Polymer fractionation in aqueous two-phase polymer systems.

We consider the effects of the addition of poly(ethylene glycol) (PEG) of different molecular weights to aqueous two-phase system of PEG 8000 and dextran 500. The first purpose of this study was to determine the molecular weight partitioning of the polymers themselves so that, for example, aqueous two-phase separations using affinity ligands can be improved. The second purpose was to examine whether this molecular weight partitioning could be predicted by using solution thermodynamic models so that it would be possible to optimize affinity partitioning without extensive laboratory work. Experimentally, we find that, by increasing the PEG concentration of any molecular weight in the feed, the high molecular weight PEG concentration in the dextran-rich phase is reduced. This observation can be used to reduce the loss of expensive ligated PEG used in affinity partitioning. Further, there is generally good agreement between our experimental data and the predictions of a solution thermodynamic model.     

Continuous counter-current two-phase aqueous extraction.

Continuous counter-current column operation provides operating convenience for contacting two-phase aqueous partitioning systems for protein extraction. We discuss in detail the important parameters for designing spray, packed, plate and York-Scheibel columns for protein recovery using both polymer-polymer and polymer-salt two-phase aqueous systems. We compare the various contractors for their operating and extraction efficiency. The work also provides a step-by-step design procedure and specific recommendations for future data needs.     

Purification of rat C6 glioma plasma membranes by affinity partitioning

We have studied the feasibility of purifying rat C6 glioma plasma membranes by a phase partitioning approach. The purification procedure involves cell homogenization and fractionation with an aqueous two-phase polymer system followed by selective affinity purification of plasma membranes by a wheat germ agglutinin-coupled polymer system. We demonstrate that the two-phase affinity partitioning technique is a simple and efficient method of isolating cell plasma membranes with high purity and yield. Furthermore, the isolated plasma membranes retain their functional integrity, as shown by the high-affinity insulin-like growth factor-I (IGF-I) binding capacity of IGF-I receptors.     

Aqueous micellar two-phase system composed of hyamine-type hydrophobically modified ethylene oxide and application for cytochrome P450 BM-3 separation

The hydrophobically modified ethylene oxide polymer, HM-EO, was modified with an alkyl halide to prepare a hyamine-type HM-EO, named N-Me-HM-EO, which could be used for forming N-Me-HM-EO/buffer aqueous micellar two-phase system. The critical micelle concentration of N-Me-HM-EO solution and the phase diagrams of N-Me-HM-EO/buffer systems were determined. By using this novel aqueous micellar two-phase system, the separation of cytochrome P450 BM-3 from cell extract was explored. The partitioning behavior of P450 BM-3 in N-Me-HM-EO/buffer systems was measured. The influences of some factors such as total proteins concentration, pH, temperature and salt concentration, on the partitioning coefficients of P450 BM-3 were investigated. Since the micellar aggregates in the N-Me-HM-EO enriched phase were positively charged, it was possible to conduct the proteins with different charges to top or bottom phases by adjusting pH and salt concentration in the system. A separation scheme consisting of two consecutive aqueous two-phase extraction steps was proposed: the first extraction with N-Me-HM-EO/buffer system at pH 8.0, and the second extraction in the same system at pH 6.0. The recovery of P450 BM-3 was 73.3% with the purification factor of 2.5. The results indicated that the aqueous micellar two-phase system composed of hyamine modified polysoap has a promising application for selective separation of biomolecules depending on the enhanced electrostatic interactions between micelles and proteins.     

The effect of KSCN on the partition of proteins in polyethylene glycol/(NH4)2SO4 aqueous two-phase system

The effect of potassium thiocyanate on the partitioning of lysozyme and BSA in polyethylene glycol 2000/ammonium sulfate aqueous two-phase system has been investigated. As a result of the addition of potassium thiocyanate to the PEG/ammonium sulfate system, the PEG/mixed salts aqueous two-phase system was formed. It was found that the potassium thiocyanate could alter the pH difference between the two phases, and, thus, influence the partition coefficients of the differently charged proteins. The relationship between partition coefficient of the proteins and pH difference between two phases has been discussed. It was proposed that the pH difference between two phases could be employed as the measurement of electrostatic driving force for the partitioning of charged proteins in polyethylene 2000/ammonium sulfate aqueous two-phase system.     

Recovery of endo-polygalacturonase using polyethylene glycol-salt aqueous two-phase extraction with polymer recycling

The partitioning behaviour of endo-polygalacturonase (endo-PG) and total protein from a clarified Kluyveromyces marxianus fermentation broth in polyethylene glycol (PEG)-ammonium sulfate and PEG-potassium phosphate (pH=7) aqueous two-phase systems was experimentally investigated. Both the enzyme and total protein partitioned in the bottom phase for these two kinds of systems. The enzyme partitioning coefficient can be lower than 0.01 in PEG8000-(NH₄)₂SO₄ ATPS with a large phase volume ratio and a moderate tie-line length, which implies the possibility of concentration operation using aqueous two phase partitioning. An ion-exchange separation of high purification efficiency was applied to analyze the clarified and dialyzed fermentation broth. A total purification factor of only 2.3 was obtained, which indicated the high enzyme protein content in the total protein of the fermentation broth. Consequently, the main purpose for separating endo-PG is concentration rather than purification. A separation scheme using an aqueous two-phase extraction process with polymer recycling and a dialysis was proposed to recover endo-PG from the fermentation supernatant of K. marxianus for commercial purpose. A high enzyme recovery up to 95% and a concentration factor of 5 to 8 with a purification factor of about 1.25 were obtained using the single aqueous two-phase extraction process. More than 95% polymer recycled will not affect the enzyme recovery and purification factor. Dialysis was used mainly to remove salts in the bottom phase. The dialysis step has no enzyme loss and can further remove small bulk proteins. The total purification factor for the scheme is about 1.7.     

High throughput characterization of structural differences between closely related proteins in solution

Partitioning of a protein in an aqueous two-phase system (ATPS) is governed by interactions of the protein with aqueous media in the two phases. Here we describe how partitioning of proteins in a set of ATPS of different compositions can be used to quantify differences between 3D structures of closely related proteins. We also provide perspective on practical applications of the technology when comparative analysis of the higher-order structure of proteins is desired.     

Multi-stage laccase extraction and separation using aqueous two-phase systems: Experiment and model

This work presents results of experimental and model investigation of continuous multi-stage enzyme extraction using aqueous two-phase systems for the first time. The aqueous two-phase system comprised polyethylene glycol 3000 and phosphate with additional sodium chloride buffered to pH 7. Two different laccases served as model enzymes. One of the laccases was directly taken from fungal culture supernatant, while the other laccase was solubilized lyophilisate. The modeling is based on an equilibrium stage approach. Equilibrium data were taken from single-stage experiments and approximated by different correlation equations. The model describes densities, phase equilibrium, enzyme activity partitioning between the phases. Moreover it allows to consider activity changes due to the aqueous two-phase system. Eight multi-stage mixer-settler experiments under varying operation conditions were performed to validate the proposed model; whereas the total throughput of all multi-stage extraction experiments was about 350 g h⁻¹. The average relative deviation of modeled activities from experimentally measured activities was 23%. Therefore, the model is able to calculate the behavior of the phases as well as the partitioning of the two enzymes between the two phases for a multi-stage process based on single-stage data.     

Partitioning of host and recombinant cells in aqueous two-phase polymer systems

The separation of host and recombinant Escherichia coli bacterial cells has been studied using the surface-sensitive technique of partitioning in aqueous two-phase polymer systems. Experiments were designed to probe charge-and hydrophobicity-related property differences of antibiotic-resistant recombinant cells and their antibiotic-sensitive hosts. Differential partitioning was observed in both charge-sensitive and non-charge-sensitive phase systems for three host-recombinant cell systems, but the non-charge-related effects appear to have a greater impact on partitioning behavior. This result suggests that plasmid-encoded products related to antibiotic resistance modify the surface hydrophobicity of the E. coli bacterial cell and that these differences can be exploited for cell separation.     

Aqueous two-phase: the system of choice for extractive fermentation

Extractive fermentation in aqueous two-phase systems is a meaningful approach to overcome low product yield in a conventional fermentation process, and by proper design of the two-phase system it is possible to obtain the product in a cell-free stream. The characteristics of an aqueous two-phase system, various polymers used for forming an aqueous two-phase system, the physicochemical parameters of the aqueous two-phase system, partitioning of biomolecules and cell mass and the effect of individual phase forming polymers on cell growth and product formation are reviewed in this article. The various extractive fermentation processes are also summarised here. At the end, the economic viability and scope of aqueous two-phase fermentation are briefly discussed in relation to the wider application of this topic. Received: 16 June 1999 / Received revision: 29 December 1999 / Accepted: 4 January 2000     

Differences in the surface properties of the mating types of Physarum polycephalum

In the slime mold Physarum polycephalum, formation of a diploid plasmodium occurs when compatible haploid amoebae fuse. To study cell surface changes associated with the fusion process, a non-destructive method known as aqueous, two-phase partitioning was employed. Using a two-phase system of dextran and polyethylene glycol, we observed that the two mating types (RSD4 and MA185) have different partition coefficients and hence different surface properties. Based on their partitioning behavior, MA185 cells appear to have a more hydrophobic surface than RSD4 amoeba. The partition coefficient of both cell types decreased with time. If amoebae were maintained in culture until they encysted, differences in their surface were not detectable.     

An aqueous hydroxypropylstarch-poly(ethylene glycol) two-phase system for extraction of alpha-lactalbumin and beta-lactoglobulin from bovine whey.

The partitioning of alpha-lactalbumin and beta-lactoglobulin from bovine whey has been studied in an aqueous poly(ethylene glycol) (PEG)-hydroxypropylstarch two-phase system. The influence of several parameters including concentrations of polymers, sodium phosphate buffer, KSCN, and of PEG palmitate, with and without the presence of Ca2+, on the partitioning of the proteins has been investigated. The separation of the two proteins was demonstrated by counter-current distribution. A purification procedure for both proteins has been developed by using PEG-hydroxypropylstarch two-phase system. This system is compared with the more costly standard system based on PEG and dextran. The possible use of the aqueous two-phase systems for batch extraction for large scale purification of these whey proteins is discussed.     

Development of a novel bioreactor system for treatment of gaseous benzene

A novel, continuous bioreactor system combining a bubble column (absorption section) and a two-phase bioreactor (degradation section) has been designed to treat a gas stream containing benzene. The bubble column contained hexadecane as an absorbent for benzene, and was systemically chosen considering physical, biological, environmental, operational, and economic factors. This solvent has infinite solubility for benzene and very low volatility. After absorbing benzene in the bubble column, the hexadecane served as the organic phase of the two-phase partitioning bioreactor, transferring benzene into the aqueous phase where it was degraded by Alcaligenes xylosoxidans Y234. The hexadecane was then continuously recirculated back to the absorber section for the removal of additional benzene. All mass transfer and biodegradation characteristics in this system were investigated prior to operation of the integrated unit, and these included: the mass transfer rate of benzene in the absorption column; the mass transfer rate of benzene from the organic phase into the aqueous phase in the two-phase bioreactor; the stripping rate of benzene out of the two-phase bioreactor, etc. All of these parameters were incorporated into model equations, which were used to investigate the effects of operating conditions on the performance of the system. Finally, two experiments were conducted to show the feasibility of this system. Based on an aqueous bioreactor volume of 1 L, when the inlet gas flow and gaseous benzene concentration were 120 L/h and 4.2 mg/L, respectively, the benzene removal efficiency was 75% at steady state. This process is believed to be very practical for the treatment of high concentrations of gaseous pollutants, and represents an alternative to the use of biofilters.     

Substitutions of surface amino acid residues of cutinase probed by aqueous two-phase partitioning

The surface properties of a protein are often crucial for recognition and interaction with other molecules. Important functional residues can be identified by mutational analysis. There is a need for rapid methods to study protein surfaces and surface changes due to mutations. Partitioning in aqueous two-phase systems has the potential to be used in this respect since protein partitioning depends on the surface properties of the protein. The influence of surface-exposed amino acid residues in protein partitioning has been studied with cutinase variants, which differed in one or several amino acid residues as a result of site-directed mutagenesis. The solvent accessibility of the mutated residues was determined with a computer program, Graphical Representation and Analysis of Surface Properties. The aqueous two-phase system was composed of dextran and a random copolymer of ethylene oxide and propylene oxide. It was shown, for the first time, to what extent surface-exposed amino acid residues influence the partition coefficient in an aqueous two-phase system. The effect on partitioning could be described only taking into account solvent accessibility and type of residue substitution. The results demonstrate that the system can be used to detect conformational changes in mutant proteins since the expected effect on partitioning due to a mutation can be calculated. The aqueous two-phase system used here does indeed provide a rapid and convenient method to study protein surfaces and slight surface changes due to mutations.     

Treatment of high-concentration gaseous benzene streams using a novel bioreactor system

A continuous treatment system combining a packed-bed column and a two-phase partitioning bioreactor has been designed to treat high-concentration benzene-containing gas streams. 1-Octadecene was used in a closed loop as an absorbant to scrub benzene in the counter-current column, after which it was transferred to the two-phase partitioning bioreactor to partition benzene into the 1 l aqueous phase for degradation by Klebsiella sp. The solvent was then recirculated back to the absorber. A gas stream containing 20 mg l^–1 benzene at a flow rate of 60 l h^–1 was introduced to the system, and the benzene was degraded at a biological removal efficiency of 87% at steady state.     

Production of immobilized penicillin acylase using aqueous polymer systems for enzyme purification and in situ immobilization

A novel approach for the isolation and purification of penicillin acylase (PA), which couples aqueous two-phase partitioning and enzyme immobilization has been investigated.A PA yield of 90% was achieved by treating E. coli cells with 4% butyl acetate, freeze-thawing step, and pressure homogenization. PA purification (93% recovery) was achieved by (1) removing cell debris via precipitation with polyethylene glycol (PEG 2000); (2) aqueous two-phase partitioning using a PEG 2000 + phosphate system (87% recovery).An in situ enzyme immobilization approach, using oxirane acrylic or aldehyde-agarose beads dispersed in the PEG-rich phase, was explored for the conversion of penicillin G to 6-aminopenicillanic acid. An appropriate immobilization reaction time was found. The catalytic performance of the enzyme, when immobilized, was found not to be affected by recycling of the phase-forming components.     

Protein separation using metal ion-bound particles in aqueous two-phase system

Metal ion affinity partitioning of protein in aqueous two-phase systems was studied using Sepharose as ligand carrier as an integrated adsorption partitioning. Cu(II)-bound Sepharose was mixed with protein solution and an aqueous two-phase system. The affinity sorbent was distributed quantitatively to the upper side or the interface. The binding studies of lysozyme to copper-bound gel in PEG/dextran two-phase systems demonstrate the feasibility of this bioseparation process. PEG/dextran system did not affect binding and elution of lysozyme to and from the Cu(II)-Sepharose particles.     

Partitioning of α-lactalbumin and β-lactoglobulin in aqueous two-phase systems of polyvinylpyrrolidone and potassium phosphate

In the present study, the partitioning of α-lactalbumin, β-lactoglobulin, and cheese whey proteins in aqueous two-phase system of polyvinylpyrrolidone-potassium phosphate is investigated. The partitioning of proteins in this system depends on the polymer and salt weight percents in feed, temperature, and pH. The orthogonal central composite design is used to study the effects of different parameters on partitioning of α-lactalbumin and β-lactoglobulin. A second order model is proposed to determine the impact of these parameters. The results of the model show that the weight percent of the salt in feed has a large effect on the protein partitioning. The weight percent of polyvinylpyrrolidone in the feed increases the partitioning coefficients. By increasing the temperature, the viscosity of polyvinylpyrrolidone is reduced and the protein can easily be transferred from one phase to the other phase. The pH of the aqueous two phase system can alter the protein partitioning coefficient through the variation of the protein net charge.