The influence of electrostatic interactions on partition in aqueous polyethylene glycol/dextran biphasic systems: Part II

In aqueous polyethylene glycol/dextran two-phase systems, the hydrophobicity, free volume, surface tension, and interfacial tension of the phases in equilibrium were measured as a function of pH and ionic strength. These parameters were found to change with pH, but the pattern and magnitude cannot explain the unusual partition of charged macromolecules, observed previously. The electrostatic potential difference was determined by a new experimental approach based on the measurement of the pH difference between the phases at equilibrium. In polyethylene glycol/dextran systems containing sodium chloride as ionized species, the electrostatic potential is not constant in the pH range 2 to 11. The partition behavior of charged macromolecules and its dependence on pH can be explained by the combined action of charge and phase potential. This conclusion was tested with poly-L-glutamate, which partitioned as predicted and in a pattern opposite to positively charged macro- molecules. (c) 1995 John Wiley & Sons, Inc.     

Metal ion separations in polyethylene glycol-based aqueous biphasic systems: correlation of partitioning behavior with available thermodynamic hydration data

Solvent extraction, utilizing an oil-water mixture (e.g, chloroform-water) and a suitable complexant, is a proven technology for the selective removal and recovery of metal ions from aqueous solutions. Aqueous biphasic systems (ABS), formed by mixing certain inorganic salts and water-soluble polymers, or by mixing two dissimilar water-soluble polymers, have been studied for more than 40 years for the gentle, non-denaturing separation of fragile biomolecules, yet ABS have been virtually ignored as a possible extraction technology for metal ions. In this report we review our metal ion partitioning work and discuss the three major types of partitioning: (1) those rare instances that the metal ion species present in a given solution partitions to the PEG-rich phase without an extractant; (2) the use of halide salts which produce a metal anion complex that partitions to the PEG-rich phase; and (3) the use of a water-soluble extractant which distributes to the PEG-rich phase. In addition, we correlate the partitioning behavior we observed with available thermodynamic data for metal ions and their complexes.     

Effects of speciation on partitioning of iodine in aqueous biphasic systems and onto ABEC resins

Polyethylene glycol (PEG)-aqueous biphasic systems (ABS) and PEG-grafted aqueous biphasic extraction chromatographic (ABEC) resins have been shown to remove inorganic species from environmental and nuclear wastes. The partitioning behavior of several iodide species (iodide, iodine, triiodide, iodate, and 4-iodo-2,6-dimethylphenol (I-DMP)) have been studied for PEG (MW 2000)-salt systems and ABEC resins. Iodide partitioning to PEG-rich phases or onto ABEC resins can be enhanced by derivatization with 2,6-dimethylphenol to form 4-iodo-2,6-dimethylphenol or by addition of I(2) to form triiodide. Conversely, iodide partitioning to the PEG-rich phase or onto ABEC resins is reduced by oxidation of iodide to IO(3)(-). Partitioning studies of iodide, iodate, and iodine in a PEG-ABS are compared to results using ABEC resins.     

Effect of some inorganic salts on the partitioning of pectinase of Polyporus squamosus in polyethylene glycol/crude dextran aqueous two-phase system

The effect of two inorganic salts, ammonium sulphate and potassium dihydrogenphosphate, on the partitioning of pectinases produced by Polyporus squamosus in polyethylene glycol/crude dextran aqueous two-phase system is reported. Presence of both salts at different concentration did not affect partition of biomass, so fungal growth was occurring exclusively in the bottom phase. At 30 mmol (NH₄)₂SO₄/l in two-phase medium, the partition coefficient of endo-pectinase was 3.9, and it was 80% improved in comparison to that obtained at twofold lower salt concentration. On the other hand, higher (NH₄)₂SO₄ concentration increased total exo-pectinase activity produced, but did not affect substantially its partition parameters. Increasing phosphate concentration stimulated partition of both enzymes to the top phase: at 0.2 mol KH₂PO₄/l the partition coefficient for exo-pectinase was about 20% higher than at 0.1 mol/l, and one-sided partition of endo-pectinase was accomplished, and consequently maximal top phase yield.     

Production,extraction, and thermodynamics protease partitioning from Aspergillus tamarii Kita UCP1279 using PEG/sodium citrate aqueous two-phase systems

Abstract

The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 2⁴, was investigated. Then, the variables studied were polyethylene glycol (PEG) molar mass (M_PEG), concentrations of PEG (C_PEG) and citrate (C_CIT), and pH. The responses analyzed were the partition coefficient (K), activity yield (Y) and purification factor (PF). The thermodynamic parameters of the ATPS partition were estimated as a function of temperature. ATPS was able to pre-purify the protease (PF = 1.6) and obtained 84% activity yield. The thermodynamic parameters ΔG°_m (?10.89?kJ mol^?1), ΔH_m (?5.0?kJ?mol^?1) and partition ΔS_m (19.74?J mol^?1 K^?1) showed that the preferential migration of almost all protein contaminants of the crude extract to the salt-rich phase, while the preferred protease was the PEG rich phase. The extracted enzyme presents optimum temperature and pH at range of 40–50?°C and 9.0–11.0, respectively. Moreover, the enzyme was identified as serine protease based on inhibition profile. ATPS showed the satisfactory performance as the first step for Aspergillus tamarii Kita UCP1279 protease pre-purification.     

Enhanced metal-affinity partitioning of genetically engineered hirudin variants in polyethylene glycol/dextran two-phase systems

Hirudin variants were constructed to exhibit an increased metal-binding affinity in an attempt to apply a metal-affinity partitioning process in a primary separation step for purification of hirudin. The hirudin variants were genetically engineered to contain additional surface-accessible histidines and produced by recombinant Saccharomyces cerevisiae. The partitioning behavior of these variants was compared with that of the wild type with a single surface-accessible histidine at position 51. Upon the addition of a small amount of Cu(II)IDA-PEG (Cu(II)iminodiacetic acid-polyethylene glycol) ligand to PEG/dextran two-phase systems, the hirudin variants with two or three surface-accessible histidines were more selectively partitioned into the PEG-rich phase than the wild type. Integrating protein engineering to metal-affinity partitioning offers the potential for general application of this technique to facilitate protein isolation, but the genetically engineered protein variants should be carefully constructed in a manner to minimize reduction of native protein activity.     

On the use of mild hydrophobic interaction chromatography for "method scouting" protein purification strategies in aqueous two-phase systems: a study using model proteins

The behavior of a series of pure proteins partitioned in aqueous two-phase systems is compared with their behavior during mild hydrophobic interaction chromatography (HIC). A simple theoretical rationale for this comparison is presented based upon solvophobic theory. Similarities were found in the behavior of the model proteins in the two forms of partition chromatography. This indicates that HIC may be employed as a rapid instrumental technique for the broad characterization of protein behavior, which may be of benefit in the development of liquid-liquid partitioning strategies. However, it has proved difficult to completely account for this behavior on the basis of the known physical and structural properties of the proteins used. The variety in the detailed partitioning behavior of this small sample of protein types suggests that partition in aqueous two-phase systems is uniquely sensitive to subtle differences in surface properties of complex macromolecules. (c) 1994 John Wiley & Sons, Inc.     

Pectinase partitioning in polyethylene glycol 1000/Na2SO4 aqueous two-phase system: statistical modeling of the experimental results

Statistical models concerning partitioning of pectinase in polyethylene glycol 1000/Na₂SO₄ aqueous two-phase system were established with response surface methodology. Concentrations of polyethylene glycol 1000 and Na₂SO₄ were selected as independent variables to evaluate their impact on parameters of partitioning in aqueous two-phase system—the partition coefficient of pectinase, purification factor and pectinase yield. An experimental space where over 2.5-fold purification was achieved, followed by over 90% yield of pectinase. The established models showed good prediction of partitioning parameters.     

Genetically engineered charge modifications to enhance protein separation in aqueous two-phase systems: Electrochemical partitioning

We have examined the effect of genetically engineered charge modifications on the partitioning behavior of proteins in dextran/polyethylene glycol two-phase systems containing potassium phosphate. By genetically altering a protein's charge, the role of charge on partitioning can be assessed directly without the need to modify the phase system. The charge modifications used are of two types: Charged tails of polyaspartic acid fused to beta-galactosidase and charge-change point mutations of T4 lysozyme which replace positive lysine residues with negative glutamic acids. The partition coefficient K(p) for these proteins was related to measured interfacial potential differences Deltaphi using the simple thermodynamic model, In K(p) = In K(o) + (F/RT)Z(p) deltaphi. The protein net charge Z(p) was determined using the Henderson-Hasselbalch relationship with modifications based on experimentally determined titration and isoelectric point data. It was found that when the electropartitioning term Z(p) deltaphi was varied by changing the pH, the partitioning of T4 lysozyme was quantitatively described by the thermodynamic model. The beta-galactosidase fusions displayed qualitative agreement, and although less than predicted, the partitioning increased more than two orders of magnitude for the pH range examined. Changes in the partitioning of lysozyme due to the various mutations agreed qualitatively with the thermodynamic model, but with a smaller than expected dependence on the estimated charge differences. The beta-galactosidase fusions, on the other hand, did not display a consistent charge based trend, which is likely due either to the enzyme's large size and complexity or to nonelectrostatic contributions from the tails. The lack of quantitative fit with the model described above suggests that the assumptions made in developing this model are oversimplified. (c) 1994 John Wiley & Sons, Inc.     

Quantitation of plasmid DNA in aqueous two-phase systems by fluorescence analysis

The development of aqueous two-phase systems for plasmid purification from Escherichia coli cell lysates requires a reliable DNA quantitation method. Plasmid DNA was quantified by fluorescence using PicoGreen nucleic acid stain. Linearity was obtained up to 40 ng plasmid ml^–1. Two polyethyleneglycol (PEG)/salt systems were studied, PEG 600/K₂HPO₄ and PEG 300/K₂HPO₄. The average plasmid recovery was 41% in the bottom phase of the first system and 35% in the top phase of the second system. This method has proved to be simple and reproducible.     

Genetically engineered charge modifications to enhance protein separation in aqueous two-phase systems: Charge directed partitioning

This report continues or examination of the effect of genetically engineered charge modifications on the partitioning behavior of proteins in aqueous two-phase extration. The genetic modifications consisted of the fusion of charged peptide tails to beta-galactosidase and charge-change point mutations to T4 lysozyme. Our previous article examined the influence of these charge modifications on partitioning as a function of interfacial potential difference. In this study, we examined charge directed partitioning behavior in PEG/dextran systems containing small amounts of the charged polymers diethylaminoethyl-dextran (DEAE-dextran) or dextran sulfate. The best results were obtained when attractive forces between the protein and polymer were present. Nearly 100% of the beta-galactosidase, which carries a net negative charge, partitioned to the DEAE-dextran-rich phase regardless of whether the phase was dextran or PEG. In these cases, cloudiness of the protein-rich phases suggest that strong charge interactions resulted in protein/polymer aggregation, which may have contributed to the extreme partitioning. Unlike the potentialdriven partitioning reported previously, consistent partitioning trends were observed as a result of the fusion tails, with observed shifts in partition coefficient (K(p)) of up to 37-fold. However, these changes could not be solely attributed to charge-based interactions. Similarly, T4 lysozyme, carrying a net positive charge, partitioned to the dextran sulfate-containing phase, and displayed four- to sevenfold shifts in K(p) as a result of the point mutations. These shifts were two to four times stronger than those observed for potential driven partitioning. Little effect on partitioning was observed when the protein and polymer had the same charge, with the exception of beta-galactosidase with polyarginine tails. The high positive charge density of these tails provided for a localized interaction with the dextran sulfate, and resulted in 2- to 15-fold shifts in K(p). (c) 1995 John Wiley & Sons, Inc.     

Continuous purification of antibodies from cell culture supernatant with aqueous two-phase systems: From concept to process

An aqueous two-phase extraction (ATPE) process based on a PEG/phosphate system was developed for the capture of human immunoglobulin G and successfully applied to a Chinese hamster ovary and a PER.C6® cell supernatant. A continuous ATPE process incorporating three different steps (extraction, back-extraction, and washing) was set up and validated in a pump mixer-settler battery. Most of the higher molecular weight cell supernatant impurities were removed during the extraction step, while most of the lower molecular weight impurities were removed during the subsequent steps. A global recovery yield of 80% and a final protein purity of more than 99% were obtained for the IgG purification from a CHO cell supernatant, representing a 155-fold reduction in the protein/IgG ratio. For the purification of IgG from a PER.C6® cell supernatant, a global recovery yield of 100%, and a host cell protein purity were attained, representing a 22-fold reduction in the host cell protein/IgG ratio. These results, thus, open promising perspectives for the application of the developed ATPE process as a platform for the capture of antibodies. In fact, this new process has shown the ability to successfully recover and purify different antibodies from distinct cell culture supernatants. This technology can also overcome some of the limitations encountered using the typical chromatographic processes, besides inherent advantages of scalability, process integration, capability of continuous operation, and economic feasibility.     

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.     

Understanding viral partitioning in two-phase aqueous nonionic micellar systems: 1. Role of attractive interactions between viruses and micelles

The partitioning behavior of viruses in the two-phase aqueous nonionic n-decyl tetra(ethylene oxide) (C10E4) micellar system cannot be fully explained by considering solely the repulsive, steric, excluded-volume interactions that operate between the viruses and the nonionic C10E4 micelles. Specifically, an excluded-volume theory developed recently by our group is not able to quantitatively predict the observed viral partition coefficients, even though this theory is capable of providing reasonable quantitative predictions of protein partition coefficients. To shed light on the discrepancy between the theoretically predicted and the experimentally measured viral partition coefficients, a central assumption underlying the excluded-volume theory that the viruses and the C10E4 micelles interact solely through repulsive, excluded-volume interactions was challenged in this study. In particular, utilizing bacteriophage P22 as a model virus, a competitive inhibition test and a partitioning study of the capsids of bacteriophage P22 were conducted. Based on the results of these two experimental studies, it was concluded that any attractive interactions between the tailspikes of bacteriophage P22 and the C10E4 micelles are negligible. Another experimental study was carried out wherein the partition coefficients of the model viruses, bacteriophages P22 and T4, were measured at various temperatures, and compared with those previously obtained for bacteriophage phiX174. This comparison also indicated that possible attractive, electromagnetic-induced interactions between the bacteriophage particles and the C10E4 micelles cannot be invoked to rationalize the observed discrepancy between the theoretically predicted and the experimentally measured viral partition coefficients.     

The application of aqueous two-phase systems to the purification of pharmaceutical proteins from transgenic sheep milk

Transgenic sheep milk containing the protein human₁-Antitrypsin (AAT) was partitioned in Poly(ethyleneglycol) (PEG)-Sulphate and PEG-Phosphate biphasic systems. Individual partition coefficients for AAT and some of the milk proteins were determined in these systems. The effects of PEG molecular weight, pH and the inclusion of NaCl on the partitioning of the proteins were also studied. It was found that increasing the concentration of NaCl and decreasing the molecular weight of the PEG resulted in an increase of the partition coefficients of the proteins to the upper (PEG) phase. This partitioning effect was greater for the more hydrophobic proteins and particularly in systems having a pH close to the isoelectric point of the protein. Solubilities of the proteins in increasing concentrations of ammonium sulphate were measured in order to investigate the effects of hydrophobic and electrostatic interactions on the partitioning of these proteins in aqueous two-phase systems. Those proteins that precipitated at low levels of ammonium sulphate showed an increase in partition coefficient at low concentrations of NaCl, or they were precipitated at the interface of the phases at low concentrations of NaCl. Proteins that had low salting out constants in ammonium sulphate solutions were relatively unaffected by NaCl in ATPS. It is probable however that conformational changes and the state of aggregation of proteins are also important and should be invoked in describing the partitioning behavior observed for -Lg for example. Comparison of theoretical and experimental values for AAT yield and purity showed clearly that partition coefficients are influenced by the degree of purity and values obtained with purified standards are not necessarily the same as for the same protein present in a complex mixture. Under the most favourable conditions using a 4% w/w loading of transgenic ovine milk, we obtained a 91% yield of AAT in the PEG phase with a purity of 73%.This revised version was published online in October 2005 with corrections to the Cover Date.     

Effect of salts and surfactants on the partitioning of Fusarium solani pisi cutinase in aqueous two-phase systems of thermoseparating ethylene oxide/propylene oxide random copolymer and hydroxypropyl starch

An aqueous two-phase system composed by a thermoseparating random copolymer of ethylene oxide/propylene oxide 50/50 (%w/w), Breox, and hydroxypropyl starch – Reppal PES 100 was evaluated for the partitioning of Fusarium solani pisi recombinant cutinase. The effect of several additives on the partitioning of pure cutinase was evaluated. Micelles of sodium dodecanoate provided a ten-fold increase of the partitioning coefficient (K=9) and recovery yields of 60-75%. The phase diagrams of the systems composed of Breox, Reppal and sodium dodecanoate were determined and it was found that in systems with high surfactant concentrations, the binodal was moved to lower polymer concentrations, enabling a two-phase system with 6% (w/w) of each polymer.     

Affinity gel electrophoresis as a predictive technique in the fractionation of transgenic sheep milk proteins by affinity aqueous two-phase partitioning

Affinity electrophoresis in the presence of various triazine dyes of sheep milk proteins, including transgenically-introduced human ₁-antitrypsin, has been evaluated as a predictive technique for possible large scale affinity-driven aqueous two-phase purifications. The success of the approach suggested it has potential as a general method for the rapid screening of ligands using only g amounts of sample, that could be applied to many complex mixtures before embarking on more costly and time-consuming two-phase partitioning experiments.     

Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Thermostable a-amylase with temperature optimum at 80 °C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000—20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000—20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of a-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60—75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na₂SO₄.     

Glucose-6-phosphate dehydrogenase partitioning in two-phase aqueous mixed (nonionic/cationic) micellar systems

The enzyme glucose-6-phosphate dehydrogenase (G6PD) plays an important role in maintaining the level of NADPH and in producing pentose phosphates for nucleotide biosynthesis. It is also of great value as an analytical reagent, being used in various quantitative assays. In searching for new strategies to purify this enzyme, the partitioning of G6PD in two-phase aqueous mixed (nonionic/cationic) micellar systems was investigated both experimentally and theoretically. Our results indicate that the use of a two-phase aqueous mixed micellar system composed of the nonionic surfactant C(10)E(4) (n-decyl tetra(ethylene oxide)) and the cationic surfactant C(n)TAB (alkyltrimethylammonium bromide, n = 8, 10, or 12) can improve significantly the partitioning behavior of G6PD relative to that obtained in the two-phase aqueous C(10)E(4) micellar system. This improvement can be attributed to electrostatic attractions between the positively charged mixed (nonionic/cationic) micelles and the net negatively charged enzyme G6PD, resulting in the preferential partitioning of G6PD to the top, mixed micelle-rich phase of the two-phase aqueous mixed micellar systems. The effect of varying the cationic surfactant tail length (n = 8, 10, and 12) on the denaturation and partitioning behavior of G6PD in the C(10)E(4) /C(n)TAB/buffer system was investigated. It was found that C(8)TAB is the least denaturing to G6PD, followed by C(10)TAB and C(12)TAB. However, the C(10)E(4)/C(12)TAB/buffer system generated stronger electrostatic attractions with the net negatively charged enzyme G6PD than the C(10)E(4)/C(10)TAB/buffer and the C(10)E(4)/C(8)TAB/buffer systems, when using the same amount of cationic surfactant. Overall, the two-phase aqueous mixed (C(10)E(4)/C(10)TAB) micellar system yielded the highest G6PD partition coefficient of 7.7, with a G6PD yield in the top phase of 71%, providing the optimal balance between the denaturing effect and the electrostatic attractions for the three cationic surfactants examined. A recently developed theoretical framework to predict protein partition coefficients in two-phase aqueous mixed (nonionic/ionic) micellar systems was implemented, and the theoretically predicted G6PD partition coefficients were found to be in reasonable quantitative agreement with the experimentally measured ones.     

Partitioning of pristinamycins in aqueous two-phase systems: A first step toward the development of antibiotic production by extractive fermentation

The partitioning of pristinamycins was studied in dextran and polyethylene glycol (PEG) aqueous two-phases systems. Pristinamycins partitioned preferentially into the PEG-rich top phase. The partition coefficient was independent of molar mass of PEG and dextran and of antibiotic concentration, but, increased exponentially with the tieline length of the system. Partition of pristinamycins was greatly improved when fatty acids esters of PEG were mixed with PEG. In such mixtures, the partition of coefficient increased up to a value of 24, dependent on the carbon chain length of fatty acids and the modified PEG concentrations. Moreover, in such system, the two groups of pristinamycins, I and II, were extracted in accordance with their hydrophobicity. Recovery of pristinanamycins produced by Streptomyces pritinaespiralis in a fermentation broth was achieved with a dextran/PEG system. Cells were confined into the bottom phase and pristinamycins partitioned in the top phase. However, due to binding of the pristinamycins to the cells, the partition coefficient was slightly lower than of pure antibiotics solutions. (c) 1994 John Wiley & Sons, Inc.