When the product is synthesized intracellularly (inside the cell) or if the product has to be extracted from plant/animals/fungal tissues, it is necessary to disrupt the cell/tissue by force. Different parameters influence the degree of cell disruption which are the nature of the microorganism, operating pressure (for mechanical method), temperature, and the number of passes.
Cell disruption methods mainly classified into two types, which are;
1. Physical methods
a) Heat shock or Thermolysis
It is only used when the product is stable to heat shock and this is an economical method. It disrupts the cell walls of an inactive organism without affecting the bioproducts.
b) Osmotic shock
We know that every cell maintains a particular osmotic gradient. If the extracellular solute concentration is reduced drastically, then it will tend to burst the cells. osmotic shock mainly used to disrupt Red blood cells(RBC)
How does osmotic shock is carried out?
Osmotic shock to the cell is carried out by simply dipping a given volume of cells that contain the bioproduct into pure water of about twice the volume of cells.
How can we find out the osmotic pressure inside the cell or cytoplasmic solution?
We can calculate the osmotic pressure using the formula :
π = C*R*T =R*T (Ci-Co)
Where Ci= inside concentration
Co= outside concentration
T= temperature
R= Gas constant
c) Ultrasonication
By name, we can guess that this method should be related to sound waves or ultrasonic waves and this method is expansive and mainly used in laboratories.
How does sonication work to disrupt the cell wall?
When ultrasound waves of frequency greater than 20KHz come with contact with the cell wall, the cell wall becomes ruptures and these phenomena are called cavitation (formation of cavities in the cell wall). When ultrasound waves pass in liquid, it creates an alternative area of compression and rarefaction. The cavities are mainly formed in the region of rarefaction.
The bubbles are formed in the cavities and these bubbles are compressed to generate several thousand atmospheric pressure. Due to the collapse of bubbles, it creates short waves and disrupts the cell walls of surrounding regions.
2.CHEMICAL METHODS
a) Alkali treatment
Alkali treatment acts on the cell wall in several ways which include saponification of lipids. This treatment is carried out at pH 11-12 for 20-30 minutes and this method is widely used due to its cheap and high effectiveness. By using this method enzyme asparaginase can be isolate.
b) Detergent solubilization
In this method, a concentrated solution of detergent is added to half the solution volume of cells to disrupt the cell walls. In dilute solution, detergent is not able to dissolve the lipid but at higher concentration, lipid solubilization begins suddenly and linearly increases with the concentration of detergent.
The point at which lipid solubility and the surface tension of the medium intersects, that point of concentration is called critical micelle concentration
Example- anionic detergent - SDS, sodium sulphonate
cationic detergent - cetyl trimethyl ammonium bromide (CTAB)
non ionic detergent- Triton X-100
C) Organic solvents
Organic solvents are absorbed by the cell wall which tends to swell and ultimate rupture occurs of the cell wall. At a lower concentration of organic solvent, it is permeable through a cell wall.
Example - Toluene
D) Enzyme digestion
In this method, the digestion of the cell wall is carried out by enzymes like cellulase and pectinase for plant cells. The enzyme lysozymes hydrolyze mucopeptide moiety of bacterial walls, mainly of gram-positive bacteria. In the case of gram-negative bacteria, digestion of the cell wall is done by lysozymes but pretreatment is required with a detergent.
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