Cell Disruption
Nur Istianah,ST,MT,M.Eng
© THP UB 2017
Definition
Cell Disruption is the method or process for disrupting or lysing the cell in order to
release the contents out of the cell.
Mechanism
1
Fermentation (rx) (intracellular
product still exist inside of cell)
2
Homogenised cell
Cell separation
3
Cell disruption
Cell
1. Gram positive bacterial 2. Gram negative bacterial 3. Yeast cell
4. Mould cells
5. Cultured mammalian 6. Cultured plant cells 7. Ground tissue
Cell wall
• Cell wall wherever present is the main barrier which needs to be disrupted to recover intracellular products.
• A range of mechanical methods can be used to disrupt the cell wall.
• Chemical methods when used for cell disruption are based on specific targeting of key cell wall components
lysozyme is used to degrades
peptidoglycan
which is a key cell wall constituent.
the peptidoglycan layer is less
susceptible to lysis by lysozyme,
helped by osmotic shock
Cell wall
• The plasma membrane can be easily destabilized by detergents, acid, alkali and organic solvents.
• The plasma membrane is also quite fragile when compared to the cell wall and can easily be disrupted using osmotic shock i.e. by suddenly changing the osmotic pressure across the membrane.
Methods
1. Disruption in bead mill
2. Disruption using a rotor-stator mill
3. Disruption using French press
4. Disruption using ultrasonic vibrations
Physical methods
1. Disruption using detergents
2. Disruption using enzymes e.g.
lysozyme
3. Disruption using solvents
4. Disruption using osmotic shock
Chemical and physicoc
hemical
The physical methods: cell wall disruption, chemical and physicochemical: destabilizing the cell membrane
Cell disruption using bead mill
• The cell disruption takes place due to the grinding
action of the rolling beads as well as the impact resulting from the cascading beads.
Cell disruption using bead mill
• Bead milling can generate enormous amounts of heat so can be carried out at low temperatures, i.e. by adding a little liquid nitrogen into the vessel. This is referred to as cryogenic bead milling. An alternative approach is to use glycol cooled equipment.
Cell disruption using bead mill
• A bead mill can be operated in a batch mode or in a continuous mode and is commonly used for disrupting yeast cells and for grinding animal tissue.
• Using a small scale unit operated in a continuous mode, a few kilograms of yeast cells can be disrupted per hour. Larger unit can handle hundreds of kilograms of cells per hour.
Cell disruption using bead mill
• Cell disruption primarily involves breaking the barriers around the cells followed by release of soluble and particulate sub- cellular components into the external liquid medium.
• Empirical models are therefore more often used for cell disruption:
rotor-stator mill
10,000 to 50,000 rpm
rotor-stator mill
• These mills are more commonly used for disruption of plant and animal tissues based material and are operated in the multi-pass mode, i.e. the disrupted material is sent back into the device for more complete disruption.
• The cell disruption caused within the rotor- stator mill can be described using the equations discussed for a bead mill.
French press
rotor-stator mill
• commonly used for small-scale recovery of intracellular proteins and DNA from bacterial and plant cells
• The cell disruption takes place primarily due to the high shear rates influence by the cells within the orifice.
• Typical operating pressure ranges from 10,000 to 50,000 psig.
Ultrasonic cell disraption
Ultrasonic cell disraption
• A frequency of 25 kHz is commonly used for cell disruption. The duration of ultrasound needed depends on the cell type, the sample size and the cell concentration.
• These high frequency vibrations cause cavitations, i.e. the formation of tiny bubbles within the liquid medium
Ultrasonic cell disraption
• When these bubbles reach resonance size, they collapse releasing mechanical energy in the form of shock waves equivalent to several thousand atmospheres of pressure. The shock waves disrupts cells present in suspension.
• For bacterial cells such as E. coli, 30 to 60 seconds may be sufficient for small samples. For yeast cells, this duration could be anything from 2 to 10 minutes.
Equation
The time constant θ depends on the processing conditions, equipment and the properties of the cells being disrupted
• In a multi-pass operation:
• A batch of yeast cells was disrupted using ultrasonic vibrations to release an intracellular product. The concentration of released product in the solution was measured during the process:
• If the ultrasonic cell disruption were carried out for 240 seconds, predict the product concentration.
Time (s) Concentration (mg/ml) 60
120
3.49 4.56
Solution
Cell disruption using detergents
• Detergents disrupt the structure of cell membranes by solubilizing their phospholipids. These chemicals are mainly used to rupture mammalian cells.
• For disrupting bacterial cells, detergents have to be used in conjunction with lysozyme. With fungal cells (i.e. yeast and mould) the cell walls have to be similarly weakened before detergents can act.
Detergent
cationic anionic and non-
ionic
Cell disruption using detergents
• Non-ionic detergents are preferred in bioprocessing since they cause the least amount of damage to sensitive biological molecules such as proteins and DNA.
Commonly used non-ionic detergents include the Triton-X series and the Tween series.
Cell disruption using detergents
• However, it must be noted that a large number of proteins denature or precipitate in presence of detergents.
• Also, the detergent needs to be subsequently removed from the product and this usually involves an additional purification/polishing step in the process.
• Hence the use of detergents is avoided where possible.
Cell disruption using enzymes
• Lysozyme (an egg based enzyme) lyses bacterial cell walls, mainly those of the gram positive type.
• Lysozyme on its own cannot disrupt bacterial cells since it does not lyse the cell membrane.
The combination of lysozyme and a detergent is frequently used since this takes care of both the barriers. Lysozyme is also used in combination with osmotic shock or mechanical cell disruption methods.
• Main limitation: high cost
Cell disruption using organic solvents
• Organic solvents like acetone mainly act on the cell membrane by solubilizing its phospholipids and by denaturing its proteins (toluene are to disrupt fungal cell)
• The limitations of using organic solvents are similar to those with detergents,
• However, organic solvents on account of their volatility are easier to remove than detergents.
Cell disruption by osmotic shock
Concentration difference
Rapid influx of water into the cell (osmotic)
rapid expansion in cell volume Cell rupture
Cell disruption by osmotic shock
• Osmotic shock is used to remove periplasmic substances (mainly proteins) from cells without physical cell disruption.
• In a large number of recombinant as well as non-recombinant gram negative bacteria, target proteins are secreted into the periplasmic space.
Exercise
• An intracellular antibiotic is being recovered by ultrasonication from 5 litres of bacterial cell suspension having a cell concentration of 15 g/1. Past experiences have shown that 50% of the antibiotic can be recovered in 40 minutes. Predict the time required for 90% recovery of the antibiotic.
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