Precipitation.

Precipitation, often used as a method for the concentration o f protein and enzymes can also provide a certain degree o f clarification. Some basic theory and principles o f precipitation as relevant to their application in this study are presented in the following section.

5.1.3.1 Introduction.

Protein precipitation from aqueous solution, with subsequent recovery o f the precipitate, constitutes one of the most important processes available for the industrial recovery and purification o f proteins. The various methods for causing the needed reduction in the solubility o f the proteins include:

• high salt concentration, to give precipitation by “salting-out”.

• pH adjustment to a protein’s isoelectric point (pi), at which the protein has a minimum solubility

• reduction of the medium dielectric constant to enhance electrostatic interactions, by, for example, the addition of miscible organic solvents

• addition of non-ionic polymers, which reduce the amount of water available for protein solvation

• addition of polyvalent metal ions to form (reversibly) protein precipitates

5.1.3.2 Precipitation by salting-out.

The salting-out o f proteins follows the empirical relationship (Cohn, 1925);

\ogS = n + p 5.1

where S is the solubility o f the protein at ionic strength I. Different proteins exhibit different values for the constants K and p which often permit fractionation o f a mixture o f proteins. A theoretical study has shown that the salting-out o f a protein can be described as a balance between a salting-in process due to electrostatic effects o f the salt and a salting-out process due to hydrophobic effects (Melander and Horvath, 1977). They viewed protein molecules are spheres covered with hydrophobic and charged hydrophilic residues and reported that the attractive forces between the hydrophobic regions on the molecules, increased with increased salt concentration, thereby inducing greater dipoles. At the same time the development of a layer o f like charges on the molecule would give a salting-in effect due to increased molecular repulsion. Generally the magnitude o f the salting out curve is much greater than the salting-in effect and a maximum solubility is reached at relatively low salt concentrations, with the solubility decreasing at higher salt concentrations.

Ammonium sulphate has a very high solubility and gives high values o f K, that is, a high degree o f protein precipitation over a relatively narrow range o f ionic strength. It also has a protective effect on protein structure and at high concentration prevents the growth o f micro-organisms. As such it is one o f the more popular salts for salting-out (Hoare, 1982).

5.1.3.3 Kinetics of precipitate formation.

An in depth consideration of the theory behind precipitate formation is beyond the scope o f this work. However in simple terms, when solution conditions are changed to favour precipitate formation, in stagnant fluids the formation of protein precipitates is a diffusion driven or perikinetic process governed by Brownian motion. Fluid shear

can increase the precipitation rate and also produce a smaller, more compact particle which will survive passage through a centrifuge. Increasing the frequency of particle- particle interaction can increase the rate o f particle precipitation. Growth of precipitates under such shear driven conditions is known as orthokinetic growth. A precipitation operation must accomplish both initial nucléation and growth of individual particles, as well as their subsequent aggregation to give a precipitate size convenient for subsequent particle recovery though centrifugation or filtration. However the use o f excessive shear forces can break up weak precipitates. The Camp number, Gt, has been defined where G is the average shear rate in and t is time of exposure to shear (Bell and Dunnill, 1982). Studies have shown that fresh precipitates subjected to a Camp number o f at least 10^ resulted in the most compact precipitates and rapid formation at low rates of shear. However it was recognised that the shear rate in a stirred vessel is not homogeneous and that the use of the Camp number is perhaps an oversimplification o f the mixing environment (Hoare, 1982).

5.1.3.4 Centrifugal recovery of protein precipitates.

Several thorough papers have reviewed the theory behind centrifuge operation and centrifugal recovery o f precipitates (Bell et a l, 1983) and should be consulted for a detailed description of all related factors. However, worthy o f note was the insight that centrifugal use for precipitate recovery requires sufficient time prior to centrifugation to allow for both precipitate formation and stabilisation. The requirements for time and shear rate described by Bell et a l, (1983) can be summarised as follow;

• Centrifugation requires a precipitate of at least a certain minimum diameter.

• A combined shear rate and time to provide a sufficiently strong floe

• Sufficient stirring to allow good bulk mixing

5.1.3.5 Precipitation of alcohol dehydrogenase.

As already discussed, Alcohol dehydrogenase (ADH) has been selected for use as the target enzyme for the assessment of packed and expanded bed recovery routes. ADH may be precipitated using a variety of methods including addition of polyethyleneglycol (Foster et a l, 1973) and salting out through addition of ammonium sulphate (Foster et a l, 1971). Richardson et a l, (1990) reported on the fractional precipitation of alcohol dehydrogenase to achieve purification of the enzyme using ammonium sulphate. Foster et a l, (1976) discussed the precipitation of the same enzyme in a continuous stirred tank reactor as opposed to precipitation in a batch reactor.

The wealth of information on the precipitation and centrifugal recovery o f ADH using ammonium sulphate, made this clarification and concentration method very attractive for comparison with both the PEI flocculation route and the expanded bed adsorption route. Precipitation using ammonium sulphate has the added advantage that the salt does not have to be removed prior to hydrophobic interaction chromatography.