简单节杆菌3-甾酮-△1 -脱氢酶基因的克隆表达及定点突变研究

目的:将来源于简单节杆菌的3-甾酮-△~1-脱氢酶(3-ketosteroid-Delta(1)-dehydrogenase,KSDD)在大肠杆菌中进行表达,获得具有活性的脱氢酶;利用计算机预测KSDD的三级结构,并通过定点突变确定酶的关键位点以期优化脱氢酶的活性及性质。方法:克隆简单节杆菌编码KSDD的基因ksdd构建原核表达载体,以Escherichia coli BL21(DE3)为表达宿主构建重组菌并诱导表达,HPLC法检测重组酶催化4-AD脱氢的转化率;通过SWISS-MODEL同源建模分析KSDD结构,对预测的催化关键位点氨基酸残基进行定点突变并研究突变后重组酶的活性变化。结果:成功构建了表达脱氢酶KSDD的重组菌E.coli pET-22-ksdd,21℃下诱导表达后,重组酶对4-AD的转化率为27%;通过SWISS-MODEL同源建模预测出脱氢酶结构并对4个关键位点进行定点突变设计,获得突变子Y120R、Y320L、Y488F和G492Y。突变子Y120R和Y488F失活,证明其为酶的活性位点;突变子Y320L的转化率与野生型基本一致,但37℃反应条件下稳定性有所提高;突变子G492Y对4-AD的转化率是野生型的1.2倍,37℃条件下稳定性有所提高,是突变后氨基酸位点疏水性增加和周围静电作用改变所导致。结论:目前对简单节杆菌3-甾酮-△~1-脱氢酶结构分析及催化机理相关的研究较少,本研究验证了KSDD的活性位点,优化了酶的稳定性,为进一步对酶的性质进行定向改造打下了基础。     

单增李斯特菌核糖核酸酶Rnase Ⅲ RncS氨基酸突变对其降解RNA活性的影响

为研究单增李斯特菌(LM)核糖核酸酶Rnase Ⅲ RncS氨基酸突变对RNA降解活性的影响。利用生物信息学软件分析单核细胞增生李斯特菌(LM)野毒株SB5中rncS基因编码的Rnase Ⅲ的结构域,并选择关键氨基酸利用基因重叠延伸PCR(SOE-PCR)技术对其进行了基因突变;然后将rncS突变基因片段D50A、E122A克隆至表达载体pET-32a(+),在大肠杆菌中利用IPTG进行诱导表达;应用SDS-PAGE和Western Blot鉴定重组蛋白的表达情况及其抗原特异性;通过体外酶活试验研究其对RNA降解活性的影响。结构域分析结果显示,LM-Rnase Ⅲ氨基酸序列含有1个双链RNA结合结构域(DSRM)和1个核酸酶结构域(RIBOc),其中结构域RIBOc含有5个活性位点。SDS-PAGE检测结果显示,表达的重组突变型Rnase Ⅲ -D50A和Rnase Ⅲ -E122 A蛋白相对分子质量均为42.5 kD,与理论值相符;Western blot分析表明重组突变型Rnase Ⅲ -D50A和Rnase Ⅲ -E122A蛋白可与LM阳性血清发生免疫学反应。体外酶活实验表明,Rnase Ⅲ发挥降解活性依赖于Mn2+或Mg2+,将其第50位天冬氨酸突变后,Rnase Ⅲ RncS的降解活性有所降低(P0.001);第122位谷氨酸突变后,Rnase Ⅲ RncS降解活性极显著下降(P0.0001),提示第122位谷氨酸是维持LM Rnase Ⅲ RncS酶活性的关键位点。     

定点突变提高醇脱氢酶LkTADH催化制备他汀关键手性砌块的酶活

(S)-6-氯-3-羰基-5-羟基己酸叔丁酯[(S)-CHOH]是他汀类药物合成的关键手性中间体。利用醇脱氢酶催化6-氯-3,5-二羰基己酸叔丁酯不对称合成(S)-CHOH是很有潜力的制备路线,目前存在的主要问题是醇脱氢酶催化活性较低。首先对来源于Lactobacillus kefir DSM 20587的醇脱氢酶的四点突变体LkTADH(A94T/F147L/A202L/L199H)进行回复突变,确定了关键位点147和202,并获得比酶活提高1倍的突变体MF147L-A202L。对这两个位点进行饱和突变,获得比酶活比LkTADH提高1.47倍的突变体MF147I-A202L。其比酶活为10.17U/mg,为目前文献报道最高水平。通过动力学分析和分子对接,分析了突变位点对酶活影响的机制,为后续研究奠定了良好的基础。     

硫氧还蛋白Lmo1903介导单增李斯特菌抵抗氧化应激的生物学功能研究

【目的】以单增李斯特菌(Listeria monocytogenes, LM)硫氧还蛋白Lmo1903为研究对象,研究其在细菌环境适应过程中的抗氧化应激生物学作用。【方法】使用生物信息学方法分析Lmo1903的进化关系和关键活性位点,使用酶切连接的方法构建Lmo1903蛋白表达载体,获得纯化的重组蛋白,以胰岛素为底物分析其氧化还原酶学活性;同时制备鼠源多克隆抗体,分析其在细胞内的定位;采用核苷酸定点突变技术构建CX1X2C基序中的半胱氨酸点突变蛋白,分析关键位点半胱氨酸对Lmo1903酶活的影响;采用同源重组原理构建lmo1903基因缺失株Δlmo1903和回补株CΔlmo1903,研究lmo1903在单增李斯特菌生长、运动和抗氧化应激方面发挥的功能。【结果】生物信息学分析显示,Lmo1903含有CX1X2C基序,与枯草芽孢杆菌(Bacillussubtilis)的TrxA的亲缘关系较近,属于硫氧还蛋白家族成员,主要定位在细菌细胞质中,具有较强的还原酶学活性,突变CX1X2C基序中的半胱氨酸残基会显著降低Lmo1903的还原酶活能力。缺失lmo1903不影响单增李斯特菌的生长能力,但显...     

深海细菌Pseudomonas sp.IOFA1醇脱氢酶酶学性质研究

[目的]对来自印度洋深海的一株假单胞菌醇脱氢酶(Adh)进行序列分析和酶活性质分析。[方法]首先以同源比对和进化树聚类为手段分析该酶序列信息。其次,在E.coli宿主中进行重组表达和镍柱亲和纯化后,对重组醇脱氢酶的酶活性质进行进一步地研究。[结果]结果显示Adh蛋白与其它物种已知醇脱氢酶的氨基酸序列最高相似性为81%,分属于第三类醇脱氢酶蛋白。酶学性质分析表明,重组酶Adh的最适作用温度为42℃,表现出良好的中低温适应性;最适p H值为5.0,在p H 4~6时具有较高的活性,表明Adh为酸性醇脱氢酶。Zn~(~(2+))、Na+在终浓度为0.5 mmol/L时对Adh有明显的激活作用,尤其是Zn~(2+)可使Adh的酶活显著提高11%。[结论]实现了adh基因在大肠杆菌的高效表达,为Adh的应用提供了理论依据。     

重组旋毛虫plancitoxin-1-like活性位点突变体蛋白的核酸酶活性

旋毛虫plancitoxin-1-like(Ts-Pt)是旋毛虫125种DNaseⅡ家族蛋白中唯一具有典型DNaseⅡ活性区域HKD基序的核酸酶,且普遍认为,组氨酸位点是DNaseⅡ的活性氨基酸位点。为研究Ts-Pt活性位点突变体蛋白的核酸酶活性,利用重叠PCR方法获得Ts-Pt活性位点突变体片段,以p ET-28a(+)为载体构建重组表达质粒并在大肠杆菌中诱导表达。重组Ts-Pt突变体蛋白经亲和层析纯化后进行SDS-PAGE分析。利用琼脂糖凝胶电泳法和核酸酶酶谱分析重组Ts-Pt突变体蛋白的核酸酶活性。成功构建含Ts-Pt突变体重组质粒的基因工程菌,SDS-PAGE和亲和层析纯化结果显示,重组Ts-Pt突变体蛋白呈包涵体表达。重组蛋白经复性后并没有表现出核酸酶活性,但核酸酶酶谱分析结果显示,包涵体表达的重组Ts-Pt突变体蛋白表现出降解DNA的能力。同时,N端和C端活性位点H及HCK和DHSK突变并不影响Ts-Pt的核酸酶活性,研究结果为进一步研究庞大的DNaseⅡ家族蛋白在旋毛虫发育和感染方面的作用提供一定的参考。     

ΦC31位点特异性整合酶DNA结合功能域的鉴定

DNA结合功能域的确定是阐明位点特异性重组酶整合机制的关键,而对酶DNA结合功能域进行突变研究是提高酶整合效率和整合特异性的重要方法.为了鉴定ΦC31位点特异性整合酶的DNA结合功能域,依据对ΦC31整合酶序列的生物信息学分析结果,利用PCR和克隆技术在pET22b原核表达载体上构建ΦC31整合酶重组截短突变体表达质粒,将获得的表达质粒转化入大肠杆菌BL21(DE3)菌株扩大培养并用IPTG诱导融合蛋白的表达,经镍柱纯化获得了纯度达90%以上的重组蛋白,分子量也与预期大小一致,Western印迹确定了重组蛋白的特异性.凝胶迁移滞后实验显示野生型以及截短突变体蛋白ΦC311-528、ΦC311-472、ΦC311-413能与细菌附着位点DNAattB和噬菌体附着位点DNAattP结合的条带,而截短突变体ΦC311-353、ΦC311-279、ΦC311-120观察不到相应的结合条带.6个截短突变体质粒在体内重组活性蓝白斑实验中均表现为蓝斑,显示出皆丧失体内重组活性.研究证实,ΦC31整合酶半胱氨酸富集域(第353～413位氨基酸)具有DNA结合的功能,而C末端缬氨酸富集区(第528～613位氨基酸)也与其重组活性相关.这为进一步了解ΦC31整合酶的结构与功能,最终引导其结构进化,提高其特异性和整合效率奠定了基础.     

酿酒酵母(Saccharomyces cerevisiae)半乳糖可诱导表达系统特性的研究

把大肠杆菌β-半乳糖苷酶基因克隆到带有酵母半乳糖可诱导启动子GAL1的穿梭表达质粒pYESZ中,并把得到的重组质粒分别转化到两种不同遗传性状的宿主菌中,其中一株菌为蛋白酶活性缺失90％以上的pep4－3突变菌株。通过比较两株重组菌产生的β-半乳糖苷酶活性水平发现在所述实验条件下,蛋白酶缺失突变菌株中产生的β-卜半乳糖苷酶活水平不仅均要高于另一对照菌株,并且pep4-3突变菌株表现出受葡萄糖阻遏的严紧程度高及对诱导反应迅速等特点。此外,带有重组质粒的pep4－3突变菌株在葡萄糖阻遏培养基中最大生长量和重组对照菌株基本相同,但β-半乳糖苷酶在pep4－3突变菌株中的表达对细胞生长的影响明显小于对照菌株。     

大肠杆菌BL21(DE3)lpxM突变株的构建

目的:lpxM基因失活可以产生极低内毒素活性的脂多糖。构建大肠杆菌BL21(DE3)的lpxM突变株,并考察该突变株的生长状态和表达重组蛋白的能力。方法:构建同源臂长500bp左右的打靶载体,借助Red同源重组系统,使E.coliBL21(DE3)的lpxM基因发生插入失活,再导入编码FLP位点特异性重组酶的质粒pCP20去除抗性基因。PCR鉴定发生插入突变的菌株,应用SDS-PAGE分析突变前后的脂多糖,考查对重组蛋白表达的影响。结果:PCR鉴定结果说明lpxM基因发生了插入突变。与出发株比较,突变株的脂多糖电泳图谱发生了明显变化,但其生长状态与表达重组蛋白的能力与出发株基本一致。结论:大肠杆菌BL21(DE3)的lpxM突变株可以用于重组蛋白的表达。     

Pseudomonas putida KT2440低特异性L-苏氨酸醛缩酶的表达、酶学性质及温度稳定性提高

【目的】从Pseudomonas putida KT2440基因组中,钓取低特异性L-苏氨酸醛缩酶基因(lta E),构建重组大肠杆菌。研究目标酶的酶学性质,和关键氨基酸位点突变对酶活和温度稳定性的影响。【方法】以P. putida KT2440基因组DNA为模板,PCR扩增出lta E基因,构建重组表达质粒p ET28a-KT2440并转化Escherichia coli BL21 (DE3),获得重组菌E. coli BL21 (DE3)/p ET-KT2440,利用Ni~(2+)柱亲和层析纯化低特异性L-苏氨酸醛缩酶(LTA),对关键氨基酸位点Thr206和Lys207实施定点突变。【结果】SDS-PAGE结果表明LTA在大肠杆菌中获得高效表达,分子量为40k Da左右,与理论值大小相符。Ni~(2+)柱亲和层析纯化LTA,获得单一条带。利用双酶耦联法测得LTA酶活为5577.3U/mg,最适反应温度为50°C,最适p H为8.0。在温度低于45°C,p H 5.0-9.0时,重组酶较稳定。LTA酶的Km和kcat值为23.95 mmol/L和19216.6 s–1。Mg~(2+)、Ca~(2+)金属离子对LTA有明显的促进作用,而Ni~(~(2+))、Cu~(2+)、Zn~(2+)、Fe~(2+)等对酶有明显的抑制作用。该酶在叔丁基甲基醚溶剂中具有良好的耐受性,在叔丁基甲基醚中保存1h后仍保留90%以上的酶活。Thr206Ser突变明显提高了酶对温度的稳定性。Lys207对酶催化功能是必需的,该位点突变对酶活都是致死的。【结论】克隆并表达P. putida KT2440的LTA酶,研究了酶学性质,通过定点改造提高了酶的温度稳定性,筛选获得一种酶耐受性好的有机溶剂,为LTA酶在有机溶剂中高效稳定催化β-羟基-α-氨基酸奠定了较坚实的研究基础。     

Molecular analysis of alcohol dehydrogenase electromorphs in wild type and transformed Drosophila melanogaster

The protein expressed by the alcohol dehydrogenase locus (Adh) in D. melanogaster comprises a small group of electromorphs. We are able to study the expression of these electromorphs by electrophoretic separation and subsequent probing of blots of the separated polypeptides with antiserum for alcohol dehydrogenase (ADH). In the present study we have utilized this technique to study and compare the ADH electromorphs in wild type D. melanogaster with D. melanogaster transformants which carry an Adh gene from D. grimshawi, D. hawaiiensis or D. affinidisjuncta and produced functional ADH (10, 19). We have determined that polypeptides are produced by the donor loci in the transformed flies and further show that although the molecular weight of the expressed polypeptides is similar to D. melanogaster electromorphs, the isoelectric points are not similar. Thus this methodology offers the potential to study naturally occurring ADH electromorphs and null alleles independent of enzymatic activity assays.     

Developmental profile and tissue distribution of Drosophila alcohol dehydrogenase: an immunochemical analysis with monoclonal antibodies.

To analyze Drosophila alcohol dehydrogenase gene (Adh) expression and tissue distribution at various developmental stages, we devised several immunochemical techniques making use of monoclonal antibodies against Drosophila alcohol dehydrogenase (ADH), which had been obtained previously. We here report their application to analyze the expression of Adh in a wild-type strain of D. melanogaster. s-ELISA tests were performed to evaluate fluctuations in ADH content and specific activity during development in individual organs as well as in whole individuals. In all cases, ADH specific activity appeared to be quite constant, which implies that variations in enzyme activity reflect differences in protein content. Immunoblottings of crude homogenates revealed immunoreactive low relative molecular mass peptides in addition to the 27 KD monomeric band, showing a conserved banding pattern in different organs and developmental stages. Immunohistochemical assays on whole organs were used to analyze the general pattern of ADH distribution. Immunoperoxidase staining of cryosections proved to be of crucial relevance, as it yielded full details of the tissue localization of ADH within the ADH-positive organs. We have shown not only that ADH displays a specific distribution in some organs but also that the enzyme is restricted to certain cell types.     

Diplotype trend regression analysis of the ADH gene cluster and the ALDH2 gene: multiple significant associations with alcohol dependence

The set of alcohol-metabolizing enzymes has considerable genetic and functional complexity. The relationships between some alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) genes and alcohol dependence (AD) have long been studied in many populations, but not comprehensively. In the present study, we genotyped 16 markers within the ADH gene cluster (including the ADH1A, ADH1B, ADH1C, ADH5, ADH6, and ADH7 genes), 4 markers within the ALDH2 gene, and 38 unlinked ancestry-informative markers in a case-control sample of 801 individuals. Associations between markers and disease were analyzed by a Hardy-Weinberg equilibrium (HWE) test, a conventional case-control comparison, a structured association analysis, and a novel diplotype trend regression (DTR) analysis. Finally, the disease alleles were fine mapped by a Hardy-Weinberg disequilibrium (HWD) measure (J). All markers were found to be in HWE in controls, but some markers showed HWD in cases. Genotypes of many markers were associated with AD. DTR analysis showed that ADH5 genotypes and diplotypes of ADH1A, ADH1B, ADH7, and ALDH2 were associated with AD in European Americans and/or African Americans. The risk-influencing alleles were fine mapped from among the markers studied and were found to coincide with some well-known functional variants. We demonstrated that DTR was more powerful than many other conventional association methods. We also found that several ADH genes and the ALDH2 gene were susceptibility loci for AD, and the associations were best explained by several independent risk genes.     

Genetic determination of the developmental program for maize scutellar alcohol dehydrogenase: Involvement of a recessive,trans-acting,temporal-regulatory gene

The developmental program of alcohol dehydrogenase (ADH) activity in the scutellum of maize strain R6-67 is different from that of W64A. The level of scutellar ADH activity in R6-67 remains relatively high during the course of early sporophytic development as compared to the commonly observed pattern. In the typical inbred strain W64A, the activity of ADH declines substantially during that period. The variance values from the crosses between R6-67 and W64A reveal that the trait is under genetic control. Detailed genetic analysis suggests that a single gene is responsible for the altered developmental program of ADH activity in R6-67. This gene meets the criteria for temporal regulatory genes and is different from Adh2, the structural gene which codes the ADH-2 isozyme. We have designated this gene as Adr1 (alcohol dehydrogenase regulator, #1). Adr1 is unlinked to Adh2. There is no de novo synthesis of ADH in the scutellum during germination, and the difference in the activity level reflects the difference in the amount of enzyme protein as demonstrated by density labeling and rocket immunoelectrophoresis. Thus, it appears that Adr1 may regulate the degradation of ADH.     

酿酒酵母乙醇脱氢酶2(ADH2)基因的克隆表达及活性验证

为了从酿酒酵母Saccharomyces cerevisiae中克隆出乙醇脱氢酶2(Alcoholdehy drogenase2,ADH2)基因并使之在大肠杆菌中高效表达。以酿酒酵母细胞中提取的总RNA为模板,通过反转录获得酿酒酵母乙醇脱氢酶2基因,连接到表达载体pTAT上,得到重组表达质粒pTAT-ADH2,将此重组质粒转化到大肠杆菌BL21中,重组工程菌株经IPTG诱导表达得到ADH2蛋白。将该蛋白纯化后,在体外进行活性检测和小鼠体内进行毒理试验,检测ADH2的酶活性。测序结果表明克隆的基因与GenBank中所报道的adh2基因序列有90%的同源性,经SDS-PAGE电泳分析,目的蛋白得到了有效表达,蛋白条带扫描分析表明,表达量占总蛋白的50%左右,纯化得到的蛋白在小鼠体内进行毒理试验,显示出一定的活性。酿酒酵母adh2基因的克隆正确,不仅在大肠杆菌中进行了高效表达而且表现出了较好的酶活性。     

ADH enzyme activity and Adh gene expression in Drosophila melanogaster lines differentially selected for increased alcohol tolerance

In Drosophila melanogaster, alcohol dehydrogenase (ADH) activity is essential for ethanol tolerance, but its role may not be restricted to alcohol metabolism alone. Here we describe ADH activity and Adh expression level upon selection for increased alcohol tolerance in different life-stages of D. melanogaster lines with two distinct Adh genotypes: Adh(FF) and Adh(SS). We demonstrate a positive within genotype response for increased alcohol tolerance. Life-stage dependent selection was observed in larvae only. A slight constitutive increase in adult ADH activity for all selection regimes and genotypes was observed, that was not paralleled by Adh expression. Larval Adh expression showed a constitutive increase, that was not reflected in ADH activity. Upon exposure to environmental ethanol, sex, selection regime life stage and genotype appear to have differential effects. Increased ADH activity accompanies increased ethanol tolerance in D. melanogaster but this increase is not paralleled by expression of the Adh gene.     

Overexpression, purification and properties of alcohol dehydrogenase IV from Saccharomyces cerevisiae

We have purified ADHIV, a novel alcohol dehydrogenase (ADH) isozyme in the yeast Saccharomyces cerevisiae, after increasing the normally low amount of ADHIV protein in laboratory strains. This was done by overexpression of the structural gene (ADH4) on a 2micro-based multicopy vector. Characterization of the purified enzyme revealed a dimeric structure as well as a different substrate specificity and pH profile as compared to other alcohol dehydrogenase isozymes. On the other hand, we could demonstrate that ADHIV is activated by zinc ions, like the other yeast alcohol dehydrogenase isozymes, and not by ferrous ions, like a structurally similar alcohol dehydrogenase from the bacterium Zymomonas mobilis.     

Intragenic recombination in maize: pollen analysis methods and the effect of parental adh1 isoalleles

The ability to stain mature pollen grains for the presence of alcohol dehydrogenase (ADH) activity permits the quantitation of ADH( +) gametophytes at frequencies below 10(-6). This resolution allows reversion and genetic fine structure analyses. The rationale of pollen analysis follows Nelson's prototype studies with waxy. As with the waxy gene, revertant frequencies for seven Adh1-deficient ( Adh1(-)) alleles appear to be in excess of microbially derived expectations. Each of the seven Adh1(-) alleles were derived from one of three naturally occurring isoalleles. Based on Schwartz's protein level characterizations of the mutants' products, it was anticipated that the seven Adh1(-) alleles should recombine to yield ADH(+) cistrons in certain pairwise combinations. This expectation was not met. The parental "wild-type" isoalleles from which the mutants were derived appear to be structurally divergent. The discussion interprets these data in view of understanding naturally occurring cistronic variation.     

Cloning and sequence analysis of the gene encoding an NADP-dependent alcohol dehydrogenase in Mycobacterium bovis BCG.

The nucleotide sequence of a 1619-bp fragment of Mycobacterium bovis BCG containing the gene that encodes an alcohol dehydrogenase (ADH) has been determined. The M(r) calculated from the deduced amino acid (aa) sequence, as well as the N terminus, are in good accordance with those determined for the ADH purified from M. bovis BCG extracts. The M. bovis BCG cloned adh gene was expressed in Escherichia coli by its own promoter and the synthesized product shows ADH activity in the butane-1-ol-NADP system. Based on comparison of the aa sequence, this enzyme belongs to the zinc-containing, long-chain alcohol/polyol dehydrogenase family, which has been primarily described in eukaryotes. Of the 22 strictly conserved residues in this group, 19 are also conserved in M. bovis BCG ADH (BCGADH).