The aqueous two-phase system provides a technically simple, easily scalable, energy-efficient, and mild separation technique for product recovery in biotechnology.
1. Most biotechnological products, soluble molecules, and particles are obtained in very dilute solutions. Hence, the first desirable step for their recovery is concentration. A two-phase system is able to carry out such a concentration provided it is constructed in a way that most of the desired substance is transferred to a phase with a small volume compared to the original solution (1). The particles may also be concentrated at the interface. Since impurities may be concentrated to a lesser extent or not at all, a concomitant purification may also be achieved. A one-step or multistep procedure may be applied depending on the partitioning of the product and the contaminants. Aqueous two-phase separation is today the preferred method for concentration and purification of viruses (see Chapter 12). Increasing use of the technique may be foreseen for preparation of different types of cells targetted for various applications.
2. The increasing use of recombinant DNA technology for protein production has brought focus on the downstream processing operations for product recovery. By tradition, protein purification is performed in discrete stages involving clarification by solid/liquid separation techniques and concentration into small volumes, followed by fractionation by high resolution chromatography techniques. A major problem is that a higher number of processing steps results in higher product loss. Also, many of the separation techniques are not easily scalable. Extraction in ATPS is in many cases a better alternative to existing technology in the early processing stages of large-scale isolation of proteins from crude homogenates, because it circumvents many of the shortcomings of centrifugation and filtration that arise because of high viscosity and heterogeneous distribution of particle size. Moreover, if properly optimized, it also provides integration of clarification, concentration, and partial purification. This has an influence on reducing the number of downstream processing steps and hence in improving the yields and costs of the recovery process.
Besides the intracellular enzymes from microbial cells, proteins from more complex raw materials, like animal tissues (see Chapter 24) and mucilagenous plant material (17), have been conveniently purified by extraction in ATPS. Protein isolation in ATPS is designed so as to suit the system under study. Two extraction steps have been commonly used (13). In the primary extraction, the cell material and the bulk of nucleic acids, polysaccharides, and contaminating proteins are collected in the denser lower phase, whereas the target protein partitions to the upper phase. A second extraction step may be applied to transfer the target protein into a fresh lower phase. This two-stage extraction procedure can be made continuous and automated. The proteins may alternatively be recovered directly from the PEG-rich phase by ultrafiltration (18), or direct application of the phase to a chromatography matrix (19).
3. Isolation of membrane proteins, normally a rather difficult and time-consuming task, has been successfully achieved by the convenient two-phase extraction procedure. Use of nonionic detergents, e.g., Triton X-114, which separate at rather mild temperatures like 20°C into a detergent-rich and detergent-poor phase, provide novel means for obtaining enriched preparations of integral membrane proteins from complex biological systems (20,21).
4. Two-phase partitioning also holds promise for rapid isolation of DNA. By using novel phase systems made of PEG-salt containing chaotropic agents and detergents, it has been shown that nucleic acids partition with high yields to the salt-rich phase, whereas proteins and other cellular material are concentrated in the other phase or precipitates at the interface (22).
5. The potential of ATPS for extraction of small-mol-wt bioproducts like amino acids has been demonstrated (see Chapter 9). In an unusual two-phase system obtained at high temperatures using PEG and sodium chloride, the amino acids are efficiently extracted into the PEG-rich phase (23). The product is then recovered in clear solution by precipitating PEG by cooling.
6. Low productivity in biotechnological processes is often a result of inhibition or toxicity of the product itself to the producer organism. Degradation of the product could be yet another cause for low productivity. ATPS has shown potential for improving the productivity of biotechnological processes by creating integration of product removal with that of bioconversion, a concept known as "extractive bioconversion" (see Chapter 37). Here the bioconversion takes place in one phase and the product is extracted into the other. Although no large-scale applications are known, a number of examples have been reported in the literature including production of proteins as well as small molecules.
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