一株乙醇降解菌株的分离和鉴定

从湖北农田土壤中筛选得到一株ALDH活性较高的菌株,该菌株在含0．64％乙醇的培养基中生长较佳,且耐受0．9％的乙醛。经菌种形态学和生理生化特征,以及16S rRNA基因序列分析,鉴定该菌株为不动杆（Acinetobacter sp．）。该菌株在乙醇和乙醛解毒研究中有重要价值。     

Growth of Acinetobacter calcoaceticus on Ethanol

A soil microorganism, identified as Acinetobacter calcoaceticus, was cultivated on ethanol as a sole source of carbon. This organism grew with a maximum specific growth rate of 0.7/h. The pH optimum for growth was between 6.5 and 7.5, and the temperature optimum was between 32 and 35 C. Ethanol metabolism by this organism was inducible by ethanol, and the presence of acetate led to the repression of ethanol dehydrogenase. At higher cell densities the cessation of growth on ethanol was accompanied by the accumulation of acetate or acetaldehyde, or both. These accumulations were attributed to a reduction in the magnesium or sulfur content of the medium and a lack of feedback inhibition by acetate of alcohol dehydrogenase.     

Peculiarities of ethanol metabolism in an Acinetobacter sp. mutant strain defective in exopolysaccharide synthesis

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.     

Coproduction of acetaldehyde and hydrogen during glucose fermentation by Escherichia coli

Escherichia coli K-12 strain MG1655 was engineered to coproduce acetaldehyde and hydrogen during glucose fermentation by the use of exogenous acetyl-coenzyme A (acetyl-CoA) reductase (for the conversion of acetyl-CoA to acetaldehyde) and the native formate hydrogen lyase. A putative acetaldehyde dehydrogenase/acetyl-CoA reductase from Salmonella enterica (SeEutE) was cloned, produced at high levels, and purified by nickel affinity chromatography. In vitro assays showed that this enzyme had both acetaldehyde dehydrogenase activity (68.07 ± 1.63 μmol min(-1) mg(-1)) and the desired acetyl-CoA reductase activity (49.23 ± 2.88 μmol min(-1) mg(-1)). The eutE gene was engineered into an E. coli mutant lacking native glucose fermentation pathways (ΔadhE, ΔackA-pta, ΔldhA, and ΔfrdC). The engineered strain (ZH88) produced 4.91 ± 0.29 mM acetaldehyde while consuming 11.05 mM glucose but also produced 6.44 ± 0.26 mM ethanol. Studies showed that ethanol was produced by an unknown alcohol dehydrogenase(s) that converted the acetaldehyde produced by SeEutE to ethanol. Allyl alcohol was used to select for mutants with reduced alcohol dehydrogenase activity. Three allyl alcohol-resistant mutants were isolated; all produced more acetaldehyde and less ethanol than ZH88. It was also found that modifying the growth medium by adding 1 g of yeast extract/liter and lowering the pH to 6.0 further increased the coproduction of acetaldehyde and hydrogen. Under optimal conditions, strain ZH136 converted glucose to acetaldehyde and hydrogen in a 1:1 ratio with a specific acetaldehyde production rate of 0.68 ± 0.20 g h(-1) g(-1) dry cell weight and at 86% of the maximum theoretical yield. This specific production rate is the highest reported thus far and is promising for industrial application. The possibility of a more efficient "no-distill" ethanol fermentation procedure based on the coproduction of acetaldehyde and hydrogen is discussed.     

Regulation of acetate metabolism in a strain of Acinetobacter sp., growing on ethanol

Ethanol metabolism in Acinetobacter sp. is limited by the rate of acetate assimilation in a reaction catalyzed by acetyl-CoA synthetase (EC 6.2.1.1). Effects of ions (sodium, potassium, and magnesium), byproducts of ethanol and acetaldehyde oxidation (NADH and NADPH), and pantothenic acid on this enzyme have been studied (sodium, NADH, and NADPH inhibit acetyl-CoA synthetase; pantothenic acid, potassium, and magnesium act as the enzyme activators). Conditions of culturing were developed, under which ethanol, acetaldehyde, and acetate in Acinetobacter cells were oxidized at the same rates, producing a threefold increase in the activity of acetyl-CoA synthetase in the cell-free extract. The results of studies of acetyl-CoA synthetase regulation in a mutant strain of Acinetobacter sp., which is incapable of forming exopolysaccharides, provide a basis for refining the technology of ethapolan production, involving the use of C2 substrates.     

Autoconditioning factor relieves ethanol-induced growth inhibition of Saccharomyces cerevisiae.

Viable Saccharomyces cerevisiae suspended in medium containing growth-inhibiting concentrations of ethanol produce a metabolite that relieves growth inhibition. This autoconditioning of the medium by yeasts is due to the formation of small amounts (0.01%, vol/vol) of acetaldehyde. The effect is duplicated precisely in fresh medium by the addition of acetaldehyde. Acetaldehyde does not increase the yield of or accelerate ethanol production by the organism. Ethanol-induced modifications of membrane order in the plasma membranes, as measured by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, were not resolved by exogenously added acetaldehyde.     

Autoconditioning factor relieves ethanol-induced growth inhibition of Saccharomyces cerevisiae

Viable Saccharomyces cerevisiae suspended in medium containing growth-inhibiting concentrations of ethanol produce a metabolite that relieves growth inhibition. This autoconditioning of the medium by yeasts is due to the formation of small amounts (0.01%, vol/vol) of acetaldehyde. The effect is duplicated precisely in fresh medium by the addition of acetaldehyde. Acetaldehyde does not increase the yield of or accelerate ethanol production by the organism. Ethanol-induced modifications of membrane order in the plasma membranes, as measured by steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, were not resolved by exogenously added acetaldehyde.     

Effect of ethanol and acetaldehyde on the release of arginine-vasopressin and oxytocin from the isolated hypothalamo-hypophyseal system of rats

Effects of ethanol and acetaldehyde on the release of arginine-vasopressin (AVP) and oxytocin (OXT) were examined using a superfusion system of the isolated hypothalamo-hypophyseal complex of rats. The release of both hormones was significantly suppressed by exposing the tissue samples to Eagle MEM medium containing 1.75 and 2.5% ethanol (the maximal suppression: AVP, 30% and 70%; OXT, 30% and 70%, respectively). However, perfusion with medium containing 3.75 and 5.0% ethanol enhanced the release of OXT during exposure to ethanol (the maximal increase, 1,000%) and the release of AVP was increased markedly just after exposure to ethanol was stopped (the maximal increase, 800%). Perfusion with medium containing 50, 100 and 250 microM acetaldehyde did not affect the release.     

Aldehyde dehydrogenase is essential for both adult and larval ethanol resistance in Drosophila melanogaster

The enzyme aldehyde dehydrogenase (ALDH) is essential for ethanol metabolism in mammals, converting the highly toxic intermediate acetaldehyde to acetate. The role of ALDH in Drosophila has been debated, with some authors arguing that, at least in larvae, acetaldehyde detoxification is carried out mainly by alcohol dehydrogenase (ADH), the enzyme responsible for converting ethanol to acetaldehyde. Here, we report the creation and characterization of four null mutants of Aldh, the putative structural locus for ALDH. Aldh null larvae and adults are poisoned by ethanol concentrations easily tolerated by wild-types; their ethanol sensitivity is in fact comparable to that of Adh nulls. The results refute the view that ALDH plays only a minor role in ethanol detoxification in larvae, and suggest that Aldh and Adh may be equally important players in the evolution of ethanol resistance in fruit-breeding Drosophila.     

A novel ethanol-hypersensitive mutant of Arabidopsis

A novel ethanol-hypersensitive mutant, geko1 (gek1), was isolated from Arabidopsis thaliana. The gek1 mutant displays an enhanced sensitivity (10-100 times greater than the wild type) to ethanol in growth medium, while it grows normally in the absence of ethanol, and responds normally to other alcohols and to environmental stresses such as heat shock and high salinity. The ethanol-hypersensitive phenotype of gek1 requires alcohol dehydrogenase activity, indicating that gek1 is sensitive not to ethanol itself but to the metabolites of ethanol. Consistent with this, gek1 shows enhanced sensitivity to acetaldehyde in the medium. The endogenous acetaldehyde levels were not different between gek1-2 and wild-type seedlings treated with ethanol. These results indicate that the ethanol hypersensitivity of gek1 is due to an enhanced sensitivity to acetaldehyde toxicity, instead of abnormally elevated accumulation of toxic acetaldehyde, which has been thought to be the major cause of ethanol toxicity in mammal cells.     

Amidino-substituted aromatic heterocycles as probes of the specificity pocket of trypsin-like proteases.

The effect of pargyline on the uptake of acetaldehyde (in the presence of pyrazole) by isolated rat liver cells was studied after incubating the liver cells for 0, 10, 30, 45, and 60 min with 0.40, 1.30, and 2.6 mm pargyline. Without any incubation period, pargyline had no effect on acetaldehyde uptake. With increasing time of incubation, there was a progressive increase in the extent of inhibition of acetaldehyde uptake by pargyline. This suggests the possibility that pargyline is metabolized to the effective inhibitor or the incubation period allows pargyline to reach its site(s) of action. Pargyline was also a more effective inhibitor of the uptake of lower concentrations of acetaldehyde, e.g., 0.167 mm, than of higher concentrations (1.0 mm) of acetaldehyde, especially after short incubation periods or when pyrazole was omitted from the reaction medium. After a 20- to 30-min incubation period, pargyline inhibited the control rate of ethanol oxidation by the liver cells, as well as the accelerated rate of ethanol oxidation found in the presence of pyruvate or an uncoupling agent. Pargyline had no effect on hepatic oxygen consumption. During ethanol oxidation, a time-dependent release of acetaldehyde into the medium was observed. Pyruvate, by increasing the rate of ethanol oxidation, increased the output of acetaldehyde five- to tenfold. Pargyline increased the output of acetaldehyde two- to threefold, despite decreasing the rate of ethanol metabolism by the liver cells. These data indicate that pargyline inhibits the low K_m aldehyde dehydrogenase in intact rat liver cells and that this enzyme plays the major role in oxidizing the acetaldehyde which arises during the metabolism of ethanol. Although most of the acetaldehyde generated during the oxidation of ethanol is removed by the liver cells in an effective manner, changes in the activity of aldehyde dehydrogenase or the rate of acetaldehyde generation significantly alter the hepatic output of acetaldehyde.     

Participation of acetaldehyde dehydrogenases in ethanol and pyruvate metabolism of the yeast Saccharomyces cerevisiae.

This work was undertaken to clarify the role of acetaldehyde dehydrogenases in Saccharomyces cerevisiae metabolism during growth on respiratory substrates. Until now, there has been little agreement concerning the ability of mutants deleted in gene ALD4, encoding mitochondrial acetaldehyde dehydrogenase, to grow on ethanol. Therefore we constructed mutants in two parental strains (YPH499 and W303-1a). Some differences appeared in the growth characteristics of mutants obtained from these two parental strains. For these experiments we used ethanol, pyruvate or lactate as substrates. Mitochondria can oxidize lactate into pyruvate using an ATP synthesis-coupled pathway. The ald4Delta mutant derived from the YPH499 strain failed to grow on ethanol, but growth was possible for the ald4Delta mutant derived from the W303-1a strain. The co-disruption of ALD4 and PDA1 (encoding subunit E1alpha of pyruvate dehydrogenase) prevented the growth on pyruvate for both strains but prevented growth on lactate only in the double mutant derived from the YPH499 strain, indicating that the mutation effects are strain-dependent. To understand these differences, we measured the enzyme content of these different strains. We found the following: (a) the activity of cytosolic acetaldehyde dehydrogenase in YPH499 was relatively low compared to the W303-1a strain; (b) it was possible to restore the growth of the mutant derived from YPH499 either by addition of acetate in the media or by introduction into this mutant of a multicopy plasmid carrying the ALD6 gene encoding cytosolic acetaldehyde dehydrogenase. Therefore, the lack of growth of the mutant derived from the YPH499 strain seemed to be related to the low activity of acetaldehyde oxidation. Therefore, when cultured on ethanol, the cytosolic acetaldehyde dehydrogenase can partially compensate for the lack of mitochondrial acetaldehyde dehydrogenase only when the activity of the cytosolic enzyme is sufficient. However, when cultured on pyruvate and in the absence of pyruvate dehydrogenase, the cytosolic acetaldehyde dehydrogenase cannot compensate for the lack of the mitochondrial enzyme because the mitochondrial form produces intramitochondrial NADH and consequently ATP through oxidative phosphorylation.     

Effect of acetaldehyde on Saccharomyces cerevisiae and Zymomonas mobilis subjected to environmental shocks

The lag phase of Saccharomyces cerevisiae subjected to a step increase in temperature or ethanol concentration was reduced by as much as 60% when acetaldehyde was added to the medium at concentrations less than 0.1 g/L. Maximum specific growth rates were also substantially increased. Even greater proportional reductions in lag time due to acetaldehyde addition were observed for ethanol-shocked cultures of Zymomonas mobilis. Acetaldehyde had no effect on S. cerevisiae cultures started from stationary phase inocula in the absence of environmental shock and its lag-reducing effects were greater in complex medium than in a defined synthetic medium. Acetaldehyde reacted strongly with the ingredients of complex culture media. It is proposed that the effect of added acetaldehyde may be to compensate for the inability of cells to maintain transmembrane acetaldehyde gradients following an environmental shock. (c) 1997 John Wiley & Sons, Inc.     

Acetaldehyde coenzyme A dehydrogenase of Escherichia coli.

Mutants of Escherichia coli (adh) in which alcohol dehydrogenase is derepressed under aerobic conditions were also found to overproduce acetaldehyde coenzyme a dehydrogenase. However, acetaldehyde coenzyme A dehydrogenase was induced by ethanol or acetaldehyde and subject to strong catabolite repression, whereas alcohol dehydrogenase was little affected by these conditions. Mutants no longer able to use ethanol as carbon source were isolated from an adh strain. Some of these mutants were revertants at the adh locus and no longer produced either alcohol dehydrogenase or acetaldehyde coenzyme A dehydrogenase. Others, designated acd, were found to lack only acetaldehyde coenzyme A dehydrogenase. The acd mutation was located at min 62 of the E. coli genetic map, the gene order being thyA-lysA-acd-serA-fda. Isolation of Tn10 insertions cotransducible with acd greatly simplified the mapping procedure.     

Transport and intracellular accumulation of acetaldehyde in saccharomyces cerevisiae

The rate of acetaldehyde efflux from yeast cells and its intracellular concentration were studied in the light of recent suggestions that acetaldehyde inhibition may be an important factor in yeast ethanol fermentations. When the medium surrounding cells containing ethanol and acetaldehyde was suddenly diluted, the rate of efflux of acetaldehyde was slow relative to the rate of ethanol efflux, suggesting that acetaldehyde, unlike ethanol, may accumulate intracellularly. Intracellular acetaldehyde concentrations were measured during high cell density fermentations, using direct injection gas chromatography to avoid the need to concentrate or disrupt the cells. Intracellular acetaldehyde concentrations substantially exceeded the extracellular concentrations throughout fermentation and were generally much higher than the acetaldehyde concentrations normally recorded in the culture broth in ethanol fermentations. The technique used was sensitive to the time taken to cool and freeze the samples. Measured intracellular acetaldehyde concentrations fell rapidly as the time taken to freeze the suspensions was extended beyond 2 s. The results add weight to recent claims that acetaldehyde toxicity is responsible for some of the effects previously ascribed to ethanol in alcohol fermentations, especially Zymomonas fermentations. Further work is required to confirm the importance of acetaldehyde toxicity under other culture conditions. (c) 1993 John Wiley & Sons, Inc.     

Specific tandem GG to TT base substitutions induced by acetaldehyde are due to intra-strand crosslinks between adjacent guanine bases.

Acetaldehyde is present in tobacco smoke and automotive exhaust gases, is produced by the oxidation of ethanol, and causes respiratory organ cancers in animals. We show both the types and spectra of acetaldehyde-induced mutations in supF genes in double- and single-stranded shuttle vector plasmids replicated in human cells. Of the 101 mutants obtained from the double-stranded plasmids, 63% had tandem base substitutions, of which the predominant type is GG to TT transversions. Of the 44 mutants obtained from the single-stranded plasmids, 39% had tandem mutations that are of a different type than the double-stranded ones. The GG to TT tandem substitutions could arise from intra-strand crosslinks. Our data indicate that acetaldehyde forms intra- as well as inter-strand crosslinks between adjacent two-guanine bases. Based upon the following observations: XP-A protein binds to acetaldehyde-treated DNA, DNA excision repair-deficient xeroderma pigmentosum (XP) cells were more sensitive to acetaldehyde than the repair-proficient normal cells, and a higher frequency of acetaldehyde-induced mutations of the shuttle vectors was found in XP cells than in normal cells, we propose that the DNA damage caused by acetaldehyde is removed by the nucleotide excision repair pathway. Since treatment with acetaldehyde yields very specific GG to TT tandem base substitutions in DNA, such changes can be used as a probe to identify acetaldehyde as the causal agent in human tumors.     

Ethanol metabolism in suspension cultured carrot cells

Ethanol production in plant tissues deprived of oxygen is a well known process. Nevertheless, little information is available on the toxic effects of ethanol on plant cells and tissues, or on the possible role of acetaldehyde, the first oxidative product of ethanol, in inducing toxic effects in plants. Data on the metabolism of ethanol in suspension cultured cells of carrot ( Daucus carola L. cv. S. Valery, cell line T22), a system highly sensitive to the presence of ethanol in the culture medium, indicate that carrot cells oxidize only small amounts of ethanol to CO₂. Instead, they convert ethanol mainly to acetaldehyde, which accumulates in the culture medium. This suggests a possible role of acetaldehyde in causing ethanol-induced injury to carrot cells.     

Effects of differential alcohol dehydrogenase activity on the developmental toxicity of ethanol in Drosophila melanogaster

The role of alcohol dehydrogenase (ADH) activity in ethanol toxicity was investigated in Drosophila melanogaster. Flies from three congenic Adh strains (high, medium, and low ADH activity) were allowed to deposit eggs on medium containing 0, 4, or 8% ethanol. The resulting larvae were allowed to complete their development in the medium, and emerging flies were examined for defects. Flies with high ADH activity had malformation incidences of 0.8, 2.4, and 5.2% at 0, 4, and 8% ethanol, respectively. The comparable incidences for the low ADH strain were 1.0, 4.1, and 8.4%, while those for the medium ADH strain were intermediate in value. These results indicate that ethanol teratogenesis may be inversely related to ADH activity. When larvae were treated with ethanol for different lengths of time during development, the incidence of defects in flies from the high ADH strain was 3.9% when exposure started at the first instar and 3.09% when exposure started at the third instar. Results of the same exposures for the intermediate ADH strain were 5.2 and 3.4%, respectively, while those for the low ADH strain were 6.9 and 5.5%, respectively. Thus, length of ethanol exposure was directly related to the increased incidence of malformations in all tested Drosophila strains. For all tested strains, defect incidences appeared to be dose-related as well, regardless of length of exposure. ADH in Drosophila has a dual function and thus can catalyze oxidation of both ethanol and its toxic metabolite, acetaldehyde. This suggests that ethanol is the proximate teratogen in Drosophila.     

乙醇与乙醛对心脏的影响及其可能机制的探讨

通过研究乙醇、乙醛对离体心脏和神经干的影响,探讨乙醇、乙醛对心脏作用的可能机制.用不同浓度的乙醇和乙醛处理牛蛙蛙心灌流标本和坐骨神经标本,用BL-420 系统对给药前后心脏的心率和振幅以及神经干最小刺激强度作记录.乙醇和乙醛可以引起神经兴奋性的改变从而影响神经冲动的传导,而且其影响具有明显的量效依赖关系,低浓度的乙醇和乙醛能使神经的兴奋性增加,高浓度则降低;乙醇对心脏的心率和振幅均有抑制作用,低浓度的乙醛对心脏心率和振幅有促进作用,高浓度的乙醛对心脏造成不可恢复的损伤.乙醇、乙醛对心脏的影响效果不同,但两者均可直接影响及通过神经而间接影响心脏的活动.