04第四讲 细胞破碎 [Chapter 4 Cell Disruption]

Homogenization (orifice type)
Harsh
Moderate
Crushing in ball mill
Harsh
Cheap
表 4.0-1. 细胞物理破碎法
方法 机械法 技术 匀浆法（片型） 研磨法 超声波法 匀浆法（孔型） 原理 效果 成本 适中 便宜 昂贵 适中 细胞悬浮液小 规模处理 细胞悬浮液大 规模处理 细胞悬浮液和 植物细胞的大 规模处理 举例 动物组织及动 物细胞
※ 4.0 Summary 概述
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. 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。
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 baker’s 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. 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。
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. 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。
Chapter 4 Chapter 4
Cell Disruption Cell Disruption
第四章 细胞破碎 第四章 细胞破碎
※ 4.0 Summary
概述 细胞膜 化学破碎方法 机械破碎
※ 4.1 Cell Membranes
※ 4.2 Chemical Methods
※ 4.3 mechanical disruption
原理 渗透压破坏细胞 细胞壁被消化，使 细胞破碎 表面活性剂溶解细 胞壁 有机溶剂溶解细胞 壁并使之失稳 碱的皂化作用使细 胞壁融解
效果 温和 温和 温和 适中 剧烈
成本 便宜 昂贵 适中 便宜 便宜
举例 血红细胞的破 坏
胆盐作用于大 肠杆菌 甲苯破碎酵母 细胞
TABLE 4.0-1. Mechanical Cell Disintegration Techniques
※ 4.1 Cell Membranes 细胞膜
Enzyme digestion Solubilization
Gentle
Expensive
Gentle
Moderateexpensive
Bile salts acting on E.coli Toluene disruption of yeast
Lipid dissolution Alkali treatment
Organic solvent dissolves in cell wall Saponification of lipids solubilizes membrane
Moderate
Cheap
Harsh
Cheap
表 4.0-1. 细胞化学破碎法
方法 化学法
技术 渗透冲击 酶消化法 增溶法 脂溶法 碱处理法
TABLE 4.0-1. Chemical Cell Disintegration Techniques
Method Chemical Technique Osmotic shock Principle Osmotic rupture of membrane Cell wall digested , providing disruption Detergents solubilize cell membrane Stress Gentle Cost Cheap Examples Rupture of red blood cells Egg lysosyme
Method Mechanical Technique Homogenization (blade type) Grinding Principle Cells chopped in Waring blender Cells ruptured by grinding with abrasives Cells broken with ultrasonic cavitation Cells forced through small hole are broken by shear Cells crushed between glass or steel balls Product Moderate Moderate Cost Moderate Cheap Examples Animal tissue and cells
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. 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。
Ultrasonication
Harsh
Expensive