Microbial cell disruption by high pressure homogenization

Gentle
Moderateexpensive
Moderate
Cheap
Harsh
Cheap
表 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 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 胆盐作用于大 肠杆菌 甲苯破碎酵母 细胞 举例 血红细胞的破 坏
• Additional heat-transfer capacity can be obtained by including internal cooling coils or an in-line heat exchanger, but with considerably more difficulty in final cleaning. For most laboratory or pilot-scale applications, the external jacket should suffice. Suspension of the tank contents is important, particularly where storage before subsequent processing is required. Tanks fitted with stirrers are ideal but expensive, and cleanability is again an issue. In laboratory settings, effective suspension can be obtained using a recirculation loop on each tank. This can be conveniently implemented using, for example, a doubleheaded peristaltic pump to simplify cleanability.
Gram-negative procaryotes
革兰氏阴性原核生物（E.coli）
组织捣碎匀浆机
超声波破碎仪
Crushing in ball mill
WSK 卧式高效全能珠磨机
ZM系列 卧式密闭珠(砂)磨机
High Speed Bead Mill
在珠磨法中，细胞的破碎率也能用一级速率方程式表示：
Product
Moderate Moderate
Cost
Moderate Cheap
Examples
Animal tissue and cells
Ultrasonication
Cells broken with ultrasonic cavitation
Cells forced through small hole are broken by shear Cells crushed between glass or steel balls
• In this chapter, We consider the methods for cell rupture most likely to be useful at larger scales. These methods are conveniently split into two groups, described as “chemical” and “mechanical.” In the chemical methods, 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.
Harsh
Moderate
Crushing in ball mill
Harsh
Cheap
表 2. 细胞物理破碎法
方法
机械法
ቤተ መጻሕፍቲ ባይዱ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
举例
动物组织及动 物细胞
细胞被搅拌器劈碎 适中 细胞被研磨物磨碎 适中 用超声波的空穴作 适中 用使细胞破碎 须使细胞通过的小 剧烈 孔，使细胞受到剪 切力而破碎 细胞被玻璃珠或铁 剧烈 珠捣碎
ln[1/(1-R)]=Kt
K－破碎速率常数 t－停留时间 R－破碎率 破碎速率常数K与许多操作参数有关，如搅拌转速、料液 的循环流速、细胞悬浮液的浓度、玻璃小珠的装置和珠体的直 径以及温度等。
CHEMICAL
METHODS
• The chemical methods of cell rupture 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 generalizations about the other two methods: enzyme digestion and alkali treatment.
Fig 2. Example of a high-pressure homogenization system.
• Figure 2 shows a typical layout for a celldisruption system based on high pressure homogenization. Two storage tanks are employed as improved disruption efficiency is achieved by operating a discrete-pass strategy, where the homogenizer feed is drawn from one tank, whereas the homogenate is passed to the other. The location of the three-way valves enables feed to be drawn from, and fed to, either tank. The feed and homogenate tanks, therefore, alternate for multiplepass strategies (the normal operational mode). As homogenization generates considerable heating of the suspension (typically 2.5 ℃ per 10 MPa of operating pressure), the tanks are jacketed and cooled at 5 ℃.
TABLE 1. Chemical Cell Disintegration Techniques
Method
Chemical Technique Osmotic shock Principle Stress Cost Cheap Examples Rupture of red blood cells Osmotic rupture of Gentle membrane
Harsh
Expensive
Cell suspensions on a small scale
Large scale treatment of cell suspensions, Large scale treatment of cell suspensions and plant cells
Homogenization (orifice type)
Microbial cell disruption by high pressure homogenization
• The disruption of a cell’s wall is often a primary step in product isolation, particularly when hosts such as Escherichia coli and Saccharomyces cerevisiae, which generally do not excrete product, are employed. Of the available methods, high-pressure homogenization is dominant at moderate or large process volumes.
TABLE 2. Mechanical Cell Disintegration Techniques
Method
Mechanical
Technique
Homogenization (blade type) Grinding
Principle
Cells chopped in Waring blender Cells ruptured by grinding with abrasives