第四讲细胞破碎 2学时 、通过本章学习应掌握的内容 1、细胞破碎的意义 常用的细胞破碎方法 3、渗透压计算方法 4、了解机械破碎法所用设备 5、渗透压的计算 二、细胞破碎的目的 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, tl 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 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. Some equipment is borrowed from the dairy industry, where it was developed for the homogenization of milk; other equipment is taken from the paint industry, where it is used for the size reduction of pigments 由于有许多生化物质存在于细胞内部,必须在纯化以前将细胞破碎,使胞内 物质释放到液相中,然后方可进行提取 三、细胞膜 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 procaryotes
第四讲 细胞破碎 2 学时 一、通过本章学习应掌握的内容 1、细胞破碎的意义 2、常用的细胞破碎方法 3、渗透压计算方法 4、了解机械破碎法所用设备 5、渗透压的计算 二、细胞破碎的目的 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. 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. Some equipment is borrowed from the dairy industry, where it was developed for the homogenization of milk; other equipment is taken from the paint industry, where it is used for the size reduction of pigments. 由于有许多生化物质存在于细胞内部，必须在纯化以前将细胞破碎，使胞内 物质释放到液相中，然后方可进行提取 三、细胞膜 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
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 三、细胞破碎的主要方法 1、化学法 The chemical methods of cell rupture listed in Table 4.0-l are dominated by osmotic shock, detergent solubilization, and lipid dissolution. These three methods are described in the following paragraphs. Before this description, we want to give some brief general izations about the other two methods enzyme digestion and alkali treatment 渗透冲击:无论动物还是植物细胞,胞内物质都是由膜组织包裏起来的,因此, 破坏细胞膜系统是胞内物质释放的关键步骤 通常细胞膜系统强度较差,易受渗透压冲击而破碎 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 (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。) 渗透压的计算方法
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 三、细胞破碎的主要方法 1、化学法 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. 渗透冲击：无论动物还是植物细胞，胞内物质都是由膜组织包裹起来的，因此， 破坏细胞膜系统是胞内物质释放的关键步骤 通常细胞膜系统强度较差，易受渗透压冲击而破碎 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. （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。） 渗透压的计算方法
RT Pout-Pin -x 酶消化:酶法破坏细胞膜系统 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 enzy mes 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消耗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λ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。)增溶:利用表面活性剂的增 溶作用,将体积为细胞体积2倍的某浓度的表面活性剂溶液加入到细胞中 去,表面活性剂能将细胞破碎,制成的悬浮液可通过离心分离除去细胞碎 片 The second major method of chemically rupturing cells is solubilization detergents. Typically, a concentrated detergent solution is added to about half solutions volume of cells. The detergent disrupts the cell mem-brane. The resulting ion can be centrifuged to remove cell frag-ments, and then run through an adsorption column or an extractor to isolate the product (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。) 脂溶:在细胞悬浮液中加入一定量的有机溶剂,细胞壁脂质层吸收后,导致胞壁 膨胀,最后裂开,细胞质中的物质就释放到周围溶液中了 碱处理
酶消化：酶法破坏细胞膜系统 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消耗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。）增溶：利用表面活性剂的增 溶作用，将体积为细胞体积 2 倍的某浓度的表面活性剂溶液加入到细胞中 去，表面活性剂能将细胞破碎，制成的悬浮液可通过离心分离除去细胞碎 片 The second major method of chemically rupturing cells is solubilization by detergents. Typically,a concentrated detergent solution is added to about half the solution’s volume of cells. The detergent disrupts the cell mem-brane. The resulting suspension can be centrifuged to remove cell frag-ments, and then run through an adsorption column or an extractor to isolate the product. (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。) 脂溶：在细胞悬浮液中加入一定量的有机溶剂，细胞壁脂质层吸收后，导致胞壁 膨胀，最后裂开，细胞质中的物质就释放到周围溶液中了 碱处理： ln(1 )1 2 x V RT p p H O out − in = − −
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 with the cell walls in a number of ways, including the saponification of lipids 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。) 2、械法 匀浆法 研磨法 超声波法∶利用频率高、波长短的超声波迸行细胞破碎,效率更高 球磨破碎法 3、其它破碎方法 冻结-融化法(亦称冻融法) 干燥法 四、本章作业 1、简述细胞破碎的意义 2、细胞破碎方法的大致分类 3、渗透压的计算方法
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。 ) 2、械法 匀浆法 研磨法 超声波法：利用频率高、波长短的超声波进行细胞破碎，效率更高 球磨破碎法 3、其它破碎方法 冻结－融化法（亦称冻融法） 干燥法 四、本章作业 1、简述细胞破碎的意义 2、细胞破碎方法的大致分类 3、渗透压的计算方法