Cell disruption methods

1. Cell disruption methods:
By
Dr. Harinatha Reddy A
ASSISTANT PROFESSOR
Department of Biotechnology
Bangalore

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3. Release of Intracellular Products:
 Several biological-products (proteins, vitamins, enzymes) which are
located within the cells.
 Such compounds have to be first released for their further
processing and final isolation.

4. The microorganisms or other cells can be disintegrated or
disrupted by physical, chemical or enzymatic methods.

5. Physical methods: Ultra sonication:

6. Ultra sonication:
 This method consist of ultrasonicator it produced ultrasonic waves.
 Ultrasonic shock waves break the microbial cell walls. The
intracellular compounds (protein, enzyme and fat) are released in to
aqueous phase.
 Ultrasonic disintegration is widely employed in the laboratory.
 However, due to high cost, it is not suitable for large-scale use in
industries.

7. Osmotic shock:

8. Osmotic shock:
 Osmotic shock or osmotic stress is a sudden change in the solute
concentration around a cell, causing a rapid change in the
movement of water across its cell membrane.
 This method involves the cells first treated with 20% sucrose
solution.
 The cells are then transferred to water at about 4°C.
 Osmotic shock is used for the release of hydrolytic enzymes and
binding proteins from Gram-negative bacteria.

9. Heat shock (thermolysis):
 Breakage of cells by subjecting them to heat is relatively easy and
cheap.
 Heat shock is the effect of subjecting a cell to a temperature that
is greater than the optimal temperature range of function of the
cell.
 Heat shock disrupt proper protein folding.

10. High pressure homogenization:
The high pressure
homogenizer
(machine) consists of
a high pressure
piston pump .
The piston creates (800 or 2000 bar) pressure.

11. High pressure homogenization:
 This technique involves forcing of cell suspension at high
pressure through a very narrow valve to come out to
atmospheric pressure.
 This sudden release of high pressure creates a liquid shear
that can break the cells.
 The high pressure homogenizer (machine) consists of a
high pressure piston pump .
 The piston creates (800 or 2000 bar) pressure.

12. Grinding with glass beads:

13.  It contains a cylindrical body with an inlet, outlet and a central
motor-driven shaft.
 To this shaft are fitted radial agitators.
 The cylinder is fitted with glass beads.
 The cells mixed with glass beads are subjected to a very high speed
in a reaction vessel.
 The cells break as they are forced against the wall of the vessel by
the beads.

14. In this method Several factors influence the cell breakage:
1. Size and quantity of the glass beads.
2. Concentration of bacteria
3. Age of cells,
4. Speed of the agitators.
5. Temperature.

15. Chemical methods of cell disruption:
 Alkalies,
 Organic Solvents
 Detergents used to lyse cell wall of microorganism to release
intracellular products.

16. NaOH, SDS used to lyse bacterial cell wall.

17. Alkalies:
 A highly alkaline solution consisting of NaOH and SDS is then
used to break down the cell walls.
 Alkali treatment has been used for the extraction of some bacterial
proteins.
 Recombinant growth hormone can be efficiently released from E.
coli by treatment with sodium hydroxide at pH 11.

18. Organic solvents:
 Methanol, ethanol, isopropanol, butanol organic solvents can be
used to disrupt the cells.
 The organic solvent dissolves membrane phospholipids and
creates membrane pores for release of intracellular contents.

19. Detergents:
 Detergents: Triton X-100, Tween, SDS and disrupts cell membranes (cell
lysis) and the release of intracellular materials in a soluble form.
 Detergents distrub the protein-protein, protein-lipid and lipid-lipid
associations.

20. Enzymatic methods of cell disruption:
 Lysozyme is the most frequently used enzyme to lyse bacterial cell
wall.
 It hydrolyses β-1, 4-glycosidic bonds between NAM-NAG of
bacterial cell walls.
 The Gram- positive bacteria are more susceptible for the action of
lysozyme.

21.  For Gram-negative bacteria, lysozyme in association with EDTA
can break the cells.
 As the cell wall gets digested by lysozyme, the osmotic effects
break the periplasmic membrane to release the intracellular
contents.
 .

22.  Certain other enzymes are also used, for cell disruption.
 For the lysis of yeast cell walls, glucanase and proteases are used.

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