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2,3-butanediol (2,3-BD) is a versatile bio-based platform chemical. An artificial four-enzyme synthetic biosystem composed of ethanol dehydrogenase, NADH oxidase, formolase and 2,3-butanediol dehydrogenase was designed for upgrading ethanol to 2,3-BD in our previous study. However, a key challenge in developing in vitro enzymatic systems for 2,3-BD synthesis is the relatively sluggish catalytic efficiency of formolase, which catalyzes the rate-limiting step in such systems. Herein, this study reports how engineering the tunnel and substrate binding pocket of FLS improved its catalytic performance. A series of single-point and combinatorial variants were successfully obtained which displayed both higher catalytic efficiency and better substrate tolerance than wild-type FLS. Subsequently, a cell-free biosystem based on the FLS:I28V/L482E enzyme was implemented for upgrading ethanol to 2,3-BD. Ultimately, this system achieved efficient production of 2,3-BD from ethanol by the fed-batch method, reaching a concentration of 1.39 M (124.83 g/L) of the product and providing both excellent productivity and yield values of 5.94 g/L/h and 92.7%, respectively. Taken together, this modified enzymatic catalysis system provides a highly promising alternative approach for sustainable and cost-competitive production of 2,3-BD.

A high ethanol usage of alcohol oxidase (AOX) was required in industry. In this study, a "expand substrate pocket" strategy achieved a high activity AOX from Hansenula polymorpha (H. polymorpha) by Phe to Val residue (F/V) site-directed mutation to enlarge ethanol channel. Although H. Polymorpha AOX (HpAOX) possessed respectively 71.3% and 76.1% similarity with AOX (PpAOX) from Pichia pastoris (P. pastoris) in DNA and protein sequences, their active site structures including catalytic site and substrate channel were similar according to computer-aided analysis. After 3D structure analysis, Phe99 residue of their substrate channels was the most important residue to impact enzyme activity because of its large aromatic side chains. F99V mutation of HpAOX (HpAOXF99V) was designed and executed based on the enzyme catalytic mechanism and molecular computation in order to allow more larger size ethanol into active site. The highest enzyme activity of the fourth strains of HpAOXF99V mutant strain exhibited 12.06-folds increase than that of the host GS115 strain. Furthermore, kinetic studies indicated that the HpAOXF99V significantly promoted catalytic efficiency of ethanol than HpAOX, including Km, Vmax, kcat and kcat/Km. We also provided a new insight that the cofactor FAD irritated both active AOX octamer biosynthesis production and enzyme-catalysed ability due to help enzyme assembly and redox potential., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Eccrine sweat is a rich and largely unexplored biofluid that contains a range of important biomarkers, from electrolytes, metabolites, micronutrients and hormones to exogenous agents, each of which can change in concentration with diet, stress level, hydration status and physiologic or metabolic state. Traditionally, clinicians and researchers have used absorbent pads and benchtop analyzers to collect and analyze the biochemical constituents of sweat in controlled, laboratory settings. Recently reported wearable microfluidic and electrochemical sensing devices represent significant advances in this context, with capabilities for rapid, in situ evaluations, in many cases with improved repeatability and accuracy. A limitation is that assays performed in these platforms offer limited control of reaction kinetics and mixing of different reagents and samples. Here, we present a multi-layered microfluidic device platform with designs that eliminate these constraints, to enable integrated enzymatic assays with demonstrations of in situ analysis of the concentrations of ammonia and ethanol in microliter volumes of sweat. Careful characterization of the reaction kinetics and their optimization using statistical techniques yield robust analysis protocols. Human subject studies with sweat initiated by warm-water bathing highlight the operational features of these systems.

Law enforcement and the general public do not yet have adequate means of assessing and preventing drunk driving. Blood alcohol concentration (BAC) is unable to be determined on-site, as it typically requires the use of complex chromatographic methods. Breathalyzers have been well established in law enforcement for correlating breath alcohol concentrations (BrAC) to BAC estimations, as they involve portable equipment with rapid analysis times. Although these BrAC measurements allow police officers to determine probable cause and to arrest an intoxicated driver at the scene, the results are preliminary and are not often considered as evidence in court. A new, noninvasive method was developed to assess an individual's level of intoxication based on the presence of ethanol in sweat on the skin surface. This intuitive system uses two enzymes, alcohol oxidase and horseradish peroxidase, to correlate ethanol sweat concentrations to the production of a color that is visible to the naked eye. The results of the controlled drinking study demonstrate the ability of both the spectrophotometric and the visualization system to quantify the amount of ethanol within authentic sweat samples collected from individuals who had consumed an alcoholic beverage. The pictorial analysis allows for the system to be analyzed without the use of a UV-vis spectrophotometer. With this method, a smartphone application would be capable of documenting and evaluating the intoxication levels of an individual based on sweat ethanol levels. The developed alcohol sensing system has the potential to impact both the general public and law enforcement, as well as the fields of forensic and biomedical science.

A newly designed amperometric biosensor for the determination of ethanol through one-step electrochemical coating of (4,7-di(thiophen-2-yl)benzo[c][1,2,5]selenadiazole-co-1H-pyrrole-3-carboxylic acid) (TBeSe-co-P3CA) on a graphite electrode is presented. It was aimed to propose a newly synthesized copolymer with enhanced biosensing properties as a novel sensor for the quantification of ethanol. The conjugated copolymer (TBeSe-co-P3CA) was prepared through electrochemical polymerization by potential cycling. After polymer modification, alcohol oxidase (AOx) was immobilized on a modified electrode surface for ethanol sensing. In the analytical investigation, the calibration plot is linear above large concentration range (0.085 to 1.7 mM), where sensitivity is around 16.44 μA/mMcm 2 with a very low detection limit (LOD) of 0.052 mM based on the signal-to-noise ratio in short response time. Moreover, interfering effect of some possible compounds were examined and the capability of the biosensor in estimating ethanol content in commercial alcoholic beverages was also demonstrated. The results showed satisfactory accuracy of the developed sensor and confirm the proposed sensor has a potential for ethanol quantification compared to the currently used techniques., (Copyright © 2019. Published by Elsevier B.V.)

Fifty years ago, in 1968, the pioneering scientists Charles S. Lieber and Leonore M. DeCarli discovered the capacity for liver microsomes to oxidize ethanol (EtOH) and named it the microsomal ethanol-oxidizing system (MEOS), which revolutionized clinical and experimental alcohol research. The last 50 years of MEOS are now reviewed and highlighted. Since its discovery and as outlined in a plethora of studies, significant insight was gained regarding the fascinating nature of MEOS: (i) MEOS is distinct from alcohol dehydrogenase and catalase, representing a multienzyme complex with cytochrome P450 (CYP) and its preferred isoenzyme CYP 2E1, NADPH-cytochrome P450 reductase, and phospholipids; (ii) it plays a significant role in alcohol metabolism at high alcohol concentrations and after induction due to prolonged alcohol use; (iii) hydroxyl radicals and superoxide radicals promote microsomal EtOH oxidation, assisted by phospholipid peroxides; (iv) new aspects focus on microsomal oxidative stress through generation of reactive oxygen species (ROS), with intermediates such as hydroxyethyl radical, ethoxy radical, acetyl radical, singlet radical, hydroxyl radical, alkoxyl radical, and peroxyl radical; (v) triggered by CYP 2E1, ROS are involved in the initiation and perpetuation of alcoholic liver injury, consequently shifting the previous nutrition-based concept to a clear molecular-based disease; (vi) intestinal CYP 2E1 induction and ROS are involved in endotoxemia, leaky gut, and intestinal microbiome modifications, together with hepatic CYP 2E1 and liver injury; (vii) circulating blood CYP 2E1 exosomes may be of diagnostic value; (viii) circadian rhythms provide high MEOS activities associated with significant alcohol metabolism and potential toxicity risks as a largely neglected topic; and (ix) a variety of genetic animal models are useful and have been applied elucidating mechanistic aspects of MEOS. In essence, MEOS along with its CYP 2E1 component currently explains several mechanistic steps leading to alcoholic liver injury and has a promising future in alcohol research., (© 2019 by the Research Society on Alcoholism.)

Herein, we report the first example of a paper-based screen-printed biosensor for the detection of ethanol in beer samples. Common office paper was adopted to fabricate the analytical device. The properties of this paper-based screen-printed electrode (SPE) were investigated by cyclic voltammetry, electrochemical impedance spectroscopy, and scanning electron microscopy, and they were compared with the well-established polyester-based SPEs as well. Paper demonstrated similar properties when compared with polyester, highlighting suitability towards its utilization in sensor development, with the advantages of low cost and simple disposal by incineration. A nanocomposite formed by Carbon Black (CB) and Prussian Blue nanoparticles (PBNPs), namely CB/PBNPs, was utilized as an electrocatalyst to detect the hydrogen peroxide generated by the enzymatic reaction between alcohol oxidase (AOx) and ethanol. After optimizing the analytical parameters, such as pH, enzyme, concentration, and working potential, the developed biosensor allowed a facile quantification of ethanol up to 10 mM (0.058 % vol ), with a sensitivity of 9.13 μA/mM cm 2 (1574 μA/% vol cm 2 ) and a detection limit equal to 0.52 mM (0.003% vol ). These satisfactory performances rendered the realized paper-based biosensor reliable over the analysis of ethanol contained in four different types of beers, including Pilsner, Weiss, Lager, and alcohol-free. The proposed manufacturing approach offers an affordable and sustainable tool for food quality control and for the realization of different electrochemical sensors and biosensors as well., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Acute alcohol intoxication (AAI) is a frequent emergency, but therapeutic drugs with superior efficacy and safety are lacking. Panax ginseng (PG) and Hippophae rhamnoides (HR) respectively has a wide application as a complementary therapeutic agent in China for the treatment of AAI and liver injury induced by alcohol. We investigated the effects of aqueous extracts from PG and HR (AEPH) on AAI mice and identified its underlying mechanisms., Materials and Methods: Models of AAI were induced by intragastric administration of ethanol (8g/kg). Seventy-two Specific pathogen-free (SPF) male Kunming mice were randomly divided into six groups: normal group, positive control group, AEPH of low dosage (100mg/kg) group, AEPH of medium dose (200mg/kg) group, AEPH of high dosage (400mg/kg) group and model group. The mice were treated with metadoxine (MTD, 500mg/kg) and AEPH. Thirty minutes later, the normal group was given normal saline, while the other groups were given ethanol (i.g., 8g/kg). The impact of AEPH was observed. In the same way, another seventy-two Kunming mice were randomly divided into six groups equally. The blood ethanol concentration at 0.5, 1, 1.5, 2, 3 and 6h after ethanol intake was determined by way of gas chromatography. The activity of alcohol dehydrogenase (ADH), aldehyde dehydrogenase (ALDH) and microsomal ethanol oxidase (EO) in liver, and the concentration of β-endorphin (β-EP), leucine-enkephalin (LENK) in the brain were determined by enzyme-linked-immunosorbent serologic assay (ELISA)., Results: AEPH markedly prolonged alcohol tolerance time and shortened sober-up time after acute ethanol administration. AEPH decreased blood ethanol levels in six tests after ethanol intake. The 7-day survival rate of AEPH group was obviously superior to model group. AEPH increased the activities of ADH, ALDH, and decreased EO activity in liver. The crucial find was that AEPH markedly decreased β-EP and LENK concentration in the brain., Conclusions: AEPH can markedly increase the levels of ADH, ALDH, decrease EO activity in liver and decrease the concentration of β-EP and LENK in the brain to against acute alcohol intoxication in mice., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Lanthanides are utilized by microbial methanol dehydrogenases, and it has been proposed that lanthanides may be important for other type I alcohol dehydrogenases. A triple mutant strain (mxaF xoxF1 xoxF2; named MDH-3), deficient in the three known methanol dehydrogenases of the model methylotroph Methylobacterium extorquens AM1, is able to grow poorly with methanol if exogenous lanthanides are added to the growth medium. When the gene encoding a putative quinoprotein ethanol dehydrogenase, exaF, was mutated in the MDH-3 background, the quadruple mutant strain could no longer grow on methanol in minimal medium with added lanthanum (La 3+ ). ExaF was purified from cells grown with both calcium (Ca 2+ ) and La 3+ and with Ca 2+ only, and the protein species were studied biochemically. Purified ExaF is a 126-kDa homodimer that preferentially binds La 3+ over Ca 2+ in the active site. UV-visible spectroscopy indicates the presence of pyrroloquinoline quinone (PQQ) as a cofactor. ExaF purified from the Ca 2+ -plus-La 3+ condition readily oxidizes ethanol and has secondary activities with formaldehyde, acetaldehyde, and methanol, whereas ExaF purified from the Ca 2+ -only condition has minimal activity with ethanol as the substrate and activity with methanol is not detectable. The exaF mutant is not affected for growth with ethanol; however, kinetic and in vivo data show that ExaF contributes to ethanol metabolism when La 3+ is present, expanding the role of lanthanides to multicarbon metabolism., Importance: ExaF is the most efficient PQQ-dependent ethanol dehydrogenase reported to date and, to our knowledge, the first non-XoxF-type alcohol oxidation system reported to use lanthanides as a cofactor, expanding the importance of lanthanides in biochemistry and bacterial metabolism beyond methanol dehydrogenases to multicarbon metabolism. These results support an earlier proposal that an aspartate residue near the catalytic aspartate residue may be an indicator of rare-earth element utilization by type I alcohol dehydrogenases., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

We previously reported the metabolic analysis of butanol and acetone production from exogenous acetate by (13)C tracer experiments (Gao et al., RSC Adv., 5, 8486-8495, 2015). To clarify the influence of acetate on acetone-butanol-ethanol (ABE) production, we first performed an enzyme assay in Clostridium saccharoperbutylacetonicum N1-4. Acetate addition was found to drastically increase the activities of key enzymes involved in the acetate uptake (phosphate acetyltransferase and CoA transferase), acetone formation (acetoacetate decarboxylase), and butanol formation (butanol dehydrogenase) pathways. Subsequently, supplementation of acetate during acidogenesis and early solventogenesis resulted in a significant increase in ABE production. To establish an efficient ABE production system using acetate as a co-substrate, several shot strategies were investigated in batch culture. Batch cultures with two substrate shots without pH control produced 14.20 g/L butanol and 23.27 g/L ABE with a maximum specific butanol production rate of 0.26 g/(g h). Furthermore, pH-controlled (at pH 5.5) batch cultures with two substrate shots resulted in not only improved acetate consumption but also a further increase in ABE production. Finally, we obtained 15.13 g/L butanol and 24.37 g/L ABE at the high specific butanol production rate of 0.34 g/(g h) using pH-stat co-feeding method. Thus, in this study, we established a high ABE production system using glucose and acetate as co-substrates in a pH-stat co-feeding system with C. saccharoperbutylacetonicum N1-4., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A two-dimensional imaging system (Sniffer-camera) for visualizing the concentration distribution of ethanol vapor emitting from wine in a wine glass has been developed. This system provides image information of ethanol vapor concentration using chemiluminescence (CL) from an enzyme-immobilized mesh. This system measures ethanol vapor concentration as CL intensities from luminol reactions induced by alcohol oxidase and a horseradish peroxidase (HRP)-luminol-hydrogen peroxide system. Conversion of ethanol distribution and concentration to two-dimensional CL was conducted using an enzyme-immobilized mesh containing an alcohol oxidase, horseradish peroxidase, and luminol solution. The temporal changes in CL were detected using an electron multiplier (EM)-CCD camera and analyzed. We selected three types of glasses-a wine glass, a cocktail glass, and a straight glass-to determine the differences in ethanol emission caused by the shape effects of the glass. The emission measurements of ethanol vapor from wine in each glass were successfully visualized, with pixel intensity reflecting ethanol concentration. Of note, a characteristic ring shape attributed to high alcohol concentration appeared near the rim of the wine glass containing 13 °C wine. Thus, the alcohol concentration in the center of the wine glass was comparatively lower. The Sniffer-camera was demonstrated to be sufficiently useful for non-destructive ethanol measurement for the assessment of food characteristics.

A simple visual ethanol biosensor based on alcohol oxidase (AOX) immobilised onto polyaniline (PANI) film for halal verification of fermented beverage samples is described. This biosensor responds to ethanol via a colour change from green to blue, due to the enzymatic reaction of ethanol that produces acetaldehyde and hydrogen peroxide, when the latter oxidizes the PANI film. The procedure to obtain this biosensor consists of the immobilization of AOX onto PANI film by adsorption. For the immobilisation, an AOX solution is deposited on the PANI film and left at room temperature until dried (30 min). The biosensor was constructed as a dip stick for visual and simple use. The colour changes of the films have been scanned and analysed using image analysis software (i.e., ImageJ) to study the characteristics of the biosensor's response toward ethanol. The biosensor has a linear response in an ethanol concentration range of 0.01%-0.8%, with a correlation coefficient (r) of 0.996. The limit detection of the biosensor was 0.001%, with reproducibility (RSD) of 1.6% and a life time up to seven weeks when stored at 4 °C. The biosensor provides accurate results for ethanol determination in fermented drinks and was in good agreement with the standard method (gas chromatography) results. Thus, the biosensor could be used as a simple visual method for ethanol determination in fermented beverage samples that can be useful for Muslim community for halal verification.

A non-invasive, passive and simple to use skin surface based sensing device for determining the blood's ethanol content (BAC) by monitoring transdermal alcohol concentration (TAC) is designed and developed. The proposed prototype is based on bienzyme amperometric composite biosensors that are sensitive to the variation of ethanol concentration. The prototype correlates, through previous calibration set-up, the amperometric signal generated from ethanol in sweat with its content in blood in a short period of time. The characteristics of this sensor device permit determination of the ethanol concentration in isolated and in continuous form, giving information of the BAC of a subject either in a given moment or its evolution during long periods of time (8h). Moreover, as the measurements are performed in a biological fluid, the evaluated individual is not able to alter the result of the analysis. The maximum limit of ethanol in blood allowed by legislation is included within the linear range of the device (0.0005-0.6 g L(-1)). Moreover, the device shows higher sensitivity than the breathalyzers marketed at the moment, allowing the monitoring of the ethanol content in blood to be obtained just 5 min after ingestion of the alcoholic drink. The comparison of the obtained results using the proposed device in the analysis of 40 volunteers with those provided by the gas chromatographic reference method for determination of BAC pointed out that there were no significant differences between both methods., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Transgenic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alterations in cell wall composition. Cell walls of CAD-RNAi stems contain a lignin polymer with a slight reduction in the S-to-G ratio without affecting the total lignin content. In addition, these cell walls accumulate higher levels of cellulose and arabinoxylans. In contrast, cell walls of CAD-RNAi midribs present a reduction in the total lignin content and of cell wall polysaccharides. In vitro degradability assays showed that, although to a different extent, the changes induced by the repression of CAD activity produced midribs and stems more degradable than wild-type plants. CAD-RNAi plants grown in the field presented a wild-type phenotype and produced higher amounts of dry biomass. Cellulosic bioethanol assays revealed that CAD-RNAi biomass produced higher levels of ethanol compared to wild-type, making CAD a good target to improve both the nutritional and energetic values of maize lignocellulosic biomass.

Furfural is an important fermentation inhibitor in hemicellulose sugar syrups derived from woody biomass. The metabolism of furfural by NADPH-dependent oxidoreductases, such as YqhD (low K(m) for NADPH), is proposed to inhibit the growth and fermentation of xylose in Escherichia coli by competing with biosynthesis for NADPH. The discovery that the NADH-dependent propanediol oxidoreductase (FucO) can reduce furfural provided a new approach to improve furfural tolerance. Strains that produced ethanol or lactate efficiently as primary products from xylose were developed. These strains included chromosomal mutations in yqhD expression that permitted the fermentation of xylose broths containing up to 10 mM furfural. Expression of fucO from plasmids was shown to increase furfural tolerance by 50% and to permit the fermentation of 15 mM furfural. Product yields with 15 mM furfural were equivalent to those of control strains without added furfural (85% to 90% of the theoretical maximum). These two defined genetic traits can be readily transferred to enteric biocatalysts designed to produce other products. A similar strategy that minimizes the depletion of NADPH pools by native detoxification enzymes may be generally useful for other inhibitory compounds in lignocellulosic sugar streams and with other organisms.

Degradation of lignocellulose with pressurised hot water is an efficient method of bioethanol production. However, the resultant solution inhibits ethanol fermentation by Saccharomyces cerevisiae. Here, we first report that glycolaldehyde, which is formed when lignocellulose is treated with pressurised hot water, inhibits ethanol fermentation. The final concentration of glycolaldehyde formed by the treatment of lignocellulose with pressurised hot water ranges from 1 to 24 M, and 1-10 mM glycolaldehyde was sufficient to inhibit fermentation. This result indicates that glycolaldehyde is one of the main substances responsible for inhibiting fermentation after pressurised hot water degradation of lignocellulose. Genome-wide screening of S. cerevisiae revealed that genes encoding alcohol dehydrogenase, methylglyoxal reductase, polysomes, and the ubiquitin ligase complex are required for glycolaldehyde tolerance. These novel findings will provide new perspectives on breeding yeast for bioethanol production from biomass treated with pressurised hot water.

Activity of key-enzymes of C2-metabolism was determined in the cells of strain-producer of surface-active substances Acinetobacter calcoaceticus K-4 grown on ethanol. It is shown that ethanol and acetaldehyde oxidation in the strain K-4 is performed by pyroquinolinquinon (PQQ) and 4-nitroso-N,N-dimethylaniline (NDMA)-dependent dehydrogenases. Activity of NDMA-dependent enzymes was maximum (100-300 nmol min(-1) mg(-1) of protein) in the early exponential growth phase of A. calcoaceticus K-4. Availability of NDMA-dependent alcohol and acetaldehyde dehydrogenases in gram-negative bacteria was established for the first time. Acetate is involved in metabolism in the strain K-4 with participation of both acetate kinase and acetate-KoA-synthetase; replenishment of the pool of C4-dicarbonic acids is performed in glioxylate cycle (activity of isocytrate lyase is 600 nmol min(-1) mg(-1) of protein) and in phosphoenolpyruvate carboxylase reaction (1600 nmol min(-1) mg(-1) of protein). Both key enzymes of gluconeogenesis take place in synthesis of carbohydrates: FEP-carboxykinase and FEP-synthetase (1200 and 4400 nmol min(-1) mg(-1) of protein, respectively). Enzymatic investigations have confirmed the capacity of strain K-4 to synthesis of surface-active trehalosemycolates (activity of trehalosephosphate synthase is 150-160 nmol min(-1) mg(-1) of protein).

The effects of catalase regulators (aminotriazole, lead acetate, taurine, di-2-ethylhexylphthalate) on the preference for ethanol, its pharmacokinetics, and activities of rat liver and brain ethanol and acetaldehyde-metabolizing enzymes were studied. Lead acetate (100 mg/kg, i.p., 7 days), aminotriazole (1 g/kg, i.p., 7 days), and taurine (650 mg/kg, i.g., 14 days) decreased ethanol consumption under conditions of free choice (10% ethanol water), whereas di-2-ethylhexylphthalate (300 mg/kg, i.g., 7 days) did not exert any effect on this parameter. Taurine, lead acetate and di-2-ethylhexylphthalate significantly activated liver ADH, MEOS and catalase peroxidase activity. Aminotriazole also activated ADH and MEOS, but inhibited liver catalase. The activities of liver and brain A1DH as well as catalase were insignificantly changed by this treatment. The 7-day administration of lead acetate, di-2-ethylhexylphthalate and aminotriazole administrations significantly influenced the ethanol (2 g/kg., i.p.) pharmacokinetic parameters: the area under the pharmacokinetic curve and the elimination half-life time were significantly reduced, whereas the elimination constant and clearance were increased. This unequivocally indicates accelerated ethanol elimination. The 14-day ingestion of taurine insignificantly changed the parameters of ethanol pharmacokinetics in rats.

A highly stable and sensitive amperometric alcohol biosensor was developed by immobilizing alcohol oxidase (AOX) through Polyamidoamine (PAMAM) dendrimers on a cysteamine-modified gold electrode surface. Ethanol determination is based on the consumption of dissolved oxygen content due to the enzymatic reaction. The decrease in oxygen level was monitored at -0.7 V vs. Ag/AgCl and correlated with ethanol concentration. Optimization of variables affecting the system was performed. The optimized ethanol biosensor showed a wide linearity from 0.025 to 1.0 mM with 100 s response time and detection limit of (LOD) 0.016 mM. In the characterization studies, besides linearity some parameters such as operational and storage stability, reproducibility, repeatability, and substrate specificity were studied in detail. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 67% of its initial sensitivity was kept after 1 month storage at 4 degrees C. The analytical characteristics of the system were also evaluated for alcohol determination in flow injection analysis (FIA) mode. Finally, proposed biosensor was applied for ethanol analysis in various alcoholic beverage as well as offline monitoring of alcohol production through the yeast cultivation., (Copyright 2010 American Institute of Chemical Engineers)

The adhS gene which encodes the smallest subunit, subunit III, of quinoprotein alcohol dehydrogenase (PQQ-ADH) from Acetobacter pasteurianus SKU1108 has been cloned and characterized. The role of this subunit on the function of PQQ-ADH was investigated by construction of adhS gene disruptant and mutants. The adhS gene disruptant completely lost its PQQ-ADH activity and acetate-producing ability but retained acetic acid toleration. In contrast, this disruptant grew well, even better than the wild type, in the ethanol containing medium even though its PQQ-ADH activity and ethanol oxidizing ability was completely lost, while NAD(+)-dependent ADH (NAD(+)-ADH) was induced. Heme staining and immunoblot analysis of both membrane and soluble fractions with anti-ADH subunit III suggested that ethanol did not affect the adhS gene expression but induced PQQ-ADH activity. Over-expressed adhS did not enhance acetic acid production in both the wild type and the adhS disruptant. In addition, deletion analysis of upstream region of adhS gene suggested that its tentative promoter(s) might be located at around 118-268 bp upstream from an initiation codon. Random mutagenesis of adhS gene revealed that complete loss of PQQ-ADH activity and ethanol oxidizing ability were observed in the mutants' lack of the 140 and 73 amino acid residues at the C-terminal, whereas the lack of 22 amino acid residues at the C-terminal affected neither the PQQ-ADH activity nor ethanol oxidizing ability. In addition, some amino acid substitutions such as Leu18Gln, Ala26Val, Val36Ile, Val54Ile, Gly55Asp, Val70Ala and Val107Ala did not show any affect on PQQ-ADH activity and ethanol oxidizing ability. Interestingly, alteration of Thr104Lys led to a complete loss of ethanol oxidizing ability. However, point mutation at the possible promoter region also exhibited low PQQ-ADH activity and ethanol oxidizing ability. This result suggests that 104Thr might be involved in molecular coupling with subunit I in order to construct active ADH complex, whereas 22 amino acid residues at C-terminal may be not necessary for PQQ-ADH activity., (Copyright 2009 Elsevier B.V. All rights reserved.)

Phanerochaete chrysosporium is a wood-rot fungus that is capable of degrading lignin via its lignolytic system. In this study, an environmentally friendly fungal pretreatment process that produces less inhibitory substances than conventional methods was developed using P. chrysosporium and then evaluated by various analytical methods. To maximize the production of manganese peroxidase, which is the primary lignin-degrading enzyme, culture medium was optimized using response surface methodologies including the Plackett-Burman design and the Box-Behnken design. Fermentation of 100 g of rice straw feedstock containing 35.7 g of glucan (mainly in the form of cellulose) by cultivation with P. chrysosporium for 15 days in the media optimized by response surface methodology was resulted in a yield of 29.0 g of glucan that had an enzymatic digestibility of 64.9% of the theoretical maximum glucose yield. In addition, scanning electronic microscopy, confocal laser scanning microscopy, and X-ray diffractometry revealed significant microstructural changes, fungal growth, and a reduction of the crystallinity index in the pretreated rice straw, respectively. When the fungal-pretreated rice straw was used as a substrate for ethanol production in simultaneous saccharification and fermentation (SSF) for 24 h, the ethanol concentration, production yield and the productivity were 9.49 g/L, 58.2% of the theoretical maximum, and 0.40 g/L/h, respectively. Based on these experimental data, if 100 g of rice straw are subjected to fungal pretreatment and SSF, 9.9 g of ethanol can be produced after 96 h, which is 62.7% of the theoretical maximum ethanol yield.

Objective: To address the question of whether increased ethanol elimination in alcoholics can be ascribed to increased metabolism via alcohol dehydrogenase (ADH; K(m) around 0.2 mM) or the microsomal ethanol-oxidizing system (MEOS; K(m) 10 mM) by kinetic analysis of hepatic ethanol elimination in recently drinking patients with alcoholic cirrhosis and healthy subjects. A further objective was to investigate whether systemic clearance of ethanol at low arterial ethanol concentrations can be used as a measure of hepatic blood flow., Material and Methods: Six patients with alcoholic cirrhosis were enrolled after 2 days of abstinence, along with 6 healthy subjects. Ethanol was administered as 6 successive infusions in increasing doses. Arterial and hepatic venous blood concentrations of ethanol were measured; hepatic blood flow was measured simultaneously. Kinetic parameters were calculated according to the sinusoidal perfusion model of enzymatic elimination by the intact liver., Results: Mean hepatic K(m) for ethanol was 0.16 mM (range 0.09-0.36) in healthy subjects and 0.36 mM (range 0.16-0.69) in patients with cirrhosis (p>0.3), both compatible with the K(m) for ADH. The two groups of subjects had similar V(max) values (p>0.3). Extrahepatic elimination of ethanol accounted for more than 50% of total elimination in both groups, which precludes the use of systemic clearance as a measure of hepatic blood flow., Conclusions: The results support the hypothesis that ADH remains the main pathway for hepatic elimination of ethanol in recently drinking patients with alcoholic cirrhosis. We interpret this as evidence against a significant contribution of MEOS in vivo.

Both antipodes of 2-azido-1-arylethanols were synthesized with excellent optical purity via enzymatic reduction of the corresponding alpha-azidoacetophenone derivatives catalyzed by a recombinant carbonyl reductase from Candida magnoliae ( CMCR) or an alcohol dehydrogenase from Saccharomyces cerevisiae ( Ymr226c). This provides an effective route to this class of important compounds in optically pure form. ( S)-2-Azido-1-( p-chlorophenyl)ethanols reacted with alkynes employing click chemistry to afford high yields of optically pure triazole-containing beta-adrenergic receptor blocker analogues with potential biological activity.

A large body of evidence supports the view that mitochondria are a primary target of alcohol stress. Changes in mitochondrial proteins due to moderate ethanol intake, however, have not been broadly and accurately estimated. For this study, rats were fed low doses of ethanol and the mitochondria were isolated from heart, kidney, and liver, using ultracentrifugation with Nycodenz density gradient. The mitochondrial proteins were well resolved upon two-dimensional electrophoresis (2DE), and the alcohol-responsive 2DE spots were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Compared with the control group, the proteins extracted from liver mitochondria of ethanol-fed rats exhibited the significant changes on 2DE images, whereas the 2DE images obtained from the kidney and the heart mitochondria remained almost unchanged by ethanol feeding. Significantly, over 50% of the alcohol-responsive proteins in liver mitochondria were members of aldo-keto reductase family (AKR), which were usually present in cytoplasm. The organelle distributions of AKR proteins in liver mitochondria were further confirmed by Western blot analysis as well as by confocal microscopy. In addition, translocations of AKR were examined in the CHANG cell line, which was cultured with and without ethanol. The results of Western blot strongly suggested that the abundances of AKR proteins in the mitochondria were greatly reduced by the presence of ethanol in culture medium. The results of this study show that, even with moderate ethanol feeding, the mitochondrial proteome in rat liver was more sensitive to alcohol stress than that of either the kidney or the heart. The translocation of AKR proteins may be involved in the detoxification of liver cells.

A new amperometric biosensor for ethanol monitoring has been developed and optimised. The biosensor uses poly(neutral red) (PNR), as redox mediator, which is electropolymerised on carbon film electrodes and alcohol oxidase (AlcOx) from Hansenula polymorpha as recognition element, immobilised by cross-linking with glutaraldehyde (GA) in the presence of bovine serum albumin (BSA) as carrier protein. Optimisation of variables affecting the system was performed and, for chronoamperometric measurements, a potential of -0.300 V versus saturated calomel electrode was chosen in 0.1M sodium phosphate buffer saline at pH 7.5. The optimised biosensor showed a good sensitivity of 171.8+/-14.8nAmM(-1) and the corresponding detection limit (signal-to-noise-ratio=3) of 29.7+/-1.5 microM. Stability studies showed a good preservation of the bioanalytical properties of the sensor, 57.6% of its initial sensitivity remaining after 3 weeks (the sensor was used two to three times per week). No significant interferences were found from compounds usually present in wine. The biosensor was used for the determination of ethanol in Portuguese red and white wines.

Thionine had strong interaction with carbon nanofiber (CNF) and was used in the non-covalent functionalization of carbon nanofiber for the preparation of stable thionine-CNF nanocomposite with good dispersion. With a simple one-step electrochemical polymerization of thionine-CNF nanocomposite and alcohol oxidase (AOD), a stable poly(thionine)-CNF/AOD biocomposite film was formed on electrode surface. Based on the excellent catalytic activity of the biocomposite film toward reduction of dissolved oxygen, a sensitive ethanol biosensor was proposed. The ethanol biosensor could monitor ethanol ranging from 2.0 to 252 microM with a detection limit of 1.7 microM. It displayed a rapid response, an expanded linear response range as well as excellent reproducibility and stability. The combination of catalytic activity of CNF and the promising feature of the biocomposite with one-step non-manual technique favored the sensitive determination of ethanol with improved analytical capabilities.

Background: Accurate, rapid, and economic on-line analysis of ethanol is very desirable. However, available biosensors achieve saturation at very low ethanol concentrations and thus demand the time and labour consuming procedure of sample dilution., Results: Hansenula polymorpha (Pichia angusta) mutant strains resistant to allyl alcohol in methanol medium were selected. Such strains possessed decreased affinity of alcohol oxidase (AOX) towards methanol: the KM values for AOX of wild type and mutant strains CA2 and CA4 are shown to be 0.62, 2.48 and 1.10 mM, respectively, whereas Vmax values are increased or remain unaffected. The mutant AOX alleles from H. polymorpha mutants CA2 and CA4 were isolated and sequenced. Several point mutations in the AOX gene, mostly different between the two mutant alleles, have been identified. Mutant AOX forms were isolated and purified, and some of their biochemical properties were studied. An amperometric biosensor based on the mutated form of AOX from the strain CA2 was constructed and revealed an extended linear response to the target analytes, ethanol and formaldehyde, as compared to the sensor based on the native AOX., Conclusion: The described selection methodology opens up the possibility of isolating modified forms of AOX with further decreased affinity toward substrates without reduction of the maximal velocity of reaction. It can help in creation of improved ethanol biosensors with a prolonged linear response towards ethanol in real samples of wines, beers or fermentation liquids.

Alcoholic myopathy is characterized by biochemical and morphological lesions within muscle, ranging from impairment of muscle strength and loss of lean tissue to cellular disturbances and altered gene expression. The chronic form of the disease is five times more common than cirrhosis and is characterized by selective atrophy of type 11 (anaerobic) fibres: type I (aerobic) fibres are relatively protected. Although the causative agent is known (i.e. ethanol), the intervening steps between alcohol ingestion and the development of symptoms and lesions are poorly understood. However, acetaldehyde appears to have an important role in the aetiology of the disease. For example, alcohol is a potent perturbant of muscle protein synthesis in vivo, and this effect is exacerbated by cyanamide pre-dosage, which raises acetaldehyde concentrations. Acetaldehyde alone also reduces muscle protein synthesis in vivo and proteolytic activity in vitro. The formation of acetaldehyde protein adducts is another mechanism of putative importance in alcoholic myopathy. These adducts are formed within muscle in response to either acute or chronic alcohol exposure and the adducts are located preferentially within the sarcolemmal and sub-sarcolemmal regions. However, the significance of protein adduct formation is unclear since we do not currently know the identity of the adducted muscle proteins nor whether adduction alters the biochemical or functional properties of skeletal muscle proteins.

Estimating the equivalent whole blood ethanol level from a serum or plasma determination has been addressed by numerous articles in both the clinical and forensic literature. All previous studies have either involved sample sizes insufficient for adequate statistical evaluation or have utilized gas chromatography for both serum and whole blood analysis. In this study, based on samples from 212 consecutive patients admitted to a hospital trauma center, serum was assayed for ethanol using an enzymatic oxidation method, and the results were compared to whole blood samples taken simultaneously and analyzed by headspace gas chromatography in a forensic toxicology laboratory. Contrary to previously published conclusions, it was found that the serum/whole blood alcohol ratio (SAC/BAC) is concentration-dependent, with average values ranging from around 1.12 to as high as around 1.18, depending on SAC, thus precluding a generally applicable SAC/BAC conversion factor. However, a linear regression model was found to provide adequate prediction intervals at any desired level of confidence for whole blood alcohol from serum alcohol levels up to 300 mg/dL. For example, at a confidence level of 95%, an SAC of 103 mg/dL corresponds to a BAC of at least 0.080 g/dL.

Drosophila melanogaster has evolved the ability to tolerate and utilize high levels of ethanol and acetic acid encountered in its rotting-fruit niche. Investigation of this phenomenon has focused on ethanol catabolism, particularly by the enzyme alcohol dehydrogenase. Here we report that survival under ethanol and acetic acid stress in D. melanogaster from high- and low-latitude populations is an integrated consequence of toxin catabolism and alteration of physical properties of cellular membranes by ethanol. Metabolic detoxification contributed to differences in ethanol tolerance between populations and acclimation temperatures via changes in both alcohol dehydrogenase and acetyl-CoA synthetase mRNA expression and enzyme activity. Independent of changes in ethanol catabolism, rapid thermal shifts that change membrane fluidity had dramatic effects on ethanol tolerance. Cold temperature treatments upregulated phospholipid metabolism genes and enhanced acetic acid tolerance, consistent with the predicted effects of restoring membrane fluidity. Phospholipase D was expressed at high levels in all treatments that conferred enhanced ethanol tolerance, suggesting that this lipid-mediated signaling enzyme may enhance tolerance by sequestering ethanol in membranes as phophatidylethanol. These results reveal new candidate genes underlying toxin tolerance and membrane adaptation to temperature in Drosophila and provide insight into how interactions between these phenotypes may underlie the maintenance of latitudinal clines in ethanol tolerance.

In the course of screening medicinal plants that modulate hepatic alcohol-metabolizing enzymes and lipid peroxidation, effects of the methanol extract (ME) of Orostachys japonicus and its major bioactive compound, gallic acid (GA), were investigated in rats treated with 10% ethanol solution for 6 weeks. The ME and GA greatly enhanced the activities of hepatic alcohol dehydrogenase (ADH), the microsomal ethanol-oxidizing system (MEOS), and aldehyde dehydrogenase (ALDH) in a dose-dependent manner, but had no effect on catalase. The hepatic lipid peroxide level increased by ethanol administration was moderately reduced by treatment with ME or GA. The results suggest that the detoxification of hepatic alcohol by O. japonicus ME under our experimental conditions was due to the enhanced activities of the alcohol-oxidizing enzymes, ADH, MEOS, and ALDH. In addition, GA may be partly responsible for the effects.

Results of research into ethanol metabolism in yeast organisms with highly pronounced aerobic metabolism are reviewed. The low activity of NAD-dependent alcohol dehydrogenase (EC 1.1.1.1), observed under the conditions of aerobic yeast growth on ethanol, demonstrates that alternative enzyme systems--alcohol oxidase (EC 1.1.3.13), microsomal ethanol-oxidizing system (including cytochrome P-450), and catalase (EC 1.11.1.6)--may be involved in the alcohol oxidation. The role of these systems in alcohol oxidation and conditions favoring their operation in this processes are analyzed. It is concluded that iron ions are important regulators of ethanol metabolism the microorganisms of this group.

Background and Aim: Ethanol has been shown to inhibit retinol oxidation at the level of alcohol dehydrogenase in liver and colon but not previously in the small intestine. In the present study we investigated how chronic alcohol feeding and acute ethanol exposure affects retinol dehydrogenase activity in the colon and small intestine of the rat., Methods: Rats were fed ethanol in a liquid diet for six weeks. Control rats received a similar diet but with ethanol isocalorically replaced by carbohydrates. Retinol dehydrogenase was analyzed from cell cytosol samples from the small and the large intestine with respect to maximum activity (V(max)), Michaelis-Menten constant (K(m)), and inhibition by ethanol (2-43 mM) in vitro., Results: Both the V(max) and the catalytic efficiency (V(max)/K(m)) were found to be significantly higher in the colon than in the small intestine (2.9-3.6 and 54-70 times higher, respectively). While chronic alcohol feeding did not affect these parameters, acute ethanol exposure reduced V(max) and V(max)/K(m) dose-dependently (p < 0.001) in both intestinal segments., Conclusion: The present data demonstrate that ethanol markedly inhibits in vitro cytosolic retinol oxidation in the small intestinal mucosa, which is considerably lower than that found in the colon. Considering the vital importance of retinol on intestinal integrity, our finding suggests that this might contribute to the ethanol-induced increase in intestinal permeability.

Most tissues of the body contain enzymes capable of ethanol oxidation or nonoxidative metabolism, but significant activity occurs only in the liver and, to a lesser extent, in the stomach. Hence, medical consequences are predominant in these organs. In the liver, ethanol oxidation generates an excess of reducing equivalents, primarily as NADH, causing hepatotoxicity. An additional system, containing cytochromes P-450 inducible by chronic alcohol feeding, was demonstrated in liver microsomes and found to be a major cause of hepatotoxicity.

Enzyme activity variation was studied in a Drosophila melanogaster population from two villages (Tiszafüred and Tiszaszolos) in Hungary. Two habitats (distillery and farmyard) were sampled in both villages and 8-9 isofemale lines were established from each sample with a total of 35 lines. The activities of ADH, alphaGPDH, IDH and 6PGDH were determined on starch gel after electrophoresis in 10 F1 females of each of the 35 isofemale lines. Three sublines were established from three selected isofemale lines of all four samples (altogether 36 sublines). Alcohol tolerance of the adult flies was assayed in these sublines. The activity of ADH was similar in the two habitats; so was the sensitivity to ethanol. Accordingly, no differences in adaptation to environmental ethanol were detected between the two habitats. The deviations between the two habitats in average activities and in the total variation of enzyme activities were not consistent in the two villages. These results suggest that founder effects and genetic drift are more pronounced in distilleries than selection. The association among enzyme activities varied greatly both between the two villages and between the two habitats. The two parameters of alcohol tolerance were not significantly different between the two habitats in any of the two villages.

Membrane-associated cytotoxicity induced by hydrophobic bile salts is a major contributing factor leading to liver diseases. Administration of ursodeoxycholate reduces serum liver enzymes in chronic liver diseases but the nature of this effect is still unclear. Using alcohol metabolising enzymes as cellular markers, the hepatotoxic properties of hydrophobic bile salts and the putative hepatoprotective effect of ursodeoxycholate was examined. Two animal models of biliary retention, bile duct obstruction and choledochocaval fistula was used to investigate the effect of taurocholate on the hepatic alcohol metabolizing enzymes: cytosolic alcohol dehydrogenase, microsomal ethanol oxidizing system, catalase and aldehyde dehydrogenase before and after the infusion of taurocholic acid or tauroursodeoxycholic acid for two days period. Bile duct obstruction was found to be similar to or slightly exceeds choledochocaval fistula in the degree of retention. Following the taurocholic acid infusion, the serum alcohol dehydrogenase activity as well as microsomal ethanol oxidizing system and aldehyde dehydrogenase were greatly increased but the level of cytosolic alcohol dehydrogenase and catalase activities was found to be lower in either or both models in comparison with the control animals. However, the tauroursodeoxycholic acid infusion did not induce any significant changes in the levels of all the alcohol metabolizing enzyme activities in either or both models. These findings suggest that hydrophobic taurocholic acid (7alpha) affects the plasmalemma to allow leakage of cytosolic alcohol dehydrogenase into the blood circulation, stimulates the biosynthesis of microsomal ethanol oxidizing system and aldehyde dehydrogenase, and suppresses the biosynthesis of alcohol dehydrogenase and catalase. But in contrast, the hydrophilic tauroursodeoxycholic acid (7beta) provided hepatoprotective effect.

Activities of the key enzymes of ethanol metabolism were assayed in ethanol-grown cells of an Acinetobacter sp. mutant strain unable to synthesize exopolysaccharides (EPS). The original EPS-producing strain could not be used for enzyme analysis because its cells could not to be separated from the extremely viscous EPS with a high molecular weight. In Acinetobacter sp., ethanol oxidation to acetaldehyde proved to be catalyzed by the NAD(+)-dependent alcohol dehydrogenase (EC 1.1.1.1.). Both NAD+ and NADP+ could be electron accepters in the acetaldehyde dehydrogenase reaction. Acetate is implicated in the Acinetobacter sp. metabolism via the reaction catalyzed by acetyl-CoA-synthetase (EC 6.2.1.1.). Isocitrate lyase (EC 4.1.3.1.) activity was also detected, indicating that the glyoxylate cycle is the anaplerotic mechanism that replenishes the pool of C4-dicarboxylic acids in Acinetobacter sp. cells. In ethanol metabolism by Acinetobacter sp., the reactions involving acetate are the bottleneck, as evidenced by the inhibitory effect of sodium ions on both acetate oxidation in the intact cells and on acetyl-CoA-synthetase activity in the cell-free extracts, as well as by the limitation of the C2-metabolism by coenzyme A. The results obtained may be helpful in developing a new biotechnological procedure for obtaining ethanol-derived exopolysaccharide ethapolan.

The adhE2 gene of Clostridium acetobutylicum ATCC 824, coding for an aldehyde/alcohol dehydrogenase (AADH), was characterized from molecular and biochemical points of view. The 2,577-bp adhE2 codes for a 94.4-kDa protein. adhE2 is expressed, as a monocistronic operon, in alcohologenic cultures and not in solventogenic cultures. Primer extension analysis identified two transcriptional start sites 160 and 215 bp upstream of the adhE2 start codon. The expression of adhE2 from a plasmid in the DG1 mutant of C. acetobutylicum, a mutant cured of the pSOL1 megaplasmid, restored butanol production and provided elevated activities of NADH-dependent butyraldehyde and butanol dehydrogenases. The recombinant AdhE2 protein expressed in E. coli as a Strep-tag fusion protein and purified to homogeneity also demonstrated NADH-dependent butyraldehyde and butanol dehydrogenase activities. This is the second AADH identified in C. acetobutylicum ATCC 824, and to our knowledge this is the first example of a bacterium with two AADHs. It is noteworthy that the two corresponding genes, adhE and adhE2, are carried by the pSOL1 megaplasmid of C. acetobutylicum ATCC 824.

Several DNA regions containing genes involved in poly-beta-hydroxybutyrate (PHB) biosynthesis and degradation and also in fatty acid degradation were identified from genomic sequence data and have been characterized in the serine cycle facultative methylotroph Methylobacterium extorquens AM1. Genes involved in PHB biosynthesis include those encoding beta-ketothiolase (phaA), NADPH-linked acetoacetyl coenzyme A (acetyl-CoA) reductase (phaB), and PHB synthase (phaC). phaA and phaB are closely linked on the chromosome together with a third gene with identity to a regulator of PHB granule-associated protein, referred to as orf3. phaC was unlinked to phaA and phaB. Genes involved in PHB degradation include two unlinked genes predicted to encode intracellular PHB depolymerases (depA and depB). These genes show a high level of identity with each other at both DNA and amino acid levels. In addition, a gene encoding beta-hydroxybutyrate dehydrogenase (hbd) was identified. Insertion mutations were introduced into depA, depB, phaA, phaB, phaC, and hbd and also in a gene predicted to encode crotonase (croA), which is involved in fatty acid degradation, to investigate their role in PHB cycling. Mutants in depA, depB, hbd, and croA all produced normal levels of PHB, and the only growth phenotype observed was the inability of the hbd mutant to grow on beta-hydroxybutyrate. However, the phaA, phaB, and phaC mutants all showed defects in PHB synthesis. Surprisingly, these mutants also showed defects in growth on C(1) and C(2) compounds and, for phaB, these defects were rescued by glyoxylate supplementation. These results suggest that beta-hydroxybutyryl-CoA is an intermediate in the unknown pathway that converts acetyl-CoA to glyoxylate in methylotrophs and Streptomyces spp.

The activity and localization of alcohol oxidase (EC 1.1.3.13) have been studied in the Pichia methanolica mutant ecr1 defective in ethanol-induced catabolite repression of enzymes of methanol utilization. Ultrasctuctural, immunocytochemical, and biochemical analyses revealed the presence of peroxisomes containing active alcohol oxidase in the mutant grown in media with methanol, ethanol, and a mixture of both substrates. No alcohol oxidase was detected in the wild-type cells (ECR1) grown on ethanol-containing media. Mutant ecr1 growing in medium containing a mixture of different alcohols and the wild-type strain growing on methanol demonstrated similar buoyant density of peroxisomes (1.24-1.27 g/cm3)during isopicnic centrifugation of the organelles in sucrose density gradients. The integrated genetic, immunocytochemical, and biochemical data are in agreement with the model that synthesis, translocation into peroxisomes, and assembly of alcohol oxidase in P. methanolica may not require any regulatory signals induced by methanol.

Oxidation of ethanol via alcohol dehydrogenase (ADH) explains various metabolic effects of ethanol but does not account for the tolerance and a number of associated disorders that develop in the alcoholic. These were elucidated by the discovery of the microsomal metabolism of ethanol. The physiologic role of this system comprises gluconeogenesis from ketones, fatty acid metabolism, and detoxification of xenobiotics, including ethanol. After chronic ethanol consumption, the activity of the microsomal ethanol-oxidizing system (MEOS) increases, with an associated rise in cytochromes P-450, especially CYP2E1. This induction is associated with proliferation of the endoplasmic reticulum, both in experimental animals and in humans. The role of MEOS in vivo and its increase after chronic ethanol consumption was shown most conclusively in alcohol dehydrogenase-negative deer mice. Enhanced ethanol oxidation is associated with cross-induction of the metabolism of other drugs, resulting in drug tolerance. Furthermore, there is increased conversion of known hepatotoxic agents (such as CCl4) to toxic metabolites, which may explain the enhanced susceptibility of alcoholics to the adverse effects of industrial solvents. CYP2E1 also has a high capacity to activate some commonly used drugs, such as acetaminophen, to their toxic metabolites, and to promote carcinogenesis (e.g., from dimethylnitrosamine). Moreover, catabolism of retinol is accelerated and there also is induction of microsomal enzymes involved in lipoprotein production, resulting in hyperlipemia. Contrasting with the chronic effects of ethanol consumption, acute ethanol intake inhibits the metabolism of other drugs through competition for the at least partially shared microsomal pathway. In addition, metabolism by CYP2E1 results in a significant free radical release and acetaldehyde production which, in turn, diminish reduced glutathione (GSH) and other defense systems against oxidative stress. Acetaldehyde also forms adducts with proteins, thereby altering the functions of mitochondria and of repair enzymes. Increases of CYP2E1 and its mRNA prevail in the perivenular zone, the area of maximal liver damage. CYP1A2 and CYP3A4, two other perivenular P-450s, can also sustain the metabolism of ethanol, thereby contributing to MEOS activity and possibly liver injury. By contrast, CYP2E1 inhibitors oppose alcohol-induced liver damage, but heretofore available compounds were too toxic for clinical use. Recently, however, polyenylphosphatidylcholine (PPC), an innocuous mixture of polyunsaturated lecithins extracted from soybeans, was discovered to decrease CYP2E1 activity. PPC (and its active component dilinoleoylphosphatidylcholine) also oppose hepatic oxidative stress and fibrosis. PPC is now being tested clinically for the prevention and treatment of liver disease in the alcoholic.

Six Drosophila melanogaster strains were constructed from two isofemale lines. The strains had four allele combinations at the alcohol dehydrogenase (Adh) and octanol dehydrogenase (Odh) loci, while all alpha-glycerophosphate dehydrogenase (alpha Gpdh), malate dehydrogenase (Mdh), and aldehyde oxidase (Aldox) alleles were identical. Second-instar and early and late third-instar larvae were exposed to different concentrations of ethanol (0, 5, and 7.5%) and 3 days later fresh weights and the activities of ADH, ODH, alpha GPDH, and MDH were measured. Activity differences were observed between the two Adh genotypes: ADHF allozyme had considerably higher activity than ADHS. Exogenous ethanol resulted in the highest increase in ADH activity in the second- and early third-instar stages. This ADH induction depended on the allele combination at the Adh and Odh loci; e.g., in the strain having the AdhS-OdhS allele combination, increased ADH activity was observed only after exposure to 7.5% ethanol. ODH activities differed according to the Odh genotypes, in that the ODHS allozyme had a higher activity than ODHF. ODH activities did not appreciably respond to different ethanol treatments. All six strains had identical alleles at the Mdh and alpha Gpdh loci, but nevertheless, the responses of these enzymes to ethanol depended on the allele combinations at the Adh and Odh loci. alpha GPDH activity followed that of ADH in all experiments. MDH activities were not influenced by exogenous ethanol in the strains homozygous for the AdhS allele. In AdhF strains, however, exposure to 7.5% ethanol resulted in a considerable decrease in MDH activity in the second-instar larvae. Correlations among the response variables showed that ODH activities were strongly associated with fresh weight and the activities of all other enzymes, except for ADH. ADH activity, however, showed a significant correlation only with alpha GPDH activity throughout the larval life. Both MDH and ODH activities were found to be in strong negative correlation with ADH activity in the second-instar larvae. At this most sensitive life stage, the metabolic response to ethanol is highly correlated.

We have recently proposed the existence of a bacteriocolonic pathway for ethanol oxidation [i.e., ethanol is oxidized by alcohol dehydrogenases (ADHs) of intestinal bacteria resulting in high intracolonic levels of reactive and toxic acetaldehyde]. The aim of this in vitro study was to characterize further ADH activity of some aerobic bacteria, representing the normal human colonic flora. These bacteria were earlier shown to possess high cytosolic ADH activities (Escherichia coli IH 133369, Klebsiella pneumoniae IH 35385, Klebsiella oxytoca IH 35339, Pseudomonas aeruginosa IH 35342, and Hafnia alvei IH 53227). ADHs of the tested bacteria strongly preferred NAD as a cofactor. Marked ADH activities were found in all bacteria, even at low ethanol concentrations (1.5 mM) that may occur in the colon due to bacterial fermentation. The Km for ethanol varied from 29.9 mM for K. pneumoniae to 0.06 mM for Hafnia alvei. The inhibition of ADH by 4-methylpyrazole was found to be of the competitive type in 4 of 5 bacteria, and Ki varied from 18.26 +/- 3.3 mM for Escherichia coli to 0.47 +/- 0.13 mM for K. pneumoniae. At pH 7.4, ADH activity was significantly lower than at pH 9.6 in four bacterial strains. ADH of K. oxytoca, however, showed almost equal activities at neutral pH and at 9.6. In conclusion, NAD-linked alcohol dehydrogenases of aerobic colonic bacteria possess low apparent Km's for ethanol. Accordingly, they may oxidize moderate amounts of ethanol ingested during social drinking with nearly maximal velocity. This may result in the marked production of intracolonic acetaldehyde. Kinetic characteristics of the bacterial enzymes may enable some of them to produce acetaldehyde even from endogenous ethanol formed by other bacteria via alcoholic fermentation. The microbial ADHs were inhibited by 4-methylpyrazole by the same competitive inhibition as hepatic ADH, however, with nearly 1000 times lower susceptibility. Individual variations in human colonic flora may thus contribute to the risk of alcohol-related gastrointestinal morbidity, such as diarrhea, colon polyps and cancer, and liver injury.

Activity changes of three enzymes (ADH, ODH and AOX) of Drosophila melanogaster were followed under different environmental conditions. The influences of ethanol, starvation (no carbohydrates in the medium) and ethanol stress during starvation were studied at both 18 and 26 degrees C. Two strains that were monomorphic for different alleles at the Odh and Aldox loci but otherwise identical were used. The investigated environmental conditions affected ADH induction by exogenous ethanol differently in the two strains. The different allozymes of ODH and AOX also responded differently to the treatments. We observed that the sucrose content of the medium on which ethanol exposure took place and the temperature strongly affected the responses within any single strain. Correlations were estimated among the three enzymes in the larval and adult stages of each strain separately. At both temperatures, differences between strains were observed in the patterns of associations of the response variables, in the larval, but not in the adult stages.

One hundred years of research about the metabolism of alcohol have provided many details, but some general aspects of the physiologic value of alcohol remain uncertain or inadequately proven. Results from epidemiologic studies appear to be in conflict with interpretations based on results from indirect calorimetric studies. The apparent inability of body mass index to be maintained in women when alcohol is consumed with food may indicate impaired metabolic processes that need to be better understood. Current evidence on the effects of alcohol are summarized to identify experimental approaches that may provide information needed to resolve the current contradictions.

The effect of ethanol on the Ca(2+)-dependent activation of mitochondrial dehydrogenases in rat brain and heart mitochondria was investigated. ADP-stimulated respiration of isolated brain and heart mitochondria (state 3) was stimulated further by submicromolar concentrations of free calcium when respiring on non-saturating concentrations of NAD-linked substrates. The stimulation of oxidative phosphorylation by Ca2+ was correlated with an increase of the mitochondrial matrix free calcium concentration ([Ca2+]m), as measured by fura-2, and with an increased reduction of the mitochondrial NAD(P) pool, indicating an activation of Ca(2+)-dependent dehydrogenases. Sodium inhibited Ca(2+)-dependent stimulation of state 3 respiration and NAD(P) reduction as a result of stimulation of Ca2+ efflux through the Na+/Ca2+ antiporter which reduced the steady-state value of [Ca2+]m. Ethanol stimulated the Na+/Ca2+ antiporter both in brain and heart mitochondria. As a result of this stimulation, ethanol, at pharmacological concentrations (50-300 mM), enhanced the sodium-dependent reduction of [Ca2+]m, and thus attenuated the activation of NAD-linked dehydrogenases and the stimulation of oxidative phosphorylation, by submicromolar concentrations of Ca2+, both in brain and heart mitochondria. This pharmacological effect of ethanol, on brain and heart mitochondria, may be responsible, in part, for the acute and chronic effects of ethanol on brain and heart function and metabolism.

The influence of starvation on activities of three enzymes (ADH, ODH and alpha GPDH) was studied in Drosophila melanogaster. The changes were compared in two inbred lines which had different allelic combinations at the Odh and Aldox loci. We also studied the effect of ethanol on media which contained no sucrose ("starvation conditions"). The results show that there are large differences in the larval and adult alcohol utilization. The alcohol content of the medium, in the absence of sugar, appeared to be toxic for the larvae, while the adults appeared to utilize it as an energy source. The two strains differed little in their responses to starvation or to the ethanol treatment applied under starvation conditions. We conclude that the degree of toxicity of ethanol is highly dependent on the presence of sucrose.