A Long-Chain Secondary Alcohol Dehydrogenase from Rhodococcus erythropolis ATCC 4277

A NAD-dependent secondary alcohol dehydrogenase has been purified from the alkane-degrading bacterium, Rhodococcus erythropolis ATCC 4277. The enzyme was found to be active against a broad range of substrates, particularly long-chain secondary aliphatic alcohols. Although optimal activity was observed with linear 2-alcohols containing between 6 and 11 carbon atoms, secondary alcohols as long as 2-tetradecanol were oxidized at 25% of the rate seen with mid-range alcohols. The purified enzyme was specific for the S-(+) stereoisomer of 2-octanol and had a specific activity for 2-octanol of over 200 (mu)mol/min/mg of protein at pH 9 and 37(deg)C, 25-fold higher than that of any previously reported S-(+) secondary alcohol dehydrogenase. Linear primary alcohols containing between 3 and 13 carbon atoms were utilized 20- to 40-fold less efficiently than the corresponding secondary alcohols. The apparent K(infm) value for NAD(sup+) with 2-octanol as the substrate was 260 (mu)M, whereas the apparent K(infm) values for the 2-alcohols ranged from over 5 mM for 2-pentanol to less than 2 (mu)M for 2-tetradecanol. The enzyme showed moderate thermostability (half-life of 4 h at 60(deg)C) and could potentially be useful for the synthesis of optically pure stereoisomers of secondary alcohols.     

NAD-dependent Alcohol Dehydrogenase and NADP-dependent Alcohol Dehydrogenase from Strawberry Seeds

Strawberry seeds are shown to contain at least two alcohol dehydrogenases; one is NAD specific and reacts with ethanol and allyl alcohol, and the other is NADP specific and reacts with benzyl alcohol and geraniol. These two alcohol dehydrogenases were distinguished on disc electrophoresis. Their properties were different each other in ammonium sulfate fractionation, optimum reaction pH and thermostability.     

Alcohol Dehydrogenase from Methylobacterium organophilum

The alcohol dehydrogenase from Methylobacterium organophilum, a facultative methane-oxidizing bacterium, has been purified to homogeneity as indicated by sodium dodecyl sulfate-gel electrophoresis. It has several properties in common with the alcohol dehydrogenases from other methylotrophic bacteria. The active enzyme is a dimeric protein, both subunits having molecular weights of about 62,000. The enzyme exhibits broad substrate specificity for primary alcohols and catalyzes the two-step oxidation of methanol to formate. The apparent Michaelis constants of the enzyme are 2.9 × 10⁻⁵ M for methanol and 8.2 × 10⁻⁵ M for formaldehyde. Activity of the purified enzyme is dependent on phenazine methosulfate. Certain characteristics of this enzyme distinguish it from the other alcohol dehydrogenases of other methylotrophic bacteria. Ammonia is not required for, but stimulates the activity of newly purified enzyme. An absolute dependence on ammonia develops after storage of the purified enzyme. Activity is not inhibited by phosphate. The fluorescence spectrum of the enzyme indicates that it and the cofactor associated with it may be chemically different from the alcohol dehydrogenases from other methylotrophic bacteria. The alcohol dehydrogenases of Hyphomicrobium WC-65, Pseudomonas methanica, Methylosinus trichosporium, and several facultative methylotrophs are serologically related to the enzyme purified in this study. The enzymes of Rhodopseudomonas acidophila and of organisms of the Methylococcus group did not cross-react with the antiserum prepared against the alcohol dehydrogenase of M. organophilum.     

NADP-Dependent Alcohol Dehydrogenase from Tea Seeds

d-Coronamic acid was deaminated by 1-aminocyclopropane-1-carboxylate (ACPC) deaminase to produce α-keto-n-caproic acid. This deaminase which was purified from Pseudomonas sp. ACP was active to only d-coronamic acid among its stereoisomers. l-Coronamic acid or dl-allocoronamic acid was inactive or negligibly poor as the substrate. In addition, both deamination of ACPC and d-coronamic acid were inhibited by l-alanine, not by d-isomer and the inhibition of ACPC deamination by l-alanine was competitive. On the basis of these results, stereoselectivity of the enzymatic deamination was discussed.     

Alcohol Dehydrogenase from Cultured Rice Cells

Rice cells in suspension culture had high alcohol dehydrogenaseactivity during the logarithmic growth phase (3rd to 5th day).Ethanol was accumulated both in the cells and in the medium.The highest amount of ethanol was accumulated on the 4th dayin cells (10 µmoles/g fresh weight) and during the stationarygrowth phase (8th day) (180 mM, ca. 1%) in the medium. The enzymewas isolated from the cell extract and purified 36-fold witha 14% yield by ammonium sulfate fractional precipitation, andchromatography on DEAE-Sephadex, Sephadex G-150 and Blue Dextran-Sepharose.The purified enzyme was homogeneous, as judged by its sedimentationvelocity, and poly acrylamide gel, starch gel and SDS-polyacrylamidegel electrophoreses. Its molecular weight was 76,000 distributedin two, identical 37,000 subunits. The isoelectric point wasat pH 5.5. The enzyme contained 2.1 g atoms of zinc, 12 freeSH groups and 3 to 4 SS bonds per molecule. The pH optimum forethanol oxidatioa was pH 9.5 and for acetaldehyde reductionpH 6.0. The K_m values for ethanol, NAD^$, acetaldehyde and NADHwere 64.5 mM, 47.1 µM, 1.3 mM and 9.5 µM. The aminoacid composition, substrate specificity, and the effects ofchelators, SH reagents and sugar metabolic intermediates alsoare reported. (Received August 25, 1981; Accepted December 7, 1981)     

Gene Cloning and Biochemical Characterization of an Alcohol Dehydrogenase from Euglena gracilis1

ABSTRACT. Euglena gracilis is a freshwater free‐living organism able to grow with ethanol as carbon source; to facilitate this metabolism several alcohol dehydrogenase (ADH) activities have been detected. We report the gene cloning, over‐expression, and biochemical characterization of a medium‐chain NAD⁺‐dependent ADH from E. gracilis (EgADH). The enzyme's amino acid sequence displayed the highest percentages of similarity and identity with ADHs of bacteria and fungi. In the predicted three‐dimensional model, all the residues involved in Zn²⁺, cofactor, and substrate binding were conserved. A conventional signal peptide for import into mitochondria could not be clearly identified. The protein of 37 kDa was over‐expressed, purified to homogeneity, and kinetically characterized. The enzyme's optimal pH was 7.0 for ethanol oxidation displaying a V_m of 11.7±3.6 U/mg protein and a K_m of 3.2±0.7 mM for this substrate. Isopropanol and isopentanol were also utilized, although with less efficiency. It showed specificity for NAD⁺ with a K_m value of 0.39±0.1 mM and Mg²⁺ or Zn²⁺ were essential for activity. The recombinant EgADH reported here may help to elucidate the roles that different ADHs have on the metabolism of short‐ and long‐chain alcohols in this microorganism.     

Alcohol Dehydrogenase of Apple

The alcohol dehydrogenase prepared from apple (Malus domesticaBorkh.) possesses both NADH and NADPH-linked activities, whenassayed with acetaldehyde as substrate. The pyridine nucleotidesbind to the same catalytic site on the enzyme. The alcohol dehydrogenasecan also catalyse the reduction of C₃–C₆ aldehydes witheither NADH or NADPH as cofactor.     

Purification and Properties of Alcohol Dehydrogenase from Tea Seeds

The process of antigen presentation is not well understood. We screened for drugs that distinguish presentation of allogeneic class 2 antigens and exogenous antigens. When spleen cells were used as antigen presenting cells (APC), leupeptin and antipain preferentially inhibited allogeneic class 2 presentation, while they did not affect presentation of exogenous antigen and T cell growth. In contrast, they inhibited both presentations when spleen adherent cells (SAC) were used as APC. Our results suggest that SAC (mainly macrophages) and splenic B cells use different pathways to present exogenous antigens and that pathways to present allogeneic class 2 molecules are similar.     

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.     

Electric Field Effects with Alcohol Dehydrogenase

Extending previous work with electrostimulation of yeast dehydrogenases in cell suspension, we attempted to influence the alcohol dehydrogenase system in vitro using methylene blue as the acceptor. According to the conformational coupling model and the free radical hypothesis, the possibility of capacitive current treatment was tested by short applications of E = 10, 20, and 30 V/cm at a frequency of 50 Hz. Besides a temperature rise, no significant kinetic data from spectrophotometry or polarography could be measured, nor could inductive coupling using magnetic fluxes of 1, 5, and 10 mT. On the other hand, marked changes of relative electrofusion yield were found after interaction of dehydrogenases with membrane surfaces of barley protoplasts. It was confirmed that in unbuffered solution containing 0.5 M mannitol, the isoelectric point (PI) of enzymes determines the relative fusion yield: Fr < 1 for pI < 7 and Fr > 1 for pI > 7. We must conclude that only the position of dehydrogenases at or within cell membranes shows responses to weak ac or stronger dc treatments influencing additionally electric moments and conformations by surface potential and adsorption energy.     

乙醇脱氢酶（ADH）产酶菌株的选育

从食醋生产企业的醋醅中采集样品,以乙醇为唯一碳源,用碳酸钙透明圈平板法分离出185株菌株,然后以产酸量和耐乙醇能力为标准,瓶发酵选育出20株ADH产酶菌株;A5-2产酸量为49.85 g/L,耐乙醇能力强,A5-2的菌种形态学和16S rDNA序列分析初步鉴定为巴斯德醋酸杆菌（ Acetobacter pasteurianus）;A5-2乙醇脱氢酶酶学性质研究表明：最适作用温度和pH分别为45℃和pH 4.0,具有一定的耐热性和良好的耐酸碱性;A5-2乙醇脱氢酶粗酶制备条件为硫酸铵饱和度70%~80%,回收率84%。     

Oxidation of Two Hydroxylated Ochratoxin A Metabolites by Alcohol Dehydrogenase

(4R)-4-hydroxyochratoxin A, (4S)-4-hydroxyochratoxin A, and 10-hydroxyochratoxin A, all formed from ochratoxin A, were incubated with alcohol dehydrogenase in the presence of NAD. Only (4R)-4-hydroxyochratoxin A and 10-hydroxyochratoxin A acted as substrates for the enzyme. K_m and turnover number for 10-hydroxyochratoxin A were 110 μM and 0.1 s⁻¹, respectively.     

The Nutrition of Tetrahymena setifera HZ-1; Sterol and Alcohol Requirements

SYNOPSIS. In a chemically-defined medium Tetrahymena setifera HZ-1 required a sterol, an alcohol, eleven amino acids, a purine, a pyrimidine, and six B-complex vitamins. The sterol requirement was met by a variety of 3β-OH, C₂₇-C₂₉ sterols including cholesterol and stigmasterol, but not by precursors of cholesterol which precede desmosterol or Δ⁷-cholestenol. Some combinations of long-chain fatty acids with a synthetic dipalmitoyl phosphorylethanolamine partly substituted for sterol. Ethyl and methyl alcohols (but not a variety of other alcohols and organic acids) satisfied the alcohol requirement.     

Losses of Alcohol and Alcohol Dehydrogenase Activity in Germinating Seeds

Losses of alcohol, which had accumulated under anaerobic conditions,occurred during the germination of several species of seedswhich could not be attributed to the volatility of the alcohol.It is suggested that utilization of the alcohol by the seedsmay occur. From the seeds, an active alcohol dehydrogenase,which is mainly confined to the cotyledons in pea seeds, canbe extracted. The activity of the enzyme decreases as the cotyledonsgrow older during germination. The properties of the enzymehave been investigated.     

香蕉肉桂醇脱氢酶基因的克隆及表达分析

肉桂醇脱氢酶(CAD)在木质素合成过程中起关键作用。通过RACE(rapid-amplification of cDNA ends)方法从香蕉根系cDNA均一化全长文库中获得一个肉桂醇脱氢酶基因,命名为MaCAD1(GenBank登录号为KF582533)。MaCAD1是香蕉MYB基因编码框全长cDNA,包含一个1 077bp的最大开放阅读框(ORF),编码358个氨基酸。蛋白质序列同源比对发现,其含有完整的醇脱氧酶的典型保守结构域,属于典型的CAD蛋白。系统进化树比对分析表明,MaCAD1与水稻OsCAD6(CAD39907)的亲缘关系较近。组织特异性研究表明MaCAD1基因组成型表达于香蕉各个组织。在耐病和感病品种中,MaCAD1均上调表达,但在耐病品种中MaCAD1在所有时间点相对于对照增加的倍数均高于感病品种,表明MaCAD1基因在香蕉的抗病性中起着重要作用,MaCAD1可以作为一个新的响应枯萎病侵染的标记基因。     

MonoADP-Ribosylation of the NAD+-Dependent Alcohol Dehydrogenase from Entamoeba histolytica

The human parasite Entamoeba histolytica is an amitochondrial protozoan whose metabolism depends on glucose fermentation. Among the metabolic enzymes absolutely required for amoeba growth is the NAD⁺-dependent alcohol dehydrogenase (EhADH2). The polymeric form of EhADH2 was sedimented at 160,000g, and in this fraction we observed [³²P]-labeling of a 96-kDa protein under mono-ADP-ribosylation conditions with [³²P]NAD⁺. The [³²P]-labeled protein had the same molecular weight as the EhADH2 monomer. Because of the importance of monoADP-ribosylation in the regulation of many physiological processes, the aim of this study was to determine whether EhADH2 is ADP-ribosylated, and what would be the consequence of this modification on its alcohol and aldehyde dehydrogenase enzymatic activities. This study describes the ADP-ribosylation of EhADH2. This modification did not have an effect on the enzymatic activities, but it may regulate other functions of EhADH2.     

A New Enzymatic Microdetermination Procedure for Ethanol with Particulate Alcohol Dehydrogenase from Acetic Acid Bacteria

A new enzymatic method for microdetermination of ethanol has been established with particulate alcohol dehydrogenase from acetic acid bacteria and applied to the practical purposes. The enzyme had an optimum pH for ethanol oxidation at a fairly acidic region. Trace amounts of ethanol could be assayed by measuring the initial reaction rate as successful as by reading the end point of the reaction. Some advantages in using this enzyme for ethanol determination were pointed out comparing with NAD-linked alcohol dehydrogenase from yeast or horse liver. Impurity in the enzyme preparations, stability of reagents and coexistence of other substances in the assay mixture were not as critical as in NAD-linked enzyme. Acidic samples could also be directly determined for ethanol without preadjustment of sample pH.     

Cytolytic Activity Released from Tetrahymena1

Using a hemolytic assay system, we have detected cytolytic activities in extracellular medium from Tetrahymena thermophila and T. pyriformis. In addition, we have identified two phospholipase activities (types A and C) from the same source by thin layer chromatography analysis of the breakdown products of a phospholipid preparation. The hemolytic activity peaks at low pH values. It is inhibited by egg lecithin, supporting the view that a phospholipase activity is involved in the cytolytic processes. Cytolytic activities may play important roles in Tetrahymena, both in nutrition, especially in parasitic and scavenger forms, and in defense against predators. Tetrahymena is probably partly protected from its own released cytolytic phospholipase by having a high proportion of phosphonolipids on its surface membrane.