we report the identification and characterization of a novel glucose-1-dehydrogenase (GDH) from Paenibacillus pini that prefers NAD+ as cofactor over NADP+. The purified recombinant P. pini GDH displayed a specific activity of 247.5U/mg. The enzyme was stable in the pH range 4-8.5 and exhibited excellent thermostability till 50°C for 24h, even in the absence of NaCl or glycerol. Paenibacillus pini GDH was also tolerant to organic solvents, demonstrating its potential for recycling cofactors for biotransformation. The potential application of the enzyme was evaluated by coupling with a NAD+-dependent alcohol dehydrogenase for the reduction of acetophenone and ethyl-4-chloro-3-oxo-butanoate. Conversions higher than 95% were achieved within 2h with low enzyme loading using lyophilized cell lysate, suggesting that P. pini GDH could be highly effective for recycling NADH in redox biocatalysis.