ptt 302 downstream processing technology semester 1...
TRANSCRIPT
PTT 302 DOWNSTREAM
PROCESSING TECHNOLOGY
SEMESTER 1 2013/2014
Lecture 2:
Cell Lysis
Overview
Selection of a cell disruption method depends
completely on the cell type
A wide variety of methods for breaking, or lysing
cells and tissues, broadly classified as ―chemical‖
and ―physical‖ methods
Cell breakage
MECHANICALNON=MECHANICAL
Shear in liquidsuspension
Shear in frozensuspension
Dessication Lysis
MechanicalAgitation
SuddenPressureChange
Ultrasound
Sudden PressureChange
Grinding
Physical Chemical Enzymatic
Classification of Cell Disruption
Elements of Cell Structure
Prokaryotic Cell
Do not contain a membrane-enclosed nucleus are classified as either
Eubacteria (commonly called bacteria) and Archaea.
The characteristics of cell envelops vary with type. The envelop
generally consists of a cytoplasmic membrane (plasma membrane) and
a cell wall
The membrane composed primarily of proteins and lipids maintains
concentration gradients while the wall provides the main mechanical
strength
The bacterial cell wall protects the plasma membrane and the cytoplasm
from osmotic stress (Fig 2.1 and 2.2)
Figure 2.1: Diagrammatic representations of the structural features
of the surfaces of (a) gram-positive (b) gram-negative bacteria. The
membrane is also called the plasma membrane of the cytoplasmic
membrane
Figure 2.2: (a) Phospholipid molecule and its outline. (b) Cell plasma
membrane formed by phospholipids with their polar head groups in
contact with aqueous phases.
Elements of Cell Structure (cont‘d)
Eukaryotic cells (cells with nuclei and internal
organelles)
More complicated than prokaryotic cells and
Bioproducts may have to be released from intracellular particles –
coated with membranes and/or consist of large macromolecular
aggregates
All cell membranes (including those of bacteria) as a separate,
immiscible, liquid phase relative to the rest of the cell
animal cells do not have a cell wall while the cell wall in plants is very
thick
The cell membrane of animal cells is easily broken whereas the cell wall
of plants is strong and relatively difficult to break (fig 2.3)
Figure 2.3: Eukaryotic cells. Simplified diagrammatic representation
Of an animal cell and a plant cell. The lysates of such cells contain the
internal structures (organelles) shown.
Cell Lysis
Two principal means of lysing cells to obtain their contents: chemical cell lysis and physical destruction via mechanical force
Changes in osmotic pressure - involve modification of a chemical potential that actually results in a mechanical force
Surfactants and enzymes added to a cell suspension act by dissolving a portion of the cell membrane and/or cell wall
Because chemical lysis conditions are detrimental to some bioproducts- sometimes necessary to use pure physical force methods
The various cell lysis methods are categorized in Table 2.1
TABLE 2.1:
CELL DISINTEGRATION TECHNIQUES
Osmotic and Chemical Cell Lysis
Every cell membrane maintains a substantial osmotic gradient;
however a drastic reduction in extracellular concentration of
solute will tend to burst cells (such as animal cells and protoplasts)
If the transmembrane osmotic pressure is due to solute
concentration inside the cell and out, the van‘t Hoff law can be
used to estimate this pressure, which applies to ideal, dilute
solutions:
where
π = osmotic transmembrane pressure
R = gas constant
T = absolute temperature (K)
ci – c0 = difference btw total solute molarity inside and
outside the cell
)( oi ccRT
Chemical Cell Lysis (cont‘d)
Bacterial and plant cells are protected against
osmotic lysis by cell walls
The weakening or partial destruction of these walls
can be achieved with chemical agents (detergents,
chelators, enzymes, solvents)
Chemical Cell Lysis – Enzymes and
Antibiotics
A number of enzymes which hydrolyse specific bonds in cell walls of a limited number of microorganisms
These enzymes are including lysozyme and enzyme extracts from leucocytes, Streptomyces spp., Micromonospora spp., Penicillium spp.
In theory, selective enzyme rupture is ideal but costs are high and the presence of the enzymes may complicate further downstream purification processes
These methods - not widely used on a large scale, with the exception of lysozyme
May be used as a pretreatment to partially hydrolyse cell walls prior to cell disruption by mechanical methods
Chemical Cell Lysis - Solvents
Usually used to lyse cells, especially eukaryotes
For example, acetone – often used early in the preparations of biochemicals from animal tissue homogenates.
It dissolves cell membranes as well as excess fat and at appropriate concentrations may aid in precipitating the product if that is desirable
A number of detergents will damage the lipoproteins of the microbial cell membrane and lead to release of intracellular components
For example - quaternary ammonium compounds, sodium lauryl sulphate, sodium dodecyl sulphate (SDS) and Triton X-100
Unfortunately, detergents may cause some protein denaturation and may need to be removed before further purification stages
The use of Triton X-100 in combination with guanidine-HCl is widely and effectively used for the release of cellular protein (Naglak and Wang, 1992; Hettwer and Wang, 1989)
Gram-negative microorganisms
Use of chelating
agents
The most common, EDTA which binds the divalent cations
Mg2+ and Ca2+
EDTA destabilizes the outer membrane of gram-negative
microorganisms which contains lipopolysaccharide (LPS),
exposing the underlying peptidoglycan layer
The inner membrane is apparently not affected by
treatment with just EDTA
Divalent cations either Mg2+ and Ca2+, stabilize the
structure of the outer leaflet by binding LPS molecules to
each other as well as to outer-membrane proteins
When EDTA removes the divalent cations from the outer
membrane, a large portion of LPS molecules are also
removed
Gram-negative microorganisms
Use of solvent Toluene, organic solvent is probably acts by dissolving inner-
membrane phospholipids
Other solvents, ether – permeabilize E.Coli cells for study of
DNA synthesis. This procedure is apparently specific for small
molecules
Use of
detergents
Both anionic and nonionic detergents have been used to
permeabilize gram-negative microbial cells
The anionic sodium dodecyl sulfate (SDS) at a conc. of
0.05% released 24% of intracellular protein, 35% of
intracellular RNA and 22% of intracellular DNA from E.Coli
(Woldringh, 1970)
The nonionic detergent: Triton X-100
The main location of detergent action in gram-negative
microorganisms seems to be the inner membrane
Use of chaotropic
agents
Example: Guanidine and urea are capable of
bringing some normally hydrophobic compounds into
aqueous solution
They accomplish this by disrupting the structure of
water, making it a less hydrophilic environment and
weakening the hydrophobic interactions among solute
molecules
Yeast
Use of solvent Toluene is used to permeabilize yeast cells for in situ
enzymatic assays and in this case the structure of the
cells is left intact and the enzyme activity remain inside
Proteins can also be removed from yeast by toluene
under appropriate conditions (higher concentrations and
higher temperatures) although the cells tend to dissolve
rather than permeabilize
Use of detergents Triton X-100 has been used to permeabilize yeast cells
for enzymatic assays
Mechanical Method of Cell Lysis
The disruption of microorganism – often required in the
large-scale production of microbial products such as
enzymes, toxins and diagnostic or therapeutic proteins
The ideal technology for cell disruption may be
characterized by:
Max. release of the product of interest
No mechanical or thermal denaturation of the product
during disruption
Min. release of proteases which may degrade the product
Min. release of particulates or soluble contaminants that
may influence downstream processing
High Pressure Cell Homogenizers
A homogeniser consists of a positive-displacement
pump, which this high-pressure pump incorporates an
adjustable valve with restricted orifice through which
cells are forced at pressures up to 550atm (Fig. 2.4 a
& b)
General applicability for cell disruption although the
homogenizing valve can become blocked when used
with highly filamentous organisms
Q: What are the factors which you believe will influence
the efficiency of the homogenizer for disrupting cells?
Figure 2.4a: Details of a high pressure (Manton—Gaulin) homogenizer valve: A, handwheel for adjusting pressure; B, spring-driven valve rod; C, valve (see Figure 2.6); D valve seat (also see Fig. 2.6); and E. impact ring of hard material. The ring E is sometimes eroded by the impact of cells and debris, which can be abrasive.
Figure 2.4b: Cell disruption in a high-pressure homogenizer.
High Pressure Cell Homogenizers - Influence
of Pressure
Cell disruption follows first-order kinetics as first described by Hetrington et. al. by the equation:
Where:
Rm = max. protein release or enzyme activity;
R = measured protein release or enzyme activity after N passes
k = a first order dimensionless rate constant (1/s)
N = number of passes
∆P = pressure drop across valve seat
x = is highly dependent on the type of cell and the conditions under which the cells were grown
x
m
m PKkkN,RR
Rlog
High Pressure Cell Homogenizers -
Influence of Pressure (cont‘d)
The dimensionless rate constant (K) – principally a
function of pressure drop across the valve seat (∆P)
The constant (K) – a function of temperature and in
some instances, cell concentration
The first order expression implies that the rate of
rupture at any time is dependent on the proportion
of cells remaining undisrupted
Subsequently, it was confirmed that the rate
constant K depends on the organism
High Pressure Cell Homogenizers -
Influence of Pressure (cont‘d)
Most applications operate within the range of 500-1000 bar
The max. allowable operating pressure is often dictated by the mechanical stability of the valve design – may due to design or material of construction
Microorganism K
Pseudomonas putida 0.41
Eschericia coli 0.39
Bacillus brevis 0.28
Saccharomyces cerevisiae 0.23
Nocardia rhodochrous 0.0085
High Pressure Cell Homogenizers -
Influence of Homogenizer Valve
Valve Seat Geometry
Valve design and selection optimize the influence of
shear and impingement on cell disruption
Operational factors such as temp and pressure stability
may also influence selection (Fig. 2.6)
Figure 2.6: Configuration of high pressure homogenizer
valves used in Manton—Gaulin homogenizers
High Pressure Cell Homogenizers -
Influence of Temperature
The rate of cell disruption increases with temperature
In selecting the inlet temperature for a homogenizer – one must
consider both the temp rise that occurs during processing and the
max allowable temp of the product
High Pressure Cell Homogenizers - Cell
Physiological Factors
The amount of disruption that can be achieved in a single pass is
a function of the type of organism and its physiological state as
well as the homogenizer operating conditions.
There are wide differences in the susceptibility of different
types of organisms to disruption, but there does not appear to
be a general correlation between difficulty of disruption and
organism classification (e.g., bacteria or yeast) (Engler and
Robinson, 1981b).
Results from disruption studies indicate that cells grown at a high
specific growth rate are more easily disrupted than cells of the
same organism grown at a lower rate on the same medium.
High Pressure Cell Homogenizers –
Other Factors
Another factor that has been shown to affect disruption
characteristics of cells is the composition of the growth medium
(Gray et a 1972).
Other environmental factors that can affect cell growth, such as
aeration, pH and temperature, may also alter the susceptibility
of cells to disruption, although they have not been studied.
Bead Mills – Principle of Operation
Originally, devised as pigment mills, they may be used for cell rupture
Bead mills use a horizontal, jacketed grinding chamber filled with grinding media, such as glass beads (Figure 2.7)
A cell slurry is introduced into the supply side of the chamber on a continuous basis
Kinetic energy is imparted to the beads by a variable speed shaft equipped with multiple discs – leads to cell disruption through the combined forces of cavitation, generation of high shear forces, grinding between the beads and by direct collision with the beads
Figure 2.7:Horizontal bead mill
Bead Mills – Principle of Operation
(cont‘d) The cell homogenate – then separated from the grinding
media by mechanical means using an annular disc
Heat dissipation – removed by passing cooling water or refrigerant through the jacket
As with the high-pressure homogeniser, disruption (followed by the release of soluble protein) can be represented by a first-order rate equations:
where: Rm = max protein release R = protein release after N passes k = a first order rate constant (1/s) N = no. of passes t = mean residence time (s0 per pass
kNt RR
R
m
mlog
Bead Mills – Principle of Operation
(cont‘d)
The ratio of heat transfer area to the mill volume
can be expressed as follows:
where:
T = cylinder/chamber diameter (m)
L = length of bead mill (m)
T
4
LT4
π
πTL
(V)millvolume
a(A)surfaceareL
2
Bead Mills – Principle of Operation
(cont‘d) The power input (P):
where :
c = dimensionless constant
ρ = suspension density (kg/m3)
N = rotational speed of the impeller (s-1)
D = impeller diameter (m)
‗c‘ depends on the type of flow in the mill (laminar or turbulent) and the type of impeller
The main problem in the scaling up of bead mills – the removal of the energy dissipated in the broth
Increasing the impeller diameter – result in a considerable increase in power input (at constant speed)
53DNcP
Advantages and disadvantages of homogenisers
and bead mill
Advantages
Homogeniser Bead Mill
1. More flexible with regard to cell types 1. May achieve disruption in a single pass
2. Requires less maintenance 2. Better temperature distribution and
control
3. No process stream contamination 3. Single pass often sufficient
4. Brief residence time 4. Aerosol generation is minimized
Disadvantages
1. Aerosol generation 1. Difficult to clean and sterilize
2. Heat generation may lead to
denaturation
2. Broad residence time distribution
3. Multiple-pass operation is standard 3. Introduces colloidal silica into
homogenate from abrasion
4. Performance and capacity varies
greatly with cell type