Cell disintegration and

extraction techniques
 Overview

To get the intracellular product

Æthe disintegration of cells is need

The methods used to break cells depend largely on the fragility of the cells
ex) animal cells: burst by osmotic shock, freeze/thaw, enzyme digestion(lipase, protease), toluen
plant tissue: pectinase and cellulase treatment
microbial cells: lysozyme treatment

To achieve a good yield,

yield
1. minimize the number of steps
2. choose appropriate disruption methods
(a) can a given disruptor be used for a particular cell type?
(b) which is the best method of extracting a product?
Key questions

1. Stability of the released protein

2
2. L ti off ttargett protein
Location t i within
ithi th
the cellll
3. The yield and kinetics of the process
4. Continuous or batch disruption
5. The need to consider subsequent steps
6. Assesing the extent of disruption
7. Marker substance for cell disruption
8. Containment of the process
-avoid release of harzardous intracellular products into envronment
9. Scale-up-cost, volume, sample viscosity
1.Stability of the released protein

•The disruption methods can impose great physical and chemical stress
ex) heat generated by mechnical disruption may result in Nz denaturation
activation of proteolytic enzyme can degrade target Nz
•Consideration:
C id ti minimize i i i ththe stress
t condition
diti

3.The yield and kinetics of the process

•Yield-the quantity of enzyme released/unit starting materials
•Specific activity-the amount of enzyme(unit)/ released protein(g)
•Factors affecting the yield of enzyme
(a) location of product with in the cell
(b) degree of disintegration
(c) extent of denaturation of the product during disruption
•Disruption rate can be modelled by “First-order process”
R=RM(1-e-kkt) : time-dependent
Æconsider the “optimun disruption time”
5. The need to consider subsequent steps

•After disruption, clarification step is need for subsequent purification step

Æusually, through centrifugation
in the laboratory, high g force
on larger scale, clarifying cell lysate is difficult
Æ whether a laboratory centrifuge is capable of sedimenting cell debris
t: the centifugal time
RS:radii
dii from
f centrifuge
t if tube
t b hheadd to
t liliquid
id surface
f
In(RS/RL) *18µ RL:radii from centrifuge tube head to bottom
t= µ:liquid velocity
ω2(ρP-ρL)dP2 ρP:particle density
ρL:liquid
q dencityy
ω:angular velocity =2π(rpm)/60
dP:mean diameter of cell debris

•Mechanical disruption methods are not needed chemicals that intetfere with
subsequent
b t purification
ifi ti step
t
6. Assesing the extent of disruption

•Use a marker substance (estimate total intracellular proteins)

•specific activity could decrease during disruption
because of releasing of nonnon-product
product proteins and denaturation of enzyme during disruption

7. Marker substance for cell disruption

tto ddetermine
t i th the degree
d off cellll di
disruption,
ti marker
k ttechniques
h i are usedd
1. Biological: visible cell counting
2. Physical: measuring the Vol. of intact cells, O.D., viscosity of the sample
3. Chemical: measuring the protein concentration
Methods of disruption
•Slow the rate of enzyme damage caused by proteas(Æadd PMSF in eukariotic cell)
4 •Prevent thermal denaturation

1. Pre-treatment of material
2. General procedure notes
3. Mixers and blenders
4. Coarse grinding: pestle and mortar
5. Fine grinding:the bead mill 10. Osmotic shock
6. Homogenization 11. Lytic enzymes
7
7. Ultrasonication 12
12. Chemical treatment
8. Heat shock 13. Detergents
9. Freezing and thawing 14. Slovents
1. Pre-treatment of material

To increase the degree of cellular disruption

ÆCombine two(or more) methods

for example, freeze-thaw step or pre-treatment of solvents or detergents

before mechnical disruption
 2. General procedure notes

Table 1.The main cell disruption methods

1. Methods needing specialist equipment Disadvantage:Cells are not extensively distintegrated
(a) mixer and blender
(b) coarse grinding with pestle and mortar incubation time
(c) fine grinding in a bead mill operational parameters
(d) homogenization (conc.of lysing chemicals, bead size, conc.,
(e) ultrasonication agitator speed for bead mill, pressure for homogenization,
Vol. Of sample for ultrasonication)
2. Methods using non-specialist equipment
(a) freezing and thawing
(b) osmotic shock
(c) chaotrophic agents
(d) detergents Disadvantage: addition of extra chemicals to
(e) solvents the system may interfere with downstream processing
(f) enzyme lysis
3. Mixers and blenders

•Grind cells coarsely

•Use the buffer containing inhibitors, reducing agents(see protocol 2)

4. Coarse grinding: pestle and mortar

•Useful for disruption of tissue samples
•Samples were grinded to fine powder under liquid nitrogen
•Nacessary to maintain the frozen state

5. Fine grinding: the bead mill

•Useful for disruption of micro-orgarnisms
•Bead mills should have cooling jackets
because of heat ggeneration duringg disruption
•Optional parameters: bead size, bead volume, agitator speed, milling time
6. Homogenization

1. Hand-held piston/plunger device

Æanimal cells are easily disrupted, but very inefficient
2. High-pressure homogenizer
Æsuitable for large scale operation
Æprinciples;
sample narrow orfice High pressure cell crusing cell breaking
pumping

usually, increasing the pressure increase the amount and rate of protein release
according to k=k’Pn(first-order reaction, p98)

High-pressure homogenizer Mechanical homogenizer

7. Ultrasonication

•Very vigorous process, results in complete solubilization

•Principles;
the vibrating titanium probe creat cavities
Æcollaps of the cavities
Æpressure changes and shear foreces which cause cell disruption
•Problem: heat-generation(cause thermal denaturation, alteration of Nz activity
impossible to adjust the power input
•The release curve for protein is usually first-order(Fig. 4), but could be affected by
power input, sample volume
•Very useful for fragmentaion of cellular DNA related with increaseing of viscosity
Non-mechanical disruption methods

Heat shock
•Consider the thermal denaturation. Leading to loss of Nz activity
•Useful for purification of heat-stable proteins(e.g.ubiquitin)
Freezing and thawing
•Very simple, but suitable for cells without a cell wall
•Repeating of freezing and thawing may cause denaturation of proteins
Osmotic shock
Lytic enzymes – bacterial disruption with lysozme
useful for Gram positive cells
Chemical treatment
·dvantage: the cell will be left substantially intact
· chemicals must bo compatible with further downstrea process
· chelating agents(sequest divalent cation)and chaotropic agents(weaken hydrophobin interaction)
D t
Detergents t -increase protein solubility
Slovents – dosen’t inactivate the enzyme products
toluene, ether, isoamylalcohol, chloroform
Colclusions: choice of methods

(a) The mechanical methods of cell disruption have the widest

application for laboratory and pilot scale disruption
(b) Homogenization has proved an effective large scale process
(c) Chemical methods are, generally, cell/product specific and
thus, not applicable to all system