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Transcript
Cell disruption
Saeb Aliwaini
Saeb Aliwaini
Cell disruption
To extract a product from cells :
- The cells are usually first separated from the culture liquid
medium
- To reduce secreted extracellular substances and unutilized
media components
How ? By microfiltration or centrifugation
Saeb Aliwaini
Type of cells
Gram positive bacterial cells
Gram negative bacterial cells
Yeast cell
fungi
Cultured mammalian cells
Cultured plant cells
Ground tissue
Saeb Aliwaini
In gram positive bacteria
- The cell wall is composed of peptidoglycan, Teichoicacid and
polysaccharides and is about 0.02 to 0.04 microns
thick.
Can be destroyed by the antibacterial
enzyme lysozyme.
Saeb Aliwaini
But in
Gram negative bacteria
• These don't have distinct cell walls but instead have multilayered
envelops.
* The peptidoglycan layer is significantly
thinner than in gram positive bacteria.
* An external layer composed of lipopolysaccharides
and proteins is usually present
* The presence of the periplasm layers which are two liquid filled gaps, one
between the plasma membrane and the peptidoglycan layer and the other
between the Peptidoglycan layer and the external lipopolysaccharides
Saeb Aliwaini
• Periplasmic layers also exits in gram positive bacteria but these are
significantly thinner than those in gram negative bacteria.
• But in many cases we need this layer to be extracted
Yeasts have thick cell walls, typically 0.1 to 0.2 microns in thickness
From polysaccharides such as chitins
Moulds are similar to yeast but multicellular
Mammalian cells are easy to disrubt
Saeb Aliwaini
• Plant cells on the other hand have very thick cell walls mainly
composed of cellulose and other polysaccharides.
• Cell wall wherever present is the main barrier which needs to
be disrupted to recover intracellular products.
lysozyme is used to disrupt the cell wall of gram positive bacteria
since it degrades peptidoglycan which is a key cell wall
constituent.
Saeb Aliwaini
• In gram negative bacteria it is less susceptible to lysis by
lysozymes in since it is shielded by a layer composed of
lipopolysaccharide and protein.
• The plasma membrane can be easily destabilized by
detergents. Or even osmotic shock
This can be achieved simply by transferring
the cell fro m Isotonic medium to distilled
water
Saeb Aliwaini
Cell disruption methods
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Physical methods
1 .Disruption in bead mill
2. Disruption using a rotor-stator mill
3. Disruption using French press
4. Disruption using ultrasonic vibrations
Chemical and physicochemical methods
1 .Disruption using detergents
2. Disruption using enzymes (lysozyme)
3. Disruption using solvents
4. Disruption using osmotic shock
Saeb Aliwaini
Cell disruption using bead mill
A tubular vessel made of metal or thick glass within which the cell suspension is
placed along with small metal or Glass beads.
The cell disruption takes place due to the grinding
action of the rolling beads as well as the impact
resulting from the cascading beads.
At low temperatures as (liquid nitrogen into the vessel)
Commonly used for disrupting yeast cells and for grinding animal tissue
Saeb Aliwaini
• Cell disruption involves particle size reduction and has certain
Similarities with grinding.
According to the Kick's law of grinding, the amount of energy
required to reduce the size of material is proportional
to the size reduction ratio:
Saeb Aliwaini
Cell disruption using rotor-stator mill
Truncated cone shaped rotating object called the rotor.
Stationary block with a tapered cavity called the stator
Typical rotation speeds are in the 10,000 to 50,000 rpm
range
The high rate of shear generated in the space between
the rotor and the stator as well as the turbulence thus
generated are responsible for cell disruption
Saeb Aliwaini
• These mills are more commonly used for disruption of plant
and animal tissues based material and are operated in the
multi pass mode ,i.e. the disrupted material is sent back into
the device for more complete disruption
Saeb Aliwaini
Cell disruption using French press
. Consists of a cylinder
•
The cell suspension is placed with in the cylinder and pressurized using the
plunger
• Provided with an impact plate, where the jet impinges causing
further cell disruption
• For small –scale recovery of intracellular proteins
and DNA from bacterial and plant cells.
Saeb Aliwaini
Cell disruption using ultrasonic vibrations
• Ultrasound emitting tips of various sizes are available
A frequency of 25kHz is commonly used for cell
disruption
Saeb Aliwaini