工程类外文文献原文及翻译论文

1.1钢筋混凝土 素混凝土是由水泥、水、细骨料、粗骨料（碎石或；卵石）、空气，通常还有其他外加剂等经过凝固硬化而成。将可塑的混凝土拌合物注入到模板内，并将其捣实，然后进行养护，以加速水泥与水的水化反应，最后获得硬化的混凝土。其最终制成品具有较高的抗压强度和较低的抗拉强度。其抗拉强度约为抗压强度的十分之一。因此，截面的受拉区必须配置抗拉钢筋和抗剪钢筋以增加钢筋混凝土构件中较弱的受拉区的强度。 由于钢筋混凝土截面在均质性上与标准的木材或钢的截面存在着差异，因此，需要对结构设计的基本原理进行修改。将钢筋混凝土这种非均质截面的两种组成部分按一定比例适当布置，可以最好的利用这两种材料。这一要求是可以达到的。因混凝土由配料搅拌成湿拌合物，经过振捣并凝固硬化，可以做成任何一种需要的形状。如果拌制混凝土的各种材料配合比恰当，则混凝土制成品的强度较高，经久耐用，配置钢筋后，可以作为任何结构体系的主要构件。 浇筑混凝土所需要的技术取决于即将浇筑的构件类型，诸如：柱、梁、墙、板、基础，大体积混凝土水坝或者继续延长已浇筑完毕并且已经凝固的混凝土等。对于梁、柱、墙等构件，当模板清理干净后应该在其上涂油，钢筋表面的锈及其他有害物质也应该被清除干净。浇筑基础前，应将坑底土夯实并用水浸湿6英寸，以免土壤从新浇的混凝土中吸收水分。一般情况下，除使用混凝土泵浇筑外，混凝土都应在水平方向分层浇筑，并使用插入式或表面式高频电动振捣器捣实。必须记住，过分的振捣将导致骨料离析和混凝土泌浆等现象，因而是有害的。 水泥的水化作用发生在有水分存在，而且气温在50°F以上的条件下。为了保证水泥的水化作用得以进行，必须具备上述条件。如果干燥过快则会出现表面裂缝，这将有损与混凝土的强度，同时也会影响到水泥水化作用的充分进行。 设计钢筋混凝土构件时显然需要处理大量的参数，诸如宽度、高度等几何尺寸，配筋的面积，钢筋的应变和混凝土的应变，钢筋的应力等等。因此，在选择混凝土截面时需要进行试算并作调整，根据施工现场条件、混凝土原材料的供应情况、业主提出的特殊要求、对建筑和净空高度的要求、所用的设计规范以及建筑物周围环境条件等最后确定截面。钢筋混凝土通常是现场浇注的合成材料，它与在工厂中制造的标准的钢结构梁、柱等不同，因此对于上面所提到的一系列因素必须予以考虑。 对结构体系的各个部位均需选定试算截面并进行验算，以确定该截面的名义强度是否足以承受所作用的计算荷载。由于经常需要进行多次试算，才能求出所需的 3 截面，因此设计时第一次采用的数值将导致一系列的试算与调整工作。 选择混凝土截面时，采用试算与调整过程可以使复核与设计结合在一起。因此，当试算截面选定后，每次设计都是对截面进行复核。手册、图表和微型计算机以及专用程序的使用，使这种设计方法更为简捷有效，而传统的方法则是把钢筋混凝土的复核与单纯的设计分别进行处理。 1.2土方工程 由于和土木工程中任何其他工种的施工方法与费用相比较，土方挖运的施工方法与费用的变化都要快得多，因此对于有事业心的人来说，土方工程是一个可以大有作为的领域。在1935年，目前采用的利用轮胎式机械设备进行土方挖运的方法大多数还没有出现。那是大部分土方是采用窄轨铁路运输，在这目前来说是很少采用的。当时主要的开挖方式是使用正铲、反铲、拉铲或抓斗等挖土机，尽管这些机械目前仍然在广泛应用，但是它们只不过是目前所采用的许多方法中的一小部分。因此，一个工程师为了使自己在土方挖运设备方面的知识跟得上时代的发展，他应当花费一些时间去研究现代的机械。一般说来，有关挖土机、装载机和运输机械的唯一可靠而又最新的资料可以从制造厂商处获得。 土方工程或土方挖运工程指的是把地表面过高处的土壤挖去（挖方），并把它倾卸到地表面过低的其他地方（填方）。为了降低土方工程费用，填方量应该等于挖方量，而且挖方地点应该尽可能靠近土方量相等的填方地点，以减少运输量和填方的二次搬运。土方设计这项工作落到了从事道路设计的工程师的身上，因为土方工程的设计比其他任何工作更能决定工程造价是否低廉。根据现有的地图和标高，道路工程师应在设计绘图室中的工作也并不是徒劳的。它将帮助他在最短的时间内获得最好的方案。 费用最低的运土方法是用同一台机械直接挖方取土并且卸土作为填方。这并不是经常可以做到的，但是如果能够做到则是很理想的，因为这样做既快捷又省钱。拉铲挖土机。推土机和正铲挖土机都能做到这点。拉铲挖土机的工作半径最大。推土机所推运的图的数量最多，只是运输距离很短。拉铲挖土机的缺点是只能挖比它本身低的土，不能施加压力挖入压实的土壤内，不能在陡坡上挖土，而且挖。卸都不准确。 正铲挖土机介于推土机和拉铲挖土机的之间，其作用半径大于推土机，但小于拉铲挖土机。正铲挖土机能挖取竖直陡峭的工作面，这种方式对推土机司机来说是危险的，而对拉铲挖土机则是不可能的。每种机械设备应该进行最适合它的性能的作业。正铲挖土机不能挖比其停机平面低很多的土，而深挖坚实的土壤时，反铲挖土机最适用，但其卸料半径比起装有正铲的同一挖土机的卸料半径则要小很多。在比较平坦的场地开挖，如果用拉铲或正铲挖土机运输距离太远时，则装有轮胎式的斗式铲运机就是比不可少的。它能在比较平的地面上挖较深的土（但只能挖机械本身下面的土），需要时可以将土运至几百米远，然后卸土并在卸土的过程中把土大致铲平。在挖掘硬土时，人们发现在开挖场地经常用一辆助推拖拉机（轮式或履带式），对返回挖土的铲运机进行助推这种施工方法是经济的。一旦铲运机装满，助推拖拉机就回到开挖的地点去帮助下一台铲运机。 斗式铲运机通常是功率非常大的机械，许多厂家制造的铲运机铲斗容量为8 m³，满载时可达10 m³。最大的自行式铲运机铲斗容量为19立方米（满载时为25 m³），由430马力的牵引发动机驱动。 翻斗机可能是使用最为普遍的轮胎式运输设备，因为它们还可以被用来送混凝土或者其他建筑材料。翻斗车的车斗位于大橡胶轮胎车轮前轴的上方，尽管铰接式翻斗车的卸料方向有很多种，但大多数车斗是向前翻转的。最小的翻斗车的容量大约为0.5立方米，而最大的标准型翻斗车的容量大约为4.5m³。特殊型式的翻斗车包括容量为4 m³的自装式翻斗车，和容量约为0.5 m³的铰接式翻斗车。必须记住翻斗车与自卸卡车之间的区别。翻斗车车斗向前倾翻而司机坐在后方卸载，因此有时被称为后卸卡车。 1.3结构的安全度 规范的主要目的是提供一般性的设计原理和计算方法，以便验算结构的安全度。就目前的趋势而言，安全系数与所使用的材料性质及其组织情况无关，通常把它定义为发生破坏的条件与结构可预料的最不利的工作条件之比值。这个比值还与结构的破坏概率（危险率）成反比。 破坏不仅仅指结构的整体破坏，而且还指结构不能正常的使用，或者，用更为确切的话来说，把破坏看成是结构已经达到不能继续承担其设计荷载的“极限状态”。通常有两种类型的极限状态，即： （1）强度极限状态，它相当于结构能够达到的最大承载能力。其例子包括结构的局部屈曲和整体不稳定性；某此界面失效，随后结构转变为机构；疲劳破坏；引起结构几何形状显著变化的弹性变形或塑性变形或徐变；结构对交变荷载、火灾和爆炸的敏感性。 （2）使用极限状态，它对应着结构的使用功能和耐久性。器例子包括结构失稳之前的过大变形和位移；早期开裂或过大的裂缝；较大的振动和腐蚀。 根据不同的安全度条件，可以把结构验算所采用的计算方法分成： （1）确定性的方法，在这种方法中，把主要参数看作非随机参数。 （2）概率方法，在这种方法中，主要参数被认为是随机参数。此外，根据安全系数的不同用途，可以把结构的计算方法分为： （1）容许应力法，在这种方法中，把结构承受最大荷载时计算得到的应力与经过按规定的安全系数进行折减后的材料强度作比较。 （2）极限状态法，在这种方法中，结构的工作状态是以其最大强度为依据来衡量的。由理论分析确定的这一最大强度应不小于结构承受计算荷载所算得的强度（极限状态）。计算荷载等于分别乘以荷载系数的活载与恒载之和。 把对应于不乘以荷载系数的活载和恒载的工作（使用）条件的应力与规定值（使用极限状态）相比较。根据前两种方法和后两种方法的四种可能组合，我们可以得到一些实用的计算方法。通常采用下面两种计算方法： 确定性的方法，这种方法采用容许应力。 概率方法，这种方法采用极限状态。 至少在理论上，概率法的主要优点是可以科学的考虑所有随机安全系数，然后将这些随机安全系数组合成确定的安全系数。概率法取决于： 2.1 Reinforced Concrete Plain concrete is formed from a hardened mixture of cement ,water ,fine aggregate, coarse aggregate (crushed stone or gravel),air, and often other admixtures. The plastic mix is placed and consolidated in the formwork, then cured to facilitate the acceleration of the chemical hydration reaction lf the cement/water mix, resulting in hardened concrete. The finished product has high compressive strength, and low resistance to tension, such that its tensile strength is approximately one tenth lf its compressive strength. Consequently, tensile and shear reinforcement in the tensile regions of sections has to be provided to compensate for the weak tension regions in the reinforced concrete element. It is this deviation in the composition of a reinforces concrete section from the homogeneity of 答题实属不易，请楼主谅解，求采纳~

可以看看这个 呵呵 是 土木专业英语上的课文 building types and designA building is closely bound up with people，for it provides with the necessary space to work and live in .As classified by their use ,buildings are mainly of two types :industrial buildings and civil buildings .industrial buildings are used by various factories or industrial production while civil buildings are those that are used by people for dwelling ,employment ,education and other social activities .Industrial buildings are factory buildings that are available for processing and manufacturing of various kinds ,in such fields as the mining industry ,the metallurgical industry ,machine building ,the chemical industry and the textile industry . factory buildings can be classified into two types single-story ones and multi-story ones .the construction of industrial buildings is the same as that of civil buildings .however ,industrial and civil buildings differ in the materials used and in the way they are used .Civil buildings are divided into two broad categories: residential buildings and public buildings .residential buildings should suit family life .each flat should consist of at least three necessary rooms : a living room ,a kitchen and a toilet .public buildings can be used in politics ,cultural activities ,administration work and other services ,such as schools, office buildings, parks ,hospitals ,shops ,stations ,theatres ,gymnasiums ,hotels ,exhibition halls ,bath pools ,and so on .all of them have different functions ,which in turn require different design types as well. Housing is the living quarters for human beings .the basic function of housing is to provide shelter from the elements ,but people today require much more that of their housing .a family moving into a new neighborhood will to know if the available housing meets its standards of safety ,health ,and comfort .a family will also ask how near the housing is to grain shops ,food markets ,schools ,stores ,the library ,a movie theater ,and the community center .In the mid-1960’s a most important value in housing was sufficient space both inside and out .a majority of families preferred single-family homes on about half an acre of land ,which would provide space for spare-time activities .in highly industrialized countries ,many families preferred to live as far out as possible from the center of a metropolitan area ,even if the wage earners had to travel some distance to their work .quite a large number of families preferred country housing to suburban housing because their chief aim was to get far away from noise ,crowding ,and confusion .the accessibility of public transportation had ceased to be a decisive factor in housing because most workers drove their cars to work .people we’re chiefly interested in the arrangement and size of rooms and the number of bedrooms .Before any of the building can begin ,plans have to be drawn to show what the building will be like ,the exact place in which it is to go and how everything is to be done.An important point in building design is the layout of rooms ,which should provide the greatest possible convenience in relation to the purposes for which they are intended .in a dwelling house ,the layout may be considered under three categories : “day”, “night” ,and “services” .attention must be paid to the provision of easy communication between these areas .the “day “rooms generally include a dining-room ,sitting-room and kitchen ,but other rooms ,such as a study ,may be added ,and there may be a hall .the living-room ,which is generally the largest ,often serves as a dining-room ,too ,or the kitchen may have a dining alcove .the “night “rooms consist of the bedrooms .the “services “comprise the kitchen ,bathrooms ,larder ,and water-closets .the kitchen and larder connect the services with the day rooms .It is also essential to consider the question of outlook from the various rooms ,and those most in use should preferably face south as possible .it is ,however ,often very difficult to meet the optimum requirements ,both on account of the surroundings and the location of the roads .in resolving these complex problems ,it is also necessary to follow the local town-planning regulations which are concerned with public amenities ,density of population ,height of buildings ,proportion of green space to dwellings ,building lines ,the general appearance of new properties in relation to the neighbourhood ,and so on .There is little standardization in industrial buildings although such buildings still need to comply with local town-planning regulations .the modern trend is towards light ,airy factory buildings .generally of reinforced concrete or metal construction ,a factory can be given a “shed ”type ridge roof ,incorporating windows facing north so as to give evenly distributed natural lighting without sun-glare .翻译：建筑类型和设计建筑物与人们有着紧密的联系，他为人们提供必要的空间，用以工作和生活。根据适用类型不同，建筑物可以分为两类：工业建筑和民用建筑。工业建筑包括各个工厂或工业生产所使用建筑，民用建筑是指那些人们用以居住，就业，教育和其他社会活动的建筑场所。工业建筑的厂房可用于采矿业，冶金工业，机械制造，化学工业和纺织工业等各类领域的加工和制造。厂房可分为两种类型：单层的和多层的。工业建筑也属于建筑的一种。但是，工业建筑与民用建筑所用的材料和建筑方式不同。民用建筑按使用可分为两大类：住宅建筑和公共建筑。住宅建筑要适应家庭生活。每个单位应包括至少三个必要客房：起居室，厨房和厕所。公共建筑可在政治，文化活动，管理工作和其他服务，如学校，写字楼，公园，医院，商店，车站，剧院，体育馆，宾馆，展览馆，洗浴池，等等。他们都有着不同的职能，这反过来又需要不同的设计类型。房屋是用以住人的. 其基本功能是提供住房的内容，但今天人们需要更多的住房内容。一个家庭在进入一个新的社区后将知道，现有住房不仅要符合其安全，健康和舒适等标准。还要考虑其附近是否有相应的配套设施，如食品市场，学校，商店，图书馆，电影院，以及社区中心等。在60年代中期住房最重要的价值是足够大的空间和方便的出入交通。大多数家庭会首选约半英亩面积土地的家庭住宅，这样将提供足够的空间的用以业余活动。在高度工业化的国家，许多家庭的首选是那种尽可能远离市中心商业圈的住房，即使距离上班地点不得不有一段距离。相当多的家庭首选是郊区的住房，因为他们的主要目的是要远离噪音，拥挤和混乱。拥有方便的公共交通使得距离不再是一个决定性因素，因为大多数人都是开着自己的汽车去上班了。人们现在主要感兴趣的是户型，房间的大小和卧室的数目。在工程项目开始之前，要做好建筑设计和施工流程,让人提前知道该建筑建成后是什么样子以及下一步应该做什么。在建筑设计中要特别重视房间的布局，其目的是提供最大的便利与可能的用途。在一个住宅建筑设计中，布局可考虑以下三个方面： “白天” ， “夜晚”和“服务”。必须注意这些空间区域之间的连通交流。 “白天”房一般包括餐厅，起居室和厨房，但其他房间可能会增加，如书房，并有可能成为一个大厅。起居室通常是最大的，往往是一个餐厅，也或可能有厨房、凹室等。 “夜间”房间包括卧室、客房。“服务”用房间包括厨房，浴室，储藏室 ，和厕所等。厨房和储藏室需设置在一起，以方便其房间功能的使用。此外，还必须考虑各种客房的朝向问题，当然最好尽可能的将那些经常使用的房间朝南设置。然而，在考虑到周围的环境和地点、道路等多方面因素，往往很难达到最佳要求。在解决这些复杂的问题，还必须按照当地城市规划条例所涉及的对公共设施，人口密度，建筑物高度，绿化面积，建筑红线等的要求，还要考虑到有相邻建筑的情况，等等。尽管工业建筑需要符合当地城市规划条例但很少有标准化的工业楼宇。现代厂房建筑的趋势是轻质、通风。一般的钢筋混凝土结构或钢结构的工厂，可以得到一个“跌”型脊屋顶，把窗户开向北以便使分布均匀的自然采光不会直射进来造成刺眼。

可以去自己学习的图书馆电子阅览室寻找资源

Synthesis of optically pure ethyl (S)-4-chloro-3-hydroxybutanoateby Escherichia coli transformant cells coexpressingthe carbonyl reductase and glucose dehydrogenase genes由共表达碳酰还原酶和葡萄糖脱氢酶的大肠杆菌转化细胞合成纯光学（S）-4-氯-3-羟基丁酸乙酯Abstract The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate((S)-CHBE) was investigated. Escherichia coli cells expressing both the carbonyl reductase (S1) gene from Candida magnoliae and the glucose dehydrogenase (GDH) gene from Bacillus megaterium were used as thecatalyst. In an organic-solvent-water two-phase system,(S)-CHBE formed in the organic phase amounted to 2.58 M (430 g/l), the molar yield being 85%. E. coli transformant cells coproducing S1 and GDH accumulated 1.25 M (208 g/l) (S)-CHBE in an aqueous monophase system by continuously feeding on COBE, which is unstable in an aqueous solution. In this case, the calculated turnover of NADP+ (the oxidized form of nicotinamide adenine dinucleotide phosphate) to CHBE was 21,600 mol/mol. The optical purity of the (S)-CHBE formed was 100% enantiomeric excess in both systems. The aqueous system used for the reduction reaction involving E. coli HB101 cells carrying a plasmid containing the S1 and GDH genes as a catalyst is simple. Furthermore, the system does not require the addition of commercially available GDH or an organic solvent. Therefore this system is highly advantageous for the practical synthesis of optically pure (S)-CHBE.本本篇文献研究了利用COBE不对称合成（S）-4-氯-3-羟基丁酸乙酯（CHBE）。大肠杆菌细胞作为催化剂同时表达了来自念珠菌属magnoliae的碳酰还原酶和来自巨大芽孢杆菌的葡萄糖脱氢酶基因。在水/有机溶剂两相体系中，(S)-CHBE在有机相中的浓度可以达到2.58M（430g/l），摩尔产率达到85%。大肠杆菌的副产物S1和GDH也达到了1.25M（208g/l），COBE在水相中不稳定，所以(S)-CHBE可以在水单相中不停的生成。在这种情况下，适当的从NADP+到CHBE的转变达到了21,600 mol/mol。所形成的CHBE的旋光度在这种体系中100%对映体过量。在水相中用携带含有S1和GDH基因质粒的E. coli HB101作为催化剂不对称还原是比较简单的。并且，这种体系并不额外需要商业GDH或者有机溶剂。因此，这种体系对于实际合成纯光学活性的(S)-CHBE是非常方便的。Optically active 4-chloro-3-hydroxybutanoic acid esters are useful chiral building blocks for the synthesis of pharmaceuticals. The (R)-enantiomer is a precursor of L-carnitine (Zhou et al. 1983), and (S)-enantiomer is an important starting material for hydroxymethylglutaryl- CoA (HMG-CoA) reductase inhibitors (Karanewsky et al. 1990). Many studies have described the microbial or enzymatic asymmetric reduction of 4-chloro-3-oxobutanoic acid esters (Aragozzini and Valenti 1992; Bare et al.1991; Hallinan et al. 1995; Patel et al. 1992; Shimizu et al. 1990; Wong et al. 1985) based on the reduction by baker’s yeast (Zhou et al. 1983).We have previously showed that Candida magnoliae AKU4643 cells reduced ethyl 4-chloro-3-oxobutanoate (COBE) to (S)-CHBE with an optical purity of 96% enantiomeric excess (e.e.) (Yasohara et al. 1999). As this yeast has at least three different stereoselective reductases (Wada et al. 1998, 1999a, b), the (S)-CHBE produced by this yeast was not optically pure. From among these three enzymes, an NADPH-dependent carbonyl reductase, designated as S1, was purified and characterized in some detail (Wada et al. 1998). We cloned and sequenced the gene encoding S1 and overexpressed it in Escherichia coli cells. This E. coli transformant reduced COBE to optically pure (S)-CHBE in the presence of glucose, NADP+, and commercially available glucose dehydrogenase (GDH) as a cofactor generator (Yasoharaet al. 2000). Here, we describe the construction of three E. coli transformants coexpressing the S1 from C. magnoliae and GDH from Bacillus megaterium genes and analyze the reduction of COBE catalyzed by these strains. Previous reports on the enzymatic reduction of COBE to (R)-CHBE with an optical purity of 92% e.e. (Kataoka et al. 1999; Shimizu et al. 1990) recommended an organic- solvent two-phase system reaction for an enzymatic or microbial reduction, because the substrate (COBE) is unstable in an aqueous solvent and inactivates enzymes. We examined the reduction of COBE to optically pure (S)-CHBE by E. coli transformants in a water monophase system reaction and discuss the possible use of this type of reaction system in industrial applications。具有旋光性的（S）-4-氯-3-羟基丁酸乙酯在药物制剂的合成中是重要的手性化合物。其右旋体是L-卡尼汀的前体，其左旋体是羟甲基戊二酰辅酶A还原酶抑制剂的起始材料。许多研究描述了以面包酵母为基础微生物或者酶的COBE的不对称还原。我们先前已经知道利用来自念珠菌属magnoliae AKU4643 细胞催化COBE生成光学纯度96%的CHBE。这种酵母至少有三种立体选择性的还原酶，这种酵母产生的CHBE并非纯光学的，在这三种酶之中，NADPH-依赖碳酰还原酶，我们克隆并测序编码S1的基因，并在大肠杆菌中过表达。大肠杆菌转化细胞在葡萄糖，NADP+和商业化的葡萄糖脱氢酶作为辅酶因子的启动子催化COBE生成纯光学的CHBE。我们构建这三种大肠杆菌转化细胞共表达来自的S1和来自巨大芽孢杆菌的GDH，并分析COBE被这几种菌株催化还原的反应机理。先前的报道表明，利用酶催化还原COBE生成CHBE光学纯度可达92%，也提到了因为底物（COBE）在水相中不稳定，并且酶容易钝化，所以利用酶或者微生物在有机溶剂/水两相体系中催化反应。我们研究了在水单相体系中由COBE还原生成纯光学的CHBE，还讨论了这种反应体系在工业应用中可能的用途。Materials and methodsBacterial strain and plasmids The E. coli strains used in this study were JM109 and HB101.Plasmid pGDA2, in which the GDH gene from B. megaterium is inserted into pKK223-3, was kindly provided by Professor I. Urabe, Osaka University (Makino et al. 1989). Plasmids pSL301 and pTrc99A were purchased from Invitrogen (USA), and Amersham Pharmacia Biotech (UK), respectively. Plasmids pUC19 and pSTV28 (Homma et al. 1995; Takahashi et al. 1995) were purchased from Takara Shuzo (Japan).材料和方法菌株和质粒本次实验中使用的大肠杆菌是JM109 and HB101。来自B. megaterium的GDH基因插入到Pkk233-3质粒中，而带有GDH基因片段的pGDA2质粒由到由大阪大学的urabe教授提供。质粒pSL301和 pTrc99A是由美国的Invitrogen公司和英国的公司分别购买的。质粒pUC19和pST28是由日本takara公司购买的。The recombinant plasmid used in this study was constructed as follows (Fig. 1): Plasmid pGDA2 was double-digested with EcoRI and PstI to isolate a DNA fragment of about 0.9 kilobase pairs (kb) including the GDH gene. This fragment was inserted into the EcoRI-PstI site of plasmid pSL301 to construct plasmid pSLG. Plasmid pSLG was double-digested with EcoRI and XhoI to isolate a DNA fragment of about 0.9 kb including the GDH gene.这次实验使用的重组质粒构建如下：质粒pGDA2 被EcoRI 和 PstI双酶切从而分离出一个大小约为0.9kb的包含有GDH基因的DNA片段。这个片段被插入到质粒Psl301的EcoRI-PstI酶切位点从而构建出质粒pSLG。质粒pSLG被EcoRI和XhoI To construct plasmid pNTS1G, this 0.9-kb fragment was inserted into the EcoRI-SalI site of pNTS1, which was constructed to overproduce S1 as described previously (Yasohara et al. 2000). To construct plasmid pNTGS1, plasmid pNTG was first generated. Two synthetic primers (primer 1, TAGTCCATATGTATAAAGATTTAG,and primer 2 TCTGAGAATTCTTATCCGCGTCCT) were prepared for polymerase chain reaction (PCR) using pGDA2 as the template. The PCR-generated fragment was double- digested with NdeI and EcoRI and then inserted into the NdeI EcoRI site of plasmid pUCNT, which was constructed from pUC19 and pTrc99A, as reported (Nanba et al. 1999), to obtain pNTG. To construct plasmid pNTGS1, two synthetic primers (primer 3, GCCGAATTCTAAGGAGGTTAATAATGGCTAAGAACTTCTCCAACG, and primer 4, GCGGTCGACTTAGGGAAGCGTGTAGCCACCGTC) were prepared using pUCHE, which contains the S1 gene as the template. The PCR-generated fragment was double-digested with EcoRI and SalI and then inserted into the EcoRI-SalI site of pNTG to obtain pNTGS1. Plasmid pNTS1G, pNTGS1 or pNTG was transformed into E. coli HB101.构建pNTS1是为了过表达前文所提到的S1，这个0.9kb大小的片段被插入到pNTS1的EcoRI-SalI酶切位点从而构建pNTS1G。为了构建质粒pNTGS1，首先需要构建pNTG。两个合成引物（引物1，TAGTCCATATGTATAAAGATTTAG和引物2，TCTGAGAATTCTTATCCGCGTCCT）和作为模板的pGDA2是PCR反应需要的。PCR得到的片段是由NdeI 和EcoRI双酶切和并插入到质粒pUCNT的NdeI EcoRI酶切位点来得到pNTG。根据报道，pUCNT是由pUC19和 pTrc99A构建而来。为了构建质粒pNTGS1，两个合成引物(引物 3, GCCGAATTCTAAGGAGGTTAATAATGGCTAAGAACTTCTCCAACG, and 引物 4, GCGGTCGACTTAGGGAAGCGTGTAGCCACCGTC)，包括了S1基因作为模板。Pcr产物片段被EcoRI和SalI双酶切然后被插入到pntg的EcoRI-SalI酶切位点得到pntg1.质粒pNTS1G, pNTGS1或者 pNTG都是导入大肠杆菌HB101.Plasmid pGDA2 was double-digested with EcoRI and PstI to isolate a DNA fragment of about 0.9 kb including the GDH gene. To construct plasmid pSTVG, this fragment was inserted into the EcoRI-PstI site of plasmid pSTV28. Plasmid pSTVG was transformed into E. coli HB101. 质粒pGDA2被EcoRI 和 PstI双酶切得到包含GDH基因的0.9kb大小的DNA片段。为了构建pSTVG质粒，这个片段被插入到pSTV28质粒的EcoRI-PstI的酶切位点。pSTVG质粒被导入到E. coli HB101。Medium and cultivationThe 2×YT medium comprised 1.6% Bacto-tryptone, 1.0% yeastextract, and 0.5% NaCl, pH 7.0. E. coli HB 101 carrying pNTS1,pNTG, pNTS1G, or pNTGS1 was inoculated into a test tube containing2 ml 2×YT medium supplemented with 0.1 mg/ml ampicillin,followed by incubation at 37 °C for 15 h with reciprocal shaking.This preculture (0.5 ml) was transferred to a 500-ml shakingflask containing 100 ml 2×YT medium. The cells were cultivatedat 37 °C for 13 h with reciprocal shaking. E. coli HB101 carryingpNTS1 and pSTVG was similarly cultivated in 2×YT mediumsupplemented with 0.1 mg/ml ampicillin and 0.1 mg/ml chloramphenicol.培养基和培菌2*YT培养基 包含有1.6%细菌用胰蛋白胨，1.0%酵母提取物，0.5% NaCl，pH7.0.携带有pNTS1,pNTG, pNTS1G, 或 pNTGS1的大肠杆菌HB101被接种到有0.1mg/ml氨苄青霉素的2ml的2*YT培养基，37°C摇床15小时。将0.5ml菌液接种到100ml2*YT培养基的500ml烧瓶中。在37°C摇床培养13小时。携带有pNTS1 和 pSTVG质粒的大肠杆菌HB101在2*YT培养基中培养方法相似，只是培养基中要加入0.1 mg/ml的氨苄青霉素和 0.1 mg/ml的氯霉素。Preparation of cell-free extracts and the enzyme assay Cells were harvested from 100 ml of culture broth by centrifugation, suspended in 50 ml of 100 mM potassium phosphate buffer (pH 6.5), and then disrupted by ultrasonication. The cell debris was removed by centrifugation; the supernatant was recovered as the cell-free extract. Carbonyl reductase S1 activity (COBE-reducing activity) was determined spectrophotometically as follows: The assay mixture consisted of 100 mM potassium phosphate buffer (pH 6.5), 0.1 mM NADPH, and 1 mM COBE. The reactions were incubated at 30 °C and monitored for the decrease in absorbance at 340 nm. The assay mixture for GDH activity consisted of 1 M Tris-HCl buffer (pH 8.0), 100 mM glucose, and 2 mM NADP+. The reactions were incubated at 25 °C and monitored for the increase in absorbance at 340 nm. One unit of S1 or GDH was defined as the amount catalyzing the reduction of 1 μmol NADP+ or oxidation of 1 μmol NADPH per minute, respectively. Protein concentrations were measured with a proteinassay kit containing Coomassie brilliant blue (Nacalai Tesque, Japan),using bovine serum albumin as the standard (Bradford 1976).无细胞抽提液和酶鉴定将100ml培养液离心收获菌体，用50ml0.1mol/LpH为6.5的磷酸缓冲液悬浮，然后超声粉碎。细胞碎片通过离心可以去除，收集上层清液就是无细胞抽提物。碳酰还原酶S1的活性由分光光度计测量如下：测定的混合物包括：0.1mol/LpH6.5的磷酸二氢钾缓冲液，0.1mMNADPH和1mMCOBE。反应在30°C条件下反应，并且随时监测其在340nm处的吸光值。测GDH混合物包括：1M pH 8.0的Tris-HCl的缓冲液，100mM的葡萄糖，2mM的NADP+。反应在25°C下进行，监测其在340nm处的吸光值。一个单位S1或GDH被定义为每分钟催化还原1μmol NADP+或氧化1 μmol NADPH的量。蛋白质的测定通过含有考马斯亮蓝的蛋白质测定试剂利用牛血清白蛋白作为标准进行测定。Study of enzyme stabilityOne milliliter of 100 mM potassium phosphate buffer (pH 6.5) containing the cell-free extracts of E. coli HB101 carrying pNTS1 (S1: 20 U/ml) was mixed with an equal volume of each test organic solvent in a closed vessel. After the mixture was shaken at 30 °C for 48 h, the remaining enzyme activities in an aqueous phase were assayed as described above. The mixture, containing 100 mM potassium phosphate buffer (pH 6.5), S1 (20 U/ml), and various concentrations of CHBE, was incubated at 30 °C for 24 h in order to study the enzyme’s stability in the presence of CHBE.The remaining enzyme activities were assayed as described above.酶稳定性的研究一毫升含有含有pNTS1质粒的E. coli HB101的无细胞抽提液的100mM磷酸氢二钾缓冲液（pH6.5）与等体积的有机溶剂混合。混合物在30 °C震摇48小时后，水相中残留的酶活力即是上述的酶活力。COBE reduction with E. coli cells expressing the S1 gene and E. coli cells expressing GDH genes in a two-phase system reaction The reaction mixture comprised 15 ml culture broth of E. coli HB101 carrying pNTG, 17 ml culture broth of E. coli HB101 carrying pNTS1, 1.6 mg NADP+, 4 g glucose, 2.5 g COBE, 25 ml n-butyl acetate, and about 25 mg Triton X-100. The pH of the reaction mixture was controlled at 6.5 with 5 M sodium hydroxide. At 2 h, 1.25 g COBE and 2.5 g glucose were added to the reaction mixture. To compare the reaction by E. coli transformant coexpressing the GDH and S1 genes, 30 ml culture broth of E. coliHB101 carrying pNTS1G was used instead of culture broth of E. coli HB101 carrying pNTG and E. coli HB101 carrying pNTS1. Other components and the procedure were the same as described above.表达S1基因和GDH基因的大肠杆菌细胞在两相反应体系中的还原反应混合物包含有带有pNTG质粒的大肠杆菌HB101的菌液15ml，pNTS1质粒的大肠杆菌HB101的菌液17ml，1.6 mg NADP+，4 g葡萄糖，2.5g的COBE，25ml的n-butyl acetate丁酰醋酸盐和大约25mg的聚乙二醇辛基苯基醚Triton X-100。用5M的NaOH溶液将pH控制在6.5。在反应两小时后，加入1.25gCOBE和2.5g葡萄糖到该混合物中。比较大肠杆菌转化细胞共表达GDH和S1基因，携带有pNTS1G质粒的大肠杆菌HB10130ml菌液取代了携带有pNTG和pNTS1质粒的大肠杆菌HB101菌液。其他的成分和步骤和上述的方法相似。 COBE reduction to (S)-CHBE in a two-phase system reaction The reaction mixture contained 50 ml of culture broth of an E. coli HB101 transformant, 3.2 mg NADP+, 11 g glucose, 10 g COBE, 50 ml n-butyl acetate, and about 50 mg Triton X-100. The reaction mixture was stirred at 30 °C, and the pH was controlled at 6.5 with 5 M sodium hydroxide. Five grams of COBE/5.5 g glucose and 10 g COBE/11 g glucose were added to the reaction mixture at 3 h and 7 h, respectively; 3.2 mg NADP+ was added at 26 h.COBE在两相系统中还原生成（S）-CHBE反应混合物包含50ml E. coli HB101转化细胞的培养液，3.2mgNADP+,11g葡萄糖，10gCOBE，50ml丁酰醋酸，和大概50mg聚乙二醇辛基苯基醚Triton X-100.在30°C温度下将其混合均匀，并用5M的NaOH溶液将pH控制在6.5。在第3小时加入5gCOBE和5.5g葡萄糖或者在第7小时加入10gCOBE和11g葡萄糖，分别在第26小时加入3.2gNADP+。 COBE reduction to (S)-CHBE in an aqueous system reaction The reaction mixture was made up of 50 ml of culture broth of an E. coli HB101 transformant, 3.1 mg NADP+, 11 g glucose, and about 50 mg Triton X-100. The reaction mixture was stirred at 30 °C. Fifteen grams of COBE was fed continuously by means of a micro-feeding machine at a rate of about 0.02 g/min for about 12 h. The pH of the reaction mixture was controlled at 6.5 with 5 M sodium hydroxide. The reaction mixture was extracted with 100 ml ethyl acetate. The organic layer was dried over anhydrous sodium sulfate and then evaporated in vacuo. COBE在水相中还原成(S)-CHBE的反应反应的体系是由50ml大肠杆菌HB101转化细胞的菌液，3.1mgNADP+，11g葡萄糖和大约50mg聚乙二醇辛基苯基醚Triton X-100。反应混合物在30°C15mg的COBE通过微量添加机器以0.02 g/min的速率连续12小时恒定的加入到体系中。用5M的NaOH溶液将pH控制在6.5。反应混合物用100ml乙酸乙酯萃取。有机层用无水硫酸钠吸干，并在真空中脱水。Analysis The organic layer was obtained on centrifugation of the reaction mixture and was assayed for CHBE and COBE by gas chromatography. Optical purity of CHBE was analyzed by high-performance liquid chromatography (HPLC), as described previously (Yasohara et al. 1999).Enzymes and chemicals Restriction enzymes and DNA polymerase were purchased fromTakara Shuzo (Japan). COBE (molecular weight: 164.59) was purchasedfrom Tokyo Kasei Kogyo (Japan). Racemic CHBE (molecularweight: 166.60) was synthesized by reduction of COBE withNaBH4. All other chemicals used were of analytical grade andcommercially available.分析离心反应混合物得到的有机层通过气相色谱法测定其CHBE和COBE。COBE的光学纯度如前所述通过高效液相色谱法进行分析。酶和化学试剂限制性内切酶和DNA聚合酶由takara公司购得，COBE（分子量：164.59）由东京Tokyo Kasei Kogyo公司购得，消旋体CHBE（分子量166.6）通过COBE及NaBH4合成。所有其他化学试剂都是分析等级和商业化的试剂。Construction of E. coli transformants overproducing S1 and GDHTo express the carbonyl reductase S1 and GDH genes in the same E. coli cells, four expression vectors were constructed (Fig. 1). Plasmids pNTS1G and pNTGS1 contain the S1 gene from C. magnoliae, the GDH gene from B. megaterium, the lac promoter derived from pUC19, and the terminator derived from pTrc99A. Plasmid pNTS1 contains the S1 gene, the lac promoter derived from pUC19, and the terminator derived from pTrc99A. The enzyme activities in cell-free extracts of the E. coli transformants are shown in Table 1. E. coli HB101 cells carrying the vector plasmid pUCNT had no detectable S1 or GDH activity. E. coli HB101 carrying either pNTS1G or pNTGS1 showed S1 and GDH activity without isopropyl-β-D-thiogalactopyranoside (IPTG) induction. The S1 activities of these two transformants were lower than the GDH activities. To obtain a transformant whose S1 activity was equal to or greater than the level of GDH activity, we used a lower copy vector, pSTV28 (Homma et al. 1995; Takahashi et al. 1995), to express the GDH gene. It may be possible to raise the S1 activity by lowering the GDH activity. Plasmid pSTVG contains the GDH gene, the lac promoter, the chloramphenicol resistance gene, and the replicative origin derived from pACYC184 for compatibility with the plasmid pNTS1. In E. coli HB101 carrying pNTS1 and pSTVG, the S1 activity was higher than the GDH activity, but this GDHlevel may be too low to regenerate in a COBE reduction reaction as described below.过产生S1和GDH的大肠杆菌转化细胞的构建为了在同一大肠杆菌细胞中表达碳酰还原酶S1和GDH基因，要构建四个表达型载体。质粒pNTS1G 和 pNTGS1包含有来自C. magnoliae的S1基因，来自B. megaterium的GDH基因，来自pUC19的LAC启动子，从pTrc99A的来的终止子，质粒pNTS1包含有S1基因，来自pUC19的LAC启动子，从pTrc99A的来的终止子。在大肠杆菌转化细胞的无细胞抽提物的酶活力如表一所示。携带有运输质粒pUCNT的大肠杆菌细胞无法检测到其S1和GDH活性。携带有pNTS1G 或 pNTGS1质粒在没有IPTG的诱导下有S1和GDH的活性。在这两个转化菌种中，S1的活力小于GDH的活力。为了得到S1活性等于或者大于GDH的大肠杆菌转化菌株，我们使用低拷贝的载体pSTV28，来表达GDH基因。它可能可以通过降低GDH的活性从而提高S1的活性。质粒pSTVG包含有GDH基因，lac启动子，和氯霉素抗性基因，以及与pNTS1具有相容性的从pACYC184得来的复制起始位点。在携带有pNTS1和pSTVG的大肠杆菌转化细胞中，S1的活性要高于GDH的活性，但是GDH的活性可能会太低而在COBE还原反应中不能再生。 太长了，字数有限制，所以不能发完。分数我无所谓啦，我很少登录的。这应该算是基因工程的吧，是我以前自己翻的，不是很好。如果你要的话可以联系我的邮箱。