Aqueous Two-phase Systems Research Articles

The phase split forced by salts or carbohydrates in nonaqueous systems

More than 2500 ternary systems containing binary nonaqueous solvents and a salt or carbohydrate were checked experimentally under ambient conditions to demonstrate the liquid–liquid phase split. The binary solvents consisted of one strongly polar component and another significantly less polar. Phase separation has been observed in more than 300 systems. These systems are direct analogues of the aqueous two-phase systems (ATPS) that have been extensively studied and found numerous applications. The obtained data were analyzed and some correlations were observed. Possibilities of creating the liquid–liquid miscibility gap depending on the solvents’ polarity and the solute properties were discussed. This phenomenon most often occurs with binary solvents containing a strongly polar component, such as formamide, dimethyl sulfoxide, ethane-1,2-diol, or N-methylformamide. Carbohydrates have been found to be better liquid–liquid phase split inducers than salts. Among the latter, calcium and lithium chlorides, and sodium iodide turned out to be the most effective. A strict correspondence of the decreasing probability of creating a miscibility gap by different ions with the reversed Hofmeister series was confirmed.

Structural characteristics and biological activity of lactic acid bacteria exopolysaccharides separated by ethanol/(NH4)2SO4 ATPS

Exopolysaccharides (EPS) from lactic acid bacteria (LAB) as edible and safe bioproducts with health benefits have become an interesting topic. In this study, aqueous two-phase system (ATPS) was established using ethanol and (NH4)2SO4 as phase-forming substances to separate and purify LAB EPS from Lactobacillus plantarum 1.0665. The operating conditions were optimized by a single factor and response surface method (RSM). The results indicated that an effectively selective separation of LAB EPS was achieved by the ATPS consisted of 28 % (w/w) ethanol and 18 % (w/w) (NH4)2SO4 at pH 4.0. Under optimized conditions, the partition coefficient (K) and recovery rate (Y) were well matched with the predicted value of 3.83 ± 0.019 and 74.66 ± 1.05 %. The physicochemical properties of purified LAB EPS were characterized by various technologies. According to the results, LAB EPS was a complex polysaccharide with a triple helix structure mainly composed of mannose, glucose and galactose in the molar ratio of 1.00: 0.32: 0.14, and it proved that the ethanol/(NH4)2SO4 system had good selectivity for LAB EPS. In addition, LAB EPS displayed excellent antioxidant activity, antihypertension activity, anti-gout capacity and hypoglycemic activity in vitro analysis. The results suggested that LAB EPS could be a dietary supplement applied in functional foods.

Specific Extraction of Aspartic Acid and Tyrosine with Aqueous Two-phase Systems Formed by Hydrophilic Organic Solvent and Monomeric Sugars

Specific Extraction of Aspartic Acid and Tyrosine with Aqueous Two-phase Systems Formed by Hydrophilic Organic Solvent and Monomeric Sugars

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.

Seawater desalination by forward-osmosis-assisted temperature swing solvent extraction

This study investigates seawater desalination by a combined process consisting of forward osmosis and temperature swing solvent extraction (TSSE). While direct desalination by temperature swing solvent extraction suffers from low extraction efficiencies, forward osmosis enables to transfer water into a more favourable salt solution expanding the amount of suitable solvents and increasing the subsequent extraction yield. In this work, tie lines of aqueous two-phase systems consisting of the thermoresponsive solvent polypropylene glycol 600 and different salts (NaCl, Na2SO4, MgSO4) were determined to investigate the influence of salt type, concentration, and temperature on the liquid-liquid equilibrium. Based on these results, MgSO4 (20 wt%) was selected as draw solute achieving water fluxes up to 7.5 Lm−2 h−1 in forward osmosis against artificial seawater. The diluted draw solution is regenerated by TSSE achieving high extraction yields (0.38 kgwater/kgsolvent). Eventually, the extracted water is separated from the solvent by temperature-induced phase separation at low temperatures enabling the use of low-grade thermal energy sources. In comparison to the direct use of the thermoresponsive polymer as draw agent, the proposed process benefits from significantly higher water fluxes due to reduced internal concentration polarization when using a salt based draw solution.

Extraction of Bovine Serum Albumin by Aqueous Two-Phase System Using PEG4000/Sodium Citrate and PEG8000/Sodium Phosphate

Aqueous Two Phase System (ATPS) or liquid-liquid extraction is used in biotechnology to recover valuable compounds from raw sources. In Aqueous Two-Phase Systems, many factors influence the Partition coefficient, K, (which is the ratio of protein concentration in the top phase to that in the bottom phase) and the Recovery percentage (Rec%). In this research, two systems of ATPS were used: first, polyethylene glycol (PEG) 4000/Sodium citrate (SC), and the second, PEG8000/ Sodium phosphate (SPH), for the extraction of Bovine Serum Albumin (BSA). The behavior of Rec% and K of pure (BSA) in ATPS has been investigated throughout the study by the effects of five parameters: temperature, concentration of polyethylene glycol (PEG4000 and PEG8000), the concentration of Sodium citrate or Sodium phosphate, pH, and the addition of sodium chloride as a supporting agent. The recovery percentage of BSA and its partition coefficient are significantly influenced by these factors to various degrees. The most influential variable in this study is PEG concentration for both systems. In addition to the PEG concentration, the stabilizing impact of NaCl is a crucial factor. The interaction between biomolecules and PEG gets more hydrophobic as the PEG concentration is raised. In the first system (PEG4000/SC), the maximum recovery percentage and partition coefficient were 98.99% and 97.69, respectively, at 31°C, PEG4000 concentration 1.5g/10 ml, Sodium citrate concentration 2.7 g/10 ml, pH 10, and 0.5 M NaCl concentration. While in the second system (PEG8000/SPH), the maximum recovery percentage and partition coefficient was 98.93% and 92.12, respectively, at 31oC, PEG8000 concentration 1.5 g/10 ml, Sodium phosphate concentration 2.4 g/10 ml, pH 10, and concentration of NaCl 0.5 M.

Efficient preparation of hyperoside and quercitrin from Zanthoxylum bungeanum Maxim leaves using an integrated surfactant-based aqueous two-phase system, back-extraction and adsorption separation

Zanthoxylum bungeanum leaves (ZBL) are of great medicinal value for being rich in hyperoside and quercitrin. In this study, a novel, efficient and economical continuous process was established. First, aqueous two-phase system (ATPS) composed of Triton X-100/(NH4)2SO4 was employed to enrich hyperoside and quercitrin from ZBL extracts and the recoveries reached 98.53% and 99.12%. Then back-extraction with dichloromethane-water system was adopted to separate hyperoside and quercitrin from Triton X-100 micelles which were recycled and the recoveries reached 86.58% and 85.19%. Finally, S-8 macroporous resin was used for removing the salt introduced in ATPS and the final recoveries reached 82.38% and 81.81%, much higher than the total flavonoids recovery as 69.08%. Furthermore, scale-up experiment certified that the continuous process was feasible for industrial production. Efficiently and economically, this method achieved a great breakthrough in purity and provided a novel reference for further purification and phase-forming component recycle.

A sulfonated ligand-aqueous two-phase system for selective extraction of thorium from mining wastewater: Process optimization, structural characterization and mechanism exploration

As a potential nuclear fuel, the recovery of thorium with high efficiency, high selectivity and high extraction capacity from thorium-containing wastewater is considered to be one of the effective ways to reduce pollution and solve the shortage of thorium resources. However, the problem of efficient and selective separation of thorium from the complex environment has not been completely solved. Herein, a functional ligand called arsenazo (ASA), polyethylene glycol, and sodium sulphate were used to construct an aqueous two-phase system (ATPs) for the selectivity extraction and separation of thorium from mining wastewater. Furthermore, the complex agent, pH value, temperature, equilibrium time, coexisting substances, and other conditions for the extraction and separation of thorium were optimized, indicating that 98.7 % of thorium was efficiently and precisely separated from the acid-leaching solution to the pH of 2–3 or 5–6 with 0.08 g/L of ASA within 10 min, and the interference with multiple coexisting ions and organic substances was avoided. Importantly, the analysis of water quality parameters, thermogravimetry, infrared spectroscopy, and ultraviolet–visible spectroscopy revealed the coordination relationship between thorium ions and complex agents, indicating that the extraction of thorium in the aqueous two-phase system was a cation exchange mechanism. More importantly, SEM, TEM, EDX, and XRD verified that thorium was efficiently and selectively separated by a sulfone acid ligand-aqueous two-phase system, providing quantitative information on thorium-containing product distribution of relatively high accuracy and avoiding the interference with coexisting substance. Therefore, this work provided theoretical guidance on treating and reusing thorium in wastewater and enriched the content of functional ligand-aqueous two-phase extraction systems in green production technology.

Heavy metals removal from soil/sediments washing effluents via biocompatible aqueous two-phase systems

The generation of high amounts of soil/sediment washing solutions is one of the main concerns faced in ex situ treatment techniques, so in the present research, we have demonstrated the suitability of an organic salt like sodium potassium tartrate to efficiently promote phase segregation in a model soil washing solution containing biodegradable non-ionic-surfactants (Tween 80 and Tergitol 15S9) and heavy metals (copper, zinc, and cadmium), after a preliminary screening of the demixing effect of different inorganic and organic salts like ammonium nitrate, ammonium acetate and sodium potassium tartrate. The aqueous two-phase systems were characterized in depth at room temperature by defining the immiscibility region and correlating the experimental data with three empirical equations, and the results were discussed in the light of a thermodynamic function like the Gibbs free energy of hydration. Then, the applicability to extract the abovementioned metal ions was researched after TLs determination, observing high heavy metals extraction percentage (over 80 % in some cases). The extraction process was included in the soil washing process at real scale and it was simulated by means of the software SuperPro Designer, that makes up a valuable tool to ease the analysis of the technical viability of the proposed strategy.

Extraction of hydroxycinnamic acids (ferulic and p-coumaric) from rice straw alkaline black liquor using Pluronic F-127 for potential applications in the cosmetics industry

In the present study, ferulic (FA) and p-coumaric (pCA) acids were extracted from rice straw (Oryza sativa) alkaline black liquor by an aqueous two-phase system (ATPS) based on cloud point of surfactant/copolymer Pluronic F-127 (PF-127). An acid precipitation process was studied as a primary purification step of FA and pCA and next, an extraction of these hydroxycinnamic acids (HAs) using PF-127 in different concentrations (1.1–17.3% w/w) was performed. The results showed that in PF-127 concentration of 9.4% w/w, yield extractions of 75.8% for FA and 74.3% for pCA were achieved. In addition, FA and pCA were concentrated 2.3 and 2.5-fold, respectively, compared to their original concentrations in the liquor, resulting in a PF-127 gel containing 0.12% w/w HAs. This material will be further investigated for potential applications in the cosmetics industry as an alternative for obtaining high value-added products in a rice straw-based biorefinery. Data availabilityData will be made available on request.

Ultrasonic-assisted aqueous two-phase extraction of polysaccharides from the fruits of Ilex cornuta (Lindl. Et Paxt.): Process optimization, structural characterization and antioxidant activity study

The aqueous two-phase extraction process (APTE) was utilized for the extraction of polysaccharides from the fruits of Ilex cornuta (Lindl. et Paxt.) (PFIC). After screening and selection of isopropanol/ammonium sulfate as the aqueous two-phase system, various parameters of crude extract amount, ultrasonic time, extraction temperature and extraction time on the extraction process was investigated in detail and optimized by response surface methodology (RSM). Under the optimal conditions in which crude extract amount was 2.23 g, ultrasonic time was 17.5 min, extraction temperature was 69 °C and extraction time was 1.55 h, the yield of polysaccharides reached 50.87% which was close to the predicted value of 50.78%. Structural features of PFIC were investigated by GPC, GC–MS and FT-IR analysis. And the results indicated that PFIC with a molecular weight of 3.89 kDa was composed of monosaccharides including rhamnose (1.83%), arabinose (11.70%), xylose (3.30%), mannose (3.55%), glucose (61.88%) and galactose (17.74%). The bioactivity experiments showed that PFIC had strong scavenging ability on DPPH radical, fine scavenging ability on hydroxyl radical and certain scavenging ability on superoxide anion radical. Overall, these results suggested that PFIC exhibiting fine to good antioxidant capacities could be potentially developed as a natural antioxidant.

Fermentative valorisation of xylose-rich hemicellulosic hydrolysates from agricultural waste residues for lactic acid production under non-sterile conditions

Lactic acid (LA) is a platform chemical with diverse industrial applications. Presently, commercial production of LA is dominated by microbial fermentation using sugary or starch-based feedstocks. Research pursuits emphasizing towards sustainable production of LA using non-edible and renewable feedstocks have accelerated the use of lignocellulosic biomass (LCB). The present study focuses on the valorisation of xylose derived from sugarcane bagasse (SCB) and olive pits (OP) through hydrothermal and dilute acid pretreatment, respectively. The xylose-rich hydrolysate obtained was used for LA production by homo-fermentative and thermophilic Bacillus coagulans DSM2314 strain under non-sterile conditions. The fed-batch mode of fermentation resulted in maximum LA titers of 97.8, 52.4 and 61.3 g/L with a yield of 0.77, 0.66 and 0.71 g/g using pure xylose, xylose-rich SCB and OP hydrolysates, respectively. Further, a two-step aqueous two-phase system (ATPS) extraction technique was employed for the separation and recovery of LA accumulated on pure and crude xylose. The LA recovery was 45 – 65% in the first step and enhanced to 80–90% in the second step.The study demonstrated an efficient integrated biorefinery approach to valorising the xylose-rich stream for cost-effective LA production and recovery.

Characterization of polymer‐polymer aqueous two‐phase system droplets for 3D culture future applications

AbstractBACKGROUNDCell‐based high‐throughput platforms using aqueous two‐phase systems (ATPS) have led to the construction of 3D cultures using ATPS in which stem cells are confined into polymer–polymer droplets, giving rise to viable spheroids for their further proliferation and differentiation. This work aims to characterize polymer–polymer ATPS droplet generation based on diameter size, morphology and uniformity, using basic laboratory equipment.RESULTSDextran (DEX), including DEX 500 000 and 70 000 g mol−1, as well as UCON were used as the droplet phase (0.5, 1, 2 μL). Polyethylene glycol (PEG), including PEG 8 000, 10 000 and 35 000 g mol−1, as well as DEX 75 000 g mol−1 and Ficoll were used as the bulk phase (50, 100 μL). Different ATPS compositions (molecular weights and concentrations) with phosphate‐buffered saline as ATPS solvent and construction strategies (droplet added, immersed or covered) were evaluated in 96‐well plates. ATPS droplets were generated in PEG‐DEX ATPS. Diameter size and morphology were susceptible to ATPS compositions, construction strategies and DEX droplet phase volumes with no significant difference in PEG bulk phase volumes, whereas higher uniformity of ATPS droplets was correlated to lower PEG bulk volumes. Therefore, 1 μL DEX droplets immersed at the bottom of a well filled with 50 μL PEG resulted in uniform ATPS droplets with a mid‐range diameter of 1.35 ± 0.23 mm.CONCLUSIONSAccording to the results, this work demonstrates simplified and practical alternatives for the generation of reproducible PEG‐DEX ATPS droplets that could be adapted for the encapsulation of different cell types for further 3D culture applications. © 2023 Society of Chemical Industry (SCI).

Formation of Aqueous Two‐Phase Systems Using Potassium Salt and Deep Eutectic Mixtures for Extraction of Proteins

AbstractLiquid‐liquid extraction such as an aqueous two‐phase system (ATPS) is considered a promising method in the biotechnology field for extraction of biomolecules. The binary mixture of polyethylene glycol (PEG)/salt is the most common ATPS. The ability of eutectic mixture‐based ATPS to fractionate protein macromolecules remains not well understood. Here, the role of hydrogen bond donors (HBDs) in eutectic mixtures was investigated in relation to the phase formation ability and extraction efficiency of protein. Both potassium salt and an eutectic combination were used to prepare the ATPSs. Choline chloride/urea aqueous two‐phase separation proved to be the most effective method for protein extraction. The creation of a two‐phase system and the efficiency with which a protein can be extracted from a crude solution are both affected by the choice of HBD.

Liquid-liquid extraction of molybdenum(VI) using aqueous two-phase systems (ATPS) consisting of polyethylene glycol (PEG) and sodium, ammonium and copper(II) salts: Comparison with conventional solvent extraction

In this work, the stripping of molybdenum from aqueous two-phase aqueous systems (ATPS) formed by a mixture of polyethylene glycol (PEG) 4000, salt and water was studied at different operational conditions of pH (7, 8 and 9) and temperature (308.15, 323.15 and 338.15 K), using two molybdenum salts (Na2MoO4 and (NH4)6Mo7O24). These salts were used for the experimental tests of extraction and stripping of the two-phase aqueous system of PEG 4000, CuSO4 and water, to evaluate the best percentage of stripping using different stripping agents: NaOH, Na2CO3, CuSO4 and a mixture of (NH4)2SO4 and NH4OH (Khayati et al., 2011). According to the results, the best stripping agent was the 20% (NH4)2SO4 solution as phase former plus 25% NH4OH as pH modifier. The experimental results indicate that the increase in temperature is directly proportional to the percentage of stripping. For the system of PEG 4000, CuSO4, water, Na2MoO4, (NH4)2SO4 and NH4OH system (ATPS 1) the percentage of stripping was 96.3%. In the case of PEG 4000, CuSO4, (NH4)6Mo7O24, water (NH4)2SO4 and NH4OH (ATPS 2) the stripping was 99%, these being the maximum values of stripping at pH = 2 and T = 308.15 K.It was determined that for ATPS 1, as the pH increases the stripping efficiency tends to decrease, while as the pH increases in ATPS 2, the stripping efficiency increases; this behavior is attributed to the hydration energy of the ions. Finally, a pre-feasibility study of the studied processes, ATPS 1, ATPS 2 and traditional solvent extraction (SX), was carried out, which included calculation of the profit factors.

Optimization of production and partial purification of inulinase from Bacillussubtilis

The use of inulinase enzyme for production of high fructose syrups (HFS) found more attention during that last few decades due to the less efforts and cost required compared with the traditional two-step method. In the current study, a bacterial isolate have been selected for its potential inulinase productivity. It was identified using 16S rRNA as Bacillus subtilis Inu and given the accession number “OK444102″ in the database of NCBI. Statistical methods; Taguchi Orthogonal Array and Box-Behnken, have been applied for the improvement inulinase production and the data revealed maximum enzyme activity titer (70.8 U/ml) at 15.61 g/l, sucrose; 15.94 g/l, molasses; 5.2 g/l, peptone; 5.2, pH; 32.5 °C, incubation temperature and 6 days, incubation period). The produced enzyme was purified using aqueous two phase system (ATPS) composed of Poly ethylene glycol (PEG) conjugated with salt solution (PEG/salt). The two phases in the system were studied and the data revealed that PEG 6000 (15%)/Sodium sulfate (15%) was the most effective system in the partitioning inulinase enzyme by 13.5 purification fold and 285% yield recovery in the top PEG rich phase. The isolated Bacillus subtilis is an efficient strain for production of inulinase compared with the other conducted studies.

Extraction and preconcentration of vanadium from food samples using aqueous two-phase systems by a multivariate study

Vanadium plays essential and toxic roles in humans and in nature, depending on its concentration. Therefore, monitoring its concentration in food samples is important, as this analyte is usually found in trace amounts in these samples. In this context, this work proposes a new methodology for the determination of vanadium in food samples using an aqueous two-phase system formed by (NH4)2SO4 + PEG400 + H2O with the complexing agent Br-PADAP to extract and pre-concentrate the analyte in the top-phase with detection by atomic absorption spectrometry. Multivariate analysis using fractional factorial design was applied to define the significant variables and a Box-Behnken matrix was used to find the critical points for these variables, which defined as pH 3.60, an ultra-thermostatted bath time of 2.12 h and complexing agent concentration of 0.00810 100w. Under optimised extraction conditions, the method presented limits of detection and quantification of 0.0220 μg⸳kg-1 and 0.665 μg.kg-1, respectively; accuracy of 2.51% and enrichment factor of 19.1. The methodology was applied to certified reference samples of apple leaves (NIST1515) and food samples with recovery percentages values of 94.1% and 93.8-108%, respectively. Therefore, a novel, efficient and organic solvent-free methodology was developed for the determination of vanadium in food samples.

Liquid sculpture and curing of bio-inspired polyelectrolyte aqueous two-phase systems

Aqueous two-phase systems (ATPS) provide imperative interfaces and compartments in biology, but the sculpture and conversion of liquid structures to functional solids is challenging. Here, inspired by phase evolution of mussel foot proteins ATPS, we tackle this problem by designing poly(ionic liquids) capable of responsive condensation and phase-dependent curing. When mixed with poly(dimethyl diallyl ammonium chloride), the poly(ionic liquids) formed liquid condensates and ATPS, which were tuned into bicontinuous liquid phases under stirring. Selective, rapid curing of the poly(ionic liquids)-rich phase was facilitated under basic conditions (pH 11), leading to the liquid-to-gel conversion and structure sculpture, i.e., the evolution from ATPS to macroporous sponges featuring bead-and-string networks. This mechanism enabled the selective embedment of carbon nanotubes in the poly(ionic liquids)-rich phase, which showed exceptional stability in harsh conditions (10 wt% NaCl, 80 oC, 3 days) and high (2.5 kg/m2h) solar thermal desalination of concentrated salty water under 1-sun irradiation.

Probing emulsion-gel transition in aqueous two-phase systems stabilized by charged nanoparticles: A simple pathway to fabricate water-in-water emulsion-filled gels

Nanoparticle-stabilized water-in-water (w/w) emulsions, an example of aqueous two-phase systems (ATPS), can produce low-fat food colloids, edible gels, bio-polymer-based bijels, scaffolds for tissue-engineering, and porous materials. This w/w emulsion is made from two thermodynamically incompatible aqueous-polymer solutions, such as polyethylene oxide (PEO) and dextran. Changing the molecular weight of PEO in one aqueous phase stabilizes w/w emulsion. The state diagram we generated using electron and light microscopy provides a novel approach to producing stable emulsion-filled gels and emulsion droplets. The visual examination, brightfield, and fluorescent microscopy studies highlight the role of molecular weight and storage duration. The production of an emulsion-filled gel is ascribed to the “active-filler-particles” arrangement, typically seen when the affinity between droplets stabilized by particles and polymer is substantial. In contrast, we expect the “inactive-filler-particles” arrangement for the samples that undergo phase inversion. The temporal evolution of shear-induced structures recorded using a rheometer demonstrates that these emulsion-filled gels’ viscoelastic properties correspond directly with time, molecular weight, and polymer composition. The emulsion-filled gels generated displayed 90-day storage stability. Our work would help us understand the complex dynamics of w/w emulsion-based formulations that need suitable size, shape, appearance, and shelf life management.

Partial purification of crude lipase extract from Yarrowia lipolytica: Precipitation, aqueous two-phase systems (ATPS), and immobilization methods

Efforts have been concentrated on developing alternative methods of enzyme purification that are less costly and highly efficient. In this work, we evaluated three different methods for lipase purification from Yarrowia lipolytica, such as precipitation using ammonium sulfate, ethanol, or acetone; aqueous two-phase systems (ATPS) based on polyethylene glycol (PEG) and potassium phosphate; and direct immobilization. It was impossible to obtain stable precipitates of the enzyme due to the low concentration of total protein and the presence of biosurfactant produced by the microorganism. Different mixture compositions were selected for the partitioning study. Three ATPS showed selective partitioning of the target enzymes, i.e., lipase and protease migrated to opposite phases. In the ATPS composed of 13 wt% PEG-4000 and 10 wt% salts, it was possible to achieve a purification factor for lipase of 4.2. Purification by immobilization performed by lipase-lipase interactions showed three lipases of distinct sizes in the crude extract. In the immobilization method by hydrophobic supports, phenyl-agarose and butyl‑agarose were more selective in immobilizing than octyl-agarose. In the ion exchange immobilization method, only the lipases identified at 66 kDa and 41 kDa have an attraction for DEAE-agarose (anionic) and sulfopropyl-agarose (cationic) matrices.