Quinoprotein alcohol dehydrogenase is involved in catabolic acetate production, while NAD-dependent alcohol dehydrogenase in ethanol assimilation in Acetobacter pasteurianus SKU1108

The relationship between quinoprotein alcohol dehydrogenase (ADH) and NAD-dependent ADH was studied by constructing quinoprotein ADH-deficient mutants. Quinoprotein ADH-deficient mutants were successfully constructed from Acetobacter pasteurianus SKU1108 by N-methyl-N'-nitro-N-nitrosoguanidine (NTG) mutagenesis and also by adhA gene disruption with a kanamycin cassette. The NTG mutant exhibited a complete loss of its acetate-producing ability and acetic acid resistance, while the disruptant also exhibited a loss of its acetic acid resistance but retained a weak ADH activity. The immunoblot analysis of quinoprotein ADH indicated that there are no appreciable ADH subunits in the membranes of both mutant strains. The NTG mutant grew better than the wild-type strain in ethanol-containing medium, despite the absence of quinoprotein ADH. In the mutant, the activities of two NAD-dependent ADHs, present in a small amount in the wild-type strain, markedly increased in the cytoplasm when cultured in a medium containing ethanol, concomitant to the increase in the activities of the key enzymes in TCA and glyoxylate cycles. The disruptant showed a poorer growth than the wild-type strain, producing a lower amount of acetic acid in ethanol culture, and it induced one of the two NAD-dependent ADHs and some of the acetate-assimilating enzymes induced in the NTG mutant. This study clearly showed that quinoprotein ADH is extensively involved in acetic acid production, while NAD-dependent ADH only in ethanol assimilation through the TCA and glyoxylate cycles in acetic acid bacteria. The differences between the NTG mutant and the disruptant are also discussed.     

巴氏醋酸杆菌AS1.41产醋酸关键酶研究

巴氏醋酸杆菌（Acetobacer pasteurianus）将乙醇氧化成醋酸的关键酶是乙醇脱氢酶（ADH）和乙醛脱氢酶（ALDH）。在不同初始乙醇含量条件下,ADH和ALDH的酶活呈现动态变化,乙醇含量为4%时,ADH和ALDH的酶活达到最大,分别为7.43 U/mg和7.18 U/mg。同时,酶活与产酸速率呈现出较高一致性：酶活越高,产酸速率越快。发酵温度为32 ℃时,菌体生长最为活跃,酶活最大,产酸最快;加入0.5%的乙酸后,ADH和ALDH的酶活分别由8.12 U/mg和7.06 U/mg提高到了9.43 U/mg和8.52 U/mg,产酸速率也得到相应提升。ALDH对乙醇、乙酸、温度的稳定性均高于ADH。     

METABOLISM OF ACETALDEHYDE—A METABOLIC BRANCHING POINT II. EFFECT OF ETHANOL

Acetaldehyde is an intermediate in alcoholic fermentation. The major part of it is reduced to ethanol but a minor fraction is normally oxidized to acetic acid. Owing to the kinetic properties of alcohol dehydrogenase and aldehyde dehydrogenase, the oxidized fraction decreased as the intracellular concentration was increased. Since alcohol dehydrogenase catalyses a reversible reaction, the distribution between oxidation and reduction of acetaldehyde was sensitive to the concentration of ethanol; an increased concentration of ethanol reduced the formation of acetic acid.     

Purification and characterization of an enzyme that has dihydroxyacetone-reducing activity from methanol-grown Hansenula ofunaensis

An intracellular enzyme having reduction activity towards dihydroxyacetone (DHA), and that was induced by DHA, was purified and characterized from a methanol-grown yeast, Hansenula ofunaensis. After harvesting cells grown in a 1% methanol medium until the early stationary phase, the enzyme was purified through ammonium sulfate fractination and a series of ion-exchange, hydrophobic, and gel-filtration column chromatographies. SDS-PAGE and HPLC showed the enzyme to be a homo dimer composed of two identical subunits, each with a molecular mass of 38 kDa. The optimum pHs for DHA reduction and glycerol oxidation were 6.0 and 7.0, respectively. The optimum temperature for enzyme activity was 55 degrees C. The enzyme reduced several other compounds, including acetaldehyde, acetol, 2-butanone and 3-methyl-2-butanone, more effectively than it did DHA, while its oxidation activity was higher towards ethanol, 2-propanol, 1,2-propanediol, 2,3-butanediol and 1,3-butanediol than towards glycerol. The K(m) values for DHA in reduction and glycerol in oxidation were 430 mM and 4 M, respectively. The K(m) values for DHA in reduction and glycerol in oxidation were 430 mM and 4 M, respectively. The purified enzyme had high K(m) values for glycerol and DHA and low K(m) values for 2-butanol and butanone, although physiologically it had a role in DHA metabolism. There were similarities between the purified enzyme and sec-alcohol dehydrogenases reported previously in their behavior towards inhibitors and metal ions, as well as in their K(m) values for 2-butanol and 2-butanone, but differences in their subunit molecular masses and activities for ethanol. At pH 9.8, the oxidative activity of the purified enzyme for l-2-butanol was about eleven times higher than that for d-2-butanol.     

米根霉乙醇脱氢酶粗酶液的特性研究

米根霉细胞中乙醇脱氢酶(ADH)催化丙酮酸向乙醇支路的转化,导致丙酮酸向乳酸转化通量减少,降低了乳酸转化率。本文初步从米根霉菌丝体中提取制备ADH粗酶液,并研究其酶学特性,结果表明,ADH粗酶液体系反应的最适温度为25℃,温度高于30℃将降低ADH活力。ADH催化体系pH值对其活力有很大影响,最适pH值在7.5左右,高于或低于此值,反应速度均很快下降。在反应体系中添加0.1μmol的EDTA、Mg2+、Ca2+或Zn2+,对该酶都有一定的抑制作用。ADH以乙醛为底物的米氏常数Km为6.90×10-4mol/L。     

Determination of the in vivo NAD:NADH ratio in Saccharomyces cerevisiae under anaerobic conditions,using alcohol dehydrogenase as sensor reaction

With the current quantitative metabolomics techniques, only whole‐cell concentrations of NAD and NADH can be quantified. These measurements cannot provide information on the in vivo redox state of the cells, which is determined by the ratio of the free forms only. In this work we quantified free NAD:NADH ratios in yeast under anaerobic conditions, using alcohol dehydrogenase (ADH) and the lumped reaction of glyceraldehyde‐3‐phosphate dehydrogenase and 3‐phosphoglycerate kinase as sensor reactions. We showed that, with an alternative accurate acetaldehyde determination method, based on rapid sampling, instantaneous derivatization with 2,4 diaminophenol hydrazine (DNPH) and quantification with HPLC, the ADH‐catalysed oxidation of ethanol to acetaldehyde can be applied as a relatively fast and simple sensor reaction to quantify the free NAD:NADH ratio under anaerobic conditions. We evaluated the applicability of ADH as a sensor reaction in the yeast Saccharomyces cerevisiae, grown in anaerobic glucose‐limited chemostats under steady‐state and dynamic conditions. The results found in this study showed that the cytosolic redox status (NAD:NADH ratio) of yeast is at least one order of magnitude lower, and is thus much more reduced, under anaerobic conditions compared to aerobic glucose‐limited steady‐state conditions. The more reduced state of the cytosol under anaerobic conditions has major implications for (central) metabolism. Accurate determination of the free NAD:NADH ratio is therefore of importance for the unravelling of in vivo enzyme kinetics and to judge accurately the thermodynamic reversibility of each redox reaction. Copyright © 2015 John Wiley & Sons, Ltd.     

醋酸菌粉末对ICR小鼠的酒精性肝脂质蓄积和氧化应激的影响研究

该研究评估了醋酸菌粉末对急性摄入酒精引起的肝脂质蓄积和氧化损伤的影响。将25只雄性ICR小鼠随机分为对照组、乙醇组、醋冻干粉高（45 mg/kg）、中（15 mg/kg）、低（7.5 mg/kg）剂量组。油红O染色观察肝脏病理变化；测定各组小鼠肝脏中甘油三酸酯（TG）、谷胱甘肽（GSH）、丙二醛（MDA)含量；分析醋酸菌粉末组小鼠的乙醇脱氢酶（ADH）和乙醛脱氢酶（ALDH）酶活性。结果表明，与乙醇组相比，高剂量组TG浓度显著降低（P＜0.05），GSH浓度极显著升高（P＜0.01），MDA含量无显著变化（P＞0.05），脂肪变性程度减轻；醋酸菌粉末组小鼠的ADH和ALDH酶活性分别为0.48 U/mg和1.38 U/mg。综上，摄入醋酸菌粉末可以减轻急性摄入酒精引起的肝脂质蓄积和氧化损伤。     

Suppression of propanoic acid,acetic acid and 3-methylbutanoic acid production by other volatiles in a Swiss cheese curd slurry system

The effect of the addition of 5–100 ppm 2,3-butanedione, butanoic acid, methyl mercaptan, ethyl butanoate, diethyl sulfide, 3-methylbutanoic acid, acetaldehyde and ethanol on production of propanoic acid, acetic acid and 3-methylbutanoic acid in a Swiss cheese slurry system was investigated. Enumeration of propionic acid bacteria (PAB) was done to monitor the microbial count in the systems. A Swiss cheese slurry system was used to simulate cheese manufacturing based on accelerated ripening for a 6-day incubation period. The headspace concentrations of propanoic acid, acetic acid and 3-methylbutanoic acid were significantly decreased by the addition of 2,3-butanedione, butanoic acid, and methyl mercaptan, but were generally unaffected by ethyl butanoate, diethyl sulfide, 3-methylbutanoic acid, acetaldehyde and ethanol. Microbial enumeration also showed that there was a significant reduction of PAB in slurry systems with 100 ppm 2,3-butanedione, butanoic acid, and methyl mercaptan.     

Substrate specificity of alcohol dehydrogenase from the yeast Hansenyls polymorpha CBS 4732 and Candida utilis CBS 621

The substrate specificity of alcohol dehydrogenase (ADH) from Hansenula polymorpha and Candida utilis has been compared with that of the classical ADH from baker's yeast. Cell-free extracts of H. polymorpha and C. utilis exhibited a much higher ratio of butanol to ethanol oxidation than baker's yeast ADH. This was also observed with the purified enzymes. The ratio of activities with ethanol and butanol was pH-dependent. With the baker's yeast enzyme the activity strongly decreased with increasing chain length, whereas the enzymes form H. polymorpha and C. utilis showed a high reactivity with long-chain alcohols. In addition, the affinity constant for ethanol was more than tenfold lower than that of the baker's yeast enzyme. The purified preparation yielded several protein bands on polyacrylamide slab gels, each of which showed activity with both ethanol and butanol.     

Purification and characterization of a novel alcohol oxidase from Paenibacillus sp. AIU 311

An oxidase catalyzing the conversion of glycolaldehyde to glyoxal was purified to the homogeneous state from Paenibacillus sp. AIU 311, and its properties were revealed. This enzyme was specific to glycolaldehyde and glyceraldehyde, and the reaction rates to other alcohols and aldehydes were less than 6% of that of glycolaldehyde. The Km values for glycolaldehyde and glyceraldehyde were estimated to be 13.2 and 7.5 mM, respectively. The glycolaldehyde oxidation was optimum at pH 6.5 and 50 degrees C. The molecular mass of this enzyme was 49 kDa, and it consisted of two identical subunits of 24 kDa. The NH2-terminal sequence was not homologous to those of alcohol oxidases. This is the first report of an oxidase exhibiting high specificity to a hydroxy group of aldehyde alcohols.     

Acetic acid bacteria spoilage of bottled red wine -- a review

Acetic acid bacteria (AAB) are ubiquitous organisms that are well adapted to sugar and ethanol rich environments. This family of Gram-positive bacteria are well known for their ability to produce acetic acid, the main constituent in vinegar. The oxidation of ethanol through acetaldehyde to acetic acid is well understood and characterised. AAB form part of the complex natural microbial flora of grapes and wine, however their presence is less desirable than the lactic acid bacteria and yeast. Even though AAB were described by Pasteur in the 1850s, wine associated AAB are still difficult to cultivate on artificial laboratory media and until more recently, their taxonomy has not been well characterised. Wine is at most risk of spoilage during production and the presence of these strictly aerobic bacteria in grape must and during wine maturation can be controlled by eliminating, or at least limiting oxygen, an essential growth factor. However, a new risk, spoilage of wine by AAB after packaging, has only recently been reported. As wine is not always sterile filtered prior to bottling, especially red wine, it often has a small resident bacterial population (<10(3) cfu/mL), which under conducive conditions might proliferate. Bottled red wines, sealed with natural cork closures, and stored in a vertical upright position may develop spoilage by acetic acid bacteria. This spoilage is evident as a distinct deposit of bacterial biofilm in the neck of the bottle at the interface of the wine and the headspace of air, and is accompanied with vinegar, sherry, bruised apple, nutty, and solvent like off-aromas, depending on the degree of spoilage. This review focuses on the wine associated AAB species, the aroma and flavour changes in wine due to AAB metabolism, discusses the importance of oxygen ingress into the bottle and presents a hypothesis for the mechanism of spoilage of bottled red wine.     

啤酒酵母乙醛代谢关键酶相关性研究

乙醛是啤酒中的主要风味物质,其代谢主要来自酵母细胞。酵母中乙醇脱氢酶及乙醛脱氢酶是乙醛代谢的关键酶,对乙醛变化起着重要作用。跟踪啤酒酵母发酵过程中相对酶活力及乙醛变化,发现两种乙醇脱氢酶和乙醛脱氢酶的相对酶活力与发酵过程乙醛含量变化具有一定相关性。同时对低产乙醛啤酒酿酒酵母kb2-4与出发菌株啤酒酵母kb进行发酵试验,跟踪检测相对酶活力及乙醛含量,其乙醇脱氢酶Ⅰ和乙醇脱氢酶Ⅱ及乙醛脱氢酶相对酶活力均高于出发菌株,平均增幅分别为15.5%,11.6%和5%。3种酶活性的变化协同作用可以使乙醛含量降幅最大为33.8%。     

猪肝乙醇脱氢酶的分离纯化及部分性质

新鲜猪肝经匀浆、缓冲液抽提、硫酸铵分级沉淀、DEAE-Sepharose离子交换层析及Superdex-200凝胶过滤层析,获得电泳纯的乙醇脱氢酶（alcohol dehydrogenase,ADH）。纯化结果显示：该酶比活力为1 622.33 U/mg,回收率为29.05%,纯化倍数为34.58;该酶分子质量约为171.79 kD,亚基分子质量约为43.68 kD。ADH酶学性质研究显示：最适反应温度和pH值分别为45 ℃和10.0;在25～45 ℃及pH 7.5～9.0范围内稳定性较好;最适条件下测得该酶对乙醇的Km值为19 mmol/L;正丁醇、氯仿、异丙醇、十二烷基硫酸钠、草酸、Zn2＋、Cu2＋、Ag＋对该酶的抑制作用最强,Mg2＋对该酶有激活作用,EDTA对该酶有双重作用。     

白酒关键微量成分对醉度及小鼠乙醇代谢和急性酒精性肝损伤的影响

模拟豉香型白酒关键微量成分构成制备配制酒,利用小鼠模型对关键微量成分缺失的配制酒进行醉度评价,检测配制酒灌 胃后小鼠体内生化指标,探究白酒关键微量成分对醉度及小鼠乙醇代谢和急性酒精性肝损伤的影响。 结果表明,酸、酯、杂醇等关键 微量成分主要通过影响乙醇代谢而导致醉度差异,对急性酒精性肝损伤不会产生显著影响,其中乙酸和乙酸乙酯可显著降低醉度（P＜0.05）,灌服后小鼠肝脏乙醇脱氢酶（ADH）和乙醛脱氢酶（ALDH）活性变化不明显,血液乙醇和乙醛含量显著增加（P＜0.05）;异丁醇和异戊醇可显著增加醉度（P＜0.05）,灌服后小鼠肝脏ADH和ALDH活性同时增加,血液乙醇和乙醛含量显著降低（P＜0.05）;灌 服缺失不同成分酒后小鼠血清谷丙转氨酶（ALT）、谷草转氨酶（AST）和肝脏超氧化物歧化酶（SOD）、丙二醛（MDA）指标无明显差异。     

粘质沙雷氏菌膜结合葡萄糖酸脱氢酶的分离纯化及其酶学特性

经超声破碎细胞、TritonX-114相分离、盐析、DEAE-sepharose离子交换层析和Hydroxyapatite吸附层析等步骤,从2-酮基葡萄糖酸（2-keogluconic acid,2KGA）生产菌株粘质沙雷氏菌JUIM03中获得比活力91.43 U/mg、纯化倍数160.4 倍的膜结合葡萄糖酸脱氢酶（gluconate dehydrogenase,GADH）。研究结果表明,粘质沙雷氏菌膜结合GADH由3 个亚基组成,其分子质量分别为65.0、45.0 kDa和23.0 kDa左右,是一种含有细胞色素C的黄素蛋白,能够催化氧化葡萄糖酸为2KGA。粘质沙雷氏菌膜结合GADH的最适反应温度为40 ℃,最适反应pH 5.0,在30 ℃以下、pH 5.0～6.0的条件下较为稳定;该酶具有严格的底物特异性,只有在D-葡萄糖酸作为底物时才具有催化活性,其Km值为1.33 mmol/L;一些金属离子（如Fe3＋、Cu2＋和Zn2＋）、有机溶剂（乙醇、异丙醇、甲醇和丙酮）、变性剂（十二烷基硫酸钠和巯基乙醇）以及有机酸（草氨酸和草酸）对膜结合GADH的催化活性具有明显的抑制作用。研究为粘质沙雷氏菌2KGA生物合成的过程优化与控制提供了一定的理论依据。     

Alcohol metabolizing and antioxidant activities of complex herbal extracts from medicinal plants

The objective of this study was to evaluate the alcohol-metabolizing and antioxidative activities of complex herbal extract (CHE). The alcohol-metabolizing activity of CHE was evaluated by assessing alcohol dehydrogenase (ADH) activity, acetaldehyde dehydrogenase (ALDH) activity, and protective effect against alcohol induced damage in in vitro and in vivo models. In this study, CHE treatment significantly increased ADH and ALDH activities and reduced cell death in alcohol-induced liver cell damage. Moreover, it also significantly reduced the serum alcohol and acetaldehyde concentrations in alcoholfed rats. To define the effect of CHE on alcohol metabolism, its antioxidative activities were estimated by measuring radical scavenging activity, ferric-reducing antioxidant potential ability, and thiobarbituric acid reactive substance, and the results demonstrated a higher antioxidative capacity of CHE than the vitamin C control at a high dose (10 mg/mL). Therefore, this study suggests that CHE can be a potential natural resource for the management of ethanol-induced liver toxicity.     

Purification and molecular characterization of a quinoprotein alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes IFO 14464

We have cloned and verified a gene for a novel quinoprotein alcohol dehydrogenase (ADH) from Pseudogluconobacter saccharoketogenes IFO 14464 that has the ability to oxidize L-sorbose to 2-keto-L-gulonic acid (2-KLGA). The enzyme was purified from the soluble fraction of the bacterium and was estimated to be a monomeric protein with a molecular weight of 65 kDa from the analyses of SDS-PAGE and gel-filtration chromatography. An open reading frame of 1824 bp for 608 amino acid residues was estimated as the gene for ADH because of the consistency of the calculated molecular mass and the elucidated partial amino acid sequences of the native enzyme. Homology search revealed that the enzyme showed close similarity to quinoprotein alcohol dehydrogenases isolated from Methylobacterium extorquens and Acetobacter aceti, particularly in the tryptophan docking motifs in the alpha-subunits of those dehydrogenases. The ability to convert L-sorbose to 2-KLGA was found when the lysate of recombinant Escherichia coli DH10B transformed with the gene for ADH was mixed with CaCl2and pyrroloquinoline quinone (PQQ). These data indicate that the cloned DNA is the desired gene for the ADH in which CaCl2 and PQQ are essential for enzymatic activity.     

浓香型白酒风味成分对乙醇代谢及关键酶活性影响的体内实验研究

选用不同产地的四种浓香型白酒样品（编号为SC1、SC2、SC3、JS）,建立小鼠灌胃模型,分别灌胃白酒、高醇白酒、高酯白酒和相同浓度的酒精溶液,随后测定小鼠的行为指标,血液中乙醇和乙醛含量以及肝脏中乙醇脱氢酶（ADH）和乙醛脱氢酶（ALDH）活性。结果表明,灌胃给药后,白酒中高浓度异丁醇、正戊醇和异戊醇显著降低了小鼠的协调能力（P＜0.05）;血液中乙醇含量（3 692～23 237 mg/L）和乙醛含量（18～84 mg/L）均升高;增加白酒中大多数醇类和酯类含量均能抑制ADH（30.93～45.73 U/L）和ALDH（87.98～104.61 U/L）活性,且对ADH抑制作用更显著（P＜0.05）;增加SC2和SC3酒样中丁酸乙酯含量可以同时促进ADH（34.73 U/L、35.11 U/L）和ALDH（104.61 U/L、103.52 U/L）的活性。体内实验结果表明,不同产地的白酒及其差异化风味成分（增量变化）对乙醇代谢和关键代谢酶有不同程度的影响。