Cell disruption is needed for the extraction of intracellular products. The method used may vary depending on the type of cell and its cell wall composition. Irrespective of the method used, the main aim is that the disruption must be effective and the method should not be too harsh so that the product recovered remains in its active form.
There are different methods available for cell disruption. In recent trends, intracellular products which are of commercial value are released from microorganisms mainly by disrupting the cells by mechanical methods which will be discussed in detail
This is the widely used method for large scale operations as well as lab scale. This method employs equipment called Homogenizer or Cell disruptor adapted from dairy industry which operates at extremely high pressures (upto 2500 bars). Cell disruptors and homogenizers are both positive displacement pumps each differs in the way that they create pressure on the sample and transfer it from pressurized chamber to another chamber which is at lower pressure. Homogenizers pressurize the sample in a chamber which is then released into a chamber of lower pressure through a homogenizing valve. Cell disruptors use a hydraulic force to accelerate the sample to high pressure and forcing them through a minute orifice to hit on a disruption head which is at a lower pressure.
The basic homogenizer consists of a positive displacement pump which forces the cell suspension through the centre of the valve seat. Pressure can be controlled by adjusting the force imparted on the valve, which is controlled either pneumatically or hydraulically. As the cell suspension is pumped through a minute orifice at high pressure it causes a shear on the cell membranes. This is followed by the sudden release of the suspension with instant expansion. Disruption of the cell is accomplished at three stages causing the explosion thereby releasing its contents.
1. Impegment on the homogenizing valve
2. High turbulence and shear combined with compression produced in the minute gap
3. Sudden pressure drop upon release
The main disruptive factor in this process is the pressure applied on the sample and consequent pressure drop across the valve. This causes the impact and shear stress on the cells making them to break which are proportional to the operating pressure.
Enzymes/Proteins are released at various rates depending on their cellular location. Proteins located in the periplasm are released faster whereas the proteins located within the cellular components are released at a slower rate. Unbound intracellular proteins may be released in a single pass whereas membrane bound enzymes or proteins may require several passes for reasonable yields to be obtained.
The rate of cell disruption is directly proportional to the third power of the turbulent velocity of the product flowing through the homogenizer channel, which in turn is directly proportional to the pressure applied on the sample. The higher the pressure, the higher the release of cell contents per pass through the machine. The release of proteins in the cell disruption process can be explained by the following equation:
Pm â€“ Maximum amount of soluble protein
PR â€“ Amount of soluble protein
k – Temperature dependent release constant
N â€“ Number of homogenizer passes
P â€“ Operating pressure
The operating parameters which affect the cell breaking efficiency of high-pressure homogenizers are as follows:
– Operating Pressure
– Process Temperature
– Number of passes
– Valve/Orifice design
– Flow rate of the sample
There are certain variables to be considered while designing a homogenizer/cell disruptor. They are:
– type of homogenizing valve/orifice
– operating pressure
– stages of disruption
– viscosity of the sample
– type of the surfactant
High-pressure homogenizers are the best available means to mechanically disrupt non-filamentous microorganisms on a large scale. The major disadvantage of homogenizer is the generation of heat during the process. The temperature rise can be prevented by cooling the sample to 40C and also providing proper cooling system for the equipment. While it is true that most of the proteins cannot withstand the high pressure inside the homogenizer, most proteins will be denatured by the temperature inside unless the device is cooled properly.
Cell disruption will release degradative enzymes like Proteases along with the protein of interest. These enzymes can cause serious loss to enzyme activity by degrading our protein of interest. This loss can be minimized by cooling the sample before and after disruption. In addition, protease inhibitors like PMSF can be added along with the sample.
(Sincere thanks to guest editor St. Mane for his contribution)