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Methods
Steps in DSP:
Primary
recover
y
Product
Purification
Recovery of Intracellular
Products:
It requires additional
processing such as cell
disruption , lysis ,
permeabilization , or
extraction.
Cytoplasm /
Intracellular
Periplasmic
Space
2. Microorganism used:
Cell wall properties
Cell Size.
Cell density
Extra
Cellular
Intracellular Products
Intracellular products
Glucose isomerase
-galactosidase
Phosphatase
Ethanol dehydrogenase
Dnase, Rnase
NADH/NAD+
rDNA intracellular
products
Chymosin (yeast/E.coli)
Insulin (E.coli,
mammalian)
Immunoglobulin
Interferons (mammalian)
Human growth hormone
(E.coli)
Human serum albumin
layer of peptidoglycan
than Gram -ve
Yeast has thick cell
wall
Outer Protein
Mannan complexs
Inner -glucans
wall
CELL DISRUPTION
Objectives:
To extract biological products of interest
the products.
Goals ?
Solubilize the product present in the cells with
Methods Of Cell
disruption:
Physico-mechanical
methods
Chemical methods
Liquid shear
Detergents
Solid shear
Osmotic shock
Alkali treatment
Freeze-thawing
Enzyme treatment
Ultrasonication
Physico-Mechanical
Methods
1. Liquid Shear:
Widely used in large scale enzyme purification
How these Homogenizer devices works ?
Cell suspension is forced under high pressure (up
to 1500 bar) through a narrow discharge valve.
followed by a pressure drop to atmospheric
Cell disruption mechanisms:
1. Impingement on the valve
2.
3.
Liquid Shear
homogenizers:
High pressure homogenizers (up to 1500 bar)
1. French Press:
laboratory level
French Press
High pressure cylinder with small
orifice and needle valve at its base.
Cell suspension is placed within the
cylinder and pressurized using the
plunger/Piston(10,000 50,000 psi)
The suspension emerges through
the orifice at very high velocity in
the form of a fine jet.
impact plate: the jet impinges
further cell disruption
Effect of Operating
Pressure on Disruption
Efficiency
Source: frenchpressurecell.com
2. Solid shear
Pressure extrusion of frozen micro-organism(-25 0
C) through a narrow orifice.
Cell disruption mechanisms:
Combination of liquid shear and presence of
ice crystals.
Not suitable for materials sensitive to freezing
and thawing.
Eg:
- Hughes press
3. Agitation with
abrasives
Grinding cells with Abrasives : Grind and
smash cells
Simple Mortar and Pestle
Ball mill/Bead mill/ Dyno mill.
Ball Mill:
Ball mill containing series of
cooling jacket.
Ideal for disruption of Yeast,
Dyno-Mill Multi-Lab
Ball mill:
4. Ultrasonication
Another liquid-shear method
High frequency vibration
(20kHz/s) at the tip of an
ultrasonication probe
cavitations cell disruption.
Ultrasonic vibrator generate
high frequency waves
Transducer - converts waves
into mechanical oscillations by
Titanium Probe.
Ultrasonication
Mechanism: Cavitation followed by shock waves
High frequency formation of tiny bubbles bubbles
collapse releasing mechanical energy (shockwave) ~
thousands atm pressure
Frequency: 25 kHz
Duration depends on cell type, sample size and cell
concentration
Bacterial cells (E. coli) 30-60 s
Yeast cells 2-10 minutes
Used in conjunction with chemical methods
Cell barriers are weakened by small amounts of
enzymes or detergents energy reduced
Ultrasonication
Rods are broken readily than
cocci
Gram Negative easily than
Gram Positive.
Not effective for molds.
Power requirements - high,
need for cooling because of
large heating effect, short
working life for probes.
Laboratory scale methodhigh cost.
Factors Effecting
Ultrasonication:
5. Freeze - Thawing
High cost
Chemical Methods
1. Detergents
Damage the lipoproteins of the cell membrane.
Detergents
Triton X-100 + Guanidine HCL( Chaotrophic
agent) widely used for release of cellular
proteins.
CTAB is widely used in the isolation of DNA
from plants.
Detergents May cause protein denaturation/
precipitation require removal before further
purification.
SDS removal was achieved by adsorption
onto Zeolite Y
Detergents
2. Osmotic shock
Sudden change in the salt concentration.
Effect on microbial cells is normally minimal
Procedure
1. Allow the cells to equilibrate in a high
sucrose medium(20%)
2. Then rapidly diluting away the sucrose.
3. Endosmosis- Cell Rapture
Used for :
3. Alkali treatment
Alkaline lysis using hydroxide and hypochlorite is
an effective and cheap method.
It acts by saponifying the cell-wall lipids
Extremely harsh technique so the product must
be resistant to degradation at high pH( 11.5-12.5
for 20-30 mins)
Uses:
Recovery of polyhydroxy alkanoates (PHA,
biodegradable polymer) from E. coli
Extraction of L-asparaginase.
Recombinant Growth Hormone by NaOH
Organic solvents:
Methanol, ethanol, Isopropanol,
butanol
Toluene: dissolves membrane
phospholipids pores
Freeze drying followed by acetone,
butanol treatment- cell wall
disruption.
Organic Solvents:
Permeabilization:
EDTA :
widely used for Gram negative
microorganisms.
Binds to the divalent cations of Ca2+ , Mg2+
that stabilize the structure of outer
membranes.
DMSO
Chemical permeabilization by antibiotics:
Penicillin or Cycloserine- interferes with cell
wall synthesis
High cost
Selective release of
product from
selected location
Removal of lysozyme
(enzyme) from the product
Release of cloned
intracellular
products.
Low energy
consumption
Small risk of product
damage
Harmless to
Lysozyme:
Cell lysis
Product renaturation
Enzymatic lysis
Algae: Cellulases
Process of lysis :
Quantifying Cell
Disruption
Microscopy (optical or SEM)
Intact vs. broken cell
Differential staining - Methylene blue dye
exclusion
automatic cell counting using a
hemocytometer
Particle size analyzers to (e.g., Coulter
counters)
Viscosity
Spectrophotometry
References:
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