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Cell Lysis and Disruption

Cell Lysis and Disruption. Cell disruption. Sonication Enzyme treatment. Laboratory techniques:. High mechanical shear. Ball mill grinding Homogenization (orifice type). Industrial technology:. Heat generation. *Chemical method + mechanical method combination.

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Cell Lysis and Disruption

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  1. Cell Lysis and Disruption

  2. Cell disruption Sonication Enzyme treatment Laboratory techniques: High mechanical shear Ball mill grinding Homogenization (orifice type) Industrial technology: Heat generation *Chemical method + mechanical method combination

  3. Product of Interest: Intracellular or Extracellular ? Table 1. Cell Disruption Technique

  4. Chemical Methods (not popular in industry) Enzyme digestion Enzyme cost Alkali treatment Harsh condition, degradation (PHB separation) • Osmotic Shock Cells are put into pure water Solutes in the cells cause an osmotic flow of water into the cell (Note: Plant cell are difficult to be burst.) • Solubilization : by detergent Concentrated detergent solution is added to disrupts the cell membrane Detergent - Ex: SDS (Sod Dodecylsulfate) Solubilize cell wall lipid

  5. Figure 1. Chemical structures of selected surfactants. • Lipid Dissolution A volume of solvent( toluene) about 10% of the biomass is added to a cell suspension The cell wall lipid is solubilized.

  6. Mechanical Disruption • Homogenizer (b) (a) 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.

  7. Apparent Particle Size Fumarase Activity Alcohol Dehydrogenase Activity Time, min 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?

  8. Ball Mill (Sand Grinder) screen Cooling Jacket Sand (bead) Feed in Figure 4. Schematic diagram for ball mill equipment. *batch, continuous type *paint, dyestuff industry *멧돌

  9. Enzymatic Lysis • dissolve the outer mannoprotein layer • production of protoplasts from yeast and bacteria • endo-β(1,3) glucanase • Lytic protease • Preparation of lytic enzymes • Lysis experiment for various enzymes -required

  10. Cell Disruption Equipment: Bead Mill Sept. 21st, 2011 Esther H. Kim

  11. Bead mill Media volume 5.5L Media volume 0.4L Laboratory bead mill (Dyno mill and DMQ-07) Production machine (DMQ-10)

  12. Scale-up & Application • Scale-up • Laboratory

  13. 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

  14. Application in research • Horizontal bead mill used for cell rupture (a) General view, (b) details of (i) stainless steel and (ii) polyurethane impellers

  15. Application in research • Bead-mill homogenization • Lysis of microbial cells in soils and sediments for large scale • relatively inexpensive • widely applicable • Compared to chemical and enzymatic method

  16. Bioseparation Engineering What is a French Press? 2011 - 9 - 21 Inae Kim

  17. Contents • What is a French Press? • Principle • Operation • Characteristics • Manufacturer

  18. What is a French Press? • 기계적으로 세포를 파괴하는 방법 • 염록체, 혈구세포및 단세포 생물의 분해와 • 동물조직 및 생물체 입자의 균등질(homogenate) 제조에 이용 • 세포핵은 손상시키지 않고 세포벽만을 분해 • Sonication보다 균일하고 완전한 분해 가능 • 고압(최대 40,000 psi)과 빠른 감압을 이용 • 단단한 세포도 쉽게 분해 가능 • Stainless steel 샘플의 오염 방지 Figure 1. French Press.

  19. Principles of the French Press Piston Cylinder body Flow valve Sample Outlet tube Figure 3. Diagram of French Press Cylinder. Figure 2. French Press Cylinder.

  20. Operations of French Press ② Figure 5. French Press . ③ ① Pump ON/OFF ② Ratio Selector valve (MED/HIGH) - 유효 피스톤 면적 결정 ③ Pressure valve (increase/decrease) ① Figure 4. Installed French Press cylinder in the press .

  21. 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

  22. Manufacturers of the French Press

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