Alcohol Dehydrogenase Reaction Research Articles

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.

Portable Smartphone Platform Based on a Single Dual-Emissive Ratiometric Fluorescent Probe for Visual Detection of Isopropanol in Exhaled Breath.

The components in the exhaled breath have been confirmed to be related to certain diseases, especially studies have shown that isopropanol (IPA) might be closely associated with illnesses such as lung cancer, and are considered as a biomarker. Herein, we designed a portable smartphone platform based on a chemically synthesized ratiometric fluorescent probe for real-time/on-site, sensitive, and quantitative visual detection of IPA in exhaled breath. The fluorescent probe was fabricated by a nicotinamide adenine dinucleotide (NAD+) functional modified onto fluorescent internal standard red carbon dots (RCDs). Whereas, IPA can convert NAD+ into reduced nicotinamide adenine dinucleotide (NADH) through an enzymatic reaction of secondary alcohol dehydrogenase (S-ADH). The electron transfer from IPA to NAD+ emitted a blue emission of NADH, which displayed consecutive color changes from red to light blue. Under optimum conditions, the fluorescent probe shows sensitive responses to IPA with a detection limit as low as 4.45 nM. Moreover, combined with the smartphone color recognizer application (APP), the ratio of fluorescence intensity response was recorded on a blue channel (B)/red channel (R), which has been employed for the visual quantitative determination of IPA with a detection limit of 8.34 nM and a recovery rate of 90.65-110.09% (RSD ≤ 4.83). The method reported here provides a convenient pathway for real-time/on-site and visual detection of IPA in exhaled air and is expected to extend the application of investigation of potential volatile biomarkers for preliminary monitoring and clinical diagnosis.

ВПЛИВ ЗАМІЩЕННОГО НІКОТИНАМІДУ ТА ЙОГО МОЖЛИВИХ МЕТАБОЛІТІВ НА АКТИВНІСТЬ ФЕРМЕНТІВ ОБМІНУ ЕТАНОЛУ

To study the influence of N-phenyl-N-(1-cyclopropylethyl)nicotinamide and its possible metabolites: hydrochlorides of N-(1-cyclopropylethyl)amine and N-phenyl-N-(1-cyclopropylethyl)amine - on the activity of main ethanol oxidation enzymes in vitro and kinetic nature of their interaction. The studies were carried out using alcohol dehydrogenase and aldehyde dehydrogenase of rat liver subcellular fractions, which were obtained by differential centrifugation. The enzyme activity was determined spectrophotometrically. The kinetic nature of alcohol dehydrogenase and isozyme form of aldehyde dehydrogenase interaction with substituted nicotinamide was investigated in the concentration range of 25-100 μM. The research results were processed by the Lineweaver-Burk method. Studies have shown that N-phenyl-N-(1-cyclopropylethyl)nicotinamide is able to reduce the rate of the reverse alcohol dehydrogenase reaction of acetaldehyde reduction to ethanol in the presence of NADH by 46% with an inhibition constant 53 μM. The activity of soluble mitochondrial aldehyde dehydrogenase was suppressed by 50% with an inhibition constant 108 μM. The kinetic nature of the substituted nicotinamide interaction with enzymes at saturating concentrations of the reaction cofactors NADH and NAD+ is quite complex. Allosteric effects can play a significant role in enzymatic activity. Possible metabolites of the compound - hydrochlorides of N-(1-cyclopropylethyl)- and N-phenyl-N-(1-cyclopropylethyl)amine – didn`t significantly influence on ethanol metabolism enzymes activity. A new inhibitor of the rate of the reverse alcohol dehydrogenase reaction and the activity of soluble mitochondrial isozyme form of aldehyde dehydrogenase, which lead to the accumulation of acetaldehyde in the body, has been discovered. N-phenyl-N-(1-cyclopropylethyl)nicotinamide can be used as a potential antialcohol sensitizing drug after research in vivo.

Biochemical Gas Sensor and Gas Concentrator for Monitoring of Ethanol in Skin Gas

A high-sensitive biochemical gas sensor (bio-sniffer) with skin gas cell and gas concentrator for monitoring of ethanol gas concentration emanated from human skin was constructed and demonstrated. This biosensor measured the concentration of ethanol according to the fluorescence intensity change of nicotinamide adenine dinucleotide (NADH), which was produced by an enzymatic reaction of alcohol dehydrogenase. By introducing a gas concentrator using liquid nitrogen, we constructed a highly sensitive system for skin gas measurements. In order to measure skin gas using the bio-sniffer, we designed a measurement system for detecting concentrated gases by connecting the sensor with a gas concentrator and introducing concentrated skin gas to the sensing surface. The monitoring of low concentration of skin ethanol gas was realized after drinking based on alcohol metabolism.

Skin ethanol gas measurement system with a biochemical gas sensor and gas concentrator toward monitoring of blood volatile compounds

We developed a biochemical gas sensor (bio-sniffer) using the enzymatic reaction of alcohol dehydrogenase (ADH) to target ethanol in skin gas. By introducing a gas concentrator using liquid nitrogen, we constructed a highly sensitive system for skin gas measurements. The ethanol bio-sniffer was built from an optical-fiber probe employing an ADH enzyme membrane, an UV-LED light source for excitation, and a photomultiplier tube. Ethanol was measured by detecting the autofluorescence of the coenzyme NADH due to the enzymatic reaction of ADH. We established a system for measuring concentrated gases by connecting the sensor with a gas concentrator and introducing concentrated skin gas to the sensing surface. This suppressed diffusion of the concentrated gases to achieve maximum fluorescence intensity by optimizing the measurement system. The calibration curve from obtained peak values showed ethanol gas can be measured over 1–3100 ppb, which included skin gas concentrations during alcohol consumption. Finally, when applied to measurements of ethanol in skin gas following alcohol consumption, the output was found to be dependent on concentration, similarly to using standard gases. Consecutive measurements were possible using periodic sampling with 6-min intervals for 180 min of monitoring. Skin ethanol concentrations rose from 20 min after consuming the alcohol, exhibited a peak value of 25 ppb skin gas ethanol at around 60 min, and gradually declined. Thus, the system can be used for non-invasive percutaneous evaluation of human volatile organic chemicals in blood.

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.

Improvement of Acetaldehyde Production in Zymomonas mobilis by Engineering of Its Aerobic Metabolism.

Acetaldehyde is a valuable product of microbial biosynthesis, which can be used by the chemical industry as the entry point for production of various commodity chemicals. In ethanologenic microorganisms, like yeast or the bacterium Zymomonas mobilis, this compound is the immediate metabolic precursor of ethanol. In aerobic cultures of Z. mobilis, it accumulates as a volatile, inhibitory byproduct, due to the withdrawal of reducing equivalents from the alcohol dehydrogenase reaction by respiration. The active respiratory chain of Z. mobilis with its low energy-coupling efficiency is well-suited for regeneration of NAD+ under conditions when acetaldehyde, but not ethanol, is the desired catabolic product. In the present work, we sought to improve the capacity Z. mobilis to synthesize acetaldehyde, based on predictions of a stoichiometric model of its central metabolism developed herein. According to the model analysis, the main objectives in the course of engineering acetaldehyde producer strains were determined to be: (i) reducing ethanol synthesis via reducing the activity of alcohol dehydrogenase (ADH), and (ii) enhancing the respiratory capacity, either by overexpression of the respiratory NADH dehydrogenase (NDH), or by mutation of other components of respiratory metabolism. Several mutants with elevated respiration rate, decreased alcohol dehydrogenase activity, or a combination of both, were obtained. They were extensively characterized by determining their growth rates, product yields, oxygen consumption rates, ADH, and NDH activities, transcription levels of key catabolic genes, as well as concentrations of central metabolites under aerobic culture conditions. Two mutant strains were selected, with acetaldehyde yield close to 70% of the theoretical maximum value, almost twice the previously published yield for Z. mobilis. These strains can serve as a basis for further development of industrial acetaldehyde producers.

Switchable sniff-cam (gas-imaging system) based on redox reactions of alcohol dehydrogenase for ethanol and acetaldehyde in exhaled breath

Measuring the volatile organic compounds (VOCs) released from a human is a promising method for noninvasive disease screening and metabolism assessment. Selectively imaging multiple VOCs derived from human breath and skin gas is expected to improve current gas analysis techniques. In this study, a gas-imaging system (sniff-cam) that can be used to simultaneously image the concentration distribution of multiple VOCs, namely, ethanol (EtOH) and acetaldehyde (AcH), was developed. The sniff-cam was based on the pH-dependent redox reactions of nicotinamide adenine dinucleotide (NAD)-dependent alcohol dehydrogenase (ADH). The sniff-cam was constructed with a camera, two ADH-immobilized meshes, and a UV-LED array sheet. The ADH-immobilized mesh containing a solution of the oxidized form of NAD (NAD+) or reduced form (NADH) was used as an EtOH-imaging mesh and an AcH-imaging mesh, respectively. The distributions of the EtOH and AcH concentrations were visualized through the fluorescence of NADH (the excitation wavelength was 340 nm; the emission wavelength was 490 nm) occurring by the ADH-mediated redox reaction. First, the influence of pH on the activity of the redox reaction of ADH was measured, and then the quantitativeness and selectivity of the sniff-cam were evaluated. The ADH-mediated reactions of EtOH and AcH showed maximum activities at pH 9.0 and pH 6.5, respectively. The sniff-cam demonstrated not only a dynamic range (0.1–1000 ppm for EtOH and 0.2–10 ppm for AcH) for measuring EtOH and AcH in breath after drinking alcohol, but also displayed a high selectivity against other breath VOCs. Finally, EtOH and AcH in breath after drinking alcohol were measured simultaneously. A group with high activity of aldehyde dehydrogenase type 2 (EtOH = 143.3 ± 13.5 ppm, AcH = 1.7 ± 0.2 ppm) and a group with low activity (EtOH = 163.3 ± 28.0 ppm, AcH = 8.4 ± 0.5 ppm) displayed differences in the concentrations of EtOH and AcH contained in their breath samples, and the effectiveness of the developed method was confirmed and compared with previous results. It is suggested that the multiplexed sniff-cam in the future may be capable of selectively and simultaneously imaging various VOCs in human breath and skin gas by using multiple NADH-dependent enzymes.

Fiber-Optic Bio-sniffer (Biochemical Gas Sensor) Using Reverse Reaction of Alcohol Dehydrogenase for Exhaled Acetaldehyde.

Volatile organic compounds (VOCs) exhaled in breath have huge potential as indicators of diseases and metabolisms. Application of breath analysis for disease screening and metabolism assessment is expected since breath samples can be noninvasively collected and measured. In this research, a highly sensitive and selective biochemical gas sensor (bio-sniffer) for gaseous acetaldehyde (AcH) was developed. In the AcH bio-sniffer, a reverse reaction of alcohol dehydrogenase (ADH) was employed for reducing AcH to ethanol and simultaneously consuming a coenzyme, reduced form of nicotinamide adenine dinucleotide (NADH). The concentration of AcH can be quantified by fluorescence detection of NADH that was consumed by reverse reaction of ADH. The AcH bio-sniffer was composed of an ultraviolet light-emitting diode (UV-LED) as an excitation light source, a photomultiplier tube (PMT) as a fluorescence detector, and an optical fiber probe, and these three components were connected with a bifurcated optical fiber. A gas-sensing region of the fiber probe was developed with a flow-cell and an ADH-immobilized membrane. In the experiment, after optimization of the enzyme reaction conditions, the selectivity and dynamic range of the AcH bio-sniffer were investigated. The AcH bio-sniffer showed a short measurement time (within 2 min) and a broad dynamic range for determination of gaseous AcH, 0.02-10 ppm, which encompassed a typical AcH concentration in exhaled breath (1.2-6.0 ppm). Also, the AcH bio-sniffer exhibited a high selectivity to gaseous AcH based on the specificity of ADH. The sensor outputs were observed only from AcH-contained standard gaseous samples. Finally, the AcH bio-sniffer was applied to measure the concentration of AcH in exhaled breath from healthy subjects after ingestion of alcohol. As a result, a significant difference of AcH concentration between subjects with different aldehyde dehydrogenase type 2 (ALDH2) phenotypes was observed. The AcH bio-sniffer can be used for breath measurement, and further, an application of breath analysis-based disease screening or metabolism assessment can be expected due to the versatility of its detection principle, which allows it to measure other VOCs by using NADH-dependent dehydrogenases.

Fluorometric Sniff-Cam (Gas-Imaging System) Utilizing Alcohol Dehydrogenase for Imaging Concentration Distribution of Acetaldehyde in Breath and Transdermal Vapor after Drinking.

Understanding concentration distributions, release sites, and release dynamics of volatile organic compounds (VOCs) from the human is expected to lead to methods for noninvasive disease screening and assessment of metabolisms. In this study, we developed a visualization system (sniff-cam) that enabled one to identify a spatiotemporal change of gaseous acetaldehyde (AcH) in real-time. AcH sniff-cam was composed of a camera, a UV-LED array sheet, and an alcohol dehydrogenase (ADH)-immobilized mesh. A reverse reaction of ADH was employed for detection of gaseous AcH where a relationship between fluorescence intensity from nicotinamide adenine dinucleotide and the concentration of AcH was inversely proportional; thus, the concentration distribution of AcH was measured by detecting the fluorescence decrease. Moreover, the image differentiation method that calculated a fluorescence change rate was employed to visualize a real-time change in the concentration distribution of AcH. The dynamic range of the sniff-cam was 0.1-10 ppm which encompassed breath AcH concentrations after drinking. Finally, the sniff-cam achieved the visualization of the concentration distribution of AcH in breath and skin gas. A clear difference of breath AcH concentration was observed between aldehyde dehydrogenase type 2 active and inactive subjects, which was attributed to metabolic capacities of AcH. AcH in skin gas showed a similar time course of AcH concentration to the breath and a variety of release concentration distribution. Using different NADH-dependent dehydrogenases in the sniff-cam could lead to a versatile method for noninvasive disease screening by acquiring spatiotemporal information on various VOCs in breath or skin gas.

Influence of Natural Solutes and Ionic Liquids on the Yield of Enzyme-Catalyzed Reactions: Measurements and Predictions

The maximum yield of enzyme-catalyzed reactions is often limited by thermodynamic equilibrium. The knowledge of influencing factors on limitations of reactions is essential for process optimization to increase yields and to reduce solvent and energy consumption. In this work the effect of solvents/cosolvents [e.g., ionic liquid (IL)] and natural solutes on thermodynamic yield limitations of two enzyme-catalyzed model reactions were investigated, namely, an alcohol dehydrogenase (ADH) reaction (acetophenone + 2-propanol ⇌ 1-phenylethanol + acetone) and an alanine aminotransferase reaction (l-alanine + 2-oxoglutarate ⇌ pyruvate + l-glutamate). Experimental results showed that the equilibrium position and the equilibrium product yield of both reactions in aqueous single-phase systems strongly depend on the type and molality of the present natural solute/IL that were present as additives in the reaction mixture. In addition, the ADH reaction was investigated in pure IL and in an IL/buffer two-phase system. Co...

Origin of the Non-Arrhenius Behavior of the Rates of Enzymatic Reactions.

The origin of the non-Arrhenius behavior of the rate constant for hydride transfer enzymatic reactions has been a puzzling problem since its initial observation. This effect has been used originally to support the idea that enzymes work by dynamical effects and more recently to suggest an entropy funnel model. Our analysis, however, has advanced the idea that the reason for the non-Arrhenius trend reflects the temperature dependence of the rearrangements of the protein polar groups in response to the change in the charge distribution of the reacting system during the transition from the ground state (GS) to the transition state (TS). Here we examine the validity of our early proposal by simulating the catalytic reaction of alcohol dehydrogenase (ADH) and determine the microscopic origin of the entropic and enthalpic contributions to the activation barrier. The corresponding analysis establishes the origin of the non-Arrhenius behaviors and quantifies our original suggestion that the classical effect is due to the entropic contributions of the environment. We also find that the quantum effects reflect in part the temperature dependence of the donor-acceptor distance.

Measuring and Predicting Thermodynamic Limitation of an Alcohol Dehydrogenase Reaction

The knowledge of thermodynamic limitations on enzymatic reactions and of influencing factors thereon is essential for process optimization to increase space–time yields and to reduce the amount of solvent or energy consumption. In this work, the alcohol dehydrogenase (ADH) catalyzed reaction from acetophenone and 2-propanol to 1-phenylethanol and acetone in aqueous solution was investigated in a temperature range of 293.15–303.15 K at pH 7. It serves as a model reaction to demonstrate the use of biothermodynamics in order to investigate and predict limitations of enzymatic reactions. Experimental molalities of the reacting agents at equilibrium were measured yielding the position of reaction equilibrium (Km) at different reaction conditions (temperature, initial reactant molalities). The maximum initial acetophenone molality under investigation was 0.02 mol·kg–1 due to solubility limitations with a 1- to 50-fold excess of 2-propanol. It was shown that Km strongly depends on the initial reactant molalities...

Bio-sniffer (gas-phase biosensor) with secondary alcohol dehydrogenase (S-ADH) for determination of isopropanol in exhaled air as a potential volatile biomarker

Exhaled breath analysis has attracted lots of researchers attention in the past decades due to its advantages such as its non-invasive property and the possibility of continuous monitoring. In addition, several volatile organic compounds in breath have been identified as biomarkers for some diseases. Particularly, studies have pointed out that concentration of isopropanol (IPA) in exhaled air might relate with certain illnesses such as liver disease, chronic obstructive pulmonary (COPD), and lung cancer. In this study, a highly sensitive and selective biochemical gas sensor (bio-sniffer) for the breath IPA concentration determination was constructed and optimized. This bio-sniffer measures the concentration of IPA according to the fluorescence intensity of oxidized nicotinamide adenine dinucleotide (NADH), which was produced by an enzymatic reaction of secondary alcohol dehydrogenase (S-ADH). The NADH detection system employed an UV–LED as the excitation light, and a highly sensitive photomultiplier tube (PMT) as a fluorescence intensity detector. A gas-sensing region was developed using an optical fiber probe equipped with a flow-cell and enzyme immobilized membrane, and connected to the NADH measurement system. The calibration range of the IPA bio-sniffer was confirmed from 1ppb to 9060ppb that was comparable to other IPA analysis methods. The results of the analysis of breath IPA concentration in healthy subjects using the bio-sniffer showed a mean concentration of 16.0ppb, which was similar to other studies. These results have demonstrated that this highly sensitive and selective bio-sniffer could be used to measure the IPA in exhaled air, and it is expected to apply for breath IPA research and investigation of biomarkers for clinical diagnosis.

Fluorometric Bio-Sniffer (Gas Phase Biosensor) for Breath Acetone as a Volatile Product of Lipid Metabolism

A highly sensitive acetone bio-sniffer (biochemical gas sensor) for assessment of lipid metabolism utilizing reverse reaction of secondary alcohol dehydrogenase was fabricated and demonstrated. This bio-sniffer consisted of a NADH fluorescence detection system, a flow-cell section, and an enzyme-immobilized membrane. This bio-sniffer presented rapid response, highly sensitivity and selectivity. The analyses of breath acetone concentrations during fasting and aerobic exercise were also demonstrated. The results showed that the breath acetone concentration would increase during long-time fasting and aerobic exercise. This acetone bio-sniffer provides a new kind of analytical tool for non-invasive evaluation of human lipid metabolism and it is also expected to use for the clinical applications such as monitoring the progression of diabetes.

A Disposable paper breathalyzer with an alcohol sensing organic electrochemical transistor.

Breathalyzers estimate Blood Alcohol Content (BAC) from the concentration of ethanol in the breath. Breathalyzers are easy to use but are limited either by their high price and by environmental concerns, or by a short lifetime and the need for continuous recalibration. Here, we demonstrate a proof-of-concept disposable breathalyzer using an organic electrochemical transistor (OECT) modified with alcohol dehydrogenase (ADH) as the sensor. The OECT is made with the conducting polymer poly(3,4-ethylenedioxythiophene):poly(styrenesulfonate) (PEDOT:PSS), and is printed on paper. ADH and its cofactor nicotinamide adenine dinucleotide (NAD+) are immobilized onto the OECT with an electrolyte gel. When the OECT-breathalyzer is exposed to ethanol vapor, the enzymatic reaction of ADH and ethanol transforms NAD+ into NADH, which causes a decrease in the OECT source drain current. In this fashion, the OECT-breathalyzer easily detects ethanol in the breath equivalent to BAC from 0.01% to 0.2%. The use of a printed OECT may contribute to the development of breathalyzers that are disposable, ecofriendly, and integrated with wearable devices for real-time BAC monitoring.

Reduced pressure gas phase bioreactor as a tool for stereoselective reduction catalyzed by alcohol dehydrogenase from Parvibaculum lavamentivorans

This study presents an immobilized alcohol dehydrogenase reaction in the gas phase under reduced pressure. The catalytic ability (activity, stereoselectivity and productivity) of alcohol dehydrogenase from Parvibaculum lavamentivorans was investigated as a function of various factors for process design. The reduction of 4-methyl-2-pentanone to (R)-4-methyl-2-pentanol with concomitant regeneration of cofactor using 2-propanol as cosubstrate was employed as a model reaction. Under reduced pressure the catalytic ability of the immobilized enzyme was efficiently exhibited, similar to that under standard pressure. On the other hand, the dependence of the water and substrate activities as well as the reaction temperature on the catalytic ability of the immobilized enzyme remained almost the same, compared to the standard pressure. Based on the kinetic parameters (maximum reaction rate and Michaelis constant) estimated, the enzyme activity was found to be effectively maintained under reduced pressure. These findings indicate that the reduced pressure gas phase reactor is capable of proceeding enzyme reaction for chiral compound production. However, as the future scope, the reaction system which proceeds with the catalytic ability of enzyme enhanced more in the reduced pressure condition needs to be widely investigated.

Salivary alcohol dehydrogenase in non-smoking and smoking alcohol-dependent persons

Increasing attention to the importance of saliva testing is not surprising because smoking and alcohol drinking act synergistically on oral tissues, and their metabolite levels, e.g., acetaldehyde, are much higher in saliva than in blood. The activity of salivary alcohol dehydrogenase (ADH) comes from oral microbiota, mucosa, and salivary glands. The purpose of this study was to investigate the involvement of ADH in the oral health pathology of smoking (AS) and non-smoking (ANS) alcohol-dependent males. The results indicated that the AS group had a more significant and longer duration (until the 30th day of alcohol abstinence) decrease in ADH activity and output than the ANS group (until the 15th day of alcohol abstinence) compared to controls (social drinkers; C). The decreased salivary flow (SF) in alcoholics was observed longer in the ANS group (until the 30th day of alcohol abstinence), whereas in the AS group SF normalized at the 15th day, probably due to the irritating effect of tobacco smoke on the oral mucosa. Because saliva was centrifuged to remove cells and debris (including microbial cells), the detected salivary ADH activity was derived from salivary glands and/or oral mucosa. A more profound and longer decrease in ADH activity/output in smoking than non-smoking alcoholics was likely due to the damaged salivary glands and/or oral mucosa, caused by the synergistic effect of alcohol drinking and smoking. The lower values of salivary ADH in smoking than non-smoking alcoholics might also be partly due to the reversed/inhibited ADH reaction by high levels of accumulated acetaldehyde.

Effects of Hydrostatic Pressure on Stereospecificity of Secondary Alcohol Dehydrogenase from Thermoanaerobacter Ethanolicus Support the Role of Solvation in Enantiospecificity

The effects of hydrostatic pressure and temperature on the reaction of secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus with enantiomers of 2-butanol, 2-pentanol, and 2-hexanol have been measured. For all substrates, increasing pressure favors the S enantiomer, whereas increasing temperature favors the R enantiomer. Fitting of the pressure and temperature data for 2-hexanol provided apparent ΔΔS‡ and ΔΔV‡ values of +46 ± 15 J/mol and +(2.0 ± 0.4) × 10–2 L/mol, respectively. These results support our previous proposal that desolvation of the enzyme active site plays an important role in stereospecificity.

A Hydrazine Coupled Cycling Assay Validates the Decrease in Redox Ratio under Starvation in Drosophila

A commonly used enzymatic recycling assay for pyridine nucleotides has been adapted to directly measure the NAD+/NADH redox ratio in Drosophila melanogaster. This method is also suitable for quantification of NADP+ and NADPH. The addition of a coupling reaction removing acetaldehyde produced from the alcohol dehydrogenase (ADH) reaction was shown to improve the linearity of NAD(H) assay. The advantages of this assay method are that it allows the determination of both NAD+ and NADH simultaneously while keeping enzymatic degradation of pyridine nucleotides minimal and also achieving better sensitivity. This method was used to determine the redox ratio of D. melanogaster and validated substantial decrease of redox ratio during starvation.