CELL DISRUPTION TECHQUINES

BY : RITIKA SHARMA

What is cell disruption????

Cell disruption is a method or process for releasing biological molecules from inside the cell. Cell disruption is the process of obtaining intracellular fluids via methods that open the cell wall without disrupting any of its components.

Factors of Cell Disruption

There are a few factors that should be considered while selecting a method for cell disruption. They are: Sample size Ability to disrupt the cells . Stability of the component needed to be isolated.

Methods of Cell Disruption

There are mainly three methods of cell disruption. They are: Physical methods Chemical methods Mechanical methods

Physical Methods
A. Thermolysis: It is relatively an easy and economical method but can be used only if the products are stable to heat shock. Principle: this technique rupture the cells by heat , although its effect depends on certain factors such as pH, ionic strength and presence of proteolytic and other hydrolytic enzymes.

B. Osmotic shock: osmotic shock is provided to

the cells by simply dumping a given volume of cells into pure water of about twice the volume of cells. The cells swell due to osmotic flow of water ultimately bursting there by releasing the products into the surrounding medium. The osmotic pressure that causes the osmotic flow is proportional to the concentration of solutes and temperature. The susceptibility of the cells to undergo disruption by osmotic shock depends on their type.

The method has been used for releasing hydrolyitc enzymes and membrane binding proteins from a number of gram positive bacteria including E.coli and Salmonella typhimurium. C . Ultrasonic waves: ultrasound waves of frequencies greater then 20kHz rupture the cell walls by a phenomenon known as cavitation. The passage of ultrasound waves in a liquid medium creates alternating areas of compression and refraction which change rapidly. The cavities formed in the areas of refraction rapidly collapse as the area changes to one of compression.

The bubbles produced in the cavities are compressed to several thousand atmosphere. The collapse of bubbles creates shock waves which disrupt the cell walls in the surrounding region. The efficiency of the method depends on various factors such as biological condition of the cell, pH, temperature, ionic strength , etc.

Chemical methods
A. Alkali Treatment: A cheap and effective method but very harsh. Treatment is carried out at pH 11-12 for about 20-30 minutes Proteases are inactivated by this treatment. The enzyme l-asparginase has been isolated by this method.

B. Detergent solubalization: Detergents are amphipathic i.e. they are capable of interacting with both water and lipid. The method involves the addition of a concentrated solution of detergent to about half the solutions volume of cells to disrupt the cell wall. Principle: key mechanism involves the action of detergents in solubilizing the lipids in the cell wall to form micelle.

Detergent do not solubilize in polar solvents but at higher concentration lipid solubalization begins suddenly and increases linearly with the concentration of the detergents. The range of detergent concentration at which the abrupt changes in lipid solubility and surface tension of the medium occur is called critical micelle concentration. Examples of detergents used in cell wall disruption: Anionic detergents such as sodium dodecyl sulphate (SDS) , sodium sulphonate . Cationic detergents such as cetyltrimethyl ammonium bromide (CTAB)

C. Cell wall permeabilization: In this method cell wall disruption is achieved by the addition of organic solvents ( for eg. addition of toluene 10% ), brings about cell wall disruption. The solvent is absorbed by the cell wall resulting in its swelling and ultimate rupture. Organic solvents at lower concentrations (1-3%) permeabilize the cell wall without disrupting it. This method is useful in retaining the contents of the cell for sequential release of desired products .

D. Enzymatic digestion:

The method is based on the digestion of the cell wall by the addition of the lytic enzymes to the cell suspension. Enzymes are highly selective , gentle and most effective. The final rupture of the cell wall often depends on the osmotic pressure of the suspending medium The method involves: Removal of extracellular products followed by addition of lytic enzymes to the microbial cells suspended in an osmotic support medium to disrupt the cell wall.

Cell wall disruption results in release of cell wall proteins leaving the protoplast intact. The protoplast is then disrupted by gentle agitation and the soluble cytoplasmic enzymes are separated from proteins and mitochondria The organelle products are finally released by the use of appropriate enzymes.

Mechanical methods
A. Bead mill disruption: The cell suspension is pumped in to the cylinder and the cell disruption occurs due to the shear forces produced between velocity gradients because of the rotary movement of the cells and beads. Also, the collision between beads and cells and grinding of cells between rolling beads also contribute to the disruptive forces.

The rate and degree of cell disruption depends on several parameters. These include: The nature of micro organism-its size, cell wall composition, thickness and concentration of microbial cells. Product location within the cell. Type of bead mill, type of impeller, its agitation speed. Bead size.

B. High Pressure Homogenizer: The cell suspension is pumped through the homogenizing valve at 200-1000 atmospheric pressure. The disrupted cell suspension is cooled to minimize the thermal denaturation of sensitive products. Cell disruption occurs due to various stresses developed in the fluid. Primary mechanism involves stress developed due to the impingement of high velocity jet of suspended cells on the stationary surface.

In addition other stresses are generated such as shear and normal stress. Different parameters influence the degree of cell disruption these include: Product location with in the cell Type of homogenizer Operating pressure Temperature

References
degradome.uniovi.es www.rpi.edu www.wikibooks.org www.piercnet.com

THANK YOU