Bio Product Recovery &
Bioseparations
DISRUPTION OF CELL
REMOVAL OF CELL DEBRIS
PRECIPITATION OF PROTEINS
ULTRAFILTRATION OF DESIRED
ENZYME
CHROMATOGRAPHIC SEPERATION
CRYSTALLISATION AND DRYING
Why disrupt cells?
• To get products we want
• Biological products can be:
a.Extracellular
b.Periplasmic
c.Intracellular
Methods of cell disruption
• Physical methods
• Chemical and enzymatic methods
• Mechanical methods
Physical methods
Thermolysis or Heat shock:-
Easy and economical method
Can be used only if the product are
thermostable
Osmotic shock:-
Most widely used method
Dramatic change in the solute concentration of
the liquid surrounding the microorganism – can
cause the cell to burst
Ultrasonication
Ultrasound Waves of frequencies greater
than 20 KHz rupture the cell wall by
phenomenon known as Cavitation
Leads to rapid increase in the temperature
It is expensive technique
Chemical Methods
Alkali Treatment
It is carried out at pH 11-12 for 20-30 min.
Alkali acts on the lipids present in the cell wall
and saponify them
Cheap and effective method but very harsh
Detergent Solubilization
Detergent Solubilization
It involves the addition of concentrated
solution of detergent
The key mechanism involves the action of
detergents in solubilizing the lipids in the cell
wall to form micelle
Most widely used method
Cell wall permeabilization
Cell wall disruption is achieved by the addition
of organic solvents
Solvent is absorbed by cell wall resulting in its
swelling and ultimate rupture
Enzyme digestion
Digestion of cell wall is achieved by the
addition of lytic enzymes to a cell
suspension.
Effective and costly process.
MECHANICAL METHODS
Bead mill
High pressure homogenizer
Choice of Disruption Method
The method selected for large scale cell
disruption will be different in every case, but
will depend on:
Susceptibility of cells to disruption
Product stability
Ease of extraction from cell debris
Speed of method
Cost of method
Precipitation of proteins
Protein precipitation types
Iso electric precipitation
Protein precipitation by addition of salts
Salting in
Salting out
Precipitation by addition of organic solvent
Precipitation by addition of non ionic
polymers
Precipitation by metal ions
Isoelectric precipitation
At pH=pI, protein is neutral and hence it
precipitates
pI is unique for each protein – desired protein can
be precipitate when the characteristic pI of it is
known.
Protein Precipitation by addition of salt:
Salting In – addition of low concentration of salts
Salting Out – Addition of high concentration of
salts
• Protein Precipitation by addition of organic
solvents – Addition of water miscible
solvent like ethanol.
• Protein Precipitation by addition of Non-
ionic polymers – water soluble polymers
such as PEG cause aggregation of proteins.
Advantages of Protein precipitation:
• Reduction in the volume.
• Concentration of desired product.
• Rapid separation.
• Less expensive and involves use of simple
equipments.
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