Extraction aqueous two-phase

Upon addition of certain polymers such as polyethylene glycol (PEG) and dextran or salt to water, a phase boundary forms even though the system consists of only one solvent, water. When a mixture of biomolecules such as a fermentation broth or a solution of lysed cells is added to such a system, each type of biomolecule partitions uniquely between the two phases, achieving separation (Kula, 1979,1990  

The parallel tie-lines in the two-phase region indicate the phase split of a system with a composition in the two-phase region into two phases with compositions at the intersections with the phase boundary line. 

Hatti-Kaul, 2001). A low degree of complexity and ease of scale-up in combination offer a potentially attractive method for initial purification of a protein mixture. In principle, only a contactor and a mixer-settler are required. 

The quality of separation depends on several factors, among them the partition coefficients of biomolecules between the phases, phase behavior, temperature, and the type and concentration of both salt and polymer. The partition coefficient of species X between the top and bottom phases is defined by Eq. (8.67). 

The partition coefficient K varies exponentially with the molecular mass of the soluble protein [Eq. (68)], where T = absolute temperature. 

ATP extraction has been used for the separation and large-scale purification of enzymes.45 47 Proteins and nucleic acids partition between the two phases differently due to differences in their surface charges and hydrophobic-hydrophilic domains. Proteins generally prefer the aqueous phase rich in 

Precipitation by Streptomycin Sulfate, Protamine Sulfate, and Manganous Sulfate from Cell Homogenate 

TABLE 3 Effect of Nucleic Acids on Partition Coefficients 

Protein Single-component system In the presence of yeast RNA In the presence of affinity ligand In the presence of affinity ligand and yeast RNA 

These measurements were conducted in ATP system consisting of 5.1% (w/w) PEG 8000 and 10% (w/w) dextran 70 in phosphate buffer at 4°C. Under these conditions, the volume ratio of PEG (light) to dextran (heavy) phases was 3 7. The composition (w/w) of PEG phase was 9.25% PEG and 0.52% dextran. The composition of the dextran phase was 0.54% PEG and 21.1% dextran. The phase composition was unaffected by NaCl concentration in the range of 0-5 M. The partition coefficient is defined as ratio of concentration in PEG phase to that in dextran phase. The triazine dye, Cibacron blue FGF, was ligated to PEG for affinity partitioning. 

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Many laboratory techniques have been described to purify proteins (25), but they are often too cosdy for industrial enzymes, especially column separations. However, aqueous two-phase extraction (26) and ion exchange are used. 

For modeling of aqueous two-phase extractions systems, see Abbott (1990,1992a-c, 1993). 

After the initial purification steps of salt precipitation of an aqueous two-phase extraction, an enzyme is often already pure enough for preparative use. If a high degree of purity or even homogeneity is required, such as for biochemical investigations of a novel enzyme, fine purification steps have to be added. The most common such step is chromatography. 

M. -R. Kula, Trends and future prospeds of aqueous two-phase extraction, Bioseparation 1990, 1, 181-189. 

B. Removal of Nucleic Acids from Cell Homogenate Using Aqueous Two-Phase Extraction... 

Novel and effective bioseparation techniques must be continuously researched and developed for profitable removal of proteins and other bioproducts of interest from very dilute solutions (A. Ramakrishnan and A. Sadana, personal communication, 1999). There appear to be two techniques that have tremendous potential for commercial applications the reverse micelle technique and aqueous two-phase extraction. Before these techniques achieve their potential, it will be necessary to further delineate the effect of mass transfer, interactions at the interfaces, and other parameters that affect both the quality and quantity of proteins separated by these techniques. It is also necessary to have a large data bank of a wide variety of proteins and bioproducts with regard to their characteristics and stability to assist future improvements in bioseparations. 

Purification can also be achieved in other ways, through precipitation with salts, crystallization, and through aqueous two-phase extraction. Some of these methods are associated with substantial capital cost, low throughput, low yields, or waste issues. Fractional precipitation, one of the oldest protein separation technologies, can be surprisingly effective to separate a compound of interest from a complex broth. For instance, the process of fractional solvent precipitation of blood plasma components has been used since World War II. 

Assays of enzymatic activity of polymer degrading enzymes are often difficult to perform using traditionally immobilized systems. Using a liquid-liquid phase system makes it easier to establish contact between the catalyst and the substrate. Products may then partition differendy as compared to the substrate. This forms the basis for the assay of dextranase using a dye substrate, blue dex-tran. The liberated color is preferentially extracted into the more hydrophobic top phase (103). Adding a ferrofluid that is a submicron magnetic preparation to an aqueous two-phase extraction system makes it possible to substantially facilitate phase separation by magnetic force (104). 

Teehnol., 44, pp. 79 4 (2005)]. Another process that exploits a phase transition to facilitate separation and recycle of solvent alter extraction utilizes ethylene oxide-propylene oxide copolymers in aqueous two-phase extraction of proteins [Persson et al.,/. Chem. Teehnol. Bioteeh-nol., 74, pp. 238-243 (1999)]. After extraction, the polymer-rich extract phase is heated above its LCST to form two layers an aqueous layer containing the majority of protein and a polymer-rich layer that can be decanted and recycled to the extraction. 

In the years since the pioneering studies of Albertsson and co-workers (1), aqueous two-phase extraction has gained wide acceptance as a method for... 

Theoretical Treatment of Aqueous Two-Phase Extraction by Using Virial Expansions... 

A theoretical treatment of aqueous two-phase extraction at the isoelectric point is presented. We extend the constant pressure solution theory of Hill to the prediction of the chemical potential of a species in a system containing soivent, two polymers and protein. The theory leads to an osmotic virial-type expansion and gives a fundamentai interpretation of the osmotic viriai coefficients in terms of forces between species. The expansion is identical to the Edmunds-Ogston-type expression oniy when certain assumptions are made — one of which is that the solvent is non-interacting. The coefficients are calculated using simple excluded volume models for polymer-protein interactions and are then inserted into the expansion to predict isoelectric partition coefficients. The results are compared with trends observed experimentally for protein partition coefficients as functions of protein and polymer molecular weights. 

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