Characterization of alcohol dehydrogenase from the haloalkaliphilic archaeon Natronomonas pharaonis

Alcohol dehydrogenase (ADH; EC: 1.1.1.1) is a key enzyme in production and utilization of ethanol. In this study, the gene encoding for ADH of the haloalkaliphilic archaeon Natronomonas pharaonis (NpADH), which has a 1,068-bp open reading frame that encodes a protein of 355 amino acids, was cloned into the pET28b vector and was expressed in Escherichia coli. Then, NpADH was purified by Ni-NTA affinity chromatography. The recombinant enzyme showed a molecular mass of 41.3 kDa by SDS-PAGE. The enzyme was haloalkaliphilic and thermophilic, being most active at 5 M NaCl or 4 M KCl and 70°C, respectively. The optimal pH was 9.0. Zn²⁺ significantly inhibited activity. The K _m value for acetaldehyde was higher than that for ethanol. It was concluded that the physiological role of this enzyme is likely the catalysis of the oxidation of ethanol to acetaldehyde.     

Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1

The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both enzymes appeared to be homotetramers with a subunit size of 45 ± 1 kDa as revealed by SDS-PAGE, which was close to the calculated value (44.8 kDa). The recombinant ADH contained 1.0 ± 0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105 ± 2 U mg⁻¹, which was very similar to that of the native enzyme (110 ± 3 U mg⁻¹). The optimal pH-values for both enzymes for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively. Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there was no activity towards methanol and secondary alcohols. Kinetic parameters of the enzymes showed lower K _m-values for acetaldehyde and NADPH and higher K _m-values for ethanol and NADP⁺. It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile.     

Heterologous Expression and Characterization of an Alcohol Dehydrogenase from the Archeon <Emphasis Type="Italic">Thermoplasma acidophilum</Emphasis>

Analysis of the Thermoplasma acidophilum DSM 1728 genome identified two putative alcohol dehydrogenase (ADH) open reading frames showing 50.4% identity against each other. The corresponding genes Ta0841 and Ta1316 encode proteins of 336 and 328 amino acids with molecular masses of 36.48 and 36.01 kDa, respectively. The genes were expressed in Escherichia coli and the recombinant enzymes were functionally assessed for activity. Throughout the study only Ta1316 ADH resulted active in the oxidative reaction in the pH range 2–8 (optimal pH 5.0) and temperatures from 25 to 90°C (optimal 75°C). This ADH catalyzes the oxidation of several alcohols such as ethanol, methanol, 2-propanol, butanol, and pentanol during the reduction of the cofactor NAD⁺. The highest activity was found in the presence of ethanol producing optically pure acetaldehyde. The specific enzyme activity of the purified Ta1316 ADH with ethanol as a substrate in the optimal conditions was 628.7 U/mg.     

The role of alcohol dehydrogenase in the fermentation of D-xylose byCandida shehatae ATCC 22984

Summary When the oxygen supply to an aerobic chemostat culture ofCandida shehatae growing on D-xylose was reduced to oxygen-limited and anoxic conditions, accumulation of ethanol occurred, the specific activity of alcohol dehydrogenase (ADH) increased up to four-fold and the number of ADH isozymes detected increased from one to three. ADH in crude extracts prepared from anoxically-grown cells had a lower affinity for but was more tolerant to ethanol than in extracts prepared from aerobically grown cells. ADH activity appeared to be sufficient for ethanol production byC. shehatae under anoxic conditions.Maintained in cooperation with the University of Wisconsin-Madison. The use of trade, firm or corporation names in this publication is for the information and convenience of the reader. Such use does not constitute an official endorsement or approval by the US Department of any product or service to the exclusion of others which may be suitable.     

Isolation of mutants of Alcaligenes eutrophus unable to derepress the fermentative alcohol dehydrogenase

Eight representative strains of Alcaligenes eutrophus, two strains of Alcaligenes hydrogenophilus and three strains of Paracoccus denitrificans were examined for their ability to use different alcohols and acetoin as a carbon source for growth. A. eutrophus strains N9A, H16 and derivative strains were unable to grow on ethanol or on 2,3-butanediol. Alcohol-utilizing mutants derived from these strains, isolated in this study, can be categorized into two major groups: Type I-mutants represented by strain AS1 occurred even spontaneously and were able to grow on 2,3-butanediol (t _d=2.7–6.4 h) and on ethanol (t _d=15–50 h). The fermentative alcohol dehydrogenase was present on all substrates tested, indicating that this enzyme in vivo is able to oxidize 2,3-butanediol to acetoin which is a good substrate for wild type strains. Type II-mutants represented by strain AS4 utilize ethanol as a carbon source for growth (t _d=3–9 h) but do not grow on butanediol. In these mutants the fermentative alcohol dehydrogenase is only present in cells cultivated under conditions of restricted oxygen supply, but a different NAD-dependent alcohol dehydrogenase is present in ethanol grown cells. Cells grown on ethanol, acetoin or 2,3-butanediol synthesized in addition two proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity and acetate thiokinase. An acylating acetaldehyde dehydrogenase (EC 1.2.1.10) was not detectable. Applying the colistin- and pin point-technique for mutant selection to strain AS1, mutants, which lack the fermentative alcohol dehydrogenase even if cultivated under conditions of restricted oxygen supply, were isolated; the growth pattern served as a readily identifiable phenotypic marker for the presence or absence of this enzyme.     

Purification and characterization of membrane-bound alcohol dehydrogenase from Acetobacter polyoxogenes sp. nov.

Summary Membrane-bound alcohol dehydrogenase (ADH) was purified from the membrane fraction of an industrial-vinegar-producing strain, A. polyoxogenes sp. nov. NBI1028 by solubilization using Triton X-100 and subsequent column chromatography on DEAE-Sepharose CL-6B and hydroxyapatite. The purified enzyme was homogeneous on polyacrylamide disc gel electrophoresis (PAGE). Upon sodium dodecyl sulphate-PAGE, the enzyme showed the presence of two subunits with a molecular mass of 72 000 daltons and 44 000 daltons, respectively. The small subunit was identified as cytochrome c. In addition, absorption and fluorescence spectra showed the the presence of pyrroloquinoline quinone in the purified ADH. The ADH preferentially oxidized aliphatic alcohols with a straight carbon chain except for methanol. Formaldehyde and acetaldehyde were also oxidizable substrates. The apparent K _m for ethanol was 1.2 mM. The optimum pH and temperature were 5.0–6.0 and 40°C, respectively. p-Chloromercuribenzoic acid and heavy metals such as CuSO₄ were inhibitory to the enzyme activity. Ferricyanide was effective as an electron acceptor.Offprint requests to: M. Fukaya     

Reconstitution of the Z-Disk

Methanol-utilizing bacteria, Klebsiella sp. No. 101 and Microcyclus eburneus could grow aerobically and statically on 1,2-propanediol. The authors examined the presence of enzyme activity of adenosyl-B₁₂ dependent diol dehydratase as well as NAD dependent diol dehydroagenase. Adenosyl-B₁₂ dependent diol dehydratase activity was not detected in these organisms, even if these grown statically.

The dehydrogenase activity was found in the extract from these methanol-utilizing bacteria cells grown on various carbon sources. The partially purified enzyme preparation from the cells of Mic. eburneus grown aerobically on 1,2-propanediol dehydrogenated 1,2-propanediol, 1,2-butanediol and 2,3-butanediol. The enzyme activity was separated into two fractions, namely enzyme I and II on DEAE-Sephadex A-25 column chromatography. The enzyme I was different from the enzyme II in the ratio of enzyme activity to 1,2-propanediol and 2,3-butanediol, heat stability, pH stability and pH optimum, and effect of 2-mercaptoethanol.     

Oxidation of ethanol by methanogenic bacteria

Methanogenium organophilum, a non-autotrophic methanogen able to use primary and secondary alcohols as hydrogen donors, was grown on ethanol. Per mol of methane formed, 2 mol of ethanol were oxidized to acetate. In crude extract, an NADP⁺-dependent alcohol dehydrogenase (ADH) with a pH optimum of about 10.0 catalyzed a rapid (5 mol/min·mg protein; 22°C) oxidation of ethanol to acetaldehyde; after prolonged incubation also acetate was detectable. With NAD⁺ only 2% of the activity was observed. F₄₂₀ was not reduced. The crude extract also contained F₄₂₀: NADP⁺ oxidoreductase (0.45 mol/min·mg protein) that was not active at the pH optimum of ADH. With added acetaldehyde no net reduction of various electron acceptors was measured. However, the acetaldehyde was dismutated to ethanol and acetate by the crude extract. The dismutation was stimulated by NADP⁺. These findings suggested that not only the dehydrogenation of alcohol but also of aldehyde to acid was coupled to NADP⁺ reduction. If the reaction was started with acetaldehyde, formed NADPH probably reduced excess aldehyde immediately to ethanol and in this way gave rise to the observed dismutation. Acetate thiokinase activity (0.11 mol/min·mg) but no acetate kinase or phosphotransacetylase activity was observed. It is concluded that during growth on ethanol further oxidation of acetaldehyde does not occur via acetylCoA and acetyl phosphate and hence is not associated with substrate level phosphorylation. The possibility exists that oxidation of both ethanol and acetaldehyde is catalyzed by ADH. Isolation of a Methanobacterium-like strain with ethanol showed that the ability to use primary alcohols also occurs in genera other than Methanogenium.Non-standard abbreviations ADH alcohol dehydrogenase - Ap₅ALi₃ P¹,P⁵-Di(adenosine-5-)pentaphosphate - DTE dithioerythritol (2,3-dihydroxy-1,4-dithiolbutane) - F₄₂₀ N-(N-l-lactyl--l-glutamyl)-l-glutamic acid phosphodiester of 7,8-dimethyl-8-hydroxy-5-deazariboflavin-5-phosphate - Mg. Methanogenium - OD₅₇₈ optical density at 578 nm - PIPES 1,4-piperazine-diethanesulfonic acid - TRICINE N-(2-hydroxy-1,1-bis[hydroxymethyl]methyl)-glycine - Tris 2-amino-2-hydroxy-methylpropane-1,3-diol - U unit (mol substrate/min)     

The activity of yeast ADH I and ADH II with long-chain alcohols and diols

The activities of yeast ADH I and ADH II towards long chain alcohols and diols were studied using rather unusual conditions (1.0 M Tris pH 8.75, approximately 0.3 mg/ml enzyme and [S]< < <K_m ) where the alcohols are oxidised quantitatively in a first-order manner. Plots of the apparent first-order rate constant versus primary alcohol chain length show double peaks with similar values for ethanol and 1-decanol and relatively low values for 1-butanol through to 1-octanol. With the α,ω diols only one peak of activity was observed with 1,14-tetradecanediol, the preferred substrate, being oxidised about the same rate as ethanol. Both enzymes were essentially inactive with short-chain diols (C₂–C₈). For all of these assays normalised rates with ADH II were about threefold faster than with ADH I.     

Characterization of Plant Alcohol Dehydrogenase

The final activity of the alcohol dehydrogenase (E.C.1.1.1.1, abbreviated ADH) from germinating pea, isolated by fractionating with ammonium sulphate, chromatography on DEAE cellulose and gel filtration, was 80,000, from bean 25,000 and from lentil 13,500 units per mg protein. Molecular weights of the ADHs are close to each other: pea and bean ADH 60,000, lentil ADH 70,000. The K_m values are mutually similar with three enzymes, i.e. of the order of 10⁻⁴M for NAD and 10⁻²M for ethanol. The pH optima lie in the alkaline region. These enzymes catalyse oxidation of a number of monovalent alcohols. At temperatures above 60°C the enzymes are thermally unstable. Stability is enhanced slowly by ethanol but not by NAD. Pyrazol, imidazol and pyridine inhibit plant ADH similarly to the enzyme from horse liver. There is a similarity between plant alcohol dehydrogenases and animal and yeast enzymes.     

Purification and characterization of an iron-containing alcohol dehydrogenase in extremely thermophilic bacterium <Emphasis Type="Italic">Thermotoga hypogea</Emphasis>

Thermotoga hypogea is an extremely thermophilic anaerobic bacterium capable of growing at 90°C. It uses carbohydrates and peptides as carbon and energy sources to produce acetate, CO₂, H₂, l-alanine and ethanol as end products. Alcohol dehydrogenase activity was found to be present in the soluble fraction of T. hypogea. The alcohol dehydrogenase was purified to homogeneity, which appeared to be a homodimer with a subunit molecular mass of 40 ± 1 kDa revealed by SDS-PAGE analyses. A fully active enzyme contained iron of 1.02 ± 0.06 g-atoms/subunit. It was oxygen sensitive; however, loss of enzyme activity by exposure to oxygen could be recovered by incubation with dithiothreitol and Fe²⁺. The enzyme was thermostable with a half-life of about 10 h at 70°C, and its catalytic activity increased along with the rise of temperature up to 95°C. Optimal pH values for production and oxidation of alcohol were 8.0 and 11.0, respectively. The enzyme had a broad specificity to use primary alcohols and aldehydes as substrates. Apparent K _m values for ethanol and 1-butanol were much higher than that of acetaldehyde and butyraldehyde. It was concluded that the physiological role of this enzyme is likely to catalyze the reduction of aldehydes to alcohols.     

Purification and properties of the coenzyme A-linked acetaldehyde dehydrogenase of Acetobacterium woodii

Coenzyme A-linked acetaldehyde dehydrogenase (ACDH) of ethanol-grown cells of Acetobacterium woodii was purified to apparent homogeneity; a 28-fold purification was achieved with 13% yield. The enzyme proved to be oxygen-sensitive and was inactive in the absence of dithioerythritol. During the purification procedure addition of 1 mM MgCl₂ was necessary to maintain enzyme activity. Alcohol dehydrogenase (ADH) activity was separated from ACDH during anion exchange chromatography using DEAE Sephacel. A part of the ACDH activity coeluted with ADH, but both could be separately eluted from a Cibacron Blue 3GA-Agarose column, revealing the same subunit structure and activity band for ACDH as found before and, thus, indicating an aggregation of the enzyme. The remaining ADH activity could be separated by gel filtration. For the native ACDH a molecular mass of 255 kDa was determined by polyacrylamide gel electrophoresis and of 272 kDa by gel filtration using Superose 12. The enzyme subunit sizes were 28 kDa and 40 kDa, respectively, indicating a ₄₄ structure for the active form. The enzyme catalyzed the oxidation of several straight chain aldehydes although it was most active with acetaldehyde. NADH strongly inhibited oxidation of acetaldehyde whereas NADPH had no effect. The inhibition was noncompetitive.Non-standard abbrevations ACDH acetaldehyde dehydrogenase - ADH alcohol dehydrogenase - CHES 2-(N-cyclohexylamino)-ethanesulfonate - DTE dithioerythritol - KP-buffer 25 mM K-PO₄, pH 7.5, containing, 4 mM DTE - MES 2-(N-morpholino)-ethanesulfonate - TAPS N-Tris-(hydroxymethyl)-methyl-3-aminopropa-nesulfonate     

Anaerobic respiration of superoxide dismutase-deficientSaccharomyces cerevisiae under oxidative stress

The ethanol productivity of superoxide dismutase (SOD)-deficient mutants ofSaccharomyces cerevisiae was examined under the oxidative stress by Paraquat. It was observed that MnSOD-deficient mutant ofS. cerevisiae had higher ethanol productivity than wild type or CuZnSOD-deficient yeast both in aerobic and in anaerobic culture condition. Pyruvate dehydrogenase activity decreased by 35% and alcohol dehydrogenase activity increased by 32% were observed in MnSOD-deficient yeast grown aerobically. When generating oxygen radicals by Paraquat, the ethanol productivity was increased by 40% in CuZnSOD-deficient or wild strain, resulting from increased activity of alcohol dehydrogenase and decreased activity of pyruvate dehydrogenase. However, the addition of ascorbic acid with Paraquat returned the enzyme activities at the level of control. These results imply that SOD-deficiency in yeast strains may cause the metabolic flux to shift into anaerobic ethanol fermentation in order to avoid their oxidative damages by Paraquat.     

Purification and Properties of Rape Alcohol Dehydrogenase

Germinating seeds with the highest specific activity (24 hour germination) were used for isolation of alcohol dehydrogenase, ADH, from rape (Brassica napus L. cv. T?ebi?ská). The rape ADH was purified by fractionation with ammonium sulphate, desalting on Sephadex G 25, chromatography on DEAE cellulose and gel filtration on Sephadex G 150. Using this isolation procedure, enzyme with a specific activity 85.6 times higher than that of the crude extract was obtained. The molecular weight of the enzyme obtained is 66.000. The enzyme is a metallo-enzyme containing sulfhydryl groups as evidence by the inhibitory effect of chelating compounds and thiol reagents. The optimum pH for the oxidation of ethanol is 8.5 and for reduction of acetaldehyde 7.0. The enzyme exhibits a relatively wide substrate specificity towards alcohols. Dimethyl-sulphoxide (DMSO), some amides and oximes and some intermediates of the carbohydrate metabolism act as ADH inhibitors, ATP as analogue of NAD also exhibits an inhibitory effect. The inhibitory effect of heterocyclic substances (pyrazol, imidazol, pyridine) is similar to the effect on liver alcohol dehydrogenase.     

Effect of yeast cell disruption on ADH activity for redox reactions with in situ cofactor regeneration in a continuous solid/gas bioreactor

It has recently been demonstrated that dried cells of Saccharomyces cerevisiae were able to produce alcohols and aldehydes in a solid/gas reactor with in situ cofactor regeneration. Since diffusion of gaseous substrates may be limited by the membrane and cell wall, cell disruption by sonication was used to improve oxidoreduction with ethanol and butyraldehyde as substrates. Results showed that partial cell disruption enhances the maximum conversion yield with the best results obtained after 2 min of sonication. Beyond this time, the ADH activity decreased. Better stability was observed in the pellet obtained after centrifugation indicating the importance of cell environment for enzyme stability. Tests on purified mitochondria showed that the ADH activity in cells was mainly cytoplasmic. The addition of oxidized cofactor did not change either the activity or the stability of the catalyst in a gaseous medium. The effect of water activity was studied on material obtained after 2 min of disruption and a reduction of critical water activity needed for revealing enzymatic activity was observed. With increasing a_w, the enzyme was active at a_w=0.3 while a water activity of 0.4 was required before disruption. Nevertheless, the best compromise between activity and stability was obtained in both cases for a water activity of 0.57.     

A quantitative description of the growth of Saccharomyces cerevisiae CBS 426 on a mixed substrate of glucose and ethanol

Saccharomyces cerevisiae CBS 426 was grown aerobically in continuous culture with a mixture of glucose and ethanol as the carbon source. The flows of biomass, glucose, ethanol, oxygen, and carbon dioxide were measured. A model for growth with two substrates was derived. Application of this model to the above-mentioned system yielded values for Y_ATP and P/O. The joint confidence regions for these parameters were calculated. The relevance to industrial production of bakers' yeast is discussed.     

Kinetics of the reaction catalysed by rape alcohol dehydrogenase

The kinetics of the enzyme reaction of ethanol oxidation and acetaldehyde reduction catalysed by alcohol dehydrogenase (ADH) (EC 1.1.1.1) isolated from germinating rape seeds obeys the bi-bi ordered mechanism of Theorell and Chance. The enzyme reaction depends on the pH and temperature. The K_m values for the basic substrates have the lowest values around the pH optimum of the reaction. The enzyme is most stable at pH 6.5–7. The K_m values for ethanol and NAD increase with increasing temperature. The maximum rate of the ethanol oxidation satisfies the Arrhenius equation. The activation energy for the given temperature range is 40.11 kJ/mol. The rape ADH is denatured by heating above 60° but the enzyme-NAD complex is thermally more stable than the enzyme alone.     

Environmental regulation of alcohol metabolism in thermotolerant methylotrophic Bacillus strains

The thermotolerant methylotroph Bacillus sp. C1 possesses a novel NAD-dependent methanol dehydrogenase (MDH), with distinct structural and mechanistic properties. During growth on methanol and ethanol, MDH was responsible for the oxidation of both these substrates. MDH activity in cells grown on methanol or glucose was inversely related to the growth rate. Highest activity levels were observed in cells grown on the C₁-substrates methanol and formaldehyde. The affinity of MDH for alcohol substrates and NAD, as well as V _max, are strongly increased in the presence of a M _r 50,000 activator protein plus Mg²⁺-ions [Arfman et al. (1991) J Biol Chem 266: 3955–3960]. Under all growth conditions tested the cells contained an approximately 18-fold molar excess of (decameric) MDH over (dimeric) activator protein. Expression of hexulose-6-phosphate synthase (HPS), the key enzyme of the RuMP cycle, was probably induced by the substrate formaldehyde. Cells with high MDH and low HPS activity levels immediately accumulated (toxic) formaldehyde when exposed to a transient increase in methanol concentration. Similarly, cells with high MDH and low CoA-linked NAD-dependent acetaldehyde dehydrogenase activity levels produced acetaldehyde when subjected to a rise in ethanol concentration. Problems frequently observed in establishing cultures of methylotrophic bacilli on methanol- or ethanol-containing media are (in part) assigned to these phenomena.Abbreviations MDH NAD-dependent methanol dehydrogenase - ADH NAD-dependent alcohol dehydrogenase - A1DH CoA-linked NAD-dependent aldehyde dehydrogenase - HPS hexulose-6-phosphate synthase - G6Pdh glucose-6-phosphate dehydrogenase     

Characterization of alcohol dehydrogenase 1 of the thermotolerant methylotrophic yeast Hansenula polymorpha

The thermotolerant methylotrophic yeast Hansenula polymorpha has recently been gaining interest as a promising host for bioethanol production due to its ability to ferment xylose, glucose, and cellobiose at elevated temperatures up to 48 °C. In this study, we identified and characterized alcohol dehydrogenase 1 of H. polymorpha (HpADH1). HpADH1 seems to be a cytoplasmic protein since no N-terminal mitochondrial targeting extension was detected. Compared to the ADHs of other yeasts, recombinant HpADH1 overexpressed in Escherichia coli exhibited much higher catalytic efficiency for ethanol oxidation along with similar levels of acetaldehyde reduction. HpADH1 showed broad substrate specificity for alcohol oxidation but had an apparent preference for medium chain length alcohols. Both ADH isozyme pattern analysis and ADH activity assay indicated that ADH1 is the major ADH in H. polymorpha DL-1. Moreover, an HpADH1-deleted mutant strain produced less ethanol in glucose or glycerol media compared to wild-type. Interestingly, when the ADH1 mutant was complemented with an HpADH1 expression cassette, the resulting strain produced significantly increased amounts of ethanol compared to wild-type, up to 36.7 g l⁻¹. Taken together, our results suggest that optimization of ADH1 expression would be an ideal method for developing H. polymorpha into an efficient bioethanol production strain.