Phosphate Aqueous Two-phase Systems Research Articles

Partitioning behavior of short DNA fragments in polymer/salt aqueous two-phase systems.

The development of liquid biopsy as a minimally invasive technique for tumor profiling has created a need for efficient biomarker extraction systems from body fluids. The analysis of circulating cell-free DNA (cfDNA) is especially promising, but the low amounts and high fragmentation of cfDNA found in plasma pose challenges to its isolation. While the potential of aqueous two-phase systems (ATPS) for the extraction and purification of various biomolecules has already been successfully established, there is limited literature on the applicability of these findings to short cfDNA-like fragments. This study presents the partitioning behavior of a 160 bp DNA fragment in polyethylene glycol (PEG)/salt ATPS at pH 7.4. The effect of PEG molecular weight, tie-line length, neutral salt additives, and phase volume ratio is evaluated to maximize DNA recovery. Selected ATPS containing a synthetic plasma solution spiked with human serum albumin and immunoglobulin G are tested to determine the separation of DNA fragments from the main plasma protein fraction. By adding 1.5% (w/w) NaCl to a 17.7% (w/w) PEG 400/17.3% (w/w) phosphate ATPS, 88% DNA recovery was achieved in the salt-rich bottom phase while over 99% of the protein was removed.

Construction of highly stable, monodisperse water-in-water Pickering emulsions with full particle coverage using a composite system of microfluidics and helical coiled tube

Water-in-water (W/W) Pickering emulsions, exhibit considerable potential in the food and pharmaceutical fields owing to their compartmentalization and high biocompatibility. However, constrained by the non-uniform distribution of shear forces during emulsification or the spatial obstruction in polydimethylsiloxane (PDMS) passive microfluidic platform, the existing methods cannot generate monodisperse W/W Pickering emulsions with high particle coverage rate, thereby limiting their applications. Herein, a novel microfluidic system is designed for the preparation of monodisperse and highly particle-covered W/W Pickering emulsions under mild conditions. pH-responsive Polyethylene glycol (PEG)/phosphate aqueous two-phase system (ATPS) is used for the emulsions' preparation. Notably, a coverage rate of 96 ± 3 % is obtained by adjusting the length of the helical coiled tube, as well as the size and contact angle of genipin cross-linked BSA (BSA-GP) particles. Moreover, these W/W Pickering emulsions, with surfaces almost completely covered, can maintain monodisperse (Ncoal = 1.18 ± 0.03) for one day. Furthermore, the results of ranitidine hydrochloride (RH) release demonstrated that the drug release rate of W/W Pickering emulsions in the simulated gastric fluid (SGF) was 10 times faster than that in the neutral solution. We believe that the highly particle-covered monodisperse W/W Pickering emulsions possess great potential applications in bioencapsulation for foods and drug delivery.

Primary recovery strategies of low‐molecular‐weight toxins from Crotalus molossus nigrescens and Crotalus atrox using aqueous two‐phase and three‐phase partition systems

AbstractBACKGROUNDRattlesnake venoms are a complex source of different toxin families with biotechnological and biomedical applications. However, the complexity of the sample and the low abundance of some toxins can restrict their study and applications. Toxin isolation usually requires multiple chromatographic steps, implying elevated operative costs and processing times. This raises the need for recovery and enrichment of those proteins and peptides. For that reason, we tested two liquid–liquid separation methods – aqueous two‐phase systems (ATPS) and three‐phase partition (TPP) – since these two alternatives have shown great potential for the primary recovery of different toxin families from rattlesnake venoms.RESULTSPolyethylene glycol (PEG)–citrate and ionic liquid (IL)–potassium phosphate ATPS and TPP based on t‐butanol and ammonium sulfate were used to partition different toxin families from Crotalus molossus nigrescens and C. atrox venoms. PEG–citrate allowed a selective partition of proteins between both phases. The best enrichment case was observed with a myotoxin band (6.6 kDa) in the PEG‐rich phase from 2% to 41.3% of abundance. In IL–potassium phosphate systems, proteins showed a preference towards the IL‐rich phase with no selective partition. In TPP, proteins were partitioned mainly to the bottom phase and interphase, but serine proteases (29.1 kDa) were enriched from 3.7% to 56.6% in the top phase.CONCLUSIONThe present study demonstrated that PEG–citrate ATPS and TPP separation methods can generate a selective partition of some toxin families from rattlesnake venoms. These enrichment methods will facilitate the isolation of toxins, from animal venoms, that are relevant for biotechnological and biomedical applications. © 2024 Society of Chemical Industry (SCI).

Aqueous two-phase extraction and characterization of thermotolerant alkaliphilic Cladophora hutchinsiae xylanase: biochemical properties and potential applications in fruit juice clarification and fish feed supplementation

Xylanase finds extensive applications in diverse biotechnological fields such as biofuel production, pulp and paper industry, baking and brewing industry, food and feed industry, and deinking of waste paper. Here, polyethylene glycol (PEG)-phosphate aqueous two-phase system (ATPS) was applied for the purification of an alkaline active and thermotolerant xylanase from a marine source, Cladophora hutchinsiae (C. hutchinsiae). In the purification process, the effects of some experimental factors such as PEG concentration and PEG molar mass, potassium phosphate(K2HP04) concentration, and pH on xylanase distribution were systematically investigated. Relative enzymatic activity and purification factor obtained were 93.21% and 7.18, respectively. A single protein band of 28 kDa was observed on SDS-PAGE. The optimum temperature and pH of xylanase with beechwood xylan were 30 °C and 9.0, respectively. The Lineweaver-Burk graph was utilized to determine the K m (4.5 ± 0.8 mg/mL), V max (0.04 ± 0.01 U) and k cat (0.001 s−1) values of the enzyme. It was observed that the purified xylanase maintained 70% of its activity at 4 °C and was found stable at pH 4.0 by retaining almost all of its activity. Enzymatic activity was slightly enhanced with Na+, K+, Ca2+ and acetone. The highest increase in the reducing sugar amount was 53.6 ± 3.8, for orange juice at 50 U/mL enzyme concentration.

Development of Enzyme-Based Cosmeceuticals: Studies on the Proteolytic Activity of Arthrospira platensis and Its Efficient Incorporation in a Hydrogel Formulation

Microalgae are a valuable source of enzymes and active ingredients due to their biochemical variability, health-beneficial properties, lower production cost and viability on an industrial scale. In addition, the growing demand for sustainable products constitutes microalgae as a viable resource for the development of a range of innovative products. In the present work, the proteolytic activity of Arthrospira platensis lysate was assessed and characterized using kinetics analysis and zymography employing different substrates, pH values and divalent ions. The results suggest the presence of two main proteolytic enzymes in the lysate. In addition, a rapid and easy purification protocol of the proteolytic activity, under mild conditions, was developed using a polyethyleneglycol (PEG)/phosphate aqueous two-phase system (ATPS). The proteolytic activity of A. platensis lysate was used to develop a hydrogel formulation as an enzyme-based cosmeceutical, with potential application as a topical exfoliating agent. The incorporation of the A. platensis extract in the developed hydrogel formulation significantly improved its operational stability over time, which is a significant advantage in enzyme-based product development.

Optimized and Scalable Green Extraction of Pristimerin, an Anticancerigen from Mortonia greggii, by Ethanol–Phosphate Aqueous Two-Phase Systems

The natural triterpene pristimerin possesses interesting potential against several types of cancer. However, its production at the industrial level requires novel environmentally friendly and effic...

Machine Learning Approaches for Prediction of Phase Equilibria in Poly (Ethylene Glycol) + Sodium Phosphate Aqueous Two-Phase Systems

In this research, liquid-liquid equilibrium (LLE) data were experimentally obtained for the ternary systems of (water + carboxylic acid + dipropyl ether) at T = 298.2 K and P = 101.3 kPa. The carboxylic acids used in this study were isobutyric acid, valeric acid, and isovaleric acid. All these systems are according to Treybal classification, Type-2 systems because the two binary subsystems are partially miscible. The lowest distribution coefficients and separation factors were calculated for isobutyric acid (40 and 329, respectively). The authenticity of the experimental equilibrium data was identified from Hand and Othmer-Tobias correlations. The experimental tie-line data were correlated by using the nonrandom two-liquid (NRTL) and universal quasi-chemical (UNIQUAC) activity coefficient models. RMSD values are between 0.0112 and 0.0155 for the NRTL model, and are between 0.0083 and 0.0153 for UNIQUAC model.

The phase behavior of n-ethylpyridinium tetrafluoroborate and sodium-based salts ATPS and its application in 2-chlorophenol extraction

In this paper, the aqueous two-phase systems (ATPS) containing n -ethylpyridinium tetrafluoroborate ([EPy]BF 4 ), sodium-based salts, and water were studied and the extraction efficiency of 2-chlorophenol was measured to study the ATPS performance in extracting phenolic compounds. The binodal curves of [EPy]BF 4 + sodium carbonate (Na 2 CO 3 ) ATPS and [EPy]BF 4 + sodium dihydrogen phosphate (NaH 2 PO 4 ) ATPS have been determined at 308.15 K, 318.15 K, and 328.15 K and atmospheric pressure. After getting good correlation with Merchuk equation, the binodal curves together with gravimetric method were used to calculate the tie-lines data. Furthermore, the reliability of tie-lines data was verified using Othmer-Tobias and Bancroft equations. Then, the salt influence and temperature influence on the phase behavior were discussed and the results show the salt-outing ability of Na 2 CO 3 is better than NaH 2 PO 4 . With the aim of studying the ATPS performance in extracting phenolic compounds, extraction efficiency for 2-chlorophenol at different temperatures were studied and the results show that [EPy]BF 4 + Na 2 CO 3 ATPS is preferred than [EPy]BF 4 + NaH 2 PO 4 ATPS in applications.

Extraction of protease from Aspergillus tamarii URM 4634 in aqueous two-phase system under continuous and discontinuous process

Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene glycol (PEG)/phosphate aqueous two-phase system under discontinuous and continuous (perforated rotative discs column) process. On the discontinuous process, it was evaluated the effect of operational conditions (PEG molar mass and its concentration, phosphate concentration and pH) over the partition coefficient, activity yield and purification factor. Protease partitioned to PEG-phase with partition coefficients up to 55.73. The best process parameters were 17.5% of PEG, with molar mass 8000 g·mol−1, 15% of phosphate salt at pH 6, with 113.15% of activity yield and purification factor of 2.62. Under continuous extraction, hold up data showed that 57.1% of the discontinuous phase was available for protein extraction. Further, separation achieved 90.0% of efficiency. The yields surpassed 100% in almost all runs, and the best purification factor was 1.84, with both flows of 2 mL·min−1. Thus, the best operational conditions reached an activity yield of 95.3% and 90.0% of separation efficiency. Hence, aqueous two-phase system PEG/phosphate extraction is an efficient process for separation of proteases produced by Aspergillus tamarii URM 4634, under continuous extraction likewise under discontinuous process.

Response Surface Methodology for the Evaluation of Lysozyme Partitioning in Poly (Vinyl Pyrrolidone) and Potassium Phosphate Aqueous Two-Phase System

The partitioning of lysozyme and extraction yield in an aqueous two-phase system containing Poly Vinyl Pyrrolidone (PVP) K25 and potassium phosphate were investigated as a function of weight percent of salt and PVP in the feed, temperature, and pH. To investigate partitioning behavior, the central composite design was considered using a quadratic model. According to the results of the model, the partitioning of lysozyme was mainly due to the impact of the weight percent of the salt in the feed. However, the partitioning was expanded a little following the increase in the weight percent of the polymer. Based on the results, there was an opposite relationship between the temperature and decrement in the viscosity of PVP as the increment in the former led to the decrement in the latter. Finally, the modification in the third factor was done by increasing the pH level. Before experimenting, some values were hypothesized such as 93.69% for the maximum extraction yield, 21.23% for PVP K25, 13.99% for the salt concentrations, 7.10 for the pH value and 35.57 for the temperature. The findings of the study suggest that the hypothesized values for different variables are in line with the experimental results.

Optimization of a Gelatin–Potassium Phosphate Aqueous Two-Phase System for the Preparation of Hydrogel Microspheres

An aqueous two-phase system provides a simple route toward the preparation of gelatin emulsions. Here, we present a simple method to generate water-in-water (w/w) emulsions from an aqueous two-phase system: gelatin and potassium phosphate (K2HPO4) salt. Liquid gelatin forms as the dispersed phase of the two-phase emulsion system, and gelatin microspheres can be retrieved after a visible light-induced crosslinking reaction. We investigated the effect of the continuous phase volume ratio on the formation of the phase-separation and emulsification process. We also studied the influence of the polymerization method on the size and morphology of gelatin hydrogel particles. The results demonstrated that K2HPO4 is an appropriate phase-forming salt, where biodegradable gelatin particles obtained through this w/w emulsion system have potential for biomedical applications. In addition, sustained release of a model molecule, methylene blue, was observed for up to 5 days from gelatin particles. This system is advantageous because if provides an inexpensive emulsion platform that avoids the use of organic solvents or auxiliary polymers to form a continuous phase.

Improved recovery of bacteriophage M13 using an ATPS-based bioprocess.

Aqueous two-phase systems (ATPS) have been widely exploited for the recovery and partial purification of biological compounds. Recently our research group characterized the primary recovery and partial purification of bacteriophage M13 using polymer-salt and ionic liquid-salt ATPS. From such study, it was concluded that PEG 400-potassium phosphate ATPS with a volume ratio (VR ) of 1 and 25% w/w TLL were the best suitable for the primary recovery of bacteriophage M13 from a crude extract, achieving a recovery yield of 83.3%. Although such system parameters were proven to be adequate for the recovery of the product of interest, it was concluded that further optimization was desirable and attainable by studying the effect of additional system parameters such as VR , concentration of neutral salt (M) and sample load (% w/w). This research work presents an optimization of a previously reported process for the recovery of bacteriophage M13 directly from a crude extract using ATPS. The increase in VR and sample load showed a positive effect in the recovery of M13 indicating an improved performance of the proposed ATPS. According to the results presented here, a system composed of PEG 400 17.2% (w/w), potassium phosphate 15.5% (w/w) and a sample load of 30% (w/w) allowed the recovery of M13 directly from a crude extract with a top phase recovery of 80.1%, representing an increase of 4.8 times in the final concentration and a reduction of 2.65 times in the processing costs. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1177-1184, 2018.

Response surface methodology for the evaluation of guanidine hydrochloride partitioning in polymer-salt aqueous two-phase system

The current study employed response surface methodology (RSM) with a face-centered central composite design (CCD) to indicate the essential variables on the partition coefficient of guanidine hydrochloride (GuHCl) in the poly (ethylene glycol) (PEG)-phosphate aqueous two-phase system (ATPS). To evaluate the partition coefficients of GuHCl in the mentioned ATPS, the pH (7.0, 8.5 and 10.0), GuHCl concentration (1.0, 3.5 and 6.0% w/w), PEG molecular weight (2,000, 4,000 and 6,000 gmol−1) and PEG/potassium phosphate concentrations ratio were selected as independent variables. A quadratic model is suggested to find the impact of these variables. The suggested model has a strong harmony with the experimental data. The results of the model display that the GuHCl concentration and weight percent of the salt in feed have a large and small influence on the GuHCl partitioning.

Rubisco separation using biocompatible aqueous two-phase systems

Mild and efficient separation processes have to be developed to convert microalgal biomass into high valuable products. Aqueous two-phase system (ATPS) was adopted as a new approach in microalgae to separate hydrophilic from hydrophobic components. In this work, three biocompatible ATPSs polyethylene glycol (PEG) 400-Potassium citrate, Iolilyte 221PG-potassium citrate and PEG 400-Cholinium dihydrogen phosphate ATPS were selected based on their interaction with Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco), a protein predominantly present in microalgae and used as ingredient in human and animal food. Binodal curves were constructed for each system and the parameters influencing phase formation were investigated. Iolilyte 221PG-potassium citrate has a stronger ability to form ATPS compared with the PEG-based systems. This stronger ability was attributed to hydrophobic and electrostatic interactions between the phase-forming components. After characterization, we investigated the performance of the ATPSs in the partitioning of Rubisco. In this study, the effect of the tie-line length (TLL), pH and type of phase-forming components on Rubisco extraction efficiency (%) was analyzed. In a single step, the appropriate parameters lead to extraction efficiencies between 80 and 100%. Additionally, stability studies were performed to see if ATPS retain the native protein structure. Iolilyte 221PG-Citrate was found to be the most efficient ATPS in Rubisco separation. However, stability studies indicated that PEG-based ATPSs have a better performance in retaining the Rubisco integrity.

Recovery and partial purification of thermophilic β-xylosidase derived from recombinant Bacillus megaterium MS941 by aqueous two-phase system

ABSTRACTA polymer–salt-based aqueous two-phase system (ATPS) was developed for the effective extraction and purification of extracellular β-xylosidase from the fermentation broth of recombinant Bacillus megaterium MS941. The effect of molecular weight (MW) of polyethylene glycol (PEG), tie-line length (TLL), volume ratio (VR), crude loading and pH on the recovery performance was evaluated. Under the optimal extraction conditions, β-xylosidase was successfully purified up to 23-fold with a recovery yield of 99% in the bottom salt-rich phase at PEG 4,000/potassium phosphate ATPS comprising TLL of 41.8, VR of 2.3, crude loading (CL) of 30% (w/w) at pH 6.

Differences in Cerrena unicolor and Pleurotus sapidus laccase partitioning in polyethylene glycol – phosphate aqueous two-phase systems

Differences in Cerrena unicolor and Pleurotus sapidus laccase partitioning in polyethylene glycol – phosphate aqueous two-phase systems

Response surface methodology and artificial neural network modelling of an aqueous two-phase system for purification of a recombinant alkaline active xylanase

A two-stage polyethylene glycol (PEG)-phosphate aqueous two-phase system was used for purification of a highly thermostable and alkaline active recombinant xylanase. Response surface methodology (RSM) and artificial neural network (ANN) have been used to develop predictive models for simulation and optimization of purification process. Effects of pH, PEG molecular weight, concentrations of phosphate, PEG and NaCl on the partitioning of the target enzyme and the contaminants were studied using a central composite design of experiments. The best first stage purification was achieved using 6% PEG 6000, 20% phosphate and pH 6. The optimum back extraction stage system consist of 10% phosphate, 10% NaCl, pH 10 and the first stage separation top phase. After the two stage phase separations, about 78% of the original enzyme activity was recovered and the specific activity of the purified enzyme was increased by a factor of 6.7. Also, the aqueous two-phase system was scaled-up 100 times. After back-extraction, the specific activity increased 6.56 times with 72% total yield. A similar design was also used to obtain a training set for ANN. A comparison between the model results and experimental data gave high correlation coefficient (R2) and showed that both models were able to predict the partitioning behavior. The results demonstrated a higher prediction accuracy of ANN compared to RSM. This superiority of ANN over other multi factorial approaches could make this estimation technique a very helpful tool for purification process.

Intensified fractionation of brewery yeast waste for the recovery of invertase using aqueous two-phase systems.

The potential recovery of high-value products from brewery yeast waste confers value to this industrial residue. Aqueous two-phase systems (ATPS) have demonstrated to be an attractive alternative for the primary recovery of biological products and are therefore suitable for the recovery of invertase from this residue. Sixteen different polyethylene glycol (PEG)-potassium phosphate ATPS were tested to evaluate the effects of PEG molecular weight (MW) and tie-line length (TLL) upon the partition behavior of invertase. Concentrations of crude extract from brewery yeast waste were then varied in the systems that presented the best behaviors to intensify the potential recovery of the enzyme. Results show that the use of a PEG MW 400gmol-1 system with a TLL of 45.0% (w/w) resulted in an invertase bottom phase recovery with a purification factor of 29.5 and a recovery yield of up to 66.2% after scaling the system to a total weight of 15.0g. This represents 15.1mg of invertase per mL of processed bottom phase. With these results, a single-stage ATPS process for the recovery of invertase is proposed.

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.

Impact of polyethylene glycol as additive on the formation and extraction behavior of ionic‐liquid based aqueous two‐phase system

Developing a novel Ionic‐liquid (IL) based aqueous two‐phase system (ATPS) with polyethylene glycol (PEG) as adjuvant for the separation of biomolecules is studied. This original work involves addition of various concentration of PEG (2000, 4000, and 6000 gr/mol) to 1‐butyl‐3‐methylimidazolium acetate+ potassium hydrogen phosphate ATPS to investigate their subsequent effect on phase diagrams and partitioning coefficient of α‐amylase. In another innovative aspect of this work, response surface methodology (RSM) based on three‐variable central composite design was employed to understand the effect of phase forming components on extraction studies of α‐amylase. The addition of small amount of PEG improved the partitioning coefficient of biomolecule. The effective excluded volume theory was applied to correlate the salting‐out ability. As a result, it can be stated that the proposed system can effectively be used in separation and purification studies instead of task specific ILs. © 2015 American Institute of Chemical Engineers AIChE J, 62: 264–274, 2016