Alcohol dehydrogenase coexisted solid-state electrochemiluminescence biosensor for detection of p53 gene

An alcohol dehydrogenase （ADH）-coexisted solidstate electrochemiluminescence （ECL） biosensor for sensitive detection of the p53 gene was developed. The electrode modified by multiwalled carbon nanotubes, Ru（bpy）]2＋3 and polypyrrole （ MWNTs-Ru （bpy） ]2＋3 -PPy ） was prepared to adsorb the ssDNA by electrostatic interactions. Then, the ssDNA recognized the gold nanoparticles （AuNPs）-labeled p53 gene and produced the AuNPs-dsDNA electrode with the AuNPs layer. The AuNPs layer adsorbed the ADH molecules for producing the ECL signal. Thus, the biosensor was based on coupling enzyme substrate reaction with solid-state ECL detection, and it displayed good sensitivity and specificity. The detection limit of the wild type p53 sequence （wtp53） is as low as 0. 1 pmol/L and the discrimination is up to 57. 1% between the wtp53 and the muted type p53 sequence （mtp53）. The amenability of this method to the analyses of p53 from normal and cancer cell lysates is demonstrated. The signal of wtp53 in the MGC-803 gastric cancer cell lysates turns out to be about 61.8% that of the wtp53 in the GES-1 normal gastric mucosal cell lysates, and the concentration of the wtp53 is found to decrease about 59 times. The method is highly complementary to enzyme-linked immunosorbent assay （ELISA）, and it holds promise for the diagnosis and management of cancer.

CAFs促进ADH1B甲基化对卵巢癌细胞增殖及侵袭的影响

目的 探讨肿瘤相关成纤维细胞(CAFs)分泌的IL-6对卵巢癌细胞增殖及侵袭的影响及机制。方法 收取新鲜离体上皮性卵巢癌及正常卵巢上皮组织,分离纯化获得CAFs及正常卵巢成纤维细胞(NFs);蛋白质印迹和免疫荧光实验检测上皮细胞和成纤维细胞标志物α-平滑肌动蛋白(α-SMA)、上皮型钙黏附素(E-cadherin)表达;收集CAFs和NFs培养上清液与卵巢癌SKOV3细胞建立间接共培养体系,细胞分为SKOV3单独培养(SKOV3)组、SKOV3与NFs上清液(NFs)组及SKOV3与CAFs上清液(CAFs)组;细胞免疫组化检测SKOV3细胞共CAFs及NFs上清液培养后乙醇脱氢酶1B(ADH1B)表达;甲基化特异性PCR (MSP)、逆转录实时荧光定量PCR(RT-qPCR)、酶联免疫吸附试验(ELISA)及蛋白质印迹实验分别检测甲基化抑制剂5-氮杂-2′-脱氧胞苷(5-Aza-dC)干预前后各组细胞ADH1B mRNA表达及甲基化状态、信号转导和激活因子3(STAT3)蛋白磷酸化水平;细胞计数试剂盒8(CCK-8)法及Transwell实验分别检测IL-6抑制剂LMT-286及重组IL-6 (rhIL-6)对细胞增殖及侵袭能力的影响。结果 肿瘤成纤维细胞中高表达α-SMA,极低表达E-cadherin;相比较SKOV3组及NFs组,CAFs组ADH1B mRNA及蛋白表达明显下调,同时CAFs组细胞上清液中IL-6水平较SKOV3组及NFs组明显升高;5-Aza-dC作用后,ADH1B甲基化部分逆转;三组细胞ADH1B mRNA和蛋白表达均增加,CAFs组STAT3磷酸化水平下降;LMT-286及rhIL-6干预均仅抑制或促进CAFs组细胞增殖和侵袭,而SKOV3组和NFs组无明显改变。结论 CAFs通过IL-6/STAT3信号通路增强ADH1B甲基化促进卵巢癌细胞增殖和侵袭。

In Vitro and In Vivo Effects and Safety Assessment of Corn Peptides on Alcohol Dehydrogenase Activities

The in vitro and in vivo effects of corn peptides（CPs） prepared from corn gluten meal by proteolysis with an alkaline protease and fractions of CPs from Sephadex G-15 and G-10 columns on activities of alcohol dehydroge-nase（ADH） were studied. The results show that CPs and fraction 3 of CPs from Sephadex G-10 column enhance in vitro ADH activity. Furthermore, the in vitro accelerating effect of the fraction 3 of CPs on ADH activity was superior to that of glutathione, which was also found even in the presence of ADH inhibitor, such as pyrazole. In the in vivo experiments, the animals were fed with different dosages of CPs and with a dose of Chinese distilled spirit orally, and sacrificed for the measurement of ADH activity. In vivo experimental results indicate that CPS enhanced hepatic ADH activities. To test the safety of CPs as health food, 30 d feeding test was performed. No obvious toxic effects were detected in treated Wistar rats.

Identification and expression patterns of alcohol dehydrogenase genes involving in ester volatile biosynthesis in pear fruit

Alcohol dehydrogenase （ADH） catalyzes the interconversion of aldehydes and their corresponding alcohols, and is a key enzyme in volatile ester biosynthesis. However, little is known regarding ADH and ADH encoding genes （ADHs） in pear. We identified 8 ADHs in the pear＇s genome （PbrADHs） by multiple sequences alignment. The PbrADHs were highly ho- mologous in their coding regions, while were diversiform in structure. 9 introns were predicted in PbrADH3-PbrADH8, while 8 introns, generated through exon fusion and intron loss, were predicted in PbrADH1 and PbrADH2. To study the genetic regulation underlying aroma biogenesis in pear fruit, we determined the PbrADH transcripts, ADH activities and volatile contents of fruits during ripening stage for Nanguoli and Dangshansuli, two cultivars having different aroma characteristics. ADH activity was strongly associated with the transcription of ADH~ in the two cultivars during fruit ripening stage. The higher ester content paralleling to a higher ADH activity was detected in Nanguoli than in Dangshansuli, so it is induced that the lower ester content in Dangshansuli fruit may be the result of weak ADH activity. The present study revealed that total ADH activity and volatile ester production correlated with increased PbrADHstranscript levels. PbrADH6 may contribute to ADH activity catalyzing aldehyde reduction and ester formation in pear fruit.

Genetic polymorphisms in cytochrome P4502E1, alcohol and aldehyde dehydrogenases and the risk of esophageal squamous cell carcinoma in Gansu Chinese males

AIM:To evaluate the association between genetic polymorphisms in CYP2E1, ALDH2 and ADH1B and the risk of esophageal squamous cell carcinoma (ESCC) in a high risk area of Gansu Province, in Chinese males. METHODS: A case-control study was conducted to investigate the genetic polymorphisms of these enzymes (CYP2E1 *c1/*c2, ALDH2 *1/*2 and ADH1B *1/*1 genotypes). A total of 80 esophageal cancer cases and 480 controls were recruited. RESULTS: Compared with controls, cases had a greater prevalence of heavier alcohol consumption (53.8% vs 16.2%) and a higher proportion of alcohol drinkers with > 30 drink-years (28.8% vs 13.5%). Heavier alcohol consumption and alcohol drinking with > 30 drink- years increased the risk of ESCC, with ORs (95% CI) of 3.20 (1.32-9.65) and 1.68 (0.96-3.21). CYP2E1 (*c1/*c1), ALDH2 (*1/*2) and ADH1B (*1/*1) genotype frequencies were higher among patients with squamous cell carcinomas, at a level close to statistical significance (P = 0.014; P = 0.094; P = 0.0001 respectively). There were synergistic interactions among alcohol drinking and ALDH2, ADH1B and CYP2E1 genotypes. The risk of the ESCC in moderate-to-heavy drinkers with an inactive ALDH2 encoded by ALDH2 *1/*2 as well as ADH1B encoded by ADH1B *1/*1 and CYP2E1 encoded by CYP2E1 *c1/*c1 was higher than that in the never/rare-to-light drinkers with an active ALDH2 (*1/*1 genotype) as well as ADH1B (*1/*2 + *2/*2) and CYP2E1 (*c1/*c2 + *c2/*c2) genotypes, with a statistically significant difference; ORs (95% CI) of 8.58 (3.28-22.68), 27.12 (8.52-70.19) and 7.64 (2.82-11.31) respectively. The risk of the ESCC in moderate-to-heavy drinkers with ALDH2 (*1/*2) combined the ADH1B (*1/*1) genotype or ALDH2 (*1/*2) combined the CYP2E1 (*c1/*c1) genotype leads to synergistic interactions, higher than drinkers with ALDH2 (*1/*1) + ADH1B (*1/*2 + *2/*2), ALDH2 (*1/*1) + CYP2E1 (*c1/*c2 + *c2/*c2) respectively , ORs (95% CI) of 7.46 (3.28-18.32) and 6.82 (1.44-9.76) respectively. Individuals with the ADH1B combined the CYP2E1 genotype showed no synergistic interaction. CONCLUSION: In our study, we found that alcohol consumption and polymorphisms in the CYP2E1, ADH1B and ALDH2 genes are important risk factors for ESCC, and that there was a synergistic interaction among polymorphisms in the CYP2E1, ALDH2 and ADH1B genes and heavy alcohol drinking, in Chinese males living in Gansu Province, China.

Relationship between genetic polymorphisms of alcohol and aldehyde dehydrogenases and esophageal squamous cell carcinoma risk in males

AIM： To investigate the association between the genetic polymorphisms of ADH2 and ALDH2, lifetime alcohol consumption and esophageal cancer risk in the Taiwan Residents men. METHODS： Between August 2000 and June 2003, 134 pathologically-proven esophageal squamous cell carcinoma male patients and 237 male controls were recruited from Kaohsiung Medical University Hospital and Kaohsiung Veterans General Hospital in southern Taiwan. ADH2 and ALDH2 polymorphisms were genotyped using PCR-RFLP. RESULTS： Compared to those with ADH2＊2/＊2, individuals with ADH2＊1/＊2 and ADH2＊1/＊1 had 2.28- and 7.14-fold, respectively, increased risk of developing esophageal cancer （95%CI = 1.11-4.68 and 2.76-18.46） after adjusting for alcohol consumption and other covariates. The significant increased risk was also noted among subjects with ALDH2＊1/＊2 （adjusted OR （AOR） = 5.25, 95%CI = 2.47-11.19）, when compared to those with ALDH2＊1/＊1. The increased risk of esophageal cancer was made greater, when subjects carried both ADH2＊1/＊1 and ALDH2＊1/＊2, compared to those with ADH2＊1/＊2 or ADH2＊2/＊2 and ALDH2＊1/＊1 （AOR = 36.79,95%a = 9.36-144.65）. Furthhermore, we found a multipticative effect of lifetime alcoholic consumption and genotypes （ADH2 and ALDH2） on esophageal cancer risk. CONCLUSION： Our findings suggest that polymorphisms of ADH2 and ALDH2 can modify the influence of alcoholic consumption on esophageal cancer risk.

Polymorphisms of alcohol dehydrogenase 2 and aldehyde dehydrogenase 2 and colorectal cancer risk in Chinese males

AIM: To evaluate the relationship between drinking and polymorphisms of alcohol dehydrogenase 2 (ADH2) and/or aldehyde dehydrogenase 2 (ALDH2) for risk of colorectal cancer (CRC) in Chinese males. METHODS: A case-control study was conducted in 190 cases and 223 population-based controls. ADH2 Arg47His (G-A) and ALDH2 Glu487Lys (G-A)genotypes were identified by PCR and denaturing high-performance liquid chromatography (DHPLC). Information on smoking and drinking was collected and odds ratio (OR) was estimated. RESULTS: The ADH2 A/A and ALDH2 G/G genotypes showed moderately increased CRC risk. The age- and smoking-adjusted OR for ADH2 A/A relative to G/A and G/G was 1.60 (95% CI=1.08-2.36), and the adjusted OR for ALDH2 G/G relative to G/A and A/A was 1.79 (95% CI=1.19-2.69). Signif icant interactions between ADH2, ALDH2 and drinking were observed. As compared to the subjects with ADH2 G and ALDH2 A alleles, those with ADH2 A/A and ALDH2 G/G genotypes had a signif icantly increased OR (3.05, 95% CI= 1.67-5.57). The OR for CRC among drinkers with the ADH2 A/A genotype was increased to 3.44 (95% CI= 1.84-6.42) compared with non-drinkers with the ADH2 G allele. The OR for CRC among drinkers with the ALDH2 G/G genotype was also increased to 2.70 (95% CI= 1.57-4.66) compared with non-drinkers with the ALDH2 A allele. CONCLUSION: Polymorphisms of the ADH2 and ALDH2 genes are significantly associated with CRC risk. There are also signifi cant gene-gene and gene- environment interactions between drinking and ADH2 and ALDH2 polymorphisms regarding CRC risk in Chinese males.

Selective Affinity Separation of Yeast Alcohol Dehydrogenase by Reverse Micelles with Unbound Triazine Dye

The reversed micelles were formed with cationic cetyltrimethylammonium bromide (CTAB) as surfactant and n-hexanol as cosolvent in the CTAB (50mmol.L-1)/hexanol (15% by volume)/hexane system. Cibacron Blue 3GA (CB) as an affinity ligand in the aqueous phase was directly introduced to the reversed micelles with electrostatic interaction between anionic CB and cationic surfactant. High molecular weight (Mr) protein, yeast alcohol dehydrogenase (YADH, Mr = 141000) from baker's yeast, has been purified using the affinity reversed micelles by the phase transfer method. Various parameters, such as CB concentration, pH and ionic strength, on YADH forward and backward transfer were studied. YADH can be transferred into and out from the reversed micelles under mild conditions (only by regulation of solution pH and salt concentration) with the successful recovery of most YADH activity. Both forward and backward extractions occurred when the aqueous phase pH＞pI with electrostatic attraction between YADH and CTAB. The recovery of YADH activity and purification factor have been improved with addition of a small amount of affinity CB. The recovery of YADH activity obtained was ～99% and the purification factor was about 4.0-fold after one cycle of full forward and backward extraction. The low ionic strength in the initial aqueous phase might be responsible for the YADH transfer into the reversed micellar phase.

The relationship between activities of hepatic and gastric alcohol dehydrogenase and occurrence of chronic alcoholic liver disease

Objective: To investigate the role of hepatic and gas-tric alcohol dehydrogenase (ADH) in different pa-thologic stages of alcoholic liver disease (ALD).Methods: Thirty-nine Wistar rats were divided ran-domly into two groups: model group (24) and con-trol group (15). The ALD model was established byinfusing alcohol into the stomach. After hepatic andgastric tissues had been stained by enzyme histo-cyto-chemistry assay, the activity varieties of hepatic andgastric ADH were observed by an optical micro-scope, and the activity alterations were also deter-mined by LUZEX-F image analysis as a semi-quanti-tative method.Results: The activity of hepatic ADH gradually in-creased, but that of gastric ADH gradually decreasedin the different pathologic stages of alcoholic liverdisease. There was a significant difference betweenthe model group and control group (P【0.05).Conclusions: Along with occurrence of ALD, the ac-tivity of hepatic ADH gradually increased, but thatof gastric ADH gradually decreased, showing thatthe activity alterations of hepatic and gastric ADHmay play an important role in the onset and develop-ment of ALD.

自杀质粒同源重组介导阴沟肠杆菌adh基因缺失突变体构建及其生物学特性

本研究旨在探究敲除阴沟肠杆菌(Enterobacter cloacae)代谢通路上乙醇脱氢酶基因对乙偶姻代谢合成的影响,进一步提高乙偶姻产量。实验基于E.cloacae SDM中的乙醇脱氢酶adh基因序列设计特异性引物,并构建adh基因自杀质粒pKR6K-∆adh。利用热激法将自杀质粒转入E.cloacae∆budCldh,成功得到多基因缺失菌株E.cloaca∆budC-ldh-adh,并进行发酵性能测试。研究结果显示,与出发菌株相比,新构建的重组菌株在敲除了负责乙醇合成的关键酶乙醇脱氢酶adh基因后,其乙偶姻的生产强度提高了20.6%,产量提高了22.6%,同时乙醇产量提高了92.8%。此外,由于通过基因改造阻断了多条分支代谢路径,流向丁二酸代谢路径的碳通量明显变多,丁二酸产量提高101.3%。本研究成果不仅为构建高效乙偶姻生产菌株提供了有价值的参考,也为乙偶姻的大规模工业生产奠定了基础。

高产乙醇肺炎克雷伯adhE缺失菌株构建及对其产乙醇能力的影响

目的探究基因adhE缺失对高产乙醇肺炎克雷伯菌株生长能力,乙醇脱氢酶活性及产乙醇能力的影响。方法以高产乙醇肺炎克雷伯菌株TH1为背景,利用温敏型质粒介导的同源重组技术构建基因adhE的缺失菌株。通过PCR扩增获取基因adhE片段并构建于pGEM-Teasy表达载体获得回补质粒,导入ΔadhE缺失菌株得到回补菌株,并将pGEM-Teasy空质粒导入高产乙醇肺炎克雷伯野生菌株及ΔadhE缺失菌株作为空载对照。在液体LB培养基中测定各菌株的生长曲线,使用试剂盒检测各菌株的乙醇脱氢酶活性并通过顶空法检测各菌株的乙醇产量。结果使用PCR技术明确ΔadhE缺失菌株与相关回补菌株构建成功。基因adhE缺失后,厌氧条件下高产乙醇肺炎克雷伯菌株的生长速度显著降低,而回补菌株的生长速度显著升高(P<0.05)。同时,相比于野生型菌株,ΔadhE缺失菌株的乙醇脱氢酶活性与乙醇产量显著降低,而回补菌株的乙醇脱氢酶活性与乙醇产量恢复到了野生型菌株的水平(P<0.05)。结论基因adhE的缺失减缓了高产乙醇肺炎克雷伯菌株厌氧条件下的生长速度,同时显著降低了高产乙醇肺炎克雷伯菌株的产乙醇能力。

超量表达 CsADH17 基因提高烟草抗旱性及香气成分分析

乙醇脱氢酶是植物香气物质合成的脂肪酸代谢等途径中的关键酶之一,对茶叶芳香物质的形成有着重要作用,因此探究CsADH17基因有利于茶树的生长发育与品质提升。以本课题组前期筛选的CsADH17基因为研究对象,克隆CsADH17基因并构建植物表达载体,遗传转化烟草,对转基因烟草非生物胁迫下CsADH17的表达量和相关生理指标进行测定与分析。结果表明,对转基因与野生型烟草进行模拟干旱胁迫后,转基因烟草植株在干旱胁迫处理下的CsADH17基因相对表达量比野生型植株高。野生型烟草植株和CsADH17转基因烟草植株在干旱胁迫处理21 d后,超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)的活性均高于野生型烟草;胁迫处理后,野生型烟草叶片中丙二醛(MDA)含量显著高于转基因烟草。香气成分分析,转基因烟草苯甲醛比野生型含量提高了44.4%,而烟碱含量比野生型提高不到5%。CsADH17基因在烟草干旱胁迫下具有抗旱作用,同时影响烟草的香气成分,为进一步揭示CsADH在茶树生长发育以及抗逆响应中提供参考。

Alcohol Dehydrogenase Activity in Cultured Cells from Different Tobacco (Nicotiana tabacum L.) Varieties

The activity of alcohol dehydrogenase (ADH) in cultured cells of various tobacco was determined. It was found that significant differences existed in cells of different varieties cultured under normal conditions and as well after treated with exogenous ethanol. The ADH activity had positive relation with the ability of the cells to catabolize exogenous ethanol, indicating that the main function of the ADH in tobacco cells was in the direction of converting ethanol to acetaldehyde.

厚壳贻贝乙醇脱氢酶激活肽的纯化、鉴定及其与ADH的相互作用

【目的】开发厚壳贻贝(Mytilus coruscus)乙醇脱氢酶(Alcohol dehydrogenase,ADH)激活肽,并探究其与ADH作用的分子机制。【方法】以体外ADH激活率为指标,通过凝胶柱层析色谱、反向液相色谱法(RP-LC)分离纯化分子质量小于1 ku的厚壳贻贝活性肽(Mytilus coruscus peptides,MCP),利用液相色谱串联质谱技术(LC-MS/MS)对高ADH激活性的肽组分进行鉴定,并通过分子对接技术探究其与ADH间的相互作用。【结果】MCP经色谱分离得到活性组分Y-I-I,鉴定并筛选出其中3条与ADH对接结合能较低的活性肽序列PPLYE、PPLYQ和APPLYQ,其中活性肽PPLYE体外ADH激活作用最佳,在PPLYE质量浓度为2.0 mg/mL时,ADH激活率达到77.35%,且呈剂量依赖性。分子对接结果显示,PPLYE可结合至ADH活性中心附近的疏水空腔内,通过疏水作用和氢键与受体蛋白ADH形成稳定复合物,其结合能最低,为-35.58 kJ/mol。【结论】PPLYE作为最佳的ADH激活肽,可通过疏水作用和氢键与ADH相互作用形成稳定的复合物来提高ADH活性,本研究为厚壳贻贝资源的开发利用及ADH激活肽的开发提供依据。

ADH1B与ALDH2在胃癌组织中的表达及其临床意义

目的探讨乙醇脱氢酶1B(alcohol dehydrogenase 1B,ADH1B)与乙醛脱氢酶2(aldehyde dehydrogenase 2,ALDH2)在胃癌组织中的表达及其临床意义。方法选取2017年05月至2019年10月期间在滁州市第一人民医院接受根治性切除的132例胃癌患者为研究对象,通过免疫组织化学染色(Immunohistochemistry,IHC)检测ADH1B与ALDH2在胃癌及癌旁组织中的表达。分析ADH1B、ALDH2表达与患者临床病理特征的关系,采用Kaplan-Meier曲线评估两者表达在胃癌中的预后价值。单、多变量Cox回归分析用于确定胃癌患者的独立预后因素。结果对132例组织样本的IHC分析显示ADH1B(41.5%vs 58.5%,χ^(2)=9.594,P=0.002)与ALDH2(38.8%vs 61.2%,χ^(2)=16.716,P<0.001)在胃癌组织中的阳性表达率显著低于其癌旁组织。与高表达组相比,ADH1B低表达在T_(3)-T_(4)期(χ^(2)=5.572,P=0.018)、pTNMⅢ期肿瘤(χ^(2)=4.675,P=0.031)中更为常见,淋巴血管侵犯率更高(χ^(2)=4.566,P=0.033)。ALDH2在低分化癌(χ^(2)=4.261,P=0.039)和pTNMⅢ期胃癌(χ^(2)=5.877,P=0.015)中表达更低,淋巴结转移率(χ^(2)=5.491,P=0.019)较高表达者更为频繁。生存分析表明ADH1B与ALDH2低表达是胃癌患者预后不良的标志。ADH1B低、高表达患者的3年总体生存(Overall survival,OS)率分别为52.3%和73.9%(χ^(2)=6.900,P=0.009),ALDH2低、高表达患者的3年OS率分别为49.8%和74.4%(χ^(2)=8.665,P=0.003)。ADH1B(HR=2.115,95%CI:1.133-3.946,P=0.019)与ALDH2低表达(HR=2.296,95%CI:1.207-4.367,P=0.011)是胃癌患者的独立预后因素。结论ADH1B与ALDH2在胃癌组织中呈明显低表达,两者可能是预测肿瘤进展与患者预后的分子标志物。

Achieving Enzyme Stability Using a Simple Fabrication Procedure： The Alcohol Dehydrogenase Example

The use of screen-printing biosensors has been updated in this article as a tool to analyze the electron transfer process involving redox proteins or enzymes. The aim of this research was to fabricate a simple apparatus which allowed the use of the enzymes （in the solid state） to maintain their stability. To prove this concept an enzyme in the solid state was mixed with the carbon ink and this mixture was used to print the working electrode. We choose as proving the alcohol dehydrogenase. The first reason is because it metabolizes the alcohol, which can be present in biological samples of blood, saliva and urine and also in the beverage; the second is that this enzyme is still a challenge to electrochemistry due to having lower stability in sensors. The results show that in this device the enzyme was active and stable during all the experiments and in the experimental conditions that could catalyze the ethanol to acetaldehyde. These devices have the advantage of being disposable, cheap and are easy to fabricate. And also, they do not need expensive tools to be fabricated, they only need 2μL of electrolyte or sample, and they need lower amounts of enzyme to permit electrochemical studies.

Efficient conversion of aromatic and phenylpropanoid alcohols to acids by the cascade biocatalysis of alcohol and aldehyde dehydrogenases

Benzyl and phenylpropanoid acids are widely used in organic synthesis of fine chemicals,such as pharmaceuticals and condiments.However,biocatalysis of these acids has received less attention than chemical synthesis.One of the main challenges for biological production is the limited availability of alcohol dehydrogenases and aldehyde dehydrogenases.Environmental microorganisms are potential sources of these enzymes.In this study,129 alcohol dehydrogenases and 42 aldehyde dehydrogenases from Corynebacterium glutamicum,Pseudomonas aeruginosa,and Bacillus subtilis were identified and explored with various benzyl and phenylpropanoid alcohol and aldehyde substrates,among which four alcohol dehydrogenases and four aldehyde dehydrogenases with broad substrate specificity and high catalytic activity were obtained.Moreover,a cascade whole-cell catalytic system including ADH-90,ALDH-40,and the NAD(P)H oxidase LreNox was established,which showed high efficiency in converting cinnamyl alcohol and p-methylbenzyl alcohol into the respective carboxylic acids.Remarkably,this biocatalytic system can be easily scaled up to gram-level production,facilitating preparation purposes.

A novel alcohol dehydrogenase in the hyperthermophilic crenarchaeon Hyperthermus butylicus

Hyperthermus butylicus is a hyperthermophilic crenarchaeon that produces 1-butanol as an end product.A thermostable alcohol dehydrogenase(ADH)must be present in H.butylicus to act as the key enzyme responsible for this production;however,the gene that encodes the ADH has not yet been identified.A novel ADH,HbADH2,was purified from a cell-free extract of H.butylicus,and its characteristics were determined.The gene that encodes HbADH2 was demonstrated to be HBUT_RS04850 and annotated as a hypothetical protein in H.butylicus.HbADH2 was found to be a primary-secondary ADH capable of using a wide range of substrates,including butyraldehyde and butanol.Butyraldehyde had the highest specificity constant,calculated as k_(cat)/K_(m),with kcat and apparent K_(m) values of 8.00±0.22 s^(-1) and 0.59±0.07 mM,respectively.The apparent Km values for other substrates,including ethanol,1-propanol,2-propanol,butanol,acetaldehyde,propanal,and acetone,were 4.36±0.42,4.69±0.41,3.74±0.46,2.44±0.30,1.27±0.18,1.55±0.20,and 0.68±0.04 mM,respectively.The optimal pH values for catalyzing aldehyde reduction and alcohol oxidation were 6.0 and 9.0,respectively,while the optimal temperature was higher than 90°C due to the increase in enzymatic activity from 60℃ to 90℃.Based on its substrate specificity,enzyme kinetics,and thermostability,HbADH2 may be the ADH that catalyzes the production of 1-butanol in H.butylicus.The putative conserved motif sites for NAD(P)^(+)and iron binding were identified by aligning HbADH2 with previously characterized Fe-containing ADHs.

Aldehyde dehydrogenase 2 overexpression inhibits neuronal apoptosis after spinal cord ischemia/reperfusion injury

Aldehyde dehydrogenase 2（ALDH2）is an important factor in inhibiting oxidative stress and has been shown to protect against renal ischemia/reperfusion injury.Therefore,we hypothesized that ALDH_2 could reduce spinal cord ischemia/reperfusion injury.Spinal cord ischemia/reperfusion injury was induced in rats using the modified Zivin＇s method of clamping the abdominal aorta.After successful model establishment,the agonist group was administered a daily consumption of 2.5%alcohol.At 7 days post-surgery,the Basso,Beattie,and Bresnahan score significantly increased in the agonist group compared with the spinal cord ischemia/reperfusion injury group.ALDH_2expression also significantly increased and the number of apoptotic cells significantly decreased in the agonist group than in the spinal cord ischemia/reperfusion injury group.Correlation analysis revealed that ALDH_2 expression negatively correlated with the percentage of TUNEL-positive cells（r=-0.485,P〈0.01）.In summary,increased ALDH_2 expression protected the rat spinal cord against ischemia/reperfusion injury by inhibiting apoptosis.

Association of genetic polymorphisms of aldehyde dehydrogenase-2 with esophageal squamous cell dysplasia

AIM:To demonstrate the possible associations between genetic polymorphisms of aldehyde dehydrogenase-2(ALDH2) and esophageal squamous cell dysplasia(ESCD).METHODS:All participants came from an area of high incidence of esophageal cancer and underwent an endoscopic staining examination;biopsies were taken from a non-staining area of the mucosa and diagnosed by histopathology.Based on the examinations,the subjects were divided into the control group with normal esophageal squamous epithelial cells and the ESCD group.ALDH2 genotypes of 396 cases were determined including 184 ESCD cases and 212 controls.The odds ratio(OR) and 95% confidence intervals(95% CI) were calculated by binary logistic regression models.RESULTS:The distribution of ALDH2 genotypes showed significant differences between the two groups.The adjustment factors were gender and age in the logistic regression models.Compared with 2*2/2*2 genotype,2*1/2*1 genotype was found to be a risk factor for ESCD,and the OR(95% CI) was 4.50(2.21-9.19).There were significant correlations between ALDH2 genotypes and alcohol drinking/smoking/history of esophageal cancer.CONCLUSION:The ALDH2 polymorphism is significantly associated with ESCD.