Ovomucoid partitioning in aqueous two-phase systems

This study evaluated the partitioning of ovomucoid from egg white, in aqueous two-phase systems (ATPS) composed of PEG 1500 and inorganic salt (lithium sulfate, sodium sulfate, magnesium sulfate, sodium carbonate or sodium citrate) at 25 °C. The results showed a great effect of the electrolyte nature on the partition coefficient. The partition coefficient value ranges from 0.02 to 6.0. The highest partition coefficients were obtained from systems composed of sodium carbonate and the lowest in systems composed of magnesium sulfate. In the system containing magnesium sulfate, a recovery percentage greater than 90% was obtained.     

Ovomucoid partitioning in aqueous two-phase systems

This study evaluated the partitioning of ovomucoid from egg white, in aqueous two-phase systems (ATPS) composed of PEG 1500 and inorganic salt (lithium sulfate, sodium sulfate, magnesium sulfate, sodium carbonate or sodium citrate) at 25 °C. The results showed a great effect of the electrolyte nature on the partition coefficient. The partition coefficient value ranges from 0.02 to 6.0. The highest partition coefficients were obtained from systems composed of sodium carbonate and the lowest in systems composed of magnesium sulfate. In the system containing magnesium sulfate, a recovery percentage greater than 90% was obtained.     

On protein partitioning in two-phase aqueous polymer systems.

The partitioning of proteins between the coexisting phases of two-phase aqueous polymer systems reflects an intricate and delicate balance of interactions between proteins, polymers, salts and water. Experimental investigations have suggested that a large number of factors influence protein partitioning, including the types of polymers, their molecular weight and concentration; the protein sizes, conformation and composition; salt type and concentration, and solution pH; and the presence of ligands attached to the polymer which may interact with surface sites of the protein. Complementary modelling attempts have been successful in illuminating several molecular-level mechanisms influencing protein partitioning using lattice-model techniques, viral expansions and a scaling-thermodynamic approach. In spite of these experimental and modelling approaches, many of the physical phenomena associated with these complex systems are not well understood. Notably, the precise nature of the protein-polymer interactions and the potent effect of inorganic salts on the partitioning of proteins in these systems remains poorly understood.     

Protein partitioning in two-phase aqueous polymer systems

Theories of protein partitioning in two-phase polymer systems which account for the effects of different aspects of system composition-such as the choice of materials, protein size, polymer molecular weight, polymer concentration, salt concentration, and affinity ligands-are reviewed. Although the present models provide some information about specific aspects of partitioning, a comprehensive and fundamental theory which can be used to predict protein partitioning behavior has not yet been developed. Some recommendations for future work are given.     

The molecular basis of partitioning in aqueous two-phase systems.

Protein purification based on partition in aqueous two-phase systems has attracted interest for many years. This approach has been advocated as a primary-stage unit operation in downstream processing. In reality, application has been strictly limited through inadequate understanding of the complex molecular forces involved in partitioning processes.     

Fundamental studies of biomolecule partitioning in aqueous two-phase systems

Phase diagram data at 4 degrees C was determined for the aqueous two-phase systems composed of polyethylene glycol, dextran, and water. The Flory-Huggins theory of polymer thermodynamics was used to correlate partitioning of biomolecules in these aqueous two-phase systems resulting in a simple linear relationship between the natural logarithm of the partition coefficient and the concentration of polymers in the two phases. This relationship was verified by partitioning a series of dipeptides which differ from one another by the addition of a CH(2) group on the c-terminal amino acid residue and by utilizing a set of low-molecular-weight proteins. The slope of the line could be expressed in terms of the interactions of the biomolecule with the phase forming polymers and water. The main result for the dipeptides was that knowledge of the partition coefficient in any of the PEG/dextran/water systems, regardless of polymer molecular weight, enabled prediction of the coefficient in all of the systems. The dipeptides were also used for determination of the Gibbs free energy of transfer of a CH(2) group between the phases. This quantity was correlated with polymer concentration, thus establishing a hydrophobicity profile for the PEG/ dextran/water systems. The methodology for predicting dipeptide partition coefficients was extended to proteins, where it was found that low-molecular-weight proteins gave a linear relationship with the tie line compositions of a phase diagram.     

Protein partitioning in weakly charged polymer-surfactant aqueous two-phase systems

The study includes partitioning of proteins in aqueous two-phase systems consisting of the polymer dextran and the non-ionic surfactant C₁₂E₅ (pentaethylene glycol mono-n-dodecyl ether). In this system a micelle-enriched phase is in equilibrium with a polymer-enriched phase. Charges can be introduced into the micelles by the addition of charged surfactants. The charge of the mixed micelles is easily varied in sign and magnitude independently of pH, by the addition of different amounts of negatively charged surfactant, sodium dodecyl sulphate (SDS), or positively charged surfactant dodecyl trimethyl ammonium chloride (DoTAC). A series of water-soluble model proteins (BSA, β-lactoglobulin, myoglobin, cytochrome c and lysozyme), with different net charges at pH 7.1, have been partitioned in non-charged systems and in systems with charged mixed micelles or charged polymer (dextran sulphate). It is shown that partition coefficients for charged proteins in dextran-C₁₂E₅ systems can be strongly affected by addition of charged surfactants (SDS, DoTAC) or polymer (dextran sulphate) and that the effects are directly correlated to protein net charge.     

Statistical thermodynamics of phase separation and ion partitioning in aqueous two-phase systems.

A general model for the phase behavior of polymer-polymer aqueous two-phase systems containing small amounts of added inorganic salts has been developed from statistical thermodynamics. The model is based on the solution theory of Hill and new electrolyte solution model based on Fluctuation Solution Theory. It includes the effect of polymer molecular weight with scaling expressions from the Renormalization Group theory of polymer solutions. The model has been used to calculate the phase diagram and the partitioning of salt for an aqueous two-phase system containing polyethylene glycol (MW = 8000) and dextran (MW = 28,700) with 0.1 mole/kg of added Na2SO4. The calculations have been compared to experimental results with good agreement.     

Purification of acid-proteases by affinity partitioning in aqueous two-phase systems

Summary An affinity polymer derivative was synthesized with the group specific acid protease inhibitor pepstatin attached to dextran (M.W. 500,0001). This derivative was used in an aqueous two-phase system with hydroxypropyldextran to purify crude solutions of chymosin and Endothia parasitica (EP) acid proteases. Chymosin was purified by a factor of 6.2 with an overall yield of 83%. EP protease was similarly purified. A new pepstatin binding protease was discovered in crude EP extracts.     

Affinity-enhanced protein partitioning in decyl beta-D-glucopyranoside two-phase aqueous micellar systems

Liquid-liquid extraction in two-phase aqueous complex-fluid systems has been proposed as a scalable, versatile, and cost-effective purification method for the downstream processing of biotechnological products. In the case of two-phase aqueous micellar systems, careful choices of the phase-forming surfactants or surfactant mixtures allow these systems to separate biomolecules based on size, hydrophobicity, charge, or specific affinity. In this article, we investigate the affinity-enhanced partitioning of a model affinity-tagged protei--green fluorescent protein fused to a family 9 carbohydrate-binding module (CBM9-GFP)--in a two-phase aqueous micellar system generated from the nonionic surfactant n-decyl beta-D-glucopyranoside (C10G1), which acts simultaneously as the phase-former and the affinity ligand. In this simple system, CBM9-GFP was extracted preferentially into the micelle-rich phase, despite the opposing tendency of the steric, excluded-volume interactions operating between the protein and the micelles. We obtained more than a sixfold increase (from 0.47 to 3.1) in the protein partition coefficient (Kp), as compared to a control case where the affinity interactions were "turned off" by the addition of a competitive inhibitor (glucose). It was demonstrated conclusively that the observed increase in Kp can be attributed to the specific affinity between the CBM9 domain and the affinity surfactant C10G1, suggesting that the method can be generally applied to any CBM9-tagged protein. To rationalize the observed phenomenon of affinity-enhanced partitioning in two-phase aqueous micellar systems, we formulated a theoretical framework to model the protein partition coefficient. The modeling approach accounts for both the excluded-volume interactions and the affinity interactions between the protein and the surfactants, and considers the contributions from the monomeric and the micellar surfactants separately. The model was shown to be consistent with the experimental data, as well as with our current understanding of the CBM9 domain.     

IgG and hybridoma partitioning in aqueous two-phase systems containing a dye-ligand

The effect of the important ATPS- and bufferparameters on IgG and hybridoma partitioning in ATPSscontaining a PEG-dye-ligand was studied. Objective wasto establish selection criteria for effective ligandsfor extractive fermentations with animal cells inATPSs.In the presence of 1% PEG-dye-ligand the binding ofIgG to the PEG-ligand was affected severely by theNa-chloride concentration. The tie-line length and pHaffected IgG partitioning to a lesser extent. Thedesired partitioning of IgG into the top phase, wasonly obtained when, in addition to the 10 mmol/kgK-phosphate buffer, no Na-chloride was present. In anATPS culture medium, with ± 35 mmol/kg Na-bicarbonateand 60 mmol/kg Na-chloride, increasing thePEG-dye-ligand concentration up to 100% did increasethe partition coefficient, but was not effective inconcentrating the IgG in the top phase of ATPS culturemedium at a pH of 7.8.Furthermore, addition of the PEG-dye-ligand to ATPSculture medium changed the hybridoma cell partitioningfrom the bottom phase to the interface.     

Amylase partitioning and extractive bioconversion of starch using thermoseparating aqueous two-phase systems.

The effectiveness of thermoseparating polymer-based aqueous two-phase systems (ATPS) in the enzymatic hydrolysis of starch was investigated. In this work, the phase diagrams of PEO-PPO-2500/ammonium sulfate and PEO-PPO-2500/magnesium sulfate systems were determined at 25 degrees C. The partition behavior of pure alpha-amylase and amyloglucosidase in four ATPS, namely, PEO-PPO/(NH(4))(2)SO(4), PEO-PPO/MgSO(4), polyethylene glycol (PEG)/(NH(4))(2)SO(4), and PEG/MgSO(4), was evaluated. The effects of phase-forming component concentrations on the enzyme activity and partitioning were assessed. Partitioning of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii was also investigated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. The PEO-PPO-2500/MgSO(4) system was extremely attractive for starch hydrolysis. Polymer-based starch hydrolysis experiments containing PEO-PPO-2500/MgSO(4) indicated that the use of ATPS had a significant effect on soluble starch hydrolysis. Batch starch hydrolysis experiments with PEO-PPO/salt two-phase systems resulted in higher production of maltose or glucose and exhibited remarkably faster hydrolysis. A 22% gain in maltose yield was obtained as a result of the increased productivity. This work is the first reported application of thermoseparating polymer ATPS in the processing of starches. These results reveal the potential for thermoseparating polymer-enhanced extractive bioconversion of starch as a practical technology.     

Purification of recombinant phenylalanine dehydrogenase by partitioning in aqueous two-phase systems

This study presents the partitioning and purification of recombinant Bacillus badius phenylalanine dehydrogenase (PheDH) in aqueous two-phase systems (ATPS) composed of polyethylene glycol 6000 (PEG-6000) and ammonium sulfate. A single-step operation of ATPS was developed for extraction and purification of recombinant PheDH from E. coli BL21 (DE3). The influence of system parameters including; PEG molecular weight and concentration, pH, (NH(4))(2)SO(4) concentration and NaCl salt addition on enzyme partitioning were investigated. The best optimal system for the partitioning and purification of PheDH was 8.5% (w/w) PEG-6000, 17.5% (w/w) (NH(4))(2)SO(4) and 13% (w/w) NaCl at pH 8.0. The partition coefficient, recovery, yield, purification factor and specific activity values were of 92.57, 141%, 95.85%, 474.3 and 10424.97 U/mg, respectively. Also the K(m) values for L-phenylalanine and NAD(+) in oxidative deamination were 0.020 and 0.13 mM, respectively. Our data suggested that this ATPS could be an economical and attractive technology for large-scale purification of recombinant PheDH.     

The partitioning of cholesterol oxidase in Triton X-114-based aqueous two-phase systems.

Cholesterol oxidase from various bacterial sources (membrane-bound and extracellular) was studied in Triton X-114R solutions above the cloud point. The influence of temperature, salt, enzyme concentration and source, and pH on phase equilibrium and enzyme partitioning was investigated in this detergent-based aqueous two-phase system. The method combines remarkable recovery (over 70% and 90% in the detergent-rich phase for the extracellular and membrane-bound forms, respectively) and 10 to 20-fold concentration of the enzyme in just one purification step. The results from cholesterol oxidase are compared with other proteins, both hydrophobic and hydrophilic. The system shows considerable promise for selectively partitioning proteins based on their surface hydrophobicity.     

Relative hydrophobicity of organic compounds measured by partitioning in aqueous two-phase systems

Partitioning of a variety of organic compounds, the majority of which represent therapeutic drugs, was examined in an aqueous dextran–polyethylene glycol (Dex–PEG) two-phase system containing 0.15 M NaCl in 0.01 M sodium phosphate buffer at pH 7.3 and in an octanol–buffer (0.15 M NaCl in 0.01 M sodium phosphate buffer, pH 7.3) system. The possibility of introducing compounds to be partitioned in an aqueous two-phase system with dimethyl sulfoxide, and the effect of this solvent on the solute partitioning was explored. Relative hydrophobicity of the compounds was estimated and expressed in equivalent numbers of methylene units. Comparison of the results obtained for several subsets of compounds in the octanol–buffer and in aqueous Dex–PEG two-phase systems clearly demonstrates the advantage of aqueous two-phase partitioning for the hydrophobicity measurements over partitioning in octanol–buffer system.     

Separation of organic dust from microorganism suspensions by partitioning in aqueous polymer two-phase systems.

Conidia of Penicillium brevi-compactum and Aspergillus fumigatus, sporangiospores of Rhizopus rhizopodiformis, spores of Streptomyces griseus, and bacterial cells of Bacillus subtilis were partitioned in two-phase systems consisting of dextran, polyethylene glycol, substituted positively charged sulfonylpolyethylene glycol, and water. At a pH of 2.8 in the system, the microorganisms showed 60 to 90% affinity for the upper, polyethylene glycol-rich phase, except for cells of B. subtilis, which were entirely located in the lower, dextran-rich phase. This partition behavior was used to separate microorganisms in aqueous suspensions of peat, wood fuel chip, and straw samples from organic dust impurities prior to total count by acridine orange staining and epifluorescence microscopy. Only one extraction of the interphase and lower phase was needed to separate approximately 98% of the conidia of Penicillium chrysogenum from a suspension containing peat dust.     

Bioconversions in aqueous two-phase systems.

Bioconversions involving enzymes and/or microbial cells in aqueous two-phase systems are reviewed. The partitioning of biocatalysts, substrates, and products is discussed in relation to their size. The efficiency of retaining biocatalysts in aqueous two-phase systems is summarized in relation to other methods of recirculating. The influence of phase components on the activity and the stability of enzymatic biocatalysts is exemplified with penicillin acylase and the cellulolytic enzyme system, and the effect of phase components on biocatalytic living cells is exemplified with the production of alpha-amylase with Bacillus sp. Process design costs in bioconversions in aqueous two-phase systems are briefly summarized.     

Aspergillus oryzae alpha-amylase partition in potassium phosphate-polyethylene glycol aqueous two-phase systems

The aim of this work is to study the partitioning of alpha-amylase from Aspergillus oryzae in polyethylene glycol-potassium phosphate systems formed by polymers of different molecular masses with different total concentrations, several NaCl concentrations and different volume ratio between the phases and at different temperatures. The enzyme was partitioned towards the top phase in the 2000-molecular-mass polyethylene glycol systems and towards the bottom phase in the other systems analyzed with higher molecular mass. The protein-medium interaction parameter (A) was determined; it increased in the same way as PEG molecular mass. The enthalpic and entropic changes found, in general, were negative and were shown to be associated by an entropic-enthalpic compensation effect suggesting that the ordered water structure in the chain of polyetyleneglycol plays a role in protein partition. The recovery in each of the phases was calculated in order to choose the best systems to be applied to enzyme isolation either from a polymer-rich or a polymer-poor phase.Enzymatic activity, circular dichroism and fluorescence were studied for the protein alone and in the presence of the different phases of the aqueous two-phase systems (ATPSs) in order to understand how they affect the enzymatic structure and the role of the protein-polymer interaction in the partitioning process. Secondary structure is not affected, in general, by the presence of the phases that do affect the enzymatic activity; therefore, there should be a change in the tertiary structure in the enzyme active site. These changes are more important for PEG 8000 than for PEG 2000 systems according to the results of the quenching of the intrinsic fluorescence. In a bio-separation process, the A. oryzae alpha-amylase could be isolated with ATPSs PEG 2000/Pi or PEG 8000/Pi with a high recovery, in the top or bottom phases, respectively.