1. Cell disruption and homogenization
of membrane bound enzymes,
Extraction
By
Mr. Sainath H. Kamble
Assistant Professor in Microbiology
D.B.F. Dayanand College of Arts and Science, Solapur
2. • Biological products synthesized by fermentation or cell
culture are either intracellular or extracellular.
• Intracellular products either occur in a soluble form in
the cytoplasm or are produced as inclusion bodies (fine
particles deposited within the cells).
• Examples of intracellular products include recombinant
insulin and recombinant growth factors.
• A large number of recombinant products form inclusion
bodies in order to accumulate in larger quantities within
the cells.
• In order to obtain intracellular products the cells first
have to be disrupted to release these into a liquid
medium before further separation can be carried out.
3. • Cells
• Different types of cell need to be disrupted in
the bio-industry:
• • Gram positive bacterial cells
• • Gram negative bacterial cells
• • Yeast cell
• • Mould cells
• • Cultured mammalian cells
• • Cultured plant cells
• • Ground tissue
4. • Cell disruption methods can be classified into three categories: Physical
methods, Chemical methods and Biological methods
• Physical methods
• • Disruption in bead mill
• • Disruption using a rotor-stator mill
• • Disruption using French press
• • Disruption using ultrasonic vibrations
• Chemical and physicochemical methods
• • Disruption using detergents
• • Disruption using solvents
• • Disruption using osmotic shock
• Biological Method
• Disruption using enzymes e.g. lysozyme
• The physical methods are targeted more towards cell wall disruption while
the chemical and physicochemical methods are mainly used for
destabilizing the cell membrane.
5. I. Physical methods for cell disruption
• Cell disruption using bead mill
• This equipment consists of a tubular vessel made of metal or thick
glass within which the cell suspension is placed along with small
metal or glass beads.
• The tubular vessel is then rotated about its axis and as a result of
this the beads start rolling away from the direction of the vessel
rotation.
• At higher rotation speeds, some the beads move up along with the
curved wall of the vessel and then cascade back on the mass of
beads and cells below.
• The cell disruption takes place due to the grinding action of the
rolling beads as well as the impact resulting from the cascading
beads.
• Bead milling can generate enormous amounts of heat.
6. • While processing thermolabile material, the milling 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.
• 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 primarily involves breaking the barriers around the cells
followed by release of soluble and particulate sub-cellular components
into the external liquid medium.
7. • Cell disruption using rotor-stator mill
• This device consists of a stationary block with a tapered
cavity called the stator and a truncated cone shaped
rotating object called the rotor.
• Typical rotation speeds are in the 10,000 to 50,000 rpm
range.
• The cell suspension is fed into the tiny gap between the
rotating rotor and the fixed stator.
• The feed is drawn in due to the rotation and expelled
through the outlet due to centrifugal action.
• The high rate of shear generated in the space between
the rotor and the stator as well as the turbulence thus
generated are responsible for cell disruption.
8. • 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.
9. • Cell disruption using French press
• The working principle of a French press which is a device commonly used
for small-scale recovery of intracellular proteins and DNA from bacterial
and plant cells.
• The device consists of a cylinder fitted with a plunger which is connected
to a hydraulic press.
• The cell suspension is placed within the cylinder and pressurized using the
plunger.
• The cylinder is provided with an orifice through which the suspension
emerges at very high velocity in the form of a fine jet.
• The cell disruption takes place primarily due to the high shear rates
influence by the cells within the orifice.
• A French press is frequently provided with an impact plate, where the jet
impinges causing further cell disruption.
• Typical volumes handled by such devices range from a few millilitres to a
few hundred millilitres.
• Typical operating pressure ranges from 10,000 to 50,000 psig.
10. • Cell disruption using ultrasonic vibrations
• Ultrasonic vibrations (i.e. having frequency greater than 18 kHz) can be used to
disrupt cells.
• The cells are subjected to ultrasonic vibrations by introducing an ultrasonic
vibration emitting tip into the cell suspension.
• Ultrasound emitting tips of various sizes are available and these are selected
based on the volume of sample being processed.
• The ultrasonic vibration could be emitted continuously or in the form of short
pulses.
• 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.
• 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.
11. II. Chemical and physicochemical methods of cell disruption
• 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.
• Detergents are classified into three categories: cationic, anionic and non-
ionic. 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. 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.
12. • Cell disruption using organic solvents
• Organic solvents like acetone mainly act on the cell membrane by
solubilizing its phospholipids and by denaturing its proteins.
• Some solvents like toluene are known to disrupt fungal cell walls.
• The limitations of using organic solvents are similar to those with
detergents, i.e. the need to remove these from products and the
denaturation of proteins.
• However, organic solvents on account of their volatility are easier to
remove than detergents.
13. • Cell disruption by osmotic shock
• As discussed early in this chapter, osmotic pressure results from a
difference in solute concentration across a semi permeable
membrane.
• Cell membranes are semi permeable and suddenly transferring a cell
from an isotonic medium to distilled water (which is hypotonic)
would result is a rapid influx of water into the cell.
• This would then result in the rapid expansion in cell volume followed
by its rupture, e.g. if red blood cells are suddenly introduced into
water, these hemolyse, i.e. disrupt thereby releasing hemoglobin.
• Osmotic shock is mainly used to lyse mammalian cells.
• With bacterial and fungal cells, the cell walls need to be weakened
before the application of an osmotic shock
14. III Biological Method
• 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.
• The main limitation of using lysozyme is its high cost.
• Other problems include the need for removing lysozyme from the product
and the presence of other enzymes such as proteases in lysozyme samples.
