Cell Lysis and Disruption. Cell disruption Laboratory techniques:Sonication Enzyme treatment...

Cell Lysis and Disruption

Cell disruption

Laboratory techniques: SonicationEnzyme treatment

Industrial technology: Ball mill grinding

Homogenization (orifice type)

High mechanical shear

Heat generation

*Chemical method + mechanical method combination

Product of Interest: Intracellular or Extracellular ?

Method Technique PrincipleStress

on ProductCost Examples

Chemical Osmotic Shock Osmotic rupture of membrane

Gentle Cheap Rupture of red blood cells

Enzyme digestion Cell wall digested,providing disruption

Gentle Expensive Micrococcus lysodeikticus treated with egg lysosyme

Solubilization Detergents solubilize cell membrane

Gentle Moderate-expensive

Bile salts acting on E. coli

Lipid dissolution Organic solvent dissolves in cell wall, and so destabilizes it

Moderate Cheap Toluene disruption of yeast

Alkali treatment Saponification of lipids solubilizes membrane

Harsh Cheap

Mechanical

Homogenization(blade type)

Cells chopped in Waring blender

Moderate Moderate Animal tissue and cells

Grinding Cell ruptured by grinding with abrasives

Moderate Cheap

Ultrasonication Cells broken withultrasonic cavitation

Harsh Expensive Cell suspensions at least on small scale

Homogenization(orifice type)

Cells forced through small hole are broken by shear

Harsh Moderate Large scale treatment of cell suspensions, except of bacteria

Crushing in ball mill

Cells crushed between glass or steel balls

Harsh Cheap Large scale treatment of cell suspensions and plant cells

Table 1. Cell Disruption Technique

Chemical Methods (not popular in industry)

Enzyme digestion Enzyme costAlkali treatment Harsh condition, degradation

(PHB separation) Osmotic Shock

Cells are put into pure waterSolutes in the cells cause an osmotic flow of water into the cell

(Note: Plant cell are difficult to be burst.)

Solubilization : by detergentConcentrated detergent solution is added to disrupts the cell membraneDetergent - Ex: SDS (Sod Dodecylsulfate)Solubilize cell wall lipid

Figure 1. Chemical structures of selected surfactants.

Lipid Dissolution

A volume of solvent( toluene) about 10% of the biomass is added to a cell suspensionThe cell wall lipid is solubilized.

Mechanical Disruption

Figure 2. Homogenizer Assembly. (a) A typical homogenizer and (b) a homogenization valve. Cell passing through this valve areruptured by both shear and mechanical stress.

Homogenizer

(a) (b)

Figure 3. Homogenization versus time. Mechanical disruption of cells reduces particle size but some may also denature some of the products in the cell.

*How long for operation?

Time, min

Alcohol DehydrogenaseActivity

Fumarase ActivityApparentParticle Size

Ball Mill (Sand Grinder)

Feed in

Cooling Jacket

screen

Sand (bead)

Figure 4. Schematic diagram for ball mill equipment.

*batch, continuous type*paint, dyestuff industry

* 멧돌

Enzymatic Lysis• dissolve the outer mannoprotein layerproduction of protoplasts from yeast and bacteria

• endo-β(1,3) glucanaseLytic protease

• Preparation of lytic enzymes• Lysis experiment for various enzymes -required

Cell Disruption Equipment: Bead Mill

Sept. 21st, 2011

Esther H. Kim

Bead mill

Laboratory bead mill (Dyno mill and DMQ-07)

Production machine (DMQ-10)

Media volume 0.4L

Media volume 5.5L

Scale-up & Application

• Laboratory Scale-up

Bead mill – Grinding media• Weaknesses of the sand mill

– Transition to closed mills– ottawa sand grinding beads

• Grinding media (beads)– Steel, zirconium oxide, aluminum oxide, Si/Al/Zr mixed oxide (SAZ),

steatite (modification of talc), glass and plastics– Diameter lies in the range from 0.1 to 3 mm.– The harder the beads, the greater the intensity of dispersion.– The number of beads is proportional to 1/d3 use smallest beads

possible.• Translational and rotational movement: compressive stress and

shear

Application in research• Horizontal bead mill used for cell rupture

(a) General view, (b) details of (i) stainless steel and (ii) polyurethane impellers

Application in researchBead-mill homogenization

Lysis of microbial cells in soils and sediments for large scale

relatively inexpensive widely applicable

Compared to chemical and enzymatic method

What is a French Press?

2011 - 9 - 21

Inae Kim

Bioseparation Engineering

Contents

1. What is a French Press?

2. Principle

3. Operation

4. Characteristics

5. Manufacturer

What is a French Press?

기계적으로 세포를 파괴하는 방법

염록체 , 혈구세포 및 단세포 생물의 분해와 동물조직 및 생물체 입자의 균등질 (homogenate) 제조에

이용

세포핵은 손상시키지 않고 세포벽만을 분해Sonication 보다 균일하고 완전한 분해 가능

고압 ( 최대 40,000 psi) 과 빠른 감압을 이용 단단한 세포도 쉽게 분해 가능

Stainless steel 샘플의 오염 방지Figure 1. French Press.

Principles of the French Press

Figure 3. Diagram of French Press Cylinder.

Flow valve

Outlet tube

Cylinder body

Piston

Figure 2. French Press Cylinder.

Sample

Operations of French Press

① Pump ON/OFF

② Ratio Selector valve (MED/HIGH)

- 유효 피스톤 면적 결정

③ Pressure valve

(increase/decrease)

Figure 4. Installed French Press cylinder in the press .

①

②

③

Figure 5. French Press .

Characteristics of the French Press

장점

-시료의 생화학적 성질 보존

-시료의 손실 위험이 적음

-시료 전처리 과정이 없음

-크기가 작은 시료는 거의 완전히 분해 가능

단점

- 시료의 부피가 클 때는 부적절

- 부품의 무게가 무거워서 조작이나 세척이 다소 어려움

- 배출구가 막힐 수 있음

Manufacturers of the French Press