| Recently, aqueous two-phase extraction (or partition of two aqueous phase system) is a compelling and a very promising technology for separation. As a novel liquid-liquid extraction technology, aqueous two-phase extraction has broad application prospects due to being widely used in bio-chemical industry, biochemistry, cell biology and other fields. The PEG,[Cnmim]Br (n=2,4,6)/potassium citrate aqueous two-phase systems were selected for separation and purification of proteins. The detailed information of the paper is summarized as follows:1. Liquid-liquid equilibrium of the polyethylene glycol (PEG) or [Cnmim]Br (n=2,4,6)/potassium citrate aqueous two-phase systemsPhase diagrams were determined by the turbidimetric titration method as the one used previously. The results showed that the ability of PEG and ionic liquid for two-phase formation followed the order:PEG6000> PEG4000> PEG2000> PEG1000;[C2mim]Br<[C4mim]Br<[C6mim]Br. To PEG/potassium citrate system, the slope of the equilibrium line and the tie line length increased as the increasing of temperature; the additional salt plays a supportive role in this system. To [Cnmim]Br (n=2,4,6)/potassium citrate system, the phase volume ratio (R) and enrichment factor (F) were significantly affected by the amount of potassium citrate near the cloud point of the system. The objects of this work are to provide the basic data and the necessary theoretical guidance.2. Bovine serum albumin partitioning in polyethylene glycol (PEG)/potassium citrate aqueous two-phase systemsThe extraction and back-extraction of bovine serum albumin (BSA) have been studied by liquid-liquid extraction with poly(ethylene glycol)(PEG)/potassium citrate aqueous two-phase system (ATPS). In this work, the ATPS was examined with regard to the effects of PEG molecular weight (PEG1000,2000,4000and6000), PEG and potassium citrate concentration, BSA concentration (CBSA), pH and temperature on BSA partition. The pH was found to have significant effects on BSA partition with low molecular weight PEG1000. The yield of the BSA,99%, was obtained in the top phase under the following conditions:19%(w/w) PEG1000,20%(w/w) potassium citrate and0.75mg/g CBSA at pH7.0and30℃. BSA can be re-extracted to a new citrate phase by decreasing the pH of the system with a92%yield. The back-extraction not only separates the BSA from the polymer, but also allows the polymer to be recycled. The global yield (Ye+Ybe) is up to91%.3. Thermodynamic studies of partitioning behavior of cytochrome c in ionic liquid-based aqueous two-phase systemThe ionic liquid/aqueous two-phase extraction systems (ATPSs) based on imidazolium ionic liquids were used to extract cytochrome c. Effects of the alkyl chain length of cation of the ionic liquids, concentration of potassium citrate, as well as the extraction temperature and pH value on the extraction efficiency have been investigated. The thermodynamic parameters (△GTo,△HTo and△STo) associated with Cyt-c partitioning in aqueous two phase systems were determined. Thermodynamic studies indicated that the partitioning of Cyt-c was driven by both hydrophobic interactions and electrostatic interactions in the extraction process. Under the optimum conditions, experiment results show that94%of the cytchrome c could be extracted into the ionic liquid-rich phase in a one-step extraction. The structural characterization of Cyt-c in the IL ATPS was investigated by UV-vis and Circular Dichroism (CD) spectra. The results demonstrated that no direct bonding interaction observed between ionic liquid and cytochrome c, while the native properties of the cytochrome c were not altered. Compared with traditional liquid-liquid extractions, which use toxic organic solvents, ionic liquid/aqueous two phase extraction offers clear advantages due to no use of volatile organic solvent and low consumption of imidazolium ionic liquids. 4. The purification of lysozyme from crude hen egg white using polyethylene glycol (PEG)/citrate aqueous two phase systemAqueous two-phase extraction is a versatile method suitable for separating biological particles and macromolecules. In the present wok, the feasibility of using PEG4000/potassium citrate aqueous two-phase system (ATPS) for recovering and purifying lysozyme was investigated. Response surface methodology was used to determine an optimized ATPS for purification of lysozyme from crude hen egg white. Mathematical models concerning the purification of lysozyme from chicken egg white in polyethylene glycol4000(PEG4000)/potassium citrate ATPS are established using response surface methodology. Screening experiment by fractional factorial designs shows that the pH of the system significantly affects the recovery and purification of the lysozyme. An optimized aqueous two phase system ATPS was proved to be at pH5.5and30℃and contained18%(w/w) PEG,16%(w/w) potassium citrate,3.75%(w/w) potassium chloride (KC1). Under those conditions, the specific activity, purification factor and activity yield for lysozyme were31100U/mg,21.11and103%, respectively. The PEG4000/potassium citrate ATPS has the potential to be applied to establish bioprocesses for the primary recovery and partial purification of lysozyme.5. Application of response surface methodology to evaluate the purification papain by aqueous two-phase systemMathematical models concerning the purification of papain from the crud papain in an aqueous two-phase system (ATPS) which are composed of polyethylene glycol (PEG)/potassium citrate are established with response surface methodology. Effects of PEG molecular weight, the concentration of PEG and potassium citrate, the concentration of additional salt (KC1), the temperature and pH on the papain purification have been investigated. Research results showed that papain tends to partitions to the more hydrophilic salt-rich phase. The steepest ascent method was used to locate the optimal domain. Papain was successfully extracted to the bottom phase of a system composed of15%(w/w) PEG4000,17%(w/w) citrate and5.0%(w/w) KC1at pH5.0and30C. Under those conditions, the specific activity, purification factor and activity yield for papain were1703.02U/mg,1.89and94%, respectively. The purity of extracted papain was confirmed by SDS-PAGE analysis. This ATPS has the potential to be applied to purification of papain.
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