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Enzyme Technology
Cell breakage
Various intracellular enzymes are used in significant
quantities and must be released from cells and purified (Table 2.1).
The amount of energy that must be put into the breakage of cells depends very
much on the type of organism and to some extent on the physiology of the
organism. Some types of cell are broken readily by gentle treatment such as
osmotic shock (e.g. animal cells and some gram-negative bacteria such as
Azotobacter species), whilst others are highly resistant to breakage.
These include yeasts, green algae, fungal mycelia and some gram-positive
bacteria which have cell wall and membrane structures capable of resisting
internal osmotic pressures of around 20 atmospheres (2 MPa) and therefore have
the strength, weight for weight, of reinforced concrete. Consequently a variety
of cell disruption techniques have been developed involving solid or liquid
shear or cell lysis.
The rate of protein released by mechanical cell disruption
is usually found to be proportional to the amount of releasable protein.
(2.5)
where P represents the protein content remaining associated
with the cells, t is the time and k is a release constant dependent on the
system. Integrating from P = Pm (maximum possible protein releasable)
at time zero to P = Pt at time t gives
(2.6)
(2.7)
As the protein released from the cells (Pr) is
given by
(2.8)
the following equation for cell breakage is obtained
(2.9)
It is most important in choosing cell disruption strategies
to avoid damaging the enzymes. The particular hazards to enzyme activity
relevant to cell breakage are summarised in Table 2.3.
The most significant of these, in general, are heating and shear.
Table 2.3. Hazards likely to
damage enzymes during cell disruption.
|
Heat |
All mechanical methods require a large input of
energy, generating heat. Cooling is essential for most enzymes. The
presence of substrates, substrate analogues or polyols may also help
stabilise the enzyme. |
|
Shear |
Shear forces are needed to disrupt cells and may
damage enzymes, particularly in the presence of heavy metal ions and/or an
air interface.] |
|
Proteases |
Disruption of cells will inevitably release
degradative enzymes which may cause serious loss of enzyme activity. Such
action may be minimised by increased speed of processing with as much
cooling as possible. This may be improved by the presence of an excess of
alternative substrates (e.g. inexpensive protein) or inhibitors in the
extraction medium. |
|
pH |
Buffered solutions may be necessary. The presence of
substrates, substrate analogues or polyols may also help stabilise the
enzyme. |
|
Chemical |
Some enzymes may suffer conformational changes in the
presence of detergent and/or solvents. Polyphenolics derived from plants
are potent inhibitors of enzymes. This problem may be overcome by the use
of adsorbents, such as polyvinylpyrrolidone, and by the use of ascorbic
acid to reduce polyphenol oxidase action. |
|
Oxidation |
Reducing agents (e.g. ascorbic acid, mercaptoethanol
and dithiothreitol) may be necessary. |
| Foaming |
The gas-liquid phase interfaces present in foams may
disrupt enzyme conformation. |
|
Heavy-metal toxicity |
Heavy metal ions (e.g. iron, copper and nickel) may be
introduced by leaching from the homogenisation apparatus. Enzymes may be
protected from irreversible inactivation by the use of chelating reagents,
such as EDTA. |
Media for enzyme extraction will be selected on
the basis of cost-effectiveness so will include as few components as possible.
Media will usually be buffered at a pH value which has been determined to give
the maximum stability of the enzyme to be extracted. Other components will
combat other hazards to the enzyme, primarily factors causing denaturation (Table
2.3).
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