Partitioning In Aqueous Twophase Systems

The separation of organic compounds by means of their differing distribution between two immiscible organic solvent phases is a routine method of chemical purification in the laboratory and on an industrial scale. This technique is not generally suitable for the purification of biological compounds such as proteins because of their low solubility and tendency to denature in the presence of organic solvents. However, it has long been known that phase separation can also occur with mixtures of certain polymers in aqueous solution (Dobry & Boyer-Kawenoki, 1947). The application of these systems to the purification of biological materials was pioneered by Per-Ake Albertsson (1956; 1958; 1986), although supported phase systems had previously been used for protein fractionation (Porter, 1955).

The most commonly studied aqueous two-phase systems (ATPS) are composed of polyethylene glycol (upper phase) and dextran or potassium phosphate (lower phase). Many other components such as methyl cellulose, ficoll and polyvinyl alcohol have also been used. At equilibrium, solutes in these systems are distributed between the two phases in accordance with their partition coefficient (K) such that

K = (top phase concentration)/(bottom phase concentration).

The partition of a solute between the phases is a surface dependent phenomenon involving various interactions, including ionic, hydrophobic and hydrogen bonding. The size, hydrophobicity and charge of a protein therefore influence its partition coefficient. The properties of the phase-forming components and the composition of the system also influence solute partition. It is therefore possible to manipulate the partition coefficients of a mixture of solutes in order to optimise their separation. For example, the inclusion of salts in the system can have a significant effect. Unequal distribution of the ions between the phases creates an electrical potential which influences protein partition according to its net charge. A comprehensive work by Albertsson (1986) describes the theory and application of ATPS in detail.

ATPS are attractive for the large-scale purification of proteins for a number of reasons. The low surface tension and mild operating conditions cause little protein denaturation. As the partition coefficient is independent of total system volume, the method is amenable to scale-up. The processes of phase mixing, separation and recovery require mixers and low-speed centrifuges which are already widely operated on a large scale in the food industry. The phase-forming compounds are non-toxic and are available in large amounts. Unlike chromatographic separations, this method is tolerant of particulate material in the feedstock and so there is little requirement for preliminary clarification processes. Indeed, partitioning in ATPS can also be used as a method of cell debris removal (Datar & Rosen, 1986). A large number of scientific papers have been published since the 1970's which describe the partitioning of proteins in ATPS and the application of this technique to protein purification. Table 5 shows some examples cited by Kroner et al (1984).

Despite the apparent advantages of this method for large-scale use, and the large body of literature on the subject which exists, extraction in ATPS is not yet widely used by industry. At least in part, this is because solute partitioning between two phases offers little scope for the extremely high degree of selectivity required to separate one protein from thousands. While large recoveries can be achieved because of the mild conditions and the ability to obtain extreme partition coefficients, purification factors are most often in single figures. However, some proteins can be purified to a high degree using ATPS, particularly those which have extreme properties or which form a large proportion of total cell protein. For example, thaumatin, which has a pi greater than 10, was purified 20-fold from an extract of E. coli with recovery greater than 90% (Cascone et al, 1991). The fluorescent pigment phycoerythrin was purified from the cyanobacterium Synechococcus DC-2 in which it forms over 50% of total cell protein (Niven et al, 1990).

The resolution of proteins by ATPS can be increased by the use of countercurrent extraction. This is effectively an automated method of carrying out multiple extractions on a single feedstock sample. After the introduction of the crude material, mixing and equilibration, the two phases are separated and re-extracted with fresh polymer/salt solutions. Repeated extractions result in the distribution of a protein between the extract fractions, dependent on its partition coefficient. Proteins with differing partition coefficients can thus be resolved. Various apparatus have been designed for this purpose in which phase mixing, separation and transfer are carried out automatically (Albertsson, 1986).

As with many other methods for the separation of biological compounds, the specificity can be increased by the use of affinity ligands. This was demonstrated in the context of ATPS by Shanbhag & Johansson (1974) who chemically modified PEG by attachment of palmitic acid as an affinity ligand for albumin. Flanagan & Barondes (1975) investigated the partition of concanavalin A in a polyethylene glycol/dextran system. Because of the affinity of concanavalin A for dextran, it partitioned into the dextran phase. On inclusion of D-mannose, an inhibitor of the affinity interaction, the partition coefficient of concanavalin A substantially increased. Since then, many other affinity interactions have been incorporated into ATPS, including the use of dye ligands such as Cibacron Blue. Kopperschlager & Johansson (1982) attached Cibacron Blue to PEG and achieved a 58-fold purification of phosphofructokinase from baker's yeast. Other chemical modifications have been made to phase-forming polymers to enhance separation such as the attachment of charged groups, thus introducing ion-exchange effects (Cheng et al, 1990).

Process economics is an important consideration in the large-scale industrial use of ATPS as a protein purification method. Some reports have suggested that the cost of the phase-forming components and waste treatment makes this method unfeasible (Schoutens & Kerkhof, 1987). Alternatively, the high biomass loading which is possible with ATPS means that lower capacity plant is required and that throughput is high compared to other methods. This results in start-up and operating cost savings (Kula et al, 1982). In a theoretical cost analysis, Kroner et al (1984) suggested that extraction in ATPS was cheaper than centrifugation or filtration. It is possible that, by the use of low-cost polymers and phase component recycling, the costs may be reduced sufficiently to make large-scale processes viable. For example, the starch-based polymer Reppal may be used in place of dextran at 10% of the cost (Mattiasson & Kaul, 1986). It is also necessary to consider effluent disposal and so, while phosphate salts may have a low initial cost, their effect on the environment makes recovery essential.

The technical feasibility of ATPS extraction on a large scale has been demonstrated in several studies. Hustedt et al (1988) described computer-controlled plant for continuous cross-current extraction of enzymes. This was operated at approximately 100 1/h with biomass loadings of 20-35% (w/w). Fumarase from Brevibacterium ammoniagenes was purified 22-fold with 75% recovery. Other enzymes, including penicillin acylase and aspartase, were processed with similar results. The same group also investigated the possibility of improving the economic feasibility of such purifications by recycling process chemicals (Papamichael et al, 1992). In studies using the separation of fumarase from baker's yeast, they estimated that chemical recycling reduced materials costs but increased labour, capital and utilities costs. The net effect on total operating costs was a saving of 14%. A similar saving was estimated by using continuous processing compared to batch processing.

In technical terms, it is apparent that extraction in ATPS is a feasible and attractive technology for large scale separations. For application in the food sector in particular, the high capacity and the use of non-toxic components and familiar equipment are advantageous. However, the economic feasibility is largely dependent on the particular process under consideration. The lack of wide-spread commercial use of this method suggests that the case for its industrial use is still not proven. The purification by ATPS of enzymes for food applications is likely to require continuous operation and phase component recycling. This increases the sophistication of the plant which is required to carry out these processes and therefore the initial capital investment required. Before this method can achieve its technical potential in the food industry, the economic case will have to be proved beyond reasonable doubt. It is possible that this will only happen after ATPS is better established in other sectors. The continued development of new processes and phase forming systems may yet contribute to the achievement of this potential.

Table 5

Purification of enzymes by extraction in ATPS

Data from Kroner et al, (1984)

Enzyme

Organism

Biomass '

Partition Yield '

Purification a-glucosidase

Glucose-6-phosphate dehydrogenase

Alcohol dehydrogenase

Hexokinase

Glucose isomerase

Pullulanase

Phosphorylase

Isoleucyl-tRNA synthase

Fumarase

Aspartase

Penicillin acylase

B-Galactosidase

Leucine dehydrogenase

Leucine dehydrogenase

Lactate dehydrogenase

L-2-hydroxy isocaproate dehydrogenase

L-2-hydroxy isocaproate dehydrogenase

Fumarase

Formaldehyde dehydrogenase

Isopropanol dehydrogenase

G1 ucose-6-phosphate dehydrogenase

Saccharomyces

Slreptomyces sp.

Klebsiella pneumoniae

Escherichia coli

Bacillus spaericus

Bacillus cereus

Lactobacillus sp.

Lactobacillus confusus

Lactobacillus casei

Brevibacterium ammoniagenes

Candida boidinii

Leuconosloc sp.

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