FALLSEM2015 16 - CP1014 - 17 Jul 2015 - RM01 - Cell Disruption Techniques

Cell Disruption
Techniques
1
Necessity
Intracellular Products - Products present in cytoplasm or
inclusion bodies (proteins)
Traditional intracellular products rDNA intracellular products

Glucose isomerase Chymosin (yeast/E.coli)
Galactosidase Insulin (E.coli, mammalian)
Phosphatase Immunoglobulin
Ethanol dehydrogenase Interferons (mammalian)
Dnase, Rnase Human growth hormone (E.coli)
NADH/NAD+ Human serum albumin
Alkaloids streptokinase
2
Choice of method : Depends on nature of cell and
the nature of the product
Types of cells
Gram positive bacterial cells
Gram negative bacterial cells
Yeast cell
fungi
Cultured mammalian cells
Cultured plant cells
Ground tissue
Prokaryotic cells : Bacteria Gram Positive and
Gram negative
4
Eukaryotic cells:
• Yeast (unicellular), mold cells (multicellular,
filamentous)
–Thick cell walls (highly crosslinked structure)- Mainly
composed of polysaccharides (glucans, mannans and
chitins)
–Plasma membranes – composed of phospholipids and
lipoproteins
• Mammalian (Animal) cells
–Animal cells do not have cell walls
• Plant cells
–Very thick cell wall (cellulose and other
polysaccharides)
5
• Physical/mechanical methods – target on cell wall
disruption
1.Bead mill/ball mill
2.Rotor-stator mill
3.French press
4.Ultrasonic vibration
• Chemical and physicochemical methods –
destabilizing the cell membrane
1.Detergents
2.Enzymes
3.Solvents
4.Osmotic shock
6
Mechanical Methods
• Cell envelope is broken physically
• Equipment for mechanical cell disruption
1.Bead mill – large scale; best for mycelial fungi

and algae
2.Homogenizer – large scale; suitable yeast and
bacteria
3.Ultrasonic
4.Blender
7
Bead Mill
• Impact of rolling and cascading beads
• Also used for grinding animal tissue
• At low temperatures as
• (liquid nitrogen into the vessel)
8
Rotor-Stator Mill
• Typical rotation speeds are in

the 10,000 to 50,000 rpm
range
• Used for Plant and animal

tissues
9
French Press
• For small –scale recovery of

intracellular proteins and
DNA from bacterial and plant
cells.
10
ultrasonic vibration
• Ultrasound emitting tips
• A frequency of 25kHz is commonly used

for cell disruption
• For bacterial cells such as E. coli, 30 to

60 Seconds maybe sufficient for small
samples
• Other methods – Homogenizers,

Blenders
Chemical Methods
Detergent - Solubulizing the plasma membrane

(Phospholipids)
Detergent – 3 categories
1.Cationic
2.Anionic
3.Non-ionic
•Triton X Series, Tween Series
•Detergent need to be removed from product
require additional purification, polishing step
•A lot of protein denature or precipitate with detergent
- try to avoid use detergent
12
Solvents
•Solvent type – acetone, toluene, ether, phenylethyl
alcohol, benzene, methanol, chloroform
•Act on cell membrane by solubilising phospholipids and

denature protein
•Toluene – can disrupt fungal cell wall
•Limitation (similar with detergents)

1.Need to remove from product
2.Denature proteins
3.Easier to remove than detergent
13
Enzymes
To disrupt cell wall

•Example 1: Lysozyme Enzyme (egg based enzyme)
–Able to hydrolyse murein (wall) in gram (-) and gram (+)
bacteria
–Cannot lyse cell membrane - thus Combine with detergent
to disrupt cell membrane
–Can also combine with osmotic or mechanical disruption
methods
–Pretreatment with EDTA will enhance effectiveness of
lysozyme
•Example 2: Glucanase and Mannase
–Combine with protease to degrade yeast cell wall
•Example 3: Cellulase and Pectinase
–To disrupt plant cells wall
14
Osmotic Shock
• When cells are dumped into pure water, cells can swell
and burst due to the osmotic flow of water into the cells.
• Procedures
1.Allow cell to equilibrate internal and external osmotic
pressure in high sucrose medium (hypertonic)
2.Then put in distilled water (hypotonic) - rapid influx of
water into the cell - volume rapid expansion - rupture cell
membrane
• Gram negative bacteria

• Mainly for mammalian cells – red blood cells
• For bacterial and fungal cells, cell walls need to be
weakened
15

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FALLSEM2015 16 - CP1014 - 17 Jul 2015 - RM01 - Cell Disruption Techniques

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Cell Disruption

Traditional intracellular products rDNA intracellular products

• Equipment for mechanical cell disruption

1.Bead mill – large scale; best for mycelial fungi

• Also used for grinding animal tissue

• Typical rotation speeds are in

• Used for Plant and animal

• For small –scale recovery of

• A frequency of 25kHz is commonly used

• For bacterial cells such as E. coli, 30 to

• Other methods – Homogenizers,

Detergent - Solubulizing the plasma membrane

•Act on cell membrane by solubilising phospholipids and

•Toluene – can disrupt fungal cell wall

•Limitation (similar with detergents)

To disrupt cell wall

• Gram negative bacteria

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