Abstract Aqueous Two-phase Systems Research Articles

Aqueous two-phase systems as a tool for bioseparation – emphasis on organic acids

Abstract Aqueous two-phase systems (ATPS) are universally recognized as an excellent alternative to the conventional separation techniques in the biotechnology, because of their undoubted advantages such as mild and biocompatible conditions, high water content, low interfacial tension, ease of process integration and scale up, etc. The formation of ATPS is due to the incompatibility of two polymers in a common solution. Other types of ATPS are formed by polymer/salt, ionic and/or non-ionic surfactants, inorganic salt/short-chain alcohols, and based on room temperature ionic liquids. ATPS are successfully used (even in large scale) for cells, enzyme and protein separation, while their application for recovery of small molecules such as organic acids, antibiotics, alcohols is more complicated as they are usually hydrophilic and tend to distribute evenly between the phases. The purpose of this paper is to overview and summarize the efforts made for the application of different types of ATPS for the separation of organic acids.

Extraction of protease from Aspergillus tamarii URM 4634 in aqueous two-phase system under continuous and discontinuous process

Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene glycol (PEG)/phosphate aqueous two-phase system under discontinuous and continuous (perforated rotative discs column) process. On the discontinuous process, it was evaluated the effect of operational conditions (PEG molar mass and its concentration, phosphate concentration and pH) over the partition coefficient, activity yield and purification factor. Protease partitioned to PEG-phase with partition coefficients up to 55.73. The best process parameters were 17.5% of PEG, with molar mass 8000 g·mol−1, 15% of phosphate salt at pH 6, with 113.15% of activity yield and purification factor of 2.62. Under continuous extraction, hold up data showed that 57.1% of the discontinuous phase was available for protein extraction. Further, separation achieved 90.0% of efficiency. The yields surpassed 100% in almost all runs, and the best purification factor was 1.84, with both flows of 2 mL·min−1. Thus, the best operational conditions reached an activity yield of 95.3% and 90.0% of separation efficiency. Hence, aqueous two-phase system PEG/phosphate extraction is an efficient process for separation of proteases produced by Aspergillus tamarii URM 4634, under continuous extraction likewise under discontinuous process.

Quantification of amino acids and peptides in an ionic liquid based aqueous two-phase system by LC\u2013MS analysis

Aqueous two-phase systems (ATPS) occur by the mixture of two polymers or a polymer and an inorganic salt in water. It was shown that not only polymers but also ionic liquids in combination with inorganic cosmotrophic salts are able to build ATPS. Suitable for the formation of ionic liquid-based ATPS systems are hydrophilic water miscible ionic liquids. To understand the driving force for amino acid and peptide distribution in IL-ATPS at different pH values, the ionic liquid Ammoeng 110™ and K2HPO4 have been chosen as a test system. To quantify the concentration of amino acids and peptides in the different phases, liquid chromatography and mass spectrometry (LC–MS) technologies were used. Therefore the peptides and amino acids have been processed with EZ:faast™-Kit from Phenomenex for an easy and reliable quantification method even in complex sample matrices. Partitioning is a surface-dependent phenomenon, investigations were focused on surface-related amino acid respectively peptide properties such as charge and hydrophobicity. Only a very low dependence between the amino acids or peptides hydrophobicity and the partition coefficient was found. Nevertheless, the presented results show that electrostatic respectively ionic interactions between the ionic liquid and the amino acids or peptides have a strong impact on their partitioning behavior.

A microdevice assisted approach for the preparation, characterization and selection of continuous aqueous two-phase systems: from micro to bench-scale.

Aqueous two-phase systems (ATPS) have emerged as an alternative strategy for the recovery and purification of a wide variety of biological products. Typical process development requires a large screening of experimental conditions towards industrial adoption where continuous processes are preferred. In this work, it was proved that under certain flow conditions, ATPS could be formed continuously inside a microchannel, starting from stocks of phase components. Staggered herringbone chaotic micromixers included within the device sequentially and rapidly prepare two-phase systems across an entire range of useful phase compositions. Two-phase diagrams (binodal curves) were easily plotted using the cloud-point method for systems of different components and compared with previously reported curves for each system, proving that phase formation inside the device correlated with the previously reported diagrams. A proof of concept for sample partitioning in such a microdevice was performed with two different experimental models: BSA and red blood cells. Finally, the microdevice was employed to obtain information about the recovery and partition coefficient of invertase from a real complex mixture of proteins (yeast extract) to design a process for the recovery of the enzyme selecting a suitable system and composition to perform the process at bench-scale.

Potassium Phosphate Salts-Based Aqueous Two-Phase Systems Applied in the Extraction of Gallic Acid from Guava

Aqueous two-phase systems (ATPS) consisting of potassium phosphate salt + alcohol or polyethylene glycol were investigated to extract gallic acid. The partitioning process was studied using purity gallic acid (GA). The constituents either promote the partition process of GA to bottom or top phase, leading to the creation of versatile systems. The salt acts as a drive force of process because it influences the value of phase pH, and consequently the deprotonation of biomolecule and its partition. The results using GA from guava are in close agreement with the model systems, validating the experimental data and supporting their applicability in real systems.

Extraction Separation of BSA in Aqueous Two-Phase Systems of Anionic and Cationic Surfactant Mixtures

Aqueous two-phase systems (ATPS) are formed in mixtures of sodium dodecylsulfate (SDS), cetyltrimethyl ammonium bromide (CTAB), and sodium bromide (NaBr). Two different kinds of ATPS appear when CTAB and SDS surfactants are in excess, respectively. Such ATPS may provide a new, useful partitioning system for separation of bovine serum albumin (BSA). The partitioning of BSA was studied in these systems. The results show that the partitioning is affected by the electrical property, the hydrophobicity and the structure of liquid crystal (LC), which exists in the top phase of the ATPS. BSA is extracted into the top phase with higher distribution coefficient when LC and BSA are oppositely charged. The hydrophobicity of LC, which can be improved by increasing the length of alkyl group, enhances the distribution coefficient. The hydrophobicity of lamellar LC and hexagonal LC is stronger than that of cubic LC, which causes extractive capability of the former is higher than the latter.

The application of aqueous two-phase systems to oligosaccharide synthesis by alpha-mannosidase catalysed glycosyl transfer reactions.

Aqueous two-phase systems formed from PEG and dextran have been applied to the synthesis of oligosaccharide by Jack bean alpha-mannosidase in reverse. Whilst rates of synthesis and percentage yields were similar in two-phase systems and one-phase aqueous buffer systems, a ten-fold increase in yield of product per unit of enzyme was seen. In addition, the use of aqueous two-phase systems offers potential process advantages over one-phase systems for the synthesis of oligosaccharides.

EXTRACTIVE FRACTIONATION OF EQUINE HYPERIMMUNE PLASMA

Aqueous two-phase partition and triphasic systems containing thiophilic and metal ligands were used to produce anti–snake venom serum using the fractionation of hyperimmune equine plasma with neutralizing activity against Bothrops spp. venom as the model. In poly(ethyleneglycol)(PEG)/salt systems, PEG molecular weight, system pH, and the amount of polyvinylpirrolidone (PVP) added were optimized in order to promote selective immunoglobulin recovery. From these studies a PEG 1500/ammonium sulfate system, pH 8.0, a tie-line length parameter (TLL) 21%, and a 2% PVP K-12 were preliminarily selected. The immunoreactive fraction (ELISA) mainly preferred the top phase, with a theoretical yield of >97% and a purification factor of 3.5. Practical recoveries were, however, hampered by limited product solubility and an erratic global process performance. In order to overcome these limitations, we moved to a system formed by derivatives of cellulose and starch, which contained an affinity solid phase. This three-phase system was compatible with the use of thiophilic or immobilized metal ion affinity (IMAC) supports, the first showing the higher selectivity. The system composed of 2% Methocel, 2% Reppal, 1.0 mol/kg ammonium sulfate, and 5–10% 2S-thioether sulfone agarose allowed recovery of 52% of the immunoreactive fraction from hyperimmune equine plasma at a total protein concentration of 0.25%. This process strategy also allowed simple operation and easy of affinity ligand recycling.

Oxidative renaturation of hen egg-white lysozyme in polyethylene glycol-salt aqueous two-phase systems.

Aqueous two-phase systems have been widely used for the separation and concentration of proteins. In this work we investigated the possibility of using aqueous two-phase system for the renaturation of inclusion body proteins by studying the effect of polyethylene glycol (PEG)-salt systems on the oxidative renaturation of hen egg-white lysozyme (HEWL) with guanidinium chloride (GdmCl) present in the system. To accomplish phase separation at moderately low concentrations of polymer and salt, the total GdmCl concentration had to be kept low (<1 M). The unfolded protein exhibited very low solubility under these conditions. In an attempt to increase the solubility of the protein, temperatures of 40, 50, and 60 degrees C were investigated. The effect of PEG molecular weight was also addressed. Best renaturation yields were obtained when using PEG 3400 and working at 50 degrees C. However, the total protein concentration had to be kept at a low level of 0.2 mg/mL. Lowering the total GdmCl concentration in the system resulted in increased aggregation.

Optimization and design considerations of two-phase continuous protein separation

Aqueous two-phase systems (ATPS) have begun to be used industrially for the isolation and separation of recombinant enzyme proteins. However, until now most studies have concentrated on investigating factors affecting the partition coefficients of molecules and even the pilot plant and scale-up developments have lacked engineering, optimization and design considerations. This paper investigates such considerations in the process of protein separation and purification in ATPS. A cost function for the process of protein extraction that takes into account the effect of variables in ATPS has been developed to study the influence of this extraction stage over the rest of the stages in the process (production, concentration and purification). The effect of the flow rate ratios, the relative sizes of the PEG and Phosphate phases and the NaCl concentration have been analysed. An extended mathematical model was used to describe the continuous, steady state operation of an aqueous two-phase system for protein extraction. The basic model is based on steady state mass balances of the main components and phase equilibrium data. Experimental data on the separation of α-amylase from Bacillus subtilis supernatant in a PEG/salt system was used. Simulations were carried out to study the effect of flow rate ratio as a function of NaCI concentration in the feed. At a flow rate ratio above 2.4 the extraction process was very sensitive to NaCl concentration. A rigorous optimization was carried out using the Sequential Quadratic Programming Method (SQP) with a fixed initial NaCI concentration of 8.8%. An optimal Flow Rate Ratio of 2.27 was obtained. Thus, the optimization strategy developed in this paper was mathematically implementable and successful. The optimal steady state process conditions corresponded to conditions of a large and continuous bottom phase in both the forward and back extractions. This is very favourable for phase separation as it results in fast settling rates which are also virtually independent of phase volume ratio, which are extremely favourable conditions for the design of appropriate settlers.

Partition of lead in a milk-pectin aqueous biphasic system with complexing agents

Aqueous two-phase systems allow partition of metal ions in nondenaturing conditions. From the fresh skim bovine milk spiked with212Pb and agitated with 4% water solution of apple pectin (degree of esterification-70%), less than 2.3% is recovered to the pectin phase and in the case of pasteurized milk less than 3.0%. Complexation with chelate forming agents was studied. Aminoacids as glycine, alanine and histidine do not exert influence on the yield, but the recovery in the pectin phase was 22% for cysteine, and 80% in the case of EdtacalTM (calcium salt of ethylenediaminetetraacetic acid). The retention of lead in milk phase is extraordinarily high as compared to strontium and corresponds to the behavior of trivalent ions.

Partition and counter-current distribution of membrane particles in aqueous dextran-poly(ethylene glycol) two-phase systems with special reference to synaptosomes

Aqueous two-phase systems composed of water, dextran and poly(ethylene glycol) can be used for the separation of biological particles. The adjustment of the partition of such particles between the two phases and the interface between them has been studied by using a preparation of synaptosomes (from calf brain cortex) also containing free mitochondria. The partition has been affected by variation of polymer concentrations and addition of salts, e.g. phosphates and chloride. The time for separation of the phases showed a bimodal behaviour with an initially rapid formation of bulk phases followed by a slow phase separation. The relative amount of mixed phases at the time of the transition was proportional to the amount of particles included. Counter-current distribution with moderate time for the phase separation was carried out in such way that the interface material travelled with approximately half the speed of the moving upper phase. In this way the distribution of the particles between the upper phase and the interface as well as between the interface and the lower phase could be studied in the same experiment. The heterogeneity of the synaptosome preparation was clearly demonstrated by counter-current distribution at low polymer concentrations while no separation was obtained when the system contained larger amounts of polymers. Possible reasons for this behaviour are discussed.

Separation of Biopolymers by Partition in Aqueous Two-Phase Systems

Abstract Aqueous two-phase systems are useful tools for fractionation and studies of cell components including proteins and nucleic acids. The systems consist of two non-miscible liquid layers which both have high content of water. The purification achieved by partition of biosubstances between the two liquid phases depends on the possibility to steer the partition in a selective way. The partition can be affected by hydrophobic and affinity ligands, as well as charged groups/ bound to soluble polymers.

Immunoassays in aqueous two-phase systems

Aqueous two-phase systems provide a novel and convenient method for separating bound from free fractions in a binding assay. The ease of automation of this type of procedure makes it particularly attractive for separations based on immobilized ligands or binders or on adsorbents.