Aqueous Twophase Extraction

Biomaterials derived from or produced by the plants, animals or the micro-organism mainly consist of proteins, fats and carbohydrates. Production of biomaterials on a commercial scale involves cell disruption followed by separation of the desired products. The cost of the separation step may be as high as 90% of the total cost of the production. For the separation of enzymes and proteins, aqueous two-phase extraction process has become a versatile and efficient method. The method can be used for the removal of cell debris as well as further purification of the biomolecules. Although, not of current interest, the developments in the field of biotechnology for separation using aqueous two-phase extraction will be of importance for macromolecules of interest to the food industry.

Aqueous two-phase systems (ATPS) are known for a long time. Beijerinck (1896) observed the formation of two liquid phases upon mixing agar with soluble starch or gelatin. However, a breakthrough in the useful aqueous two-phase systems was made by Albertsson (1956) when he obtained aqueous two-phase systems in which both phases contained very high concentration of water. This solved the problem of denaturation and precipitation of proteins.

The advantages of the aqueous two-phase extraction process are: (1) biocompatibility, (2) easy processing, (3) high capacity, (4) easy and precise scale-up, (5) high product yields, (6) high potential for continuous processing, and (7) low investment cost. The aqueous two-phase systems have extreme physical properties compared to the conventional liquid-liquid extraction systems. The interfacial tension is in the range of 10~3-10 mN/m; phase viscosities are usually 1-103 mPa.s; and density differences between phases are low, ranging from 20-100 kg/m3. The performance of extraction equipment for the aqueous two-phase systems is markedly different from that of conventional equipment.

The ATPS are usually based on polymer-polymer, PEG/Dextran being the most common, and polymer-salt systems. The salt-based systems are more economical because of low cost of the phase-forming components and are also easy to handle unlike dextran-based systems which are characterized by high viscosity. The two phase polymer-polymer systems and polymer-salt systems have been studied by many workers. Review articles by Koningsveld (1963), Cebezas et al (1990), Forciniti and Hall (1990) and Diamond and Hsu (1992) summarize the work in this field. Figure 5 presents a typical phase diagram for PEG (4000)-sodium sulfate-buffer system. Sodium orthophosphate solution in required quantity was used as buffer.

Anhydrous NajSO^,weight percent

Figure 5. Phase diagram of the PEG(4000)-sodium sulphate-buffer system

Anhydrous NajSO^,weight percent

Figure 5. Phase diagram of the PEG(4000)-sodium sulphate-buffer system

5.1. Protein partitioning

Protein partitioning in ATPS depends on many factors such as phase polymers, the ionic composition and the partitioned substance. The type of polymer as well as their molecular weights and the presence of certain chemical groups influence the partitioning of proteins. The ionic composition is of vital importance as the sign and magnitude of the interfacial electric potential are determined by the ions present. The properties such as size, charge and biospecific surface properties, presence of receptors and biospeciftc ligands and chirality govern partitioning. The partitioning of a protein between the top and bottom phases is defined by the partition coefficient (m) and is the ratio of concentrations in the top and bottom phases. The overall partition coefficient is resolved in a number of factors as given below:

m = m0.mel.mhf0b.mbi0Sp.msize.mc0nf (1)

where the suffices el, hfob, biosp, size and conf stand for electrochemical, hydrophobic, biospecific, size dependent and conformational contributions, respectively, to the partition coefficient. The m° includes all other factors such as relative solvation of the solute molecule in the phases.

Increased polymer concentration shifts the phase system away from the critical point and the physical properties of the coexisting phases become more different. The partitioning of the protein becomes more favourable. However, cell organelles adsorb more strongly and selectively to the interface when polymer concentration increases.

Molecular weight of the polymer affects the partitioning of high molecular weight proteins. The higher the molecular weight of the polymer, the lower will be the partition coefficient.

Salts have a paramount effect in the partitioning of all kinds of molecules and cell particles. Salts with different ions have different affinities for the two phases. An electric potential is created between the phases. A salt with two ions that have different affinities for the two phases will generate larger potential difference.

The isoelectric point of proteins can be determined by a cross-partition and partition coefficient is determined as a function of pH with two different salts (Albertsson et al, 1970).

Presence of a charged polymer in one of the phases has a stronger effect on the partitioning of charged macromolecules than that of the salts (Johansson et al, 1973).

Hydrophobic groups such as palmitate bound to polyethylene glycol (PEG) show increased affinity for proteins with hydrophobic binding and cause protein to be partitioned in the PEG-rich phase.

Affinity ligands attached to one of the polymers can be used to extract ligand-binding proteins and nucleic acids into the corresponding phase.

The composition of the two phases changes with temperature. Proteins partition more equally between phases of a two-polymer system when temperature is increased. This effect may be counteracted by using higher concentrations of polymer.

5.2. Aqueous two-phase separation of protein mixtures

Partitioning in two aqueous phases can be done for the separation of proteins from cell debris and for purification from other proteins. Differences in partition coefficients between different proteins are high. Therefore, the number of stages for purification is less. First stage of extraction in PEG-rich phase and a second stage for back-extraction in a salt phase may purify the enzyme (Hustedt, 1985). Partitioning may be done in a multistage unit or a continuous extraction unit such as spray tower or a rotating disc contactor. Albertsson (1986), Nguyen et al (1988), Stewart (1990) and Pathak et al (1991) have presented various aqueous systems used for extraction of biomolecules. Table 6 provides examples of partitioning of proteins in aqueous two-phase and three-phase systems. Table 7 gives examples of proteins purified by subsequent extraction steps. The table also includes information on the number of extraction steps, enrichment factor and the final yield.

A typical process for enzyme purification is shown in Figure 6. The cells are disrupted by wet milling and passed through a heat exchanger. The PEG and salts are added into this stream of broken cells. After mixing and attainment of equilibrium, the phases are separated. The product-rich top PEG phase is sent to a second mixer after addition of more salt. The bottom salt phase containing cell debris and proteins is discarded. After attaining equilibrium in the second back-extraction mixer, the process stream is sent to a separator. The top PEG phase is subjected to by-product recovery and recycled or goes to waste. The bottom salt phase is used for the product recovery. A large scale separation using this strategy was reported by Kula et al (1982).

Table 6

Examples of partitioning of proteins in aqueous two-phase and three-phase systems

Protein

Main System Components Reference

Albumins

Haemoglobins

Proteins from Baker's yeast (glycolytic enzymes)

Histone protein

Membrane proteins

Protein from micro-organisms

Cellulases

Fusion protein BetaGal

Dextran, PEG Dextran, PEG Dextran, PEG

Salt, PEG Dextran, PEG

Dextran, PEG Detergent

Salt, PEG Dextran, PEG

Dextran, PEG

Salt, PEG

Albertsson et al (1970); Johansson et al (1970a,b, 1978)

Albertsson et al (1970); Silverman et al (1979);

Johansson and Hartman (1974) Johansson et al (1973), Shanbhag et al (1972)

Bidney and Reeck (1977) Axelsson and Shanbhag (1976) Gineitis et al (1984)

Albertsson and Andersson (1981); Svensson et al (1985)

Hustedt et al (1983) Kroner et al (1982)

Tjerneld et al (1985)

Strandberg et al (1991)

Table 7

Examples of proteins purified by subsequent extraction steps

Table 7

Examples of proteins purified by subsequent extraction steps

Enzyme

Organism

Number of extraction steps

Enrichment factor

Final yield

Source

Formate dehydrogenase

Candida boidinil

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