182 J. A Asenjo and J B Chaudhuri water and the solubilised protein stabilised by a surfactant shell layer. For protein extraction, one phase is the aqueous feed solution, the other the reversed micellar phase that acts as the extractant. They have several of the advantages quoted for aqueous two- phase systems The suitability of using foam separation as well as gas aphrons as novel separation techniques for proteins are presently under investigation 7.3.1 Aqueous two-phase separation Partitioning in two aqueous phases can be used for the separation of proteins from cell debris as well as for purification from other proteins. Partitioning can be done in a single step or as a multistage process. Differences in partition coefficients, however, between the different proteins can be high, hence one step tends to be sufficient (usually one for extraction and one for elution or back-extraction). The use of affinity partitioning can eatly enhance the specificity of the extraction. A typical process for extraction of a protein into the top PEG phase in a first stage and the back extraction into a bottom salt phase(e.g. phosphate or sulphate) in a second 'back extraction' step from a cell homogenate that includes recycle of the PEG phase is shown in Fig. 7. 1(Hustedt et al 1985) PEG +salt Sal ⊙ Glass-bead mixer eat Separator Tc hase phase Separator Cell debris Fig. 7. 1. Scheme of enzyme purification by liquid-liquid extraction. The cells are disrupted by et milling, and after passing through a heat exchanger, PEG and salts are added into the process stream of broken cells. After mixing and obtaining of equilibrium the phase system is separated the outflowing bottom phase is going aining PEG-rich top a second mixer after addition of more salt to ocess stream. The product is recovered in the resulting bottom phase while the concentrated PEG solution (upper phase) goes to waste or is led. From Hustedt et al, 1985)182 water and the solubilised protein stabilised by a surfactant shell layer. For protein extraction, one phase is the aqueous feed solution, the other the reversed micellar phase that acts as the extractant. They have several of the advantages quoted for aqueous two￾phase systems. The suitability of using foam separation as well as gas aphrons as novel separation techniques for proteins are presently under investigation. 7.3.1 Aqueous two-phase separation Partitioning in two aqueous phases can be used for the separation of proteins from cell debris as well as for purification from other proteins. Partitioning can be done in a single step or as a multistage process. Differences in partition coefficients, however, between the different proteins can be high, hence one step tends to be sufficient (usually one for extraction and one for elution or back-extraction). The use of affinity partitioning can greatly enhance the specificity of the extraction. A typical process for extraction of a protein into the top PEG phase in a first stage and the back extraction into a bottom salt phase (e.g. phosphate or sulphate) in a second ‘back extraction’ step from a cell homogenate that includes recycle of the PEG phase is shown in Fig. 7.1 (Hustedt et al., 1985). J. A. Asenjo and J. B. Chaudhuri .................................................................... Fig. 7.1. Scheme of enzyme purification by liquid-liquid extraction. The cells are disrupted by wet milling, and after passing through a heat exchanger, PEG and salts are added into the process stream of broken cells. After mixing and obtaining of equilibrium the phase system is separated, the outflowing bottom phase is going to waste. The product-containing PEG-rich top phase goes to a second mixer after addition of more salt to the process stream. The product is recovered in the resulting bottom phase while the concentrated PEG solution (upper phase) goes to waste or is recycled. (From Hustedt et al., 1985)