Physico-chemical features of solvent media in the phases of aqueous polymer two-phase systems

Solvent polarity and pH in the coexisting aqueous phases of aqueous dextran-poly(ethylene glycol) and dextran-Ficoll two-phase systems of varied polymer concentrations were examined using the solvatochromic technique and potentiometric measurements, respectively. The relative solvent polarity of the phases, as measured by the solvatochromic technique, is suggested as a measure of the hydration power of water in the phases of aqueous polymer systems. Partitioning of a series of sulphonephthalein dyes in aqueous dextran-poly(ethylene glycol) and dextran-Ficoll two-phase systems of fixed polymer composition containing 0.01 mol/L universal buffer, pH 7.15, was studied. The results obtained are discussed together with those reported earlier on the physico-chemical features of aqueous media in the coexisting phases of the systems. It is suggested that the two phases of aqueous polymer systems should be viewed as two immiscible water-like solvents. The implications of the suggestion for the theoretical treatment of aqueous polymer two-phase systems are discussed.     

Aqueous two-phase systems: a novel approach for the separation of proteose peptones

Poly(ethylene glycol) and dextran aqueous two-phase systems (ATPS) were developed to facilitate the separation of components of the proteose peptone fraction of bovine milk, which are mostly large casein derived peptides or glycoproteins. These have proved difficult to purify using conventional chromatographic procedures. ATPS exploit differences in hydrophobicity, size and ionic properties of the proteose peptones with a view to developing methods for future large scale preparations of the individual components of this whey protein fraction.     

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.     

Casein hydrolysis in PEG-salt two phase system by Eudragit-chymotrypsin bioconjugate

A bioconjugate of -chymotrypsin and Eudragit S-100 was used in an aqueous two-phase system (polyethylene glycol/phosphate) for casein hydrolysis. More product was obtained by replacing the lower salt phase with a fresh one during the reaction. The bioconjugate could be reused six times for casein hydrolysis.     

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     

Bioconversions in aqueous two-phase systems.

Bioconversions involving enzymes and/or microbial cells in aqueous two-phase systems are reviewed. The partitioning of biocatalysts, substrates, and products is discussed in relation to their size. The efficiency of retaining biocatalysts in aqueous two-phase systems is summarized in relation to other methods of recirculating. The influence of phase components on the activity and the stability of enzymatic biocatalysts is exemplified with penicillin acylase and the cellulolytic enzyme system, and the effect of phase components on biocatalytic living cells is exemplified with the production of alpha-amylase with Bacillus sp. Process design costs in bioconversions in aqueous two-phase systems are briefly summarized.     

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.     

Affinity-specific protein separations using ligand-coupled particles in aqueous two-phase systems: II. Recovery and purification of pyruvate kinase and alcohol dehydrogenase from Saccharomyces cerevisiae

The novel approach of using aqueous two-phase systems for the elution of protein from ligand-coupled particles is investigated using pyruvate kinase and alcohol dehydrogenase from recombinant Saccharomyces cerevisiae and Cibacron blue F3G-A-coupled Sepharose CL6B (Blue-Sepharose) particles as a model system. The ligand-coupled particles distribute quantitatively to the polyethylene glycol-(PEG-) rich top phase and the recovered enzymes partition selectively to the dextran-(DEX-) rich bottom phase. An effective recovery and partial purification of pyruvate kinase and alcohol dehydrogenase from Blue-Sepharose particles using PEG8000-DEXT500 aqueous two-phase systems are demonstrated through a modest increase of salt concentration. The bioselective eluting agent, MgADP, which is useful in chromatographic operations, is not required for the process using aqueous two-phase systems. Recovery of pyruvate kinase, which is bound to ligand-coupled particles, in the DEX-rich bottom phase of aqueous two-phase systems can be up to 95% in one-step operations. The mixing time of ligand-coupled particles with aqueous two-phase systems is a major controlling variable. The salt concentration, the molecular weight of polymer, and the total volume of aqueous two-phase systems also influence the recovery of pyruvate kinase from ligand-coupled particles. The recovered enzymes in the DEX-rich bottom phase remain biologically stable over a long period of storage time. The concentration of product protein in a reduced volume and the easy separation from ligand-coupled particles are added advantages of the process using aqueous two-phase systems. Preliminary studies with goat polyclonal anti-pyruvate kinase-coupled Sepharose particles indicate that the process also may be applicable when a high-affinity ligand such as antibody is used. The experimental results and a theoretical derivation based on equilibrium models for binding/dissociation of ligands and proteins show that the process results in better recovery as compared to that of conventional bulk elution techniques.     

Growth and Secondary Metabolite Production by Hairy Roots of Tagetes Patula in Aqueous Two-Phase Systems

Hairy roots of Tagetes patula have been grown in aqueous two-phase systems. After selecting suitable polymers from single-phase experiments (in which salt phases were unable to support growth in the desired concentrations) several two-phase systems were tested for their influence on cell growth and thiophene production. Cell growth occurred in all aqueous two-phase systems, but the highest growth rate was achieved in normal medium. There was no difference in thiophene production between medium and aqueous two-phase systems. The partition of thiophenes favoured slightly the more hydrophobic top phase in most cases, while the cells were confined to the bottom phase. One aqueous two-phase system (15% polyethyleneglycol 10,000 and 15% Reppal PES 200) was tested in a stirred tank reactor with normal medium as a control. The growth rate in medium was higher than in the aqueous two-phase system, while the thiophene production per unit cell weight was in the same range for both systems. The excretion of thiophenes in the reactor with the aqueous two-phase system was about ten times as high as in the control reactor. The amount excreted was however still not more than 3% of the total production.     

Growth and Secondary Metabolite Production by Hairy Roots of Tagetes Patula in Aqueous Two-Phase Systems

Hairy roots of Tagetes patula have been grown in aqueous two-phase systems. After selecting suitable polymers from single-phase experiments (in which salt phases were unable to support growth in the desired concentrations) several two-phase systems were tested for their influence on cell growth and thiophene production. Cell growth occurred in all aqueous two-phase systems, but the highest growth rate was achieved in normal medium. There was no difference in thiophene production between medium and aqueous two-phase systems. The partition of thiophenes favoured slightly the more hydrophobic top phase in most cases, while the cells were confined to the bottom phase. One aqueous two-phase system (15% polyethyleneglycol 10,000 and 15% Reppal PES 200) was tested in a stirred tank reactor with normal medium as a control. The growth rate in medium was higher than in the aqueous two-phase system, while the thiophene production per unit cell weight was in the same range for both systems. The excretion of thiophenes in the reactor with the aqueous two-phase system was about ten times as high as in the control reactor. The amount excreted was however still not more than 3% of the total production.     

Partitioning of low molecular combination peptides in aqueous two-phase systems of poly(ethylene glycol) and dextran in the presence of small amounts of K2HPO4/KH2PO4 buffer at 293 K: experimental results and predictions

Buffered aqueous two-phase systems are effective extraction systems for separating amphoteric hydrocarbons like, for example, polypeptides from aqueous phases. The design and basic engineering of such processes requires the knowledge of the liquid-liquid equilibrium. The study presented here aims to contribute to the development of methods to predict the partitioning of peptides in aqueous two-phase systems. Experimental results are reported for the partitioning of small amounts ( approximately 0.001 g solute per gram of solution) of low molecular combination peptides of glycine, L-glutamic acid, L-phenylalanine, and L-lysine (9 dipeptides, gly-glu, gly-phe, gly-lys, glu-gly, phe-gly, phe-glu, lys-gly, lys-glu, lys-phe; 7 tripeptides, gly-gly-phe, gly-phe-gly, glu-gly-phe, phe-gly-gly, lys-gly-lys, lys-glu-gly, lys-phe-lys) in aqueous two-phase systems of high molecular weight dextran (molecular weight about 500,000) and poly(ethylene glycol) (molecular weight about 6,000 and 35,000, respectively) in the presence of small amounts (about 0.05 mol/kg) of K2HPO4/KH2PO4 buffer at about 293 K. The new data are compared to predictions. Partition coefficients are predicted applying a group contribution excess Gibbs energy model. The model is an osmotic virial equation. It uses surface fractions to encounter for the probability of interactions between solutes. All model parameters were taken from the literature. They were determined exclusively from experimental data for the phase forming systems and for the partitioning of amino acids and their di- and tripeptides (containing only a single amino acid), but no experimental data for the partitioning of combinations peptides were used. In most cases predicted partition coefficients agree favourably with the experimental data.     

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.     

固定化青霉素酰化酶在光-pH敏感可回用两水相中裂解青霉素G为6-APA

两水相体系在发展中存在的关键问题是相体系回收困难.由于生产成本及降低污染的原因, 用过的相体系需要回收和重复使用.用环境敏感型溶解可逆聚合物形成可回用两水相体系是当前是为可行的回收方法。本文在光敏感可回用高聚物PNBC与pH敏感型可回用高聚物PADB形成的两水相体系中进行固定化青霉素酰化酶的相转移催化青霉素G产生6-APA的反应。在这个两水相体系中,通过优化,在1% NaCl 存在下,６－ＡＰＡ的分配系数可达5.78。催化动力学显示,达平衡的时间近7h,反应最高得率约85.3%（pH 7.8, 20℃）。较相近条件下的单水相反应得率提高近20%。在反应过程中,通过底物及产物的分配系数检测,发现底物分配系数变化不大,而产物6-APA及苯乙酸的分配系数发生很大变化,从而引起产物的得率变化。在两水相中,底物及产物主要分配在上相,固定化酶分配在下相,底物青霉素G进入下相经酶催化产生的6-APA及苯乙酸又转入上相,从而解除了青霉素酰化酶催化反应的底物及产物抑制作用,达到提高产物得率的效果。此外,采用固定化酶较固定化细胞效率高,占用下相体积小,较游离酶稳定性高,且完全单侧分配在下相。因此,在两水相中进行固定化酶的催化反应具有明显的优越性。形成两水相的高聚物PNBC通过488 nm 的激光照射或经滤光的450nm 光源照射得到回收;pH敏感型成相聚合物PADB可通等电点 4.1沉淀可实现循环利用,高聚物的回收率在95%-98%之间,按此回收率计算,聚合物可使用60次以上。     

Liquid–liquid extraction of an extracellular alkaline protease from fermentation broth using aqueous two-phase and reversed micelles systems

Summary The study of recovery of an extracellular alkaline protease from fermentation broth produced by Norcadiopsis sp, was carried out with liquid–liquid extraction through sodium di-(2-ethylhexyl) sulphosuccinate/isooctane reversed micelles systems and aqueous two-phase systems (polyethylene glycol/potassium phosphate). The best conditions for extraction and back-extraction with the reversed micelles system was obtained at pH 9.0 and pH 5.0, respectively, showing a yield of protein of 6.16%, a specific activity of 4.10 U/ml and a purification factor of 1.80. The studies using aqueous two-phase systems of polyethylene glycol/potassium phosphate at pH 10.0 showed purification factors of 2 and 5, and protein yield of 11 and 4%, respectively, for polyethylene glycol 550/potassium phosphate and polyethylene glycol 8000/potassium phosphate. The results indicate that the aqueous two-phase systems are more attractive as a first step in the isolation and purification processes.     

An enzymatic method for radiolabeling vertebrate vitellogenin

Partition in aqueous two-phase systems should have great potentialities for studies of biological material. However, the general use of the method has probably been hindered by the difficulties in finding the composition of two-phase systems with desired qualities. Experimental design has proved to be of immense value for optimization in many different areas and should also be useful in selecting the best possible two-phase system. Therefore the simplex method has been applied to the search for the composition of an aqueous two-phase system in which the partition behavior of filamentous actin and human spectrin differ as much as possible.     

Changes in plasma membrane and microfilaments accompanying morphologic differentiation in Chinese hamster ovary (CHO) cells.

Studies on the application of the techniques of counter-current distribution (CCD) in aqueous two-phase systems and multiple sedimentation for the fractionation of metaphase chromosomes are presented. The two-phase systems were composed of aqueous solutions of Dextran 500 and poly(ethylene)glycol 6000 (PEG). It has been found that different groups of chromosomes differ in their distribution between the two phases and that the introduction of PEG with covalently attached positively or negatively charged groups provides a means of steering the distribution of chromosomes. A rough fractionation of chromosomes on the basis of size is possible by the technique of multiple sedimentation and this, in combination with CCD, yields 10 fractions of chromosomes. Partition and CCD in aqueous two-phase system separate chromosomes according to their surface properties and may prove useful for isolation of individual chromosomes in bulk.     

Physical properties of detergent-based aqueous two-phase systems

The physical behavior of the binary phase systems of the non-ionic polyoxyethylene detergent Agrimul NRE 1205 and water was investigated. This technical detergent can be used for the large-scale recovery of biomolecules in detergent based aqueous two-phase systems. The phase diagram was determined. It shows significant and unexpected differences to highly purified detergents. Very similar to neat detergents the phase diagram can be influenced by auxiliary chemicals thus shifting the entire phase diagram in general to lower temperatures. This was demonstrated by lowering the cloud-point by various additions. The concentration factor, as an important parameter of a first capture step in purification was investigated and modeled. Auxiliary chemicals, temperature change and change in detergent concentration also influence the viscosity and density of the phases. These experimental data are shown. They can help to explain the separation behavior of proteins. In large-scale separations aqueous two-phase systems are separated using disc-stack centrifuges. It is demonstrated that this is not a feasible method for detergent-based aqueous two-phase extraction and the physical reason is presented.     

Physical properties of detergent-based aqueous two–phase systems

The physical behavior of the binary phase systems of the non-ionic polyoxyethylene detergent Agrimul NRE 1205 and water was investigated. This technical detergent can be used for the large-scale recovery of biomolecules in detergent based aqueous two-phase systems. The phase diagram was determined. It shows significant and unexpected differences to highly purified detergents. Very similar to neat detergents the phase diagram can be influenced by auxiliary chemicals thus shifting the entire phase diagram in general to lower temperatures. This was demonstrated by lowering the cloud-point by various additions. The concentration factor, as an important parameter of a first capture step in purification was investigated and modeled. Auxiliary chemicals, temperature change and change in detergent concentration also influence the viscosity and density of the phases. These experimental data are shown. They can help to explain the separation behavior of proteins. In large-scale separations aqueous two-phase systems are separated using disc-stack centrifuges. It is demonstrated that this is not a feasible method for detergent-based aqueous two-phase extraction and the physical reason is presented. This revised version was published online in July 2006 with corrections to the Cover Date.     

高速逆流色谱技术在生物大分子分离纯化中的应用

高速逆流色谱是一种连续液-液色谱技术,具有无固相载体、样品无需严格预处理等优点。近10年来,在设备结构和溶剂体系等方面进行了大量的研究开发,已推广应用于生物技术、医药、天然产物、环境监测、食品等领域。为适应生物大分子和活性细胞的分离,采用条件温和的双水相体系,研究开发相应的高速逆流色谱设备已成为热点。针对双水相体系的特点,已经开发出了多种具有较高固定相保留率的新型高速逆流色谱设备,通过优化实验条件,成功地进行了多种蛋白质的分离纯化。本对该领域的最新进展进行了综述与评价。