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), while 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.

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 = P_m (maximum possible protein releasable) at time zero to P = P_t at time t gives

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.

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).