Biomolecules partitioning

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 major theories developed to predict phase separation and biomolecule partitioning in aqueous two-phase systems are mostly extensions of the widely known polymer solution theories of Flory and Huggins and the osmotic virial expansion. 

DIA Diamond, A.D. and Hsu, J.T., Fundamental studies of biomolecule partitioning in aqueous two-phase systems, Biotechnol. Bioeng., 34, 1000, 1989. 

Free energy calculations rely on the following thermodynamic perturbation theory [6-8]. Consider a system A described by the energy function = 17 + T. 17 = 17 (r ) is the potential energy, which depends on the coordinates = (Fi, r, , r ), and T is the kinetic energy, which (in a Cartesian coordinate system) depends on the velocities v. For concreteness, the system could be made up of a biomolecule in solution. We limit ourselves (mostly) to a classical mechanical description for simplicity and reasons of space. In the canonical thermodynamic ensemble (constant N, volume V, temperature T), the classical partition function Z is proportional to the configurational integral Q, which in a Cartesian coordinate system is... 

It is believed that solubilization is initiated when a biomolecule with charged surface groups approaches the bulk aqueous-lipophilic solvent interface, where the interactions cause the bulk interface s surfactant layer to bend, such that the protruding biomolecule becomes surrounded by the surfactant layer [105]. Ultimately, a filled w/o-ME forms, and partitions to the bulk organic phase [105]. 

The partitioning behavior of biomolecules in RME can be regulated by varying the size and shape of the reverse micelles (RMs). This can be easily ac-... 

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). 

Other applications besides purification of biomolecules are affinity partitioning (Labrou, 1994), extractive bioconversions (Andersson, 1990 Kaul, 1991 Zijlstra, 1998), liquid-liquid partition chromatography, and analytical assays. Although industrial applications of aqueous two-phase systems have not gained widespread use to date, practical and economic feasibility has been proved (Tjemeld, 1990 Cunha, 2002). 

Extraction is based on the transfer of a solute from one phase to another, according to its partition properties between two immiscible phases. In biotechnological processes, the most widely used extraction is liquid-liquid involving the partitioning of biomolecules, organelles or cells between the phases (Harrison et al., 2003). 

First, it is necessary to establish the phase diagram of the aqueous two-phase system formed by water and two water-soluble polymers. Second, a method must be established for calculating the distribution coefficient of a biomolecule that partitions between the two aqueous phases. A simple molecular-thermodynamic description is provided by the osmotic virial... 

E. Large-Scale Aqueous Two-Phase Partitioning of Biomolecules... 

TABLE I Partition Coefficients of Some Biomolecules in Solvent Extraction... 

Aqueous two-phase systems are routinely used for enzyme purification. Walter and Johansson7 edited a comprehensive book on the subject entitled Aqueous Two-Phase Systems as Vol. 228 of the series Methods in Enzy-mology published by Academic Press. Another very useful book is the one edited by Walter, Brooks, and Fisher6 entitled Partitioning in Aqueous Two-Phase Systems Theory, Methods, Uses, and Applications to Biotechnology which deals with separations of a variety of biomolecules and bioparticles. 

TABLE 11 Affinity Partitioning of Biomolecules in Aqueous Two-Phase Systems... 

---