Acoustic demixing of aqueous two-phase systems

Aqueous two-phase systems demix slowly due to similar physical properties. This is one of the major drawbacks for their adaptation for industrial scale extraction of enzymes. In the present work, a method to accelerate the demixing rates of these systems, employing a traveling acoustic wave field is reported for the first time. Phase-demixing for three systems, viz. polyethylene glycol (PEG)/sodium sulfate, PEG/potassium phosphate and PEG/maltodextrin were studied. The acoustically assisted process decreased the demixing time significantly (about 2- to 3-fold in PEG/salt systems and about 2-fold in the PEG/maltodextrin system), compared to that in gravity alone. Ultrasonication apparently enhanced the coalescence of the dispersed phase droplets due to the mild circulation currents it caused in the dispersion. This in turn enhanced the rate of demixing due to the increased migration velocity of the larger droplets. Received: 3 November 1999 / Received revision: 10 January 2000 / Accepted: 14 January 2000     

Acoustic field assisted demixing of aqueous two-phase polymer systems

Acoustic field assisted demixing was employed to decrease the demixing time in polymer–polymer (polyethylene glycol–maltodextrin) two-phase system. Application of acoustic field has decreased the demixing time in these systems up to 2-fold. Ultrasonication has induced mild circulation currents in the phase dispersion, which has enhanced the rate of droplet coalescence, eventually resulting in decreased demixing time. In polymer-polymer systems, phase demixing was found to depend greatly on which of the phases is continuous and viscosity of the continuous phase was observed to have a strong influence on the movement of the droplets and hence the phase demixing. Addition of NaCl increased the demixing time and presence of E.coli cells did not seem to have any influence on phase demixing. This revised version was published online in July 2006 with corrections to the Cover Date.     

Acoustic field assisted enhanced demixing of aqueous two-phase systems

Aqueous two-phase extraction has been recognized as a versatile downstream processing technique for the recovery of biomolecules. A major deterrent to its industrial exploitation is the slow demixing of the two aqueous phases after extraction, due to their similar physical properties. A method to decrease the demixing times of these systems, employing a travelling acoustic wave field, is reported. The effects of phase composition and microbial cells on demixing in a polyethylene glycol/potassium phosphate two-phase system are studied in detail. As phase composition increased, demixing time decreased gradually. Phase volume ratio was found to have a significant effect on demixing time at low phase compositions. However, at intermediate and high phase compositions, only a small effect on demixing time was observed. The effect of phase composition and volume ratio on demixing behavior was explained based on the droplet size of the dispersed phase, which is the resultant effect of the physical properties of the phases. At all the phase compositions studied, the acoustically assisted process decreased the demixing time by 17-60% when compared to demixing under gravity alone. Increasing the cell concentration increased the demixing time markedly in case of yeast cells. However, it remained practically constant in the case of Lactobacillus casei cells. Application of an acoustic field reduced the demixing times up to 60% and 40% in the case of yeast and L. casei cells, respectively. Visual observations indicated that ultrasonication caused mild circulation currents in the phase dispersion enhancing droplet-droplet interaction, which in turn enhanced the rate of coalescence, eventually resulting in an enhanced demixing rate.     

Enzyme purification by immobilized metal ion affinity partitioning--application to D-hydroxyisocaproate dehydrogenase.

Extraction and purification of D-2-hydroxyisocaproate dehydrogenase from Lactobacillus casei has been studied by means of immobilized metal ion affinity partitioning (IMAP) in aqueous two-phase systems. The partition of the enzyme can be influenced strongly by inclusion of iminodiacetic acid as chelating ligand coupled to polyethylene glycol and loaded with Cu2+ ions into the phase system. This applies to polyethylene glycol/dextran as well as polyethylene glycol/salt phase systems. An increase in enzyme partition coefficient of up to about 1000-fold was observed. Based on the mathematic model presented recently by Suh and Arnold (1990) approximately 6.4 histidine residues were calculated to be involved in the enzyme-metal chelate complex. Direct extraction of the enzyme from both cell homogenate and cell debris supernatant proved unsatisfactory due to disturbances caused by the presence of cell debris and low molecular weight cell components. A combination with a preceding prepurification by a fractional precipitation with polyethylene glycol resulted in a strong affinity effect accompanied by an efficient purification during IMAP (purification factor of 11 with a yield of approximately 90%). Based on this step, an efficient downstream process can be designed for D-hydroxyisocaproate dehydrogenase.     

Porcine pancreatic lipase partition in potassium phosphate-polyethylene glycol aqueous two-phase systems

This research study examined porcine pancreatic lipase partition in aqueous two-phase systems formed by polyethylene glycol-potassium phosphate at pH 6.0, 7.0 and 8.0, the effect of polymer molecular mass, and NaCl concentration. The enzyme was preferentially partitioned into the polyethylene glycol rich phase in systems with molecular mass 4000-8000, while with polyethylene glycol of 10,000 molecular mass it was concentrated in the phosphate rich phase. The enthalpic and entropic changes found due to the protein partition were negative for all the polyethylene glycol molecular mass systems assessed. Both thermodynamic functions were shown to be associated by an entropic-enthalpic compensation effect suggesting that the water structure ordered in the ethylene chain of polyethylene glycol plays a role in the protein partition. The addition of NaCl increased the lipase affinity to the top phase and this effect was most significant in the system polyethylene glycol 2000-NaCl 3%. This system yielded an enzyme recovery more than 90% with a purification factor of approximately 3.4.     

Vancomycin partitioning in aqueous two-phase systems: effects of pH, salts, and an affinity ligand

The partitioning of vancomycin in polyethylene glycol (PEG)-dextran and PEG-phosphate aqueous two-phase systems was studied at different pHs, at varying concentrations of neutral salts, and with an affinity ligand attached to methoxy polyethylene glycol (MPEG). Vancomycin is found to partition preferentially into the PEG-rich top phase, and its partition coefficient increases nearly exponentially with the addition of water structure-making salts, such as sodium sulfate and sodium chloride, but is independent of sodium phosphate concentration. In the PEG-dextran system the vancomycin partition coefficient increases 3-fold in acidic and neutral solutions, while in the PEG-phosphate system it increases about 30-fold on the addition of the same amount of sodium chloride (1. 5 mol/kg). In basic solution, above its isoelectric point, the vancomycin partition coefficient increases slightly with NaCI concentration in the PEG-dextran system. We also examined the use of the dipeptide D-ala-D-ala as an affinity ligand on MPEG to extract vancomycin into the PEG-rich phase. The vancomycin partition coefficient increased almost 7-fold upon adding the MPEG-ligand in an amount equal to approximately 3% of the total PEG in the system. Finally, fractionation of the polydisperse phase-forming polymers in the two-phase PEG-dextran system was observed. The effect of this polymer fractionation on the partition coefficient of vancomycin is discussed.     

Magnetic aqueous two-phase separation: a new technique to increase rate of phase-separation, using dextran-ferrofluid or larger iron oxide particles

A new technique to speed up the phase separation of aqueous two-phase systems is described. The technique is based on the addition of magnetically susceptible additives (ferrofluids or iron oxide particles). In a magnetic field such additives will induce a faster phase separation. In one approach, dextran-stabilized ferrofluid was added to an aqueous two-phase system containing polyethylene glycol and dextran. The ferrofluid was totally partitioned to the dextran phase. After mixing of the two-phase system, it was possible to reduce the separation time by a factor of 35 by applying a magnetic field to the system. Another approach involved the use of 1-micron iron oxide particles instead of ferrofluid. In this case also, the phase-separation time was reduced, by a factor of about 70, when the system was placed in a magnetic field. The addition of ferrofluid and/or iron oxide particles was shown to have no influence on enzyme partitioning or on enzyme activity. The partitioning of chloroplasts, on the other hand, was influenced unless the ferrofluid used had been treated with epoxysilane. A column system comprising 15 magnetic separation stages was constructed and was used for semicontinuous separation of enzyme mixtures.     

Separation of Listeria monocytogenes and Salmonella berta from a complex food matrix by aqueous polymer two-phase partitioning

In the present study, the use of aqueous polymer two-phase systems for separation of pathogenic bacteria from a complex food sample was investigated. Three different two-phase systems, a polyethylene glycol 3350/dextran T 500, a methoxy polyethylene glycol 5000/dextran T 500 and a polyethylene glycol 3350/hydroxypropyl starch system, were compared at pH 3 and pH 6 for their capacity to separate the pathogenic bacteria Listeria monocytogenes and Salmonella berta from a Cumberland sausage. In all three phase systems, the food particles partitioned to the lower phase. Best performance was obtained by the polymer combinations, polyethylene glycol 3350/dextran T 500 and polyethylene glycol 3350/hydroxypropyl starch. In these systems, Salmonella berta partitioned to the hydrophobic upper phase both at pH 3 and pH 6 with an average partitioning ratio of 80% and a recovery of 56%. Listeria monocytogenes partitioned to the upper phase at pH 3 only with an average partitioning ratio of 72% and a recovery of 45%. This method may become a valuable tool for separation of bacteria from complex food matrices.     

Phase diagrams for dextran-PEG aqueous two-phase systems at 22°C

Summary We have determined phase diagrams at 22°C for the aqueous two-phase systems composed of dextran, polyethylene glycol, and water. The effects of polyethylene glycol and dextran molecular weight on phase separation are reported. These phase diagrams provide more complete data for dextran/PEG/water system, and will be needed for the correlation of biomolecule partitioning.     

Simultaneous biosynthesis and purification of two extracellular Bacillus hydrolases in aqueous two-phase systems

Thermostable alpha-amylase with temperature optimum at 80 degrees C, molecular mass 58 kDa and pI point 6.9 was purified from a catabolite resistant Bacillus licheniformis strain. The enzyme was sensitive to inhibition by metal ions and N-bromosuccinimide. The partition behaviour of this enzyme in aqueous two-phase systems (ATPS) of the polymer-polymer-water type was investigated and some effects of type, molecular weight and concentration of phase components were studied. Up to 100% retention in the bottom phase of polyethylene glycol 10,000-20,000/dextran 200 system was reached. Best partition conditions were obtained in PEG 10,000-20,000/polyvinyl alcohol 200 systems, where the partition coefficient K increased 750 times to 7.5. Simultaneous production and purification of alpha-amylase and serine proteinase in PEG-polymer-water ATPS were examined. In the system PEG 6,000/ficoll, up to 90% of the amylase was retained in the bottom phase, whereas about 95% of the total protein (K = 22.8) and 60-75% of the proteinase were in the top phase. Similar separation of the enzymes from laboratory supernatant was obtained in system PEG/Na2SO4.     

Optimization of bovine serum albumin partition coefficient in aqueous two-phase systems

Partitioning of proteins in aqueous two-phase systems has been shown to provide a powerful method for separating and purifying mixtures of biomolecules by extraction. These 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. There are many factors which influence the partition coefficient K, the ratio of biomolecule concentration in the top phase to that in the bottom phase, in aqueous two-phase systems. The value of the partition coefficient relies on the physico-chemical properties of the target biomolecule and other molecules and their interactions with those of the chosen system. In this work, the partition behavior of pure bovine serum albumin in aqueous two-phase systems was investigated in order to see the effects of changes in phase properties on the partition coefficient K. The concentration of NaCl and pH were considered to be the factors having influence on K. Optimal conditions of these factors were obtained using the Box-Wilson experimental design. The optimum value of K was found as 0.0126 when NaCl concentration and pH were 0.14 M and 9.8, respectively, for a phase system composed of 8% (w/w) polyethylene glycol 3,350 - 9 (% w/w) dextran 37,500 - 0.05 M phosphate at 20 °C.     

Preparation of benzoyl dextran and its use in aqueous two-phase systems.

The graft modification of dextran with benzoyl groups has been studied. The factors that affect the degree of substitution of benzoyl dextran were investigated. Phase diagrams for aqueous two-phase systems composed of polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran have been determined. Phase separation was also obtained in aqueous solution of two benzoyl dextran polymers with different degrees of substitution. A four-phase system was obtained with a mixture of polyethylene glycol, dextran and two kinds of benzoyl dextrans. The partitioning of methylene blue and a Procion yellow HE-3G dextran derivative were studied in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems and in systems of two benzoyl dextrans differing in degree of substitution. The proteins bovine serum albumin and glucose-6-phosphate dehydrogenase were partitioned in polyethylene glycol/benzoyl dextran aqueous two-phase systems and the effect of the degree of substitution of benzoyl dextran was studied. Chlorella pyrenoidosa, thylakoid membrane vesicles, plasma membrane vesicles and chloroplasts were partitioned in polyethylene glycol/benzoyl dextran and dextran/benzoyl dextran two-phase systems, and in a polyethylene glycol/dextran/benzoyl dextran four-phase system.     

On the use of phase partition to isolate vacuoles from cultured cells

Vacuoles isolated from cultured rose-cell protoplasts by controlled lysis were subjected to partition in polyethylene glycol-dextran two-phase systems. The vacuoles showed a strong affinity for the polyethylene glycol phase, like plasma membrane and unlike other membraneous organelles. After phase partition at 4°C, dilution of the polyethylene glycol destroyed a large proportion of the vacuoles. After phase partition at room temperature (ca. 20°C), the vacuoles in the polyethylene glycol phase were relatively stable to dilution. Two-phase partition may be useful for purification of intact vacuoles and possibly of tonoplast membranes. Minor proportions of acid phosphatase and -mannosidase were associated with these vacuoles.     

Fractionation of proteins from human serum by counter-current distribution

Proteins of human serum have been fractionated by counter-current distribution using aqueous two-phase systems. These were composed of either polyethylene glycol and dextran or polyethylene glycol and the new water soluble starch polymer Aquaphase PPT. The distribution of serum proteins in the polyethylene glycol-Aquaphase PPT system resembles that in the polyethylene glycol-dextran system.The partition of a number of proteins could be changed by introducing polymer-bound reactive dyes into one of the phases. Due to affinity for the dyes several proteins were transferred into the phase containing the polymer-bound ligand leading to an improved separation of individual proteins.Furthermore, the effect of two different dyes, immobilised in the opposite phases, on counter-current distribution of serum proteins was demonstrated. The applicability of this method for fractionation of serum proteins is discussed.     

Activation of Cytosolic Pyruvate Kinase by Polyethylene Glycol

Homogeneous cytosolic pyruvate kinase from endosperm of germinating castor oil (Ricinus communis L. cv Hale) seeds was potently activated by polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the pyruvate kinase reaction mixture caused a 2.6-fold increase in maximal velocity and 12.5- and 2-fold reductions in Km values for phosphoenolpyruvate and ADP, respectively. Glycerol, ethylene glycol, and bovine serum albumin also enhanced pyruvate kinase activity, albeit to a lesser extent than polyethylene glycol. The addition of 5% (w/v) polyethylene glycol to the elution buffer during high-performance gel filtration chromatography of purified cytosolic pyruvate kinase helped to stabilize the active heterotetrameric native structure of the enzyme. A higher degree of inhibition by MgATP, but lower sensitivity to the inhibitors 3-phosphoglycerate and fructose- 1,6-bisphosphate, was also observed in the presence of 5% (w/v) polyethylene glycol. It is concluded that (a) plant cytosolic pyruvate kinase activity and regulation, like that of other regulatory pyruvate kinases, is modified by extreme dilution in the assay medium, probably as a result of deaggregation of the native tetrameric enzyme, and (b) ATP is probably the major metabolic effector of germinating castor endosperm cytosolic pyruvate kinase in vivo.     

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.     

双水相电泳分离蛋白质的研究

近几年来,随着生物技术的迅速发展,制备型电泳技术的研究得到了重视。然而由于技术上的原因,大规模的制备型电泳技术的研究还未能取得突破。阻碍电泳放大的一个主要问题是由于电加热作用而导致的热对流对电泳分离的破坏。为解决这一问题,人们提出了许多方法。例如,在太空的微重力环境下进行电泳,应力稳定自由流动电泳,循环等电聚焦和区带电泳,色谱电泳和等电膜等电聚焦等。这些方法在电泳放大上都取得了一定的进展,但各有其局限性。最近,Clark提出利用双水相的液液界面阻止热对流的设想,为开发大规模的制备型电泳技术开辟了一条新途径、Raghava Rao等在两种双水相体系上施加电场后成倍地缩短了分相时间。Levine和Bier采用U型管电泳装置研究了双水相体系中血红蛋白的电泳迁移率,观测到界面有阻滞作用。Clark在柱型电泳装置中进行了一组双水相萃取肌红蛋白的简单实验。在10mA的恒电流下电泳40min之后,肌红蛋白的分配系数为7.5,而当电场反向后,分配系数变为0.04,界面阻力并不显著,两者结论并不一致。     

Choice of an aqueous polymer two-phase system for cell partition

Partition of human erythrocytes in aqueous two-phase polymer systems produced by Ficoll and different molecular weight fractions of dextran and polyethylene glycol and the influence of the ionic composition on the cells' partition in the systems was studied. It is found that the Ficoll-dextran-40 system is characterized by a number of advantages as compared with the common dextran-polyethylene glycol system or the others systems under study. The main advantage of the system appears to be that it is possible to concentrate the red cells in the top phase or in the bottom phase of the system, depending on the system ionic composition. The influence of the nature and the concentration of salt additives on this two-phase system formation is examined.     

Column chromatographic separation of cells using aqueous polymeric two-phase systems

Cell separation using aqueous polymeric two-phase systems is well established. For separations of cells having similar partition coefficients a multistep countercurrent distribution procedure has to be used. However, its operation is limited by time and apparatus constraints. As an alternative strategy we have developed a chromatographic technique in which the dextran-rich phase of a dextran/polyethylene glycol (PEG) phase system is immobilized onto derivatized agarose beads. The PEG-rich phase is used as the eluent. Inclusion of PEG-fatty acid affinity ligand gradients into the eluent produces separations of mammalian erythrocytes based on the differential interaction between the fatty acid and the erythrocyte membranes. A model separation of dog and human erythrocytes has been carried out.     

Analysis of phase composition in aqueous two-phase systems using a two-column chromatographic method: Application to lactic acid production by extractive fermentation

A new chromatographic system for the simultaneous analysis of polyethylene glycol, dextran, sugars, and low-molecular-weight fatty acids was developed. The system is based on a gel exclusion column which allows a first separation between high- and low-molecular-weight compounds, and a cationic exchange column used to further separate the low-molecular-weight compounds. Two applications of the system were demonstrated: (i) after optimizing eluent conditions the gel exclusion column was used to determine the influence of lactic acid, phosphate buffer, and lactic acid bacteria on the ethylene oxide propylene oxide-dextran T40 phase diagram by HPLC; (ii) the ion exchange column was coupled in series with the gel exclusion column and the concentration of polyethylene glycol, dextran, glucose, lactate, acetate, and formate was determined in samples from the fermentative production of lactic acid in a polyethylene glycol 8000-dextran T40 aqueous two-phase system. The fermentation was operated without pH control in a repeated extractive batch mode, where the cell-free top phase was replaced four times, whereas the cell-containing bottom phase was reused repeatedly. The yield was 1.1 mol of lactic acid formed per mole of glucose added and the productivity was 4.7 mM.h(-1). The polymeric composition of the fermentation system was monitored during the five repeated extractive batches, and it showed a progressive depletion in polyethylene glycol and a progressive enrichment in dextran. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 54: 303-311, 1997.