Partitioning In Aqueous Two-phase Systems Research Articles

Tyrosinase from the peels of Musa cavendish: purification, characterization, immobilization and application in phenol biodegradation

Tyrosinase is an enzyme implicated in fruit ripening and the browning of plant parts exposed to mechanical injury. Tyrosinase has numerous reported biotechnological importance. This enzyme was extracted from the waste peels of Musa cavendish, purified via aqueous two-phase partitioning system (ATPS) and QAE-Sephadex ion-exchange chromatography. The purified tyrosinase was biochemically characterized. Tyrosinase was thereafter immobilized, characterized, and applied in the oxidation of phenols in model wastewaters. After purification, the yield and fold obtained were 23% and 8.0 respectively. The molecular weight of the enzyme was estimated to be 43.0 kDa. The purified enzyme had an optimum pH and temperature of 6.5 and 60 °C respectively. The (M−1s−1) obtained for substrates such as L-DOPA, catechol and pyrogallol were respectively. The purified enzyme was stable in organic solvents: acetone > ethanol > methanol > n-butanol. Quercetin, ascorbic acid, l-cysteine and arginine were potent inhibitors. The immobilized tyrosinase was more resistant to thermoinactivation than the free enzyme as evidenced by its kinetic ( and thermodynamic ( characteristics. The immobilized tyrosinase removed 70% of 650-µM phenol after 2 h compared to 50% removal by the free tyrosinase. After six repeated cycles of phenol biodegradation using the immobilized enzyme, about 40% of phenol was removed. The fresh peels of M. cavendish served as a green-based source of relatively thermostable immobilized tyrosinase deployable in large-scale biodegradation of phenol-containing wastewater.

Phase equilibrium and ionic liquid partitioning in aqueous two-phase system composed of alcohol and small molecule compound

Phase equilibrium and ionic liquid partitioning in aqueous two-phase system composed of alcohol and small molecule compound

Experimental Studies and Artificial Neural Networks (Ann) Modeling of Asparaginase Partitioning in Aqueous Two-Phase Systems: Polyethylene Glycol and Inorganic Salts

Experimental Studies and Artificial Neural Networks (Ann) Modeling of Asparaginase Partitioning in Aqueous Two-Phase Systems: Polyethylene Glycol and Inorganic Salts

Aqueous two-phase partitioning and characterization of xylanase produced by Streptomyces geysiriensis from low cost lignocellulosic substrates

Microbial production of xylanase is gaining the commercial importance, due to its wide range of applications from paper and pulp to food and feed industries. Streptomyces geysiriensis was used for the production of extracellular xylanase from lignocellulosic substrates such as rice bran and saw dust, under solid-state fermentation. The influence of pH, temperature and incubation period for the maximum production of xylanase was investigated with 1:2 (w/v) of substrate to moisture ratio at 100rpm shaking conditions. The maximum production was recorded after 5 days of fermentation with pH 8.0at 40°C. The scale-up was done based on the results of optimized parameters using 3L Applikon autoclavable bioreactor with maximum yield of 186 U/ml after 4 days of fermentation. Extracellular xylanase was separated by partitioning in aqueous two-phase system consisting of 20% polyethylene glycol 6000 and 12% K2HPO4 with maximum yield of 93.97%. The investigation of the effect of pH and temperature and its incubation time showed that xylanase was retained its activity in a pH range of 6.5-8.5, with thermal stability from 20°C to 60°C up to 180min. The presence of metal ions was found to inhibit the activity of xylanase especially Cu2+ and Zn2+. Xylanase was stable both at 4°C and room temperature (35°C) for 30 and 9 days respectively. The kinetic parameters Km (0.48mg/ml) and Vmax (8.33 U/mg) were determined using birchwood xylan as substrate.

A new tool for quantification of membrane protein partitioning between different cell membranes

The aqueous two-phase partition system (ATPS) is a method widely used to separate and purify plant and animal membranes carrying bound proteins. However, a common problem of this separation is a mutual contamination of obtained phases. Such contamination adversely affects the accuracy of values of the protein of interest partition between particular membranes when determined by direct measurement. In order to overcome this problem, we have developed a fairly simple mathematical algorithm and found formulas designed to quantify correctly the distribution of the protein of interest between two different membranes. This new tool makes it possible to determine the bias-adjusted ratio of protein distribution between the membranes, regardless of the efficiency of membrane separation in a two-phase system. By means of this algorithm, not only current, but also a number of previously published ATPS-based experiments were (re)analyzed and quantified. The quantitative results of this large-scale analysis of the subcellular localization of various membrane proteins from Arabidopsis, potato, melon, and corn including cytokinin and ethylene receptors, ABCG14 cytokinin transporters, LRR receptor-like protein kinases, and BAK1 co-receptors are presented and discussed here.

Optimization of aqueous two-phase partitioning of Aureobasidium pullulans α-amylase via response surface methodology and investigation of its thermodynamic and kinetic properties

Industrial enzymes such as α-amylase must be thermostable and also easily purified/concentrated. Hence, aqueous two-phase partitioning systems (ATPS) was exploited for the partitioning of α-amylase from Aureobasidium pullulans due to its numerous advantages over conventional purification strategy. A. pullulans α-amylase was partially purified using ATPS via response surface methodology (RSM). The potentials of the ATPS-purified enzyme for possible industrial application such as resistance to thermal inactivation was investigated in comparison with the crude enzyme. PEG-6000 was the polymer of choice for ATPS as it resulted in higher purification factor (PF), %yield (Y), and partition coefficient (PC). At optimum levels (% w/v) of 20, 12 and 7.5 for PEG-6000, sodium citrate and sodium chloride respectively, maximum PF, Y and PC of 4.2, 88%, and 9.9 respectively were obtained. The response model validation and reliability were established based on the closeness between the experimented and predicted values. The kinetic and thermodynamic parameters such as Q10, t1/2, kd, D − value, Ed, ΔHd∗,ΔGd∗andΔSd∗ of the ATPS-purified α-amylase indicated that it was thermostable at 50 to 60 °C compared to the crude α-amylase. A thermodynamically stable and ATPS-purified α-amylase from A. pullulans has properties easily applicable for most industrial processes.

Use of Aqueous Two-Phase and Three-Phase Partitioning Systems for Purification of Lipase Obtained in Solid-State Fermentation by Rhizopus arrhizus

Background: Purification of enzymes by conventional methods such as precipitation and chromatographic techniques is a costly and time-consuming procedure and may lead to low yields of enzyme activity. Alternative liquid-liquid extraction methods such as Aqueous Two-Phase Systems (ATPS) and Three Phase Partitioning (TPP) are characterized by the high enzyme yields and purification degree. Objective: The objective of this study was the application of partitioning systems ATPS and TPP for purification of lipase produced in solid-state fermentation by Rhizopus arrhizus. Methods: ATPS and TPP were used for purification of lipase, obtained by solid state cultivation of Rhizopus arrhizus. Results: Lipase was isolated with PEG4000/potassium sodium tartrate ATPS and the effect of the system composition, including PEG 4000 and potassium sodium tartrate concentrations on lipase partitioning was studied. When using 30% PEG4000/21% potassium sodium tartrate, lipase was distributed in the top phase, and the highest recovery yield of 217% and purification fold of 6.1 were achieved. It was found that at PEG4000 concentration of or higher than 15%, the enzyme was present in the top polymer-rich phase with a partitioning yield of over 90%. Upon application of TPP for lipase isolation, the effect of t-butanol concentration, ammonium sulfate concentration and pH on enzyme partitioning was investigated. The highest lipase recovery yield of 71% and 19.1-fold purification were achieved in the interfacial phase in the presence of 30% ammonium sulfate saturation with 1.0:0.5 crude extract/t-butanol ratio at pH 7 in a single step. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymographic analysis showed significant purification of lipase by TPP and the presence of two multiple forms of the enzyme. Conclusion: ATPS (PEG4000/ Potassium sodium tartrate) and TPP (1.0:0.5 crude extract/t-butanol ratio, 30% ammonium sulfate saturation, pH 7) proved to be rapid methods for the isolation and purification of lipase and they can be used in downstream processing for industrial preparation of the enzyme.

Study of phase behavior and Congo red dye partitioning in aqueous two-phase systems composed of hydrophilic alcohols (1-propanol/ 1-butanol) and sodium salts

ABSTRACTThe partitioning of the Congo red dye in ATPSs formed by alcohols (1-butanol, 1-propanol)/sodium salts was considered. Binodal and the LLE data were experimentally determined at 298.15 K. The salting-out abilities of the salts follow the order Na3C6H5O7 > NaH2PO4 > C2H3 O2Na. The phase-forming abilities of the alcohols follow the order: 1-butanol > 1-propanol. The four-parameter equation was applied to correlate the binodal curves data. Therefore, The Bancroft and Othmer-Tobias equations were used to prove the reliability of the corresponding LLE data. ATPS composed of 6.5% of 1-butanol and 20% of Na3C6H5O7 had the highest values of extraction by the yield of 98.54%.

Partial characterization of Bacillus pumilus catalase partitioned in poly(ethylene glycol)/sodium sulfate aqueous two-phase systems

Aqueous two-phase partitioning system (ATPS) was used to extract and purify catalase from Bacillus pumilus. The system parameters for effective purification of catalase were optimized. The best catalase recovery (123%) with a 4.6-fold purification was obtained in the bottom phase of ATPS including the mixture of 15% (w/w) PEG4000, 10% (w/w) Na2SO4 and 3% (w/w) NaCl at pH 5.0. The purified enzyme was characterized regarding its activity and stability. The highest enzyme activity was observed at pH 7.0 and 37 °C on hydrogen peroxide. The enzyme was quite stable at temperatures between 30 and 55 °C and a pH range of 7.0–9.0. The Km and Vmax values were determined from Lineweaver–Burk plot as 11 mM and 1667 µmole ml−1 min−1, respectively. Overall, it can be said that ATPS is a rapid, reasonable, straightforward and cost-effective process for catalase purification in comparison to the chromatographic methods.

Phase equilibrium and protein partitioning in aqueous two-phase systems containing imidazolium ionic liquids and surfactant at low voltage levels

In this paper, the binodal data of aqueous two-phase system (ATPS) containing imidazolium-based ionic liquids (chlorinated 1-ethyl-3-methylimidazole, [Emim]Cl), and a non-ionic surfactant (octylphenol polyethoxylene, Triton X-100) was obtained by means of the cloud point method in the temperature intervals T=(288.15–308.15) K. It was found that the [Emim]Cl/Triton X-100 ATPS was sensitive to temperature and had a long phase segregation time. Screening experiments of various additives showed that adding 7% of sodium dodecyl sulfate (SDS) to the total mass of surfactants (w/w) reduced the temperature sensitivity, and improved the phase separation rate of the system. The liquid-liquid equilibrium (LLE) data of [Emim]Cl/Triton X-100 ATPS without additive and containing 7% SDS at 298.15K was further studied according to the cloud point method. To further shorten the long phase disengagement time, a low-voltage alternating current (AC) electric field was added to the [Emim]Cl/surfactant ATPS. Through the optimization of AC electric field conditions (such as electric field type, frequency and duty ratio), a relatively fast and stable [Emim]Cl/surfactant ATPS was established. In 0.67V/cm field strength, 1000Hz frequency and 50% duty ratio of impulse waveform, the phase splitting time is shortened by 2–3 times. With and without electric field, protein partitioning of cytochrome c (Cyt-c), bovine serum albumin (BSA) and lysozyme (Lys) in [Emim]Cl/surfactant ATPS indicated that Cyt-c and BSA tended to distribute in the bottom phase, while Lys was inclined to distribute in the top phase. The application of the AC electric field can still decrease the phase separation time in the protein distribution process, and has no significant effect on the distribution behavior of proteins. Furthermore, Lys activity in the top phase of ATPS was significantly enhanced, which could be caused by the low concentration of Triton X-100 in the top phase based on the comparison of Lys fluorescence spectra analysis in aqueous solution, in the top phase and in the bottom phase of [Emim]Cl/surfactant ATPS.

Equilibrium data and thermodynamic studies of α-amylase partition in aqueous two-phase systems

Liquid-liquid equilibrium data (LLE) were obtained for aqueous two-phase systems (ATPS) composed of polyethylene glycol (average molar mass of 1500 g mol−1), sodium citrate/citric acid and water, at different pH (4.0, 5.0, 6.0 and 7.0) and temperatures T = (303.15 and 313.15) K. The thermodynamic study of the phase equilibrium was performed. The experimental results of the LLE were correlated using the UNIversal Functional Activity Coefficient (UNIFAC) model. The salting-out effect evaluated using the model of the effective excluded volume (EEV). Partitioning of the α-amylase (EC 3.2.1.1) was investigated as a function of pH, temperature, and tie line length (TLL). It was observed that the temperature had no influence on the formation of the biphasic region. Increments in pH value resulted in the raising of biphasic region. Isothermal titration calorimetry (ITC) assays was performed in order to deeply understanding the intermolecular interactions involved in the α-amylase partition and the driving forces that govern this process. It was measured the Gibbs free energy transfer (ΔtrG), transfer enthalpy variation (ΔtrH) and transfer entropy variation (ΔtrS) during the enzyme partition, as a function of the pH, temperature and TLL. For α-amylase partition, it was verified that at pH 4.0 the enzyme had a strong tendency to transfer to the polymer-rich phase. At pH 5.0 α-amylase showed affinity for the salt-rich bottom phase, while at pH 6.0, the partition coefficient of the enzyme increased as temperature was reduced to 303.15 K. The opposite behavior was noted for α-amylase at pH 7.0. Thermodynamic analysis based on isothermal titration microcalorimetry indicated the partition of α-amylase partition in the ATPS at pH 4.0 and 7.0 and 313.15 K was accompanied by endothermic heat and was entropically driven to the upper phase. Results of yield (95.373%) parameter indicated the applicability of ATPS for amylase purification.

Plasma Membrane Protein Profiling in Beta-Amyloid-Treated Microglia Cell Line.

In the responsiveness of microglia to toxic stimuli, plasma membrane proteins play a key role. In this study we treated with a synthetic beta amyloid peptide murine microglial cells metabolically differently labelled with stable isotope amino acids (SILAC). The plasma membrane was selectively enriched by a multi-stage aqueous two-phase partition system. We were able to identify by 1D-LC-MS/MS analyses 1577 proteins, most of them are plasma membrane proteins according to the Gene Ontology annotation. An unchanged level of amyloid receptors in this data set suggests that microglia preserve their responsiveness capability to the environment even after 24-h challenge with amyloid peptides. On the other hand, 14 proteins were observed to change their plasma membrane abundance to a statistically significant extent. Among these, we proposed as reliable biomarkers of the inflammatory microglia phenotype in AD damaged tissues MAP/microtubule affinity-regulating kinase 3 (MARK3), Interferon-induced transmembrane protein 3 (IFITM3), Annexins A5 and A7 (ANXA5, ANXA7) and Neuropilin-1 (NRP1), all proteins known to be involved in the inflammation processes and in microtubule network assembly rate.

Plasmid DNA affinity partitioning using polyethylene glycol - sodium sulfate aqueous two-phase systems.

Extraction of plasmid DNA (pDNA) from an alkaline bacterial cell lysate has been investigated using partitioning and affinity partitioning in aqueous two-phase systems (ATPSs) containing 12% (w/w) polyethylene glycol and 12% (w/w) sodium sulfate. In this work, a modified 20bp pyrimidine oligonucleotide acting as a triplex-forming oligonucleotide (TFO) was employed as an affinity ligand to improve the partition behavior. The pUC118 plasmid was chosen to be extracted because of its specific oligonucleotide recognition sequence. In order to find the proper conditions, operational parameters such as PEG molecular weight, pH and lysate load were also studied. The results showed that adding the affinity ligand strongly changed the partitioning behavior of pDNA without any considerable impact on the partitioning of RNA and protein content. The best achieved result was at pH=6 and PEG MW of 600Da and 60% of lysate load. In these conditions, 67% of pDNA was recovered in the top phase while the contaminants mainly accumulated in the bottom phase.

Separation of catalase from Amsonia orientalis with single step by aqueous two-phase partitioning system (ATPS)

ABSTRACT Catalase from Amsonia orientalis was purified by ATPS, and its efficiency was compared against hydrophobic interaction chromatography. Activity recovery and purification fold of purified catalase by ATPS were examined under varying experimental conditions. The effects of various factors such as type of phase-forming salts, (PEG) mass, with their different concentrations, pH and temperature effects on partitioning were investigated. The highest activity recovery (156%) and purification fold (8.67) of catalase were obtained in the ATPS system containing 10% (g/g) PEG4000, 15% (g/g) Na2SO4 at pH 6.0 and room temperature. In hydrophobic interaction chromatography, the enzyme has been purified 12.54-fold with 57.5% recovery. The molecular weight of catalase was determined as 75 kDa by SDS-PAGE.

What are the structural features that drive partitioning of proteins in aqueous two-phase systems?

Protein partitioning in aqueous two-phase systems (ATPSs) represents a convenient, inexpensive, and easy to scale-up protein separation technique. Since partition behavior of a protein dramatically depends on an ATPS composition, it would be highly beneficial to have reliable means for (even qualitative) prediction of partitioning of a target protein under different conditions. Our aim was to understand which structural features of proteins contribute to partitioning of a query protein in a given ATPS. We undertook a systematic empirical analysis of relations between 57 numerical structural descriptors derived from the corresponding amino acid sequences and crystal structures of 10 well-characterized proteins and the partition behavior of these proteins in 29 different ATPSs. This analysis revealed that just a few structural characteristics of proteins can accurately determine behavior of these proteins in a given ATPS. However, partition behavior of proteins in different ATPSs relies on different structural features. In other words, we could not find a unique set of protein structural features derived from their crystal structures that could be used for the description of the protein partition behavior of all proteins in all ATPSs analyzed in this study. We likely need to gain better insight into relationships between protein-solvent interactions and protein structure peculiarities, in particular given limitations of the used here crystal structures, to be able to construct a model that accurately predicts protein partition behavior across all ATPSs.

Analysis of the distribution of organic compounds and drugs between biological tissues in the framework of solute partitioning in aqueous two-phase systems.

Partition coefficients of non-ionic essentially nonpolar compounds between air and rat biological tissues and between blood and other tissues were examined and found to be linearly interrelated according to the previously established equation for partition coefficients of solutes in aqueous two-phase systems: log Kblood-tissue-1 = k0 + k1 log Kblood-tissue-2 + k2 log Kblood-tissue-3, where k0, k1, and k2 are constants. Analysis of partition coefficients of amphiphilic and ionizable drugs between blood and different tissues in rats in vivo showed that the above relationship holds for the blood-tissue partition coefficients of these compounds as well. The data obtained indicate that distribution of organic compounds between different biological tissues may be considered in the framework of solute partitioning in aqueous two-phase systems, and imply that aqueous media in different tissues have different solvent properties, and compound-water interactions in these media may respond to different environments governed by the tissue composition.

Solvent interaction analysis as a proteomic approach to structure-based biomarker discovery and clinical diagnostics

Proteins have several measurable features in biological fluids that may change under pathological conditions. The current disease biomarker discovery is mostly based on protein concentration in the sample as the measurable feature. Changes in protein structures, such as post-translational modifications and in protein–partner interactions are known to accompany pathological processes. Changes in glycosylation profiles are well-established for many plasma proteins in various types of cancer and other diseases. The solvent interaction analysis method is based on protein partitioning in aqueous two-phase systems and is highly sensitive to changes in protein structure and protein–protein- and protein–partner interactions while independent of the protein concentration in the biological sample. It provides quantitative index: partition coefficient representing changes in protein structure and interactions with partners. The fundamentals of the method are presented with multiple examples of applications of the method to discover and monitor structural protein biomarkers as disease-specific diagnostic indicators.

Osmotic Virial Coefficients as Access to the Protein Partitioning in Aqueous Two-Phase Systems

A promising alternative to state of the art chromatographic separations of therapeutic proteins is the extraction of the target protein using an aqueous two-phase system (ATPS). The use of an additional salt working as a displacement agent can influence the protein partitioning behavior in ATPS and thus enable a selective purification of the target protein. The selection of a suitable ATPS for protein extraction requires information concerning the protein-protein interactions (second osmotic virial coefficient B22 ) as well as the interactions between protein and solute (displacement agent and phase-forming components) (cross virial coefficient B23 ). In this work, the partitioning behavior and the precipitation affinity of immunoglobulin G (IgG) is considered within a polyethylene glycol (PEG)-phosphate ATPS. The influence on IgG partitioning upon addition of NaCl and (NH4)2 SO4 was investigated. In order to access the IgG precipitation affinity and the IgG partitioning behavior, the B22 and B23 values were determined for several combinations of solute [PEG, phosphate buffer, NaCl, and (NH4)2 SO4 ] and IgG based on static light scattering measurements. A qualitative estimation of the IgG precipitation affinity and the suitability of a solute as potential displacement agent within the PEG-phosphate ATPS on the basis of the measured B22 and B23 values is presented.

Purification of papain by metal affinity partitioning in aqueous two-phase polyethylene glycol/sodium sulfate systems.

A simple and inexpensive aqueous two-phase affinity partitioning system using metal ligands was introduced to improve the selectivity of commercial papain extraction. Polyethylene glycol 4000 was first activated using epichlorohydrin, then it was covalently linked to iminodiacetic acid. Finally, the specific metal ligand Cu(2+) was attached to the polyethylene glycol-iminodiacetic acid. The chelated Cu(2+) content was measured by atomic absorption spectrometry as 0.88 mol/mol (polyethylene glycol). The effects on the purification at different conditions, including polyethylene glycol molecular weight (2000, 4000, and 6000), concentration of phase-forming components (polyethylene glycol 12-20% w/w and sodium sulfate 12-20%, w/w), metal ligand type, and concentration, system pH and the commercial papain loading on papain extraction, were systematically studied. Under optimum conditions of the system, i.e. 18% w/w sodium sulfate, 18% w/w polyethylene glycol 4000, 1% w/w polyethylene glycol-iminodiacetic acid-Cu(2+) and pH 7, a maximum yield of 90.3% and a degree of purification of 3.6-fold were obtained. Compared to aqueous two phase extraction without ligands, affinity partitioning was found to be an effective technique for the purification of commercial papain with higher extraction efficiency and degree of purification.

Responses of proteins to different ionic environment are linearly interrelated

Protein partitioning in aqueous two-phase systems (ATPS) is widely used as a convenient, inexpensive, and readily scaled-up separation technique. Protein partition behavior in ATPS is known to be readily manipulated by ionic composition. However, the available data on the effects of salts and buffer concentrations on protein partitioning are very limited. To fill this gap, partitioning of 15 proteins was examined in dextran–poly(ethylene glycol) ATPSs with different salt additives (Na2SO4, NaClO4, NaSCN, CsCl) in 0.11M sodium phosphate buffer, pH 7.4. This analysis reveals that there is a linear relationship between the logarithms of the protein partition coefficients determined in the presence of different salts. This relationship suggests that the protein response to ionic environment is determined by the protein structure and type and concentrations of the ions present. Analysis of the differences between protein structures (described in terms of proteins responses to different salts) and that of cytochrome c chosen as a reference showed that the peculiarities of the protein surface structure and B-factor used as a measure of the protein flexibility are the determining parameters. Our results provide better insight into the use of different salts in manipulating protein partitioning in aqueous two-phase systems. These data also demonstrate that the protein responses to different ionic environments are interrelated and are determined by the structural peculiarities of protein surface. It is suggested that changes in ionic microenvironment of proteins may regulate protein transport and behavior in biological systems.