Aqueous two-phase fractionation of biological suspensions for protein recovery from bovine blood

The practical application of a two-stage aqueous two-phase systems (ATPS) for the fractionation and recovery of proteins from biological suspensions is described. A model process for the recovery of proteins from whole bovine blood was selected to study the implementation of an ATPS process. Recycling of used PEG into the initial extraction stage did not significantly influence the partition behaviour of serum albumin in subsequent cycles. © Rapid Science Ltd. 1998     

Aqueous two-phase extraction for the purification of alkaline agarases from culture extracts of Pseudomonas aeruginosa AG LSL-11

The agarases were purified for the first time an using aqueous two-phase system (ATPS) consisting of polyethylene glycol (PEG) and phosphate salt. The three extracellular, alkaline agarases produced by Pseudomonas aeruginosa AG LSL-11 were efficiently extracted into the top PEG-rich layer. The influencing factors on the partition of agarases--molecular weight of the PEG, system pH, system temperature, and NaCl concentration--were investigated. All the factors were found to have a significant effect on the partition of agarases except NaCl. The optimal ATPS parameters for the partitioning and purification of agarases were found to be 12% PEG 600 and 11.9% (w/w) phosphate salt at pH 8.0 and 4°C. All three agarases were concentrated in the top PEG phase with 6.19-fold purity and 71.21% recovery. The ATPS was found to be more convenient and economical than the conventional ion-exchange chromatography (IEC) method for extraction of three agarases and could be significantly employed for the purification of agarases from fermentation broth.     

Aqueous two-phase extraction for downstream processing of amyloglucosidase

A polymer/salt aqueous two-phase system has been successfully employed for separation and purification of amyloglucosidase. The effects of system pH, molecular weight of polymer and composition of the two-phase system on amyloglucosidase partition behaviour in polyethylene glycol (PEG 4000, 6000)/disodium hydrogen phosphate were investigated. Experimental data are explained based on Kim's theoretical model for the prediction of biomolecule partitioning in a PEG/salt system.     

Aqueous two-phase systems extraction of alpha-toxin from Clostridium perfringens type A

Two sequential half-fraction designs were applied to studying the alpha-toxin partition produced by Clostridium perfringens type A in aqueous two phase systems (ATPS), as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows, in a single step, an alpha-toxin purification of 4.6-fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase.     

Aqueous two-phase extraction coupled with ultrafiltration for purification of amyloglucosidase

Aqueous two phase extraction (ATPE) in combination with ultrafiltration was employed for concentration and purification of amyloglucosidase produced by solid state fermentation. After extraction (with water) from dry moldy bran the dilute enzyme extract was concentrated by ATPE in a polyethylene glycol (PEG)/maltodextrin (MDX) system. The enzyme in the top PEG rich phase was then extracted into a Na₂HPO₄ rich bottom phase and further concentrated by ultrafiltration. The partitioning behavior of amyloglucosidase was examined in PEG/MDX, PEG/Na₂SO₄, PEG/Na₂HPO₄, PEG/KH₂PO₄ aqueous two phase systems. Effect of buffering salts such as NaCl, Na₂HPO₄, KH₂PO₄ and Na₂SO₄ on the partitioning behavior of enzyme was studied in PEG/MDX system. Maximum partitioning of amyloglucosidase was seen with KH₂PO₄ (m = 18.1). A two stage ATPE employing PEG/MDX (buffered with KH₂PO₄) and PEG/Na₂HPO₄ systems, followed by ultrafiltration has resulted in an overall recovery of 78.4% with 3.1 fold purification and 9.4 fold concentration of the enzyme.     

Aqueous two-phase metal affinity extraction of heme proteins

An iminodiacetic acid derivative of poly(ethylene glycol) (PEG-IDA) that chelates metal cations has been synthesized and used to extract proteins in metal affinity aqueous two-phase PEG/dextran systems. With less than 1% of the PEG substituted with chelated copper, partition coefficients are shown to increase by factors of up to 37 over extraction with unsubstituted PEG. The proteins studied are preferentially extracted into the Cu(II)PEGIDA phase in proportion to the number of accessible histidine residues on their surface. The affinity contribution to partitioning is proportional to the number of exposed histidine over a very wide range. The partition coefficients of heme-containing proteins measured in the Cu(II)PEG-IDA/dextran systems increase with the pH of the extraction mixture from pH 5.5 to pH 8.0, while partition coefficients in the unsubstituted PEG/dextran systems are very nearly independent of pH. The strong pH dependence of the metalaffinity extraction can be utilized in the recovery of the extracted protein.     

Aqueous two-phase extraction and purification of animal proteins

Aqueous two-phase systems provide a rapid, easily scalable method for separation of soluble proteins from insoluble materials and other undesired proteins. The method can be operated in continuous mode. It is particularly useful for animal proteins, as it overcomes difficulties of other methods in removing bulk insoluble material, while at the same time providing purification with respect to total soluble protein. This article describes the development of methods for aqueous two-phase extraction and purification of animal proteins, at both laboratory and pilot scale. The strengths, weaknesses, and possible future prospects for the method are discussed.     

Aqueous two-phase assisted by ultrasound for the extraction of anthocyanins from Lycium ruthenicum Murr.

In this study, an efficient ultrasound-assisted aqueous two-phase extraction method was used for the extraction of anthocyanins from Lycium ruthenicum Murr. An ethanol/ammonium sulfate system was chosen for the aqueous two-phase system due to its fine partitioning and recycling behaviors. Single-factor experiments were conducted to determine the optimized composition of the system, and the response surface methodology was used for the further optimization of the ultrasound-assisted aqueous two-phase extraction. The optimal conditions were as follows: a salt concentration of 20%, an ethanol concentration of 25%, an extraction time of 33.7?min, an extraction temperature of 25°C, a liquid/solid ratio of 50:1 w/w, pH value of 3.98, and an ultrasound power of 600?W. Under the above conditions, the yields of anthocyanins reached 4.71?mg/g dry sample. For the further purification, D-101 resin was used, and the purity of anthocyanins reached 25.3%. In conclusion, ultrasound-assisted aqueous two-phase extraction was an efficient, ecofriendly, and economical method, and it may be a promising technique for extracting bioactive components from plants.     

Aqueous two-phase systems strategies to establish novel bioprocesses for stem cells recovery

Abstract

During the past decade, stem cell transplantation has emerged as a novel therapeutic alternative for several diseases. Nevertheless, numerous challenges regarding the recovery and purification steps must be addressed to supply the number of cells required and in the degree of purity needed for clinical treatments. Currently, there is a wide range of methodologies available for stem cells isolation. Nevertheless, there is not a golden standard method that accomplishes all requirements. A desirable recovery method for stem cells has to guarantee high purity and should be sensitive, rapid, quantitative, scalable, non- or minimally invasive to preserve viability and differentiation capacity of the purified cells. In this context, aqueous two-phase systems (ATPS) represent a promising alternative to fulfill the mentioned requirements, promoting the use of stem cell-based therapies for incurable diseases. This practical review focuses on presenting the bases for the development of a novel and scalable bioprocess for the purification of stem cells, with a case scenario of CD133⁺ cells. The bioengineering strategies include the application of immunoaffinity ATPS in its multiple variants, including antibody-polymer conjugation, antibody addition and antibody immobilization. Conclusions are drawn in the light of the potential generic implementation of these strategies as an initial step in the establishment of bioprocesses for the purification of stem cells.     

Aqueous two-phase recovery of bio-nanoparticles: a miniaturization study for the recovery of bacteriophage T4

Biotechnology industry has recently been demanding nanoparticulate products (20-200 nm) such as viruses, plasmids, virus-like particles and drug delivery assemblies. These products are mainly used as gene delivery systems in gene therapy protocols. During the process development for the manufacture of these products, it is crucial to optimize the recovery and purification steps. Unfortunately, the high value of some bio-nanoparticles complicates the optimization studies. The solvent extraction method with aqueous two-phase systems (ATPS) has been used to successfully recover bioproducts on a large scale. In this study, the potential miniaturization of ATPS is presented. The partition behavior of pure bovine serum albumin (BSA) in PEG-800-phosphate and bacteriophage T4 in PEG 8000-phosphate and PEG 600-sulphate systems were studied at three different scales (10 g, 2 g and 300 microl). The results obtained showed that the volume ratio (V(R)) for BSA (V(R)=1.0) was comparable to the blank systems at the scales studied. Additionally, the partition coefficient (K) was also similar (K=0.05) with more than 82% of BSA concentrated in the bottom phase. Same system was challenged with bacteriophage T4 showing a V(R)=1.0 and K greater than 5 with the infective particles concentrated in the top phase. The bacteriophage T4 was concentrated in opposite phase in the PEG-600-sulfate system with a consistent V(R)=0.8 and K<0.2 for the scales analyzed. The partition behavior the bacteriophage T4 was comparable to that reported previously for adenoviral vectors in same system at 15 ml scale. The results obtained demonstrated that the miniaturization of ATPS is feasible and reproducible for the two models selected. This provides significant information about the miniaturization process of such ATPS for their potential generic applications in the recovery of different bio-nanoparticle products.     

Aqueous two-phase partition of complex protein feedstocks derived from brain tissue homogenates

This study describes the application of aqueous two-phase partition using polyethylene glycol (PEG)-potassium phosphate systems for the direct recovery of proteins, and aggregates thereof, from mammalian brain tissue homogenates. Investigation of established methodologies for the purification of prion proteins (PrP) from bovine brain affected with transmissible spongiform encephalopathy (BSE) has identified an alternative purification regime based on aqueous two-phase partition. This circumvents energy-intensive and rate-limiting unit operations of ultracentrifugation conventionally used for isolation of PrP. Selectivity of various PEG-phosphate systems varied inversely with polymer molecular mass. The maximum protein recovery from bovine brain extracts was obtained with systems containing PEG 300. Manipulation of the aqueous environment, to back-extract protein product from the PEG-rich top phase into the phosphate-rich lower phase, enabled integration of ATPS with conventional hydrophobic interaction chromatography (HIC) which selectively removes obdurate contaminating proteins (i.e. ferritin).     

Novel polymer-polymer conjugates for recovery of lactic acid by aqueous two-phase extraction

A new family of polymer conjugates is proposed to overcome constraints in the applicability of aqueous two-phase systems for the recovery of lactic acid. Polyethylene glycol-polyethylenimine (PEI) conjugates and ethylene oxide propylene oxide-PEI (EOPO-PEI) conjugates were synthesized. Aqueous two-phase systems were generated when the conjugates were mixed with fractionated dextran or crude hydrolyzed starch. With 2% phosphate buffer in the systems, phase diagrams with critical points of 3.9% EOPO-PEI-3.8% dextran (DEX) and 3.5% EOPO-PEI-7.9% crude starch were obtained. The phase separation temperature of 10% EOPO-PEI solutions titrated with lactic acid to pH 6 was 35 degrees C at 5% phosphate, and increased linearly to 63 degrees C at 2% phosphate. Lactic acid partitioned to the top conjugate-rich phase of the new aqueous two-phase systems. In particular, the lactic acid partition coefficient was 2.1 in 10% EOPO-PEI-8% DEX systems containing 2% phosphate. In the same systems, the partitioning of the lactic acid bacterium, Lactococcus lactis subsp. lactis, was 0.45. The partitioning of propionic, succinic, and citric acids was also determined in the new aqueous two-phase systems.     

Aqueous two-phase extraction of 2,3-butanediol from fermentation broths using an ethanol/phosphate system

Separation of 2,3-butanediol from the complex fermentation broths is a difficult task and becomes a bottleneck in industrial production. Aqueous two-phase systems composed of hydrophilic solvents and inorganic salts could be used to extract 2,3-butanediol from fermentation broths. Aqueous two-phase extraction of 2,3-butanediol from fermentation broths was studied by ethanol and dipotassium hydrogen phosphate system. The influences of phase composition on partition of 2,3-butanediol, removal of cells and biomacromolecules were investigated. The partition coefficient and recovery of 2,3-butanediol reached up to 28.34 and 98.13%, respectively, and the selective coefficient of 2,3-butanediol to glucose was 615.87 when the system was composed of 24% (w/w) ethanol and 25% (w/w) dipotassium hydrogen phosphate. Simultaneously, cells and proteins could be removed from the fermentation broths and the removal ratio reached 99.63 and 85.9%, respectively. This process is convenient and economic, furthermore, the operation is easy to scale-up, that is, this method provides a new possibility for the separation and refining of 2,3-butanediol.     

Aqueous two-phase extraction of 2,3-butanediol from fermentation broths by isopropanol/ammonium sulfate system

A novel aqueous two-phase system consisted of 2-propanol/ammonium sulfate was used for the extraction of 2,3-butanediol from fermentation broths. The maximum partition coefficient and recovery of 2,3-butanediol reached 9.9 and 93.7%, respectively, and more than 99% of the cells and about 85% of the soluble proteins were removed when 34% (w/w) 2-propanol and 20% (w/w) ammonium sulfate were used. The separated cells could be re-used as inocula for subsequent fermentations. The aqueous two-phase system described in this study may have potential application in the extraction of 2,3-butanediol produced by industrial fermentation processes.     

Aqueous two-phase systems

Biphasic systems formed by mixing of two polymers or a polymer and a salt in water can be used for separation of cells, membranes, viruses, proteins, nucleic acids, and other biomolecules. The partitioning between the two phases is dependent on the surface properties and conformation of the materials, and also on the composition of the two-phase system. The mechanism of partitioning is, however, complex and not easily predicted. Aqueous two-phase systems (ATPS) have proven to be a useful tool for analysis of biomolecular and cellular surfaces and their interactions, fractionation of cell populations, product recovery in biotechnology, and so forth. Potential for environmental remediation has also been suggested. Because ATPS are easily scalable and are also able to hold high biomass load in comparison with other separation techniques, the application that has attracted most interest so far has been the large-scale recovery of proteins from crude feedstocks. As chemicals constitute the major cost factor for large-scale systems, use of easily recyclable phase components and the phase systems generated by a single-phase chemical in water are being studied.     

Aqueous two-phase extraction of 2,3-butanediol from fermentation broths using an ethanol/ammonium sulfate system

Separation of 2,3-butanediol from the fermentation broth is a difficult task that has become a bottleneck in industrial production. Aqueous two-phase systems composed of hydrophilic solvents and inorganic salts could be used to extract 2,3-butanediol from fermentation broth. The ethanol/ammonium sulfate system was investigated in detail, including phase diagram, effect of phase composition on partition, removal of cells and biomacromolecules from the broths and recycling of ammonium sulfate. The highest partition coefficient (7.10) and recovery of 2,3-butanediol (91.7%) were obtained by a system composed of 32% (w/w) ethanol and 16% (w/w) ammonium sulfate. The maximum selective coefficient of 2,3-butanediol to glucose was 30.74 in the experimental range. In addition, cells and proteins could be simultaneously removed from the fermentation broth. The removal ratio of cells and proteins reached 99.7% and 91.2%, respectively. The recovery of ammonium sulfate in the bottom phase reached 97.14% when two volumes of methanol were added to the salt-rich phase.     

Aqueous two-phase extraction of plant enzymes from sources containing large amounts of tannins and anionic mucilages.

The isolation of plant enzymes is frequently hampered by the presence of phenolic compounds, pigments and mucilages. Recently, extraction procedures based on aqueous two-phase systems were used to overcome these problems. Two-phase systems have a great advantage in respect to yield, product purity and processing time. Two-phase systems may open many new avenues in research as well as for application of enzymes from plant material, especially making available enzymes of sources avoided till now, due to the difficulties to work with.     

Aqueous two-phase extraction and polyelectrolyte precipitation combination: A simple and economically technologies for pepsin isolation from bovine abomasum homogenate

The combination of two bioseparation techniques, partition in aqueous two-phase systems and polyelectrolyte precipitation of the target enzyme from the phase where it is present, was assayed to purify pepsin from bovine abomasum homogenate. Pepsin was partitioned in favor of the polyethyleneglycol-rich phase in an aqueous two-phase system of polyethyleneglycol 600 and 1450-sodium phosphate; however, a great amount of impure proteins were present. Chitosan (a cationic natural polyelectrolyte) was added to precipitate this acid enzyme as a form of insoluble complex. The addition of this second step increased the purity of the enzyme significantly while the yield was not significantly decreased. The combination of both partition in polyethyleneglycol 1450-phosphate system and chitosan precipitation produced a pepsin recovery of 48.5% with a purification factor of 9.0. The biological activity of the recovered enzyme remained unaltered.     

Aqueous two-phase systems containing urea: influence of protein structure on protein partitioning

During recombinant E. coli fermentation with high-expression levels inclusion bodies are often formed. Aqueous two-phase systems have been successfully used in the presence of urea for the initial recovery step of inclusion bodies from E. coli. Basic studies of the complex interactions are lacking. For a systematic study of protein partitioning in the presence of urea we selected T4-lysozyme mutants with different thermal stability as a model. The stabilization of these variants by phase components was investigated measuring the fluorescence emission of tryptophan residues in the protein. Protein structure was stabilized at pH 7 in the order of S0(4)(2-) > PEG = Dextran > H(2)O. The conformation of proteins was shown to have a strong influence on the partitioning in aqueous two-phase systems. Tryptophan and its homologuous di- and tripeptdides were partitioned in similar phase systems to normalize for contribution from hydrophobic interactions.     

Aqueous two-phase systems for molecular weight analyses

Summary The potential of aqueous two-phase systems for the estimation of molecular weights of proteins and enzyme-inhibitor complexes has been demonstrated. The method is simple and rapid, and employs extremely small amounts of the test sample. It is based on the measurement of the phase transition behaviour on dilution of the system as a function of the molecular weight of the molecule or complex under study.