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.     

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 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.     

Purification of hyperthermophilic archaeal amylolytic enzyme (MJA1) using thermoseparating aqueous two-phase systems

Purification of a recombinant, thermostable alpha-amylase (MJA1) from the hyperthermophile, Methanococcus jannaschii, was investigated in the ethylene oxide-propylene oxide random copolymer (PEO-PPO)/(NH(4))(2)SO(4), and poly(ethylene glycol) (PEG)/(NH(4))(2)SO(4) aqueous two-phase systems. MJA1 partitioned in the top polymer-rich phase, while the remainder of proteins partitioned in the bottom salt-rich phase. It was found that enzyme recovery of up to 90% with a purification factor of 3.31 was achieved using a single aqueous two-phase extraction step. In addition, the partition behavior of pure amyloglucosidase in polymer/salt aqueous two-phase systems was also evaluated. All of the studied enzymes partitioned unevenly in these polymer/salt systems. This work is the first reported application of thermoseparating polymer aqueous two-phase systems for the purification of extremophile enzymes.     

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.     

Preliminary application of light-pH sensitive recycling aqueous two-phase systems to purification of lipase

One key problem of aqueous two-phase systems (ATPS) is that phase-forming polymers could not be recycled efficiently. This results in high cost and environmental pollution. In this study, we introduced novel aqueous two-phase systems which are composed by pH-sensitive polymer P_ADB and light-sensitive polymer P_NNC. P_NNC is enriched in the top phase while P_ADB is found in the bottom phase. And recoveries of two-phase-forming polymers can both reach over 96%. This aqueous two-phase system was used for purification of lipase from its crude material. The influences of various process parameters such as concentration of the phase-forming polymer, system pH, different types and concentrations of neutral salts on partitioning of lipase are evaluated. It has been found that partition coefficient of pure lipase could reach 0.061 under optimized conditions. Lipase from crude material was purified with 83.7% recovery and a purification factor of approximately 18 folds.     

Pilot-scale separation of lysozyme from hen egg white by integrating aqueous two-phase partitioning and membrane separation processes

A novel, cost-effective method of lysozyme separation from hen egg white was studied. This method integrates aqueous two-phase partitioning in the system EO₅₀PO₅₀/phosphates with membrane separation processes. The experiments were carried out in a pilot-scale on crude hen egg white.Initially, by forming an aqueous two-phase system, lysozyme was selectively extracted to the upper, polymer-rich phase while the other egg white proteins partitioned to the lower, phosphate-rich phase. Then, in order to recover lysozyme, thermoseparation of polymer-rich phase was applied. A novel approach for the simultaneous thermoseparation of the polymer-rich phase as well as for the recovery of the lysozyme was proposed, using a cross-flow microfiltration. Additionally, recovery of proteins by ultrafiltration from lower, phosphate-rich phase was also investigated.Lysozyme could be obtained after the thermal phase separation by means of microfiltration at a total recovery over the extraction steps of 47.5 and the purification factor of 10.5. The specific activity of lysozyme preparations was 34 188 U/mg of protein. Using cross-flow membrane techniques, it was found that the recovery of the polymer by microfiltration from the top phase was 83.9.     

Partition of horseradish peroxidase in aqueous two-phase systems containing polyvinylpyrrolidone

Growing peroxidase utilisation in different industries encourages the search for high benefit/cost ratio purification methods such as aqueous two-phase partition. In this way, the partitioning behaviour of peroxidase from Armoracia rusticanaroots in polyvinylpirrolidone/Reppal and polyvinylpirrolidone/salt aqueous two-phase systems was investigated. Based on these results, a two-step purification process was developed. In the first system (polyvinylpyrrolidone K30/Reppal PES 200, pH 7.0), cell debris and some contaminating proteins were shifted to the bottom phase while peroxidase concentrated in the top phase. After discarding the bottom phase, the second step involved addition of magnesium sulphate thus forming a second aqueous two-phase system. At this step, the enzyme was extracted into the salt-rich bottom phase. The overall yield was 75% and the purification factor 7.3.This revised version was published online in October 2005 with corrections to the Cover Date.     

Mechanisms of phase behaviour and protein partitioning in detergent/polymer aqueous two-phase systems for purification of integral membrane proteins

Detergent/polymer aqueous two-phase systems are studied as a fast, mild and efficient general separation method for isolation of labile integral membrane proteins. Mechanisms for phase behaviour and protein partitioning of both membrane-bound and hydrophilic proteins have been examined in a large number of detergent/polymer aqueous two-phase systems. Non-ionic detergents such as the Triton series (polyoxyethylene alkyl phenols), alkyl polyoxyethylene ethers (C(m)EO(n)), Tween series (polyoxyethylene sorbitol esters) and alkylglucosides form aqueous two-phase systems in mixtures with hydrophilic polymers, such as PEG or dextran, at low and moderate temperatures. Phase diagrams for these mixtures are shown and phase behaviour is discussed from a thermodynamic model. Membrane proteins, such as bacteriorhodopsin and cholesterol oxidase, were partitioned strongly to the micelle phase, while hydrophilic proteins, BSA and lysozyme, were partitioned to the polymer phase. The partitioning of membrane protein is mainly determined by non-specific hydrophobic interactions between detergent and membrane protein. An increased partitioning of membrane proteins to the micelle phase was found with an increased detergent concentration difference between the phases, lower polymer molecular weight and increased micelle size. Partitioning of hydrophilic proteins is mainly related to excluded volume effects, i.e. increased phase component size made the hydrophilic proteins partition more to the opposite phase. Addition of ionic detergent to the system changed the partitioning of membrane proteins slightly, but had a strong effect on hydrophilic proteins, and can be used for enhanced separation between hydrophilic proteins and membrane protein.     

Recovery of extracellular human insulin-like growth factor-I and II as a fusion protein from Escherichia coli culture broth by aqueous two-phase extraction.

The primary purification of human insulin-like growth factor-I (IGF-I) and IGF-II, produced extracellularly in Escherichia coli as a fusion to two domains (ZZ) derived from staphylococcal protein A, has been studied. First, the partitioning of IgG-affinity purified ZZ-IGF-I and ZZ-IGF-II, respectively, to the top phase in poly(ethylene glycol)/potassium phosphate aqueous two-phase systems were investigated. Thereafter, the extraction of ZZ-IGF-I with a poly(ethylene glycol) 1500/potassium phosphate system was performed directly in the bioreactor after the cultivation. This resulted in a reduction of the cultivation volume more than 3-fold with a recovery of about 90% of target protein in a poly(ethylene glycol)-rich phase. The majority of the cells partitioned to the potassium phosphate-rich bottom phase, while a smaller fraction was collected at the interface, and/or as a densely packed cake on top of the interface. Contaminating proteins were also eliminated to some extent, which resulted in an almost 2-fold protein purification. Some obvious benefits offered by the aqueous two-phase system in the primary purification have been demonstrated: Firstly, the possibility to an early process volume reduction and thereby a concentration of the target protein. Secondly, a simultaneous protein purification was achieved. From this work it can be concluded that aqueous two-phase extraction should be considered as an attractive candidate for the primary steps during the design of new purification processes for extracellular proteins.     

Thermoseparating water/polymer system: a novel one-polymer aqueous two-phase system for protein purification

In this study we show that proteins can be partitioned and separated in a novel aqueous two-phase system composed of only one polymer in water solution. This system represents an attractive alternative to traditional two-phase systems which uses either two polymers (e.g., PEG/dextran) or one polymer in high-salt concentration (e.g., PEG/salt). The polymer in the new system is a linear random copolymer composed of ethylene oxide and propylene oxide groups which has been hydrophobically modified with myristyl groups (C(14)H(29)) at both ends (HM-EOPO). This polymer thermoseparates in water, with a cloud point at 14 degrees C. The HM-EOPO polymer forms an aqueous two-phase system with a top phase composed of almost 100% water and a bottom phase composed of 5-9% HM-EOPO in water when separated at 17-30 degrees C. The copolymer is self-associating and forms micellar-like structures with a CMC at 12 microM (0.01%). The partitioning behavior of three proteins (lysozyme, bovine serum albumin, and apolipoprotein A-1) in water/HM-EOPO two-phase systems has been studied, as well as the effect of various ions, pH, and temperature on protein partitioning. The amphiphilic protein apolipoprotein A-1 was strongly partitioned to the HM-EOPO-rich phase within a broad-temperature range. The partitioning of hydrophobic proteins can be directed with addition of salt. Below the isoelectric point (pI) BSA was partitioned to the HM-EOPO-rich phase and above the pI to the water phase when NaClO(4)was added to the system. Lysozyme was directed to the HM-EOPO phase with NaClO(4), and to the water phase with Na-phosphate. The possibility to direct protein partitioning between water and copolymer phases shows that this system can be used for protein separations. This was tested on purification of apolipoprotein A-1 from human plasma and Escherichia coli extract. Apolipoprotein A-1 could be recovered in the HM-EOPO-rich phase and the majority of contaminating proteins in the water phase. By adding a new water/buffer phase at higher pH and with 100 mM NaClO(4), and raising the temperature for separation, the apolipoprotein A-1 could be back-extracted from the HM-EOPO phase into the new water phase. This novel system has a strong potential for use in biotechnical extractions as it uses only one polymer and can be operated at moderate temperatures and salt concentrations and furthermore, the copolymer can be recovered.     

Isolation of alpha-1-antitrypsin from human plasma by partitioning in aqueous biphasic systems of polyethyleneglycol-phosphate

The partitioning of alpha-1-antitrypsin was assayed in biphasic aqueous systems containing potassium phosphate and two polyethyleneglycols of molecular mass 600 and 1000, respectively. In order to isolate the alpha-1-antitrypsin from serum plasma, the partitioning behaviour of human serum albumin, its principal contaminant, was also studied. Several aqueous two-phase systems with different partitioning properties were obtained by varying the PEG1000/PEG600 mass proportion. In systems with PEG1000/PEG600 mass ratio of 8, the optimal difference between the partition coefficients of both proteins was found. Under such conditions, a satisfactory purification was carried out by a three-step extraction procedure. By applying this method the alpha-1-antitrypsin specific activity increased severalfold (nearly 10 times) with a yield of 43%.     

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).     

A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I-hydrophobin I

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I(core)-hydrophobin I (EGI(core)-HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI(core)-HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI(core)-HFBI was quantitatively back-extracted (K(EGIcore-HFBI)=150, yield=99%) into a water phase. In this second step, ethylene oxide-propylene oxide (EOPO) copolymers were added to the micelle-rich phase and temperature-induced phase separation at 55 degrees C was performed. Total recovery of EGI(core)-HFBI after the two separation steps was 90% with a volume reduction of six times. For thermolabile proteins, the back-extraction temperature could be decreased to room temperature by using a hydrophobically modified EOPO copolymer, with slightly lower yield. The addition of thermoseparating co-polymer is a novel approach to remove detergent and effectively releases the fusion protein EGI(core)-HFBI into a water phase.     

Aqueous polymer two-phase systems: effective tools for plasma membrane proteomics

Plasma membranes (PMs) are of particular importance for all living cells. They form a selectively permeable barrier to the environment. Many essential tasks of PMs are carried out by their proteinaceous components, including molecular transport, cell-cell interactions, and signal transduction. Due to the key role of these proteins for cellular function, they take center-stage in basic and applied research. A major problem towards in-depth identification and characterization of PM proteins by modern proteomic approaches is their low abundance and immense heterogeneity in different cells. Highly selective and efficient purification protocols are hence essential to any PM proteome analysis. An effective tool for preparative isolation of PMs is partitioning in aqueous polymer two-phase systems. In two-phase systems, membranes are separated according to differences in surface properties rather than size and density. Despite their rare application to the fractionation of animal tissues and cells, they represent an attractive alternative to conventional fractionation protocols. Here, we review the principles of partitioning using aqueous polymer two-phase systems and compare aqueous polymer two-phase systems with other methods currently used for the isolation of PMs.     

The use of detergent-based aqueous two-phase systems for the isolation of extracellular proteins: purification of a lipase from Pseudomonas cepacia.

The partitioning of a variety of extracellular lipases, both pro- and eucaryotic, in detergent-based aqueous two-phase systems was examined. The results revealed that all procaryotic lipases showed a clear preference for the detergent-rich coacervate phase. In contrast, all eucaryotic lipases were significantly excluded from this phase, most probably caused by their glycosylation. The potential of such detergent-based systems for the isolation of extracellular lipases directly from cell-free culture broth was analyzed using the bacterium Pseudomonas cepacia (DSM 50181). This strain was identified after a limited screening for lipase activity. About 76% of the lipase could be extracted into the coacervate phase in just one purification step, leading to a four-fold concentration of lipase and a purification factor of 24.     

Affinity extraction of lactate dehydrogenase by aqueous two-phase systems using free triazine dyes

Affinity partitioning of lactate dehydrogenase (LDH) was studied in polyethylene glycol (PEG) /salt and PEG / hydroxypropyl starch (PES) aqueous two-phase systems, using free triazine dyes as their affinity ligands. The free dyes showed one-sided partition to the top PEG-rich phase and thus enhanced the affinity partitioning effect in the systems. A two-step affinity extraction process has been discussed for large scale purification of LDH from rabbit muscle.Hu Lin is one of the cooperator of the experiment.     

Large scale extraction of alpha-lactalbumin and beta-lactoglobulin from bovine whey by precipitation with polyethylene glycol and partitioning in aqueous two-phase systems.

The milk proteins alpha-lactalbumin and beta-lactoglobulin have been isolated from bovine whey by fractional precipitation with polyethylene glycol (PEG) and hydrophobic partitioning in aqueous PEG-hydroxypropylstarch two-phase systems using PEG-bound palmitate as hydrophobic ligand. The possible use of this combination for large scale purification of these whey proteins is discussed.     

Evaluation of different primary recovery methods for E. coli-derived recombinant human growth hormone and compatibility with further down-stream purification

Due to advances in fermentation technology, it is now possible to obtain fermentation broth with over 30% solids. The high solid content makes the clarification step difficult, especially at large scale. The primary protein recovery step is challenging due to the heterogeneous solution of soluble and insoluble material. In this study, we compare different primary recovery routes and the compatibility with the initial capture chromatography step. The primary recovery routes studied are standard clarification by centrifugation and extraction in aqueous two-phase systems. The compatibility of the feed streams from the different primary recovery steps with the first chromatography step is addressed. An anion-exchange column was used as the first capture column in the purification process. The aqueous two-phase system was composed of a random copolymer of ethylene oxide and propylene oxide (EOPO) in combination with a waxy starch. The target protein in this study was human growth hormone (hGH) produced in recombinant Escherichia coli. The purity of hGH in the top phase after aqueous two-phase extraction was found to be significantly higher than in clarified homogenate supernatant and increased as the EOPO polymer concentration in the aqueous two-phase system increased. Stability of the supernatant and EOPO top phases and hGH were determined by turbidity measurements and LC-MS assay. All of the feed-streams from the primary recovery steps were compatible with the anion-exchange chromatography step; however, the capacity of the resin was strongly dependent on the purity of the load. Different process aspects, e.g., resin capacity, viscosity, purification, and yield of hGH and scalability are compared.     

Optimization of bovine serum albumin sorption and recovery by hydrogels

Aqueous two-phase systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextran (Dx), or a polymer and a salt, usually PEG and phosphate or sulfate. Partitioning of proteins in such systems provides a powerful method for separating and purifying mixtures of biomolecules by extraction. If one of the phase forming polymers is a crosslinked gel, then the solution-controlled gel sorption may be considered as a modification of aqueous two-phase extraction. Since PEG/dextran systems are widely used in aqueous two-phase extraction and dextran gels (Sephadex) are common chromatographic media, we choose a PEG/dextran gel system as a model system in this study. The partitioning behavior of pure bovine serum albumin (BSA) in PEG/dextran gel systems is investigated to see the effects of variations in PEG and NaCl concentrations on the partition coefficient K. By making use of the Box-Wilson experimental design, K is shown to be maximized at 9.8 (%, w/w) PEG and 0.2 M NaCl concentrations, respectively, as 182.