细胞破碎 [Chapter 4 Cell Disru.ppt

Chapter 4 Cell Disruption 第四章 细胞破碎 4.1 Cell Membranes 细胞膜 4.0 Summary 概述 4.2 Chemical Methods 化学破碎方法 4.3 mechanical disruption 机械破碎 4.0 Summary 概述 l Bioseparations usually begin with the separation of biomass from broth .The separation commonly uses filtration or centrifugation as described in Chapters 2 and 3. In many cases, the desired product is in the broth. Antibiotics, are commonly in the broth; so are extracellular enzymes, many polysaccharides, and most amino acids. In all these cases, the separated broth can be treated to isolate and purify the product, as a byproduct. l 生物分离的第一步是将生物机体从发酵液中分离，通常使用过滤和离 心等方法，这在第二、三章中已有陈述。大多数情况下，抗生素，胞 外酶，一些多糖，及氨基酸等目标产物存在于在发酵液中。在上述过 程中，需被分离的发酵液可被看作一种副产物来处理，以此分离和纯 化产物。 l In some cases, the products of interest are not in the broth but are in the biomass. In particular, most proteins produced in quantity by genetically manipulated bacteria are not excreted into the broth, but are precipitated within the cell. Lipids and some antibiotics are also trapped in the biomass. In a few cases, like bakers yeast, the desired product is the cell mass itself. In a few others, desired products like steroids can be extracted without rupturing the cells. In many cases, the product is trapped in the biomass: It is intracellular. l 有些目标产物不在发酵液中，而是存在于生物体中。尤其是由基因工程 菌产生的大多数蛋白质不会被分泌到发酵液中，而是在细胞内沉积。脂 类物质和一些抗生素也是包含在生物体中。还有一些目标产物就是细胞 本身，如面包酵母。还有，产物如类固醇不必通过细胞破碎提取。大多 数情况下，产物还是包裹在生物体内，属胞内产物。 l Releasing this trapped material usually involves rupturing the cell wall, and is the subject of this short chapter. The methods of cell rupture have largely been developed in biochemistry, and hence are of small scale. Their application to the larger scale operations implied by genetic engineering is speculative. The equipment which is used is not designed for biotechnology. l 使胞内产物释放出来一般需要破碎细胞壁，这也是这一章的主题。细胞 破碎的方法在生物化学领域中得到了很广泛的运用，但多数在小规模生 产中，在大规模生产尤其是基因工程中应用极少。 l In this chapter, we begin in Section 4.1 by reviewing the structure of the cell wall. We then consider the methods for cell rupture most likely to be useful at larger scales. These methods, listed in Table 4.0-1, are conveniently split into two groups, described as “chemical” and “mechanical.” In the chemical methods detailed in Section 4.2, the cell membrane is ruptured by osmotic pressure, dissolved by detergents, or enfeebled by organic solvents. These chemical cases are often gentle so that the products are not irreversibly denatured even while the cells are ruptured. Scale-up is easy: If we want to treat 10 times the biomass, we need to use 10 times as much chemical. l 4.1节回顾一下细胞壁的结构。从中可知，在大规模生产中，细胞破碎 方法是十分有用的。我们很容易将这些方法划分为两大类，化学法和机 械法，表4.0-1中已经列出。化学法我们将在4.2节作详细介绍，有渗透 冲击法、表面活性剂增溶法或有机溶剂溶解法。这些化学法较温和，细 胞破坏后产物也不会不可逆的变性，规模也容易放大：如果需要处理10 倍量的生物有机体，只需要加入10倍的化学药品剂量。 TABLE 4.0-1. Chemical Cell Disintegration Techniques MethodTechniquePrincipleStress CostExamples ChemicalOsmotic shockOsmotic rupture of membrane GentleCheapRupture of red blood cells Enzyme digestion Cell wall digested , providing disruption GentleExpensiveEgg lysosyme SolubilizationDetergents solubilize cell membrane GentleModerate- expensive Bile salts acting on E.coli Lipid dissolution Organic solvent dissolves in cell wall ModerateCheapToluene disruption of yeast Alkali treatment Saponification of lipids solubilizes membrane HarshCheap 方法技术原理效果成本举例 化学法渗透冲击渗透压破坏细胞温和便宜血红细胞的破 坏 酶消化法细胞壁被消化，使 细胞破碎 温和昂贵 增溶法表面活性剂溶解细 胞壁 温和适中胆盐作用于大 肠杆菌 脂溶法有机溶剂溶解细胞 壁并使之失稳 适中便宜甲苯破碎酵母 细胞 碱处理法碱的皂化作用使细 胞壁融解 剧烈便宜 表 4.0-1. 细胞化学破碎法 MethodTechniquePrincipleProduct CostExamples MechanicalHomogenization (blade type) Cells chopped in Waring blender ModerateModerateAnimal tissue and cells GrindingCells ruptured by grinding with abrasives ModerateCheap UltrasonicationCells broken with ultrasonic cavitation Harsh Expensive Cell suspensions at least on a small scale Homogenization (orifice type) Cells forced through small hole are broken by shear Harsh Moderate Large scale treatment of cell suspensions, Crushing in ball mill Cells crushed between glass or steel balls Harsh Cheap Large scale treatment of cell suspensions and plant cells TABLE 4.0-1. Mechanical Cell Disintegration Techniques 方法技术原理效果成本举例 机械法匀浆法（片型）细胞被搅拌器劈碎适中适中动物组织及动 物细胞 研磨法细胞被研磨物磨碎适中便宜 超声波法用超声波的空穴作 用使细胞破碎 适中昂贵细胞悬浮液小 规模处理 匀浆法（孔型）须使细胞通过的小 孔，使细胞受到剪 切力而破碎 剧烈适中细胞悬浮液大 规模处理 珠磨破碎法细胞被玻璃珠或铁 珠捣碎 剧烈便宜细胞悬浮液和 植物细胞的大 规模处理 表 4.0-1. 细胞物理破碎法 l The mechanical methods described in Section 4.3 are dominated by homogenization and by ball mill grinding. In both cases, cells are broken by high mechanical shear. Heat generation is a problem and scale-up is obscure: If we find that a method works at a laboratory scale, we often have little idea how it will work at a large scale. As a result, this part of the chapter is more a guide to planning experiments than a synopsis of engineering analysis. l 4.3节机械法主要有匀浆法和研磨法。这两种方法中，细胞通过高的 机械剪切力被破坏。热量的产生是该法的一个缺点，而且不容易规模 放大:如果一种方法可以在实验室规模下操作，我们并不知道这种方 法是否在大规模生产中可行。所以，这部分内容仅作为实验的指导， 而不是工程分析。 4.1 Cell Membranes 细胞膜 l At this point, we pause briefly to explore the physical structure of microbial membranes and the complexity of the problems which we face. At present, knowledge of this general structure does not provide a direct guide to methods of cell rupture. In the future, it may. As a result, we feel that a synopsis has merit. In this synopsis, we emphasize Gram-negative procaryotes. l 本节主要探寻细胞膜的物质结构及我们所面对的几个复杂问题。现在所 有的关于细胞膜结构的基本知识，并不能为细胞破碎方法提供基本的引 导。也许将来可以。正因如此，对此进行提纲切领的介绍很有必要。主 要强调的是革兰氏阴性原核生物。其细胞结构中没有细胞核：基因物质 位于单链DNA上。典型的生物是大肠杆菌，是生物技术研究的主体。通 过这种细胞生产了很多细胞重组的产物。 l Such cells have no nuclei: Their genetic factors are carried in a single strand of DNA. The best example of this type of cell is Escherichia coli, the host organism for many efforts in biotechnology. This is the cell which produces most recombinant products developed to date. l 此细胞结构没有细胞核：基因在单链DNA上。典型的是大肠杆菌 ，是生物技术研究的主体。通过这种细胞生产了很多细胞重组的 产物。 l The basic cell envelope for Gram-negative cells, shown in Figure 4.1- 1a, has three layers. The outer layer, about 8 nm thick, consists of a polymer containing both protein and lipopolysaccharide. The second thinner layer is peptidoglycan. Below this second layer is a gap , called the periplamic space, which is also 8nm thick. Enzymes are often located in this gap. Gram-positive procaroytes, shown in Figure 4.1-1 are missing the first outer layer, but have both the second peptidog1ycan layer and the periplasmic space. l 革兰氏阴性细胞结构如图4.1-1 所示。有三层：最外层约8mm厚， 酶大多数镶嵌在这层膜上。革兰氏阳性原核生物缺少最外层结构， 但有第二层肽聚糖层和胞浆空间。 Gram-negative procaryotes 革兰氏阴性原核生物（E.coli） l The third membrane layer, called the plasma membrane or the inner membrane, is common to both Gram-positive and Gram-negative organisms. It consists largely of phospholipids, but also contains dispersed proteins and metal ions. l 第三层为浆膜层或内膜层，革兰氏阴性菌和革兰氏阳性都有这一结构 ，该层主要由磷脂组成，还有分散的蛋白质分子和金属离子。 l These lipid molecules have two parts, a hydrophobic part and the hydrophilic part The hydrophobic part or “tail” often contains two alkyl groups; and the hydrophilic part or “head” often includes a charged group, a zwitterion, or an alcohol. In this inner membrane, the phospho1ipid tails aggregate together, and the heads are exposed to the water, as shown in Figure 4.1-2 The result is a lame11ar bi1ayer. l 磷脂分子由疏水和亲水基团组成。疏水基团尾部有两烷基基团，亲水 基团头部有两性基团，或者说是醇基，在内膜层中，磷脂的尾部聚集 在一起，头部暴露在水中，如图4.1-2所示，形成磷脂双分子层。 structure of microbial membranes 细胞膜结构 l These three layers have different functions. The outer membranes and the peptidoglycan layer provide mechanical strength; it is their rupture which is a central subject of this chapter. The weaker plasma membrane-the innermost layer-controls the permeability of the cell, including transport of nutrients into the cells interior and export of metabolites into the surrounding solution. l 这三层有不同功能。外层及肽聚糖层使细胞有一定的机械强度；破 坏该层是本章讨论的重点。浆膜层和细胞内膜控制细胞的渗透压， 即将营养物质运送到细胞内，将代谢物排出胞外。 l This cell interior, called the cytoplasm, is an aqueous solution of salts, sugars, amino acids, and bipo1ymers. The biopo1ymers include proteins, many of them enzymes; ribonucleic acid (RNA); and deoxyribonucleic acid (DNA). l 细胞膜内部包含的物质称为细胞质，它是一种液态的溶液，溶有很 多盐、糖、氨基酸和生物聚和物。生物聚和物包括蛋白质、酶、 RNA、DNA 。 l In naturally occurring procaryotes, the proteins are in solution; but in many genetically modified procaryotes, excess protein is synthesized and precipitated within the cytoplasm. Often, we seek to rupture the cell wall solely to recover this protein. In other cases, we want to remove only some of the layers to release specific enzymes. l 一些自然生长的细胞中蛋白质都溶解在细胞质中，在一些基因控 制细胞中，合成的过量蛋白质在胞浆中沉降。有时细胞壁的目的 就是为了获得蛋白质，有时则为了使细胞释放出特殊的酶。 l Eucaryotic cells, those with true nuclei, are structurally more l complicated than procaryotes. These cells, exemplified by the yeast in Figure 4.1-3, have a membrane surrounding the cell which is similar in structure to the procaryote membrane. l 真核细胞，具有真正的细胞核，其结构要比原核生物复杂的多，以图 4.1-3中所示的酵母菌为例，和原核细胞一样真核细胞也具有一层细 胞膜。 动物细胞 植 物 细 胞 模 式 图 l Eucaryote membranes apparently do contain steroids, which are absent in procaryote membranes. In addition to this surrounding membrane, the cells contain organelles l 真核生物的细胞膜中含有固醇，而原核生物中没有。除了细胞膜 ，真核细胞具有一些有特殊作用的细胞器。 l Each organelle has a specialized function: For example, the mitochondria are responsible for respirarion and the nucleus contains the DNA (as chromosomes). Each structure is surrounded by a membrane which is similar to the inner plasma membrane of procaryotes. The increased structural complexity of the eucaryotes is not mirrored by increased chemical complexity. l 每个细胞器也都由一层膜包着与原核细胞的内膜浆膜层比较类似。真核生 物的结构复杂并不反映其性质也很复杂。 4.2 CHEMICAL METHODS 化学方法 l The chemical methods of cell rupture listed in Table 4.0-1 are dominated by osmotic shock, detergent so1ubilization, and lipid dissolution. These three methods are described in the following paragraphs. Before this description, we want to give some brief generalizations about the other two methods: enzyme digestion and alkali treatment. 表4.0-1介绍了主要的几种化学方法，有渗透冲击法，表面活性 剂增溶法、脂溶法。首先简单的介绍一下酶消化法和碱处理法。 l Both enzyme digestion and alkali treatment are effective methods of cell rupture, but each has a major disadvantage. 酶消化法和碱处理法都是细胞破碎的有效方法，但 是也都有各自的缺点。 l The disadvantage of enzyme digestion is the cost of the enzymes which. Frequently makes this method prohibitive at a large scale. This is unfortunate, because the method is both gentle and selective. It is nothing more than adding to a cell suspension enzymes which react quickly with the cell walls and destroy them. Because the enzymes selectively catalyze these cell wall reactions, they react only sparingly with other solutes within the cell. 酶消化法的缺点在于酶的价格昂贵限制了在大规模生产中的使 用，虽然条件温和、具有选择性。细胞悬浮液中加入酶能迅速和 细胞壁反应并破坏它们。酶选择性的催化细胞壁反应，不破坏细 胞内的其它物质。 l Alkali treatment is the antithesis of enzyme digestion, for it is harsh rather than gentle, nonselective rather than specific, and cheap rather than expensive. Alkali added to a cell suspension reacts with the cell walls in a number of ways, including the saponification of lipids in the cell walls. 碱处理法和酶消化法相反，反应激烈，不具选择性，而且较便宜。 碱加入细胞悬浮液中后和细胞壁进行了多种反应，包括使磷脂皂化 。 l In a sense, the treatment is an extreme example of so1ubilization, for it converts cell wall components into detergents. Including protein denaturations. As a result, the method tends both to rupture cell walls and to destroy products. Even though it is cheap, alkali treatment is less useful than the three methods which are detailed next. 该操作是细胞增溶的很好的例子，因为它使细胞壁的成分溶于 表面活性剂，也使蛋白质变性。该法不仅破坏了细胞壁也破坏了 产物，因此即便很便宜，碱处理也是一种很不常用的方法。 A、Osmotic Shock(渗透冲击法) l The simplest of the three major chemical methods is osmotic shock. This is nothing more than dumping a given volume of cells into pure water- often about twice the volume of cells. The cells swell because they contain solutes which cause an osmotic flow of water into the cells. In some cases, they swell so much that they burst. Their contents, released into the surrounding solution, can now be separated using the methods in following chapters. 三种主要细胞破碎化学方法中最简单的是渗透冲击法。此法将一定 体积的细胞液加到2倍体积的水中，细胞中溶质浓度高，水不断进入 细胞，使细胞膨胀，最后导致破裂。细胞破裂后释放到周围环境中的 胞内物可用后面章节介绍的方法分离。 l The susceptibility of cells to lysis depends strongly on their type. Red blood cells are easily lysed. Animal cells often can be lysed, but only after animal tissue has been mechanically minced or homogenized, as described in Section 4.3. Plant cells are much more difficult to 1yse, for their cell walls often contain strong woody material which is relatively impermeable to osmotic flow. 细胞破碎的难易决定于其类型，红血球细胞容易溶破，动物细胞只 有当其组织被用4.3节所介绍的方法机械切碎或匀浆后才易溶破。植 物细胞很难溶破，因为植物细胞中含有大量的木质成分，通过渗透 流很难渗透。 l The osmotic flow involved comes from the osmotic pressure, which is surprisingly large. To estimate the size of this pressure, we turn to the definition of chemical equilibrium, which states that the chemical potential of water H20 must be constant： 渗透流的动力来自渗透压，渗透压可能很大。可以通过化学平 衡来估算，水的化学电势是常数，即： l H20 (outside) = H20 (inside) (4.2-1) l The chemical potential of the pure water outside must include a reference value and a pressure correction; the corresponding potential inside involves a reference value, pressure correction, and a correction for solution concentration. For an ideal incompressible solution, these corrections convert Eq. (4.2-1) into the following. 细胞外纯水的化学电势包括标准化学电势和压力修正项；与之平衡 的胞内的化学电势包括三项，即标准化学电势、压力修正相和浓度 修正项。对于理想的不可压缩溶液，这些修正相和式（4.2-1）可改 写为： l 0 H20+VH2oPout = 0 H20+VH2oPin+RT ln(1-x1) (4.2-2) l Where 0H20 is the reference value,-VH2o is the partial molar volume of water, and x1 is the total mole fraction of solutes inside the cell. If the cells contents are a dilute solution, -VH2o is almost equal to the molar volume of water -VH2o , and x1 is small l 0H20标准化学电势 VH2o水的偏摩尔体积 x1细胞内所有溶质的总摩尔分数 若包内物为稀溶液，其偏摩尔体积等于纯水的摩尔体积。且x1很 小时就有： l Thus l Pout-Pin=(RT/-VH2o)ln(1-x1) =(RT/-VH2o) (-x1-) =-RTc1+ (4.2-3) l This relation is called vant Hoffs law.(该式称为范 特苛夫定律。) l Thus at equilibrium, the pressure outside the cell must be less than that inside. If this is not the case, then water will flow into the cell and potentially lyses the cell. We can use Eq. (4.2-3) to estimate the size of the pressure. Many cells have a solute concentration roughly equivalent to 0.1 M NaCl, or 0.2 M solutes. 由该方程知细胞内压力必须小于细胞外 压力，否者，水会流入细胞内，溶破细胞。我们可以采用等式 4.2-3 估算出渗透压的大小。许多细胞内溶质浓度大约为0.1M NaCl 或0.2M溶质 。 l This osmotic pressure is, for us, always surprisingly large. This large pressure is what can lead to cell rupture. 由此可见渗透压必须很大，才导致了细胞的破碎。 B 、Solubilization 增溶法 l The second major method of chemically rupturing cells is solubilization by detergents. Typically,a concentrated detergent solution is added to about half the solutions volume of cells. The detergent disrupts the cell membrane. The resulting suspension can be centrifuged to remove cell fragments, and then run through an adsorption column or an extractor to isolate the product. 第二种是利用表面活性剂的增溶法。最典型的是将体积为细胞体 积两倍的某浓度的表面活性剂加入到细胞中。表面活性剂能将细 胞壁破碎，制成的悬浮液可用离心分离除去细胞碎片，再用吸附 柱或萃取剂分离制得产品。 l The reason that solubilization is effective lies in detergent chemistry. This chemistry depends on detergent structures like those in Figure 4.2-1. All these structures have a hydrophilic portion, which is often ionic, and a hydrophobic part, which is frequently a hydrocarbon. 方法有效在于表面活性剂的化学性质，化学性质由图4.2-1所示 的化学结构表示。结构中有一个亲水基团，通常是离子；一个疏 水基团，通常是烃基。 l As a result, these detergents are all amphipathic, capable of interacting with both water and lipid. 表面活性剂通常是两性的，既能和水作用也能和脂作 用。 l This amphipathic nature holds whether the detergents are anionic, cationic, or nonionic. All types are illustrated in the figure. Sodium dode-cylsulfate (SDS), which is the example given, is the most widely studied of the anionic detergents. The anionic materials also include the soaps, which are the salts of fatty acids. 无论表面活性剂是阴离子、阳离子还是非离子型，都是两性的。 SDS（十二烷基磺酸钠）是典型的阴离子表面活性剂。阴离子表面 活性剂还包括肥皂（脂肪酸盐）。 l Because soaps depend on a carboxylic acid group, they are effective detergents only at high pH, where this group remains ionized. They are ineffective in hard water, where calcium ions can react with them to form insoluble precipitates. 由于肥皂的增溶作用依赖于羧基，因此只有在Ph 值较 高，羧基解离的情况下，肥皂才是有效的表面活性剂。 在硬水中，由于Ca2+与羧酸基团形成不可溶的沉淀，而 使肥皂失效。 l The disadvantages of conventional soaps can be avoided by replacing the carboxylic acid group with a sulfate. The sulfate in most current laundry detergents, again those purchased at the supermarket, is attached to a linear alkyl chain. The sulfate can also be attached to a benzene ring, making a sulfonate like that shown in Figure 4.2-1. Such sulfonates are more effective at cell rupture than alkyl sulfates are; perhaps because of this, sulfonates are not easily degraded microbiologically and so are no longer commonly used in laundry detergents. 可通过将肥皂的羧基用硫酸盐代替来改变传统肥皂的缺点。硫酸盐 代替的肥皂是目前洗衣店常用及超市中常见的表面活性剂。硫酸盐 可连在苯环上，结构如图4.2-1所示。这种硫酸盐表面活性剂比碱对 细胞破碎的作用更好，磺酸盐不易被微生物降解，所以洗衣店中并 不常用。 l Cationic detergents largely are based on tetraalkylammonium salts. Cetyltrimethylammonium bromide, the example in Figure 4.2-1, is typical: It contains one long alkyl tail (the 16 carbon ”cetyl”) and 3 methyl groups, all attached to a positively charged nitrogen atom. The counterion is often a halide. In the supermarket, these detergents are most frequently found in shampoos, correctly suggesting that they will offer a more gentle means of cell rupture. 阳离子表面活性剂主要是烷基胺盐。图4.2-1中的十二烷基溴胺是典 型的例子。它有一个长烷烃链（十六烷基）和三个甲基，都连接在一 个带正电的氮原子上，负离子通常是卤素，市场上常做洗发剂出售。 细胞破碎时条件较温和。 l In passing we should note that cationic detergents with two long chains are often oil soluble, and are implicated in the formation of membrane pores. They have rarely been tested as a route to cell rupture. 过去认为带有两个长尾的阳离子表面活性剂溶于油， 和细胞膜孔的形成有关，但是很少用于细胞破碎。 l Nonionic detergents, of which the commercial material Triton X is an example, are most commonly based on water soluble polymers. They are less well defined chemically. In the supermarket, they show up in dishwash-ing liquids whose advertising is such a curse on evening television