Aqueous Micellar Systems Research Articles

Effect of electrolytes as adjuvants in GFP and LPS partitioning on aqueous two-phase systems: 2. Nonionic micellar systems

Abstract Lipopolysaccharide endotoxins (LPS) are the most common contaminant pyrogenic compounds found in intracellular recombinant biomolecules purified from Gram-negative bacteria, such as Escherichia coli. Thus, the purification downstream processing should guarantee the effective removal of LPS from the final bioproduct, particularly, therapeutic biopharmaceuticals. Aqueous two-phase micellar systems (ATPMS) appear to be an excellent strategy to purify recombinant biopharmaceuticals from the cell lysate of E. coli, reducing high LPS concentrations. In order to demonstrate the effectiveness of ATPMS as a biopharmaceutical purification platform, the influence of inorganic salt electrolytes (NaCl, Li2SO4, KI, or KNO3) on the partitioning of green fluorescent protein (GFP) and LPS removal using ATPMS composed of n-decyl tetraethylene oxide (C10E4) was evaluated. The impact of different LPS concentrations on GFP partitioning was also studied. The addition of electrolytes (i.e., NaCl or Li2SO4) to the C10E4-based ATPMS have reduced the phase forming temperatures to very mild conditions (ca. 17.00 and 13.00 °C, for NaCl and Li2SO4, respectively). The selective partitioning ability of the proposed ATPMS was further demonstrated, where a complete removal of the LPS from the micelle-poor phase (REMLPS > 98%) and a preferential GFP recovery (RECGFP = 97%, KGFP > 7) to the micelle-poor phase was obtained. The GFP partitioning was even enhanced by increasing LPS loading (104–106 EU/mL), probably due to the formation of mixed micelles between LPS and C10E4. It is here demonstrated that a C10E4/buffer + salt-based ATPMS can be a useful and straightforward platform for the removal of endotoxin contaminants and the purification of recombinant biopharmaceuticals from E. coli cell lysates.

Direct recovery of mangostins from Garcinia mangostana pericarps using cellulase-assisted aqueous micellar biphasic system with recyclable surfactant

The α- and γ-mangostins from Garcinia mangostana pericarps (GMP) exhibit antioxidant, anti-bacterial, anti-inflammatory and anti-tumor properties. The extraction yields α- and γ-mangostins are often limited by the presence of the GMP cell walls. Therefore, the extraction and recovery of mangostins from GMP with an Aspergillus niger cellulase-assisted aqueous micellar biphasic system (CA-AMBS) was developed for enhanced yield of mangostins. Effects of the concentration of cellulase, the incubation time and the temperature of the system on the recovery of mangostins were investigated. The optimum condition for the recovery of α- and γ-mangostins was obtained with the addition of 0.5% (w/w) cellulase incubated at 40°C for 2h. High log partition coefficients of α-mangostins (log Kα 4.79±0.02) and γ-mangostins (log Kγ 4.02±0.02) were achieved. High yields of α-mangostins (73.4%) and γ-mangostins (14.0%) were obtained from the micelle-rich bottom phase with final concentrations of 3.67mg/mL and 0.70mg/mL, respectively. The back-extraction of mangostins was performed with the addition of 30% (w/w) of isopropanol and 0.05M of KCl at pH 9 to the bottom phase of the CA-AMBS. The yields of the α- and γ-mangostins from GMP were considerably enhanced with the CA-AMBS and the direct recovery of mangostins was demonstrated without additional downstream processing steps.

Solubility Enhancement of Polycyclic Aromatic Hydrocarbons by an Eco-Friendly Ester-Linked Gemini Surfactant and its Mixtures with Conventional Surfactants

AbstractThe present study deals with the solubility enhancement of the two polycyclic aromatic hydrocarbons (PAHs) anthracene and pyrene in the aqueous micellar system of the cationic ester-containing cleavable gemini surfactant ethane-1,2-diyl-bis(N,N-dimethyl-N-tetradecylammoniumacetoxy) dichloride (14-E2-14 = C14H29(CH3)2N+(CH2COOCH2)2N+(CH3)2C14H29 · 2Cl−)), and its equimolar binary mixtures with some typical conventional cationic, anionic and non-ionic surfactants. The surface tension and conductivity measurements were used to evaluate the physicochemical parameters such as the critical micelle concentration (CMC), the interaction parameter (βm) and Gibbs excess free energy of micellization (ΔGexm) of the systems. The extent of solubilization of the micellar systems towards PAHs has been quantified in terms of molar solublization ratio (MSR), micellar/water partition coefficient (ln Km) and the standard Gibbs free energy of solubilization (ΔGs0). Above the CMC, all studied single as well as binary gemini-conventional surfactant systems show an increase in solubilization of the PAHs. For pure systems, the MSR value of Brij 58 was found to be significantly higher than that of the other amphiphiles. Amongst the mixed surfactant systems, the solubility enhancement of anthracene is found to be maximum in the 14-E2-14 + SDS/SDBS system whereas the system14-E2-14 + Brij 58 shows a higher solubility for pyrene.

An insight into effect of micelle-forming surfactants on aqueous solubilization and octanol/water partition coefficient of the drugs gemfibrozil and ibuprofen

Aqueous micellar solubilization of ibuprofen and gemfibrozil drugs was investigated at constant temperature (25.0±0.1) °C and atmospheric pressure using shake-flask and UV–vis spectrophotometric techniques. The solubility measurement was done in presence of three surfactants possessing different head groups, namely sodium dodecylsulfate (anionic SDS), cethyltrimethylammonium bromide (cationic CTAB) and polyethylene glycol dodecyl ether (non-ionic Brij 35) as well as their binary mixtures. The micellar solubility descriptors of χ (molar solubilization capacity), K (micelle–water partition coefficient), and ΔGs∘ (standard free energy of solubilization) were obtained to estimate quantitatively the solubilization efficiency of the surfactant systems. Moreover, the octanol-water partition coefficient of these drugs was also evaluated in the micellar solutions. The results obtained in the aqueous micellar system demonstrate that, irrespective of the surfactant type, the solubility of drugs increases with increasing the concentration of micelles. It was found that the solubility of ibuprofen and gemfibrozil in Brij 35 and CTAB solutions, respectively is higher compared to that in the other surfactants. However, the partition coefficients of these drugs show an inverse trend with solubility data. Finally, the effect of the surfactant type on solubility enhancement of the drugs is explained in terms of possible interactions between the drug and the micelle.

Aqueous micellar two-phase system as an alternative method to selectively remove soy antinutritional factors

In this work, different antinutritional factors (trypsin inhibitors, isoflavones and raffinose family oligosaccharides) were selectively removed from soy flour by using aqueous micellar two-phase systems (AMTPS). The effects of independent variables including temperature (30–60 °C), time (10–40 min) and solid to liquid ratio (0.025–0.050 g/L) on the extraction of each antinutritional factor were analyzed using a full factorial design. As general tendency, temperature and time were the most significant parameters (p < 0.05). The best condition for the selective recovery (97% of isoflavones at top phase, and more than 50% of the rest of ANFs at bottom phase) were 5 g/L of Genapol X-080, 0.2 moL/L of sodium citrate pH 5.00, 30 °C, 40 min and 0.050 g/L. Besides, in vitro gastrointestinal digestions assays demonstrated that the treated soy flour improved its protein digestibility. The findings of this work represent the introduction of a novel methodology to selectively remove soy antinutritional factors.

Application of aqueous two-phase micellar system to improve extraction of adenoviral particles from cell lysate.

Viral vectors are important in medical approaches, such as disease prevention and gene therapy, and their production depends on efficient prepurification steps. In the present study, an aqueous two-phase micellar system (ATPMS) was evaluated to extract human adenovirus type 5 particles from a cell lysate. Adenovirus was cultured in human embryonic kidney 293 (HEK-293) cells to a concentration of 1.4 × 1010 particles/mL. Cells were lysed, and the system formed by direct addition of Triton X-114 in a 23 full factorial design with center points. The systems were formed with Triton X-114 at a final concentration of 1.0, 6.0, and 11.0% (w/w), cell lysate pH of 6.0, 6.5, and 7.0, and incubation temperatures at 33, 35, and 37°C. Adenovirus particles recovered from partition phases were measured by qPCR. The best system condition was with 11.0% (w/w) of Triton X-114, a cell lysate pH of 7.0, and an incubation temperature at 33°C, yielding 3.51 × 1010 adenovirus particles/mL, which increased the initial adenovirus particles concentration by 2.3-fold, purifying it by 2.2-fold from the cell lysate, and removing cell debris. In conclusion, these results demonstrated that the use of an aqueous two-phase micellar system in the early steps of downstream processing could improve viral particle extraction from cultured cells while integrating clarification, concentration, and prepurification steps.

Simultaneous Extraction, Enrichment and Removal of Dyes from Aqueous Solutions Using a Magnetic Aqueous Micellar Two-Phase System

The magnetic aqueous micellar two-phase system (MAMTPS) has the advantages combined of magnetic solid phase extraction (MSPE) and aqueous micellar two-phase system (AMTPS). Thus, MAMTPS based on Fe3O4 magnetic nanoparticles (MNPs) and a nonionic surfactant Triton X-114 (TX-114) was developed for the extraction, enrichment and removal of three dyes (Congo red, methyl blue, and methyl violet) from aqueous solutions in this study. The MNPs Fe3O4@NH2 was screened as the optimal MNPs benefiting the extraction. Then, the influencing factors of MNPs amount, TX-114 concentration, vibration time, and extraction temperature were investigated in detail. The results showed that the extraction efficiencies of three dyes almost reached 100% using MAMTPS under the optimal conditions; MAMTPS had higher extraction ability than the individual MSPE or AMTPS. Thus, MAMTPS had the advantages of simple operation, high extraction ability, easy recycling of MNPs, and short phase-separation time, which showspotential for use in the extraction and analysis of contaminants from water samples.

Oil-in-Water Emulsion Templated and Crystallization-Driven Self-Assembly Formation of Poly(l-lactide)-Polyoxyethylene-Poly(l-lactide) Fibers.

A molecular solution of an amphiphilic block copolymer may act as an oil phase by dispersing into an aqueous micellar system of small-molecular surfactant, forming oil-in-water (O/W) emulsion droplets. In this paper, an as-synthesized triblock copolymer poly(l-lactide)-polyoxyethylene-poly(l-lactide) (PLLA-PEO-PLLA) was dissolved in tetrahydrofuran (THF) and then added to an aqueous micellar solution of nonaethylene glycol monododecyl ether (AEO-9), forming initially coalescent O/W emulsion droplets in the size range of 35 nm-1.3 μm. Along with gradual volatilization of THF and simultaneous concentration of PLLA-PEO-PLLA molecules, the amphiphilic copolymer backbones themselves experience solution-based self-assembly, forming inverted core-corona aggregates within an oil-phase domain. Anisotropic coalescence of adjacent O/W emulsion droplets occurs, accompanied by further volatilization of THF. The hydrophilic block crystallization of core-forming PEOs and the hydrophobic chain stretch of corona-forming PLLAs together induce the intermediate formation of rod-like architectures with an average diameter of 300-800 nm, and this leads to a large-scale deposition of the triblock copolymer fibers with an average diameter of ∼2.0 μm. Consequently, this strategy could be of general interest in the self-assembly formation of amphiphilic block copolymer fibers and could also provide access to aqueous solution crystallization of hydrophilic segments of these copolymers.

Impact of Surface Active Ionic Liquids on the Cloud Points of Nonionic Surfactants and the Formation of Aqueous Micellar Two-Phase Systems.

Aqueous micellar two-phase systems (AMTPS) hold a large potential for cloud point extraction of biomolecules but are yet poorly studied and characterized, with few phase diagrams reported for these systems, hence limiting their use in extraction processes. This work reports a systematic investigation of the effect of different surface-active ionic liquids (SAILs)-covering a wide range of molecular properties-upon the clouding behavior of three nonionic Tergitol surfactants. Two different effects of the SAILs on the cloud points and mixed micelle size have been observed: ILs with a more hydrophilic character and lower critical packing parameter (CPP < 1/2) lead to the formation of smaller micelles and concomitantly increase the cloud points; in contrast, ILs with a more hydrophobic character and higher CPP (CPP ≥ 1) induce significant micellar growth and a decrease in the cloud points. The latter effect is particularly interesting and unusual for it was accepted that cloud point reduction is only induced by inorganic salts. The effects of nonionic surfactant concentration, SAIL concentration, pH, and micelle ζ potential are also studied and rationalized.

Imidazolium-based ionic liquids as co-surfactants in aqueous micellar two-phase systems composed of nonionic surfactants and their aptitude for recovery of natural colorants from fermented broth

Over the last few years, synthetic colorants have been increasingly replaced by natural alternatives that cause fewer adverse health effects and can show biological activity. These biocompounds can be produced by fermentative processes using microorganisms such as bacteria and filamentous fungi. However, the biocompounds must be extracted from the fermented broth before industrial application. Thus, the development of effective techniques for the extraction and purification of biocolorants with high recovery is of great interest. The main goal of this study was to evaluate the recovery of natural colorants produced by Talaromyces amestolkiae using aqueous micellar two-phase system (AMTPS) composed of Triton X-114 and Ionic Liquids (ILs) from the imidazolium family as co-surfactants. Preliminary experiments were performed to determine the binodal curves in both presence and absence of fermented broth containing the red colorants. Subsequently, a set of partitioning experiments was performed using the developed systems. Under all conditions, the red colorants were partitioned mainly into the micelle-rich phase. The systems with low concentrations of Triton X-114 and ILs showed high partition coefficients. The system (0.1 wt% [C10mim]Cl + 3 wt% Triton X-114) with the highest partition coefficient (KRC = 6.12) was used to study the effect of temperature on partitioning. The partitioning of the red colorants was strongly influenced by the temperature, and KRC increased to 24.71 at 45 °C. The results of this study demonstrate that the partitioning of biomolecules can be controlled by the presence of ILs, mainly by electrostatic interactions, or by the proper adjustment of the partitioning temperature.

An affinity-based aqueous two-phase mixed micellar system and its purification of Yeast 3',5'-bisphosphate nucleotidase.

Aqueous two-phase micellar system (ATPMS), as an alternative liquid-liquid extraction technique, has been extensively exploited for the precise separation or large-scale concentration of biomolecules. In this article, a novel affinity-based ATPMS composed of mixed micelles was constructed by introducing a Copper-chelated Triton X-114 (TX-Cu(II)) into an aqueous solution of hydrophobically modified ethylene oxide polymer (HM-EO). Phase diagram of the HM-EO/TX-Cu(II) system was measured, and the partitioning behavior of model proteins (YND, BSA, lysozyme) were studied by using this new system. The addition of HM-EO can result in formation of the micellar network in the micelle-rich phase, making the phase separation easier and stabler. In addition, the extractive performance of ATPMS was enhanced due to the existence of the mixed micelles composed by HM-EO and Cu(II)-chelated TX. It was found in the partitioning experiments that the hexahistidine-tagged Yeast 3',5'-bisphosphate nucleotidase (YND) was selectively extracted into the micelle-rich phase, while the histidine-poor proteins (BSA and lysozyme) remained in the micelle-poor phase. Finally, HM-EO/TX-Cu(II) was used directly to process the fermentation broth. The target protein, YND could be recovered from the cell lysate with a recovery yield of 49.23% and purification factor of 2.63. The results indicated that the new affinity-based HM-EO/TX-Cu(II) system had high partitioning performance which is promising for effectively separation of the histidine-tagged proteins.

Recovery of mangostins from Garcinia mangostana peels with an aqueous micellar biphasic system

Methods of extraction and separation of antioxidants from plant sources by chromatography and solvent extraction are often time and solvent consuming, and result in low yields. A simple and mild thermo-induced aqueous micellar biphasic system (AMBS) was used to recover α- and γ-mangostins from Garcinia managostana peels. AMBS composed of non-ionic surfactants such as Triton X-114, Triton X-45, and Pluronic triblock copolymers were developed. Effects of the types of the non-ionic surfactant, its concentration, sample load, pH of the AMBS and the equilibration temperature on recovery of mangostins were studied. The optimum condition for recovery of α- and γ-mangostins was achieved with an AMBS composed of 3% (w/w) Pluronic L-81 and a sample loading of 0.5% (w/w) crude peels. At equilibration temperature of 40°C and pH 8, 2646.6±1.2μg/mL of α-mangostin and 379.4±1.2μg/mL of γ-mangostin were recovered in the micelle-rich phase of the AMBS with a log partition coefficient of α- (logKα) and γ-mangostin (logKγ) of 4.00±0.02 and 3.36±0.01, respectively. α- and γ-mangostins could be recovered simultaneously with a yield of 52.93% and 7.59%, respectively in a single-step. This study demonstrated that AMBS can serve as a tool to efficiently recover and separate various bioactive compounds from plant sources.

ChemInform Abstract: Multicomponent Cascade Synthesis of Biaryl‐Based Chalcones in Pure Water and in an Aqueous Micellar Environment.

The challenging multicomponent cascade synthesis of biaryl-based chalcones was carried out in pure water and in an aqueous micellar system. The first step of the protocol was a simple Pd-catalysed, ligand-free, and aerobic Suzuki–Miyaura reaction in aqueous medium. This proved to be extremely efficient for the coupling of aryl and heteroaryl bromides with different arylboronic acids. Subsequently, the resulting intermediates underwent an in-situ aldol condensation reaction to give biaryl(hetero)chalcones in good to excellent yields. When the protocol was applied to highly lipophilic or less reactive reagents, micellar catalysis was required for good results. To achieve this, we successfully used a new surfactant obtained from renewable resources that we recently designed. Furthermore, using this additive, the catalytic system can be repeatedly recycled without significant loss of activity.

Liquid–liquid extraction of antimicrobial peptide P34 by aqueous two-phase and micellar systems

ABSTRACTAntimicrobial peptide P34 is a promising biopreservative for utilization in the food industry. In this work, aqueous biphasic systems (ABS) and aqueous biphasic micellar systems (ABMS) were studied as prestep for purification of peptide P34. The ABS was prepared with polyethylene glycol (PEG) and inorganic salts and the ABMS with Triton X-114 was chosen as the phase-forming surfactant. Results indicate that peptide P34 partitions preferentially to PEG-rich phase and extraction with ammonium sulfate [(NH4)2SO4], yielding a 75% recovery of the antimicrobial activity, specific activity of 1,530 antimicrobial units per mg of protein, and purification fold of 2.48. Protein partition coefficient and partition coefficient for the biological activity with (NH4)2SO4 system were 0.48 and 64, respectively. Addition of sodium chloride did not affect recovery, but decreased protein amount in the PEG-rich phase, indicating a higher partition of biomolecules. ABMS did not yield good recovery of antimicrobial activity. Purification fold using PEG–(NH4)2SO4 and 1.0 mol l−1 sodium chloride was twice higher than that obtained by conventional protocol, indicating a successful utilization of ABS as a step for purification of peptide P34.

Effect of excluded-volume and hydrophobic interactions on the partition of proteins in aqueous micellar two-phase systems composed of polymer and nonionic surfactant

The partitioning behaviors of several model proteins were investigated in aqueous micellar two-phase systems (AMTPS) composed of polyethylene glycol (PEG) with different molecular weight (4,000, 8000 and 20,000) and nonionic surfactant (TX-100), respectively. The influences of proteins molecular size or hydrophobicity, tie line length (TLL), compositions concentrations and PEG molecular weight on the partition coefficients of proteins were extensively studied. In order to evaluate the contributions of excluded-volume interaction and hydrophobic interaction to proteins partitioning in PEG/TX-100 AMTPS in detail, firstly, the various proteins partition coefficients in PEG/Na2SO4 aqueous two-phase systems (ATPS) and TX-100/Na2SO4 AMTPS were investigated for the cross-sectional radius fitting in PEG-rich phase and TX-100-rich phase, respectively. The cross-sectional radius fitting results embodied that the order of cross-sectional radius in various phases are PEG4,000-rich phase, PEG8,000-rich phase, TX-100-rich phase and PEG20,000-rich phase. Secondly, the quantitative assessment of excluded-volume and hydrophobic contributions to proteins partitioning in PEG/TX-100 AMTPS were further investigated using the cross-sectional radius in PEG-rich phase and TX-100 phase, respectively. It was indicated that the excluded-volume interaction was stronger than hydrophobic interaction for proteins partitioning in PEG/TX-100 AMTPS. Finally, the effect of hydrophobic interaction on proteins partitioning was also analyzed in detail. The intrinsic hydrophobicity (P0) and the hydrophobic resolution (R) of PEG/TX-100 AMTPS with various TLL were assessed according to the model proteins partitioning and proteins' surface hydrophobicity. For proteins partitioning in PEG/TX-100 AMTPS, the molecular weight of PEG played an important role in excluded-volume interaction, not in hydrophobic interaction.

Integrated extraction and purification of soy isoflavones by using aqueous micellar systems

In this work, an integration of solid-liquid and liquid-liquid extractions by using aqueous micellar two-phase systems was evaluated as potential tool to purify soy isoflavones. Additionally, the proposed methodology aimed to preserve the protein content of the processed soy flour. The extractive assays were performed in AMTPS formed by Triton X-114 and sodium tartrate. In order to optimize the purification process, temperature and time were evaluated as independent variables. Under optimal working conditions, i.e. 100min and 33°C of incubation, IF were purified with a recovery percentage of 93 and a purification factor of almost 10. More importantly, the obtained sample presented an aglycone proportion superior to the reported by other methodologies. These results open perspectives to the use of aqueous micellar two-phase systems as an integrative methodology to extract, concentrate and purify isoflavones.

Prostaglandin H synthase kinetics in the two-phase aqueous-micellar system

Reaction mixture for PGHS (prostaglandin-H-synthase) is a two-phase system including micellar hydrophobic phase and hydrophilic aqueous phase. Reagents added to the mixture are distributed between phases, thus concentrations of reagents dissolved in phases can differ significantly from their overall contents.Using dynamic light scattering we found that the hydrophobic phase produced by tween-20 consists of micelles, which radius (4–5nm) does not depend on either tween-20 overall content (0.1%–1% v/v) or arachidonic acid (AA) addition (10–1000μM) or PGHS addition (1μM).Tween-20 overall content changing from 0.1% to 2% v/v dramatically affected COX kinetic, but accounting AA distribution between phases allowed us to estimate “true” parameters, independent of the tween-20 overall content and the concentration of another substrate: KMOx equals 9.8μM O2 in the aqueous phase or 0.0074bar in the gaseous phase, KMAA equals 5400μM AA in the phase of tween-20 micelles and 5400/PμM AA in the aqueous phase (P is the distribution ratio for the AA between the aqueous phase and the hydrophobic phase (P≫1000)). This approach allowed to evaluate PS, the distribution ratio for the AA between the hydrophobic phase and the PGHS active center (PS ~310). This coefficient indicates the AA selectivity toward the cyclooxygenase active center.

Multicomponent Cascade Synthesis of Biaryl‐Based Chalcones in Pure Water and in an Aqueous Micellar Environment

The challenging multicomponent cascade synthesis of biaryl‐based chalcones was carried out in pure water and in an aqueous micellar system. The first step of the protocol was a simple Pd‐catalysed, ligand‐free, and aerobic Suzuki–Miyaura reaction in aqueous medium. This proved to be extremely efficient for the coupling of aryl and heteroaryl bromides with different arylboronic acids. Subsequently, the resulting intermediates underwent an in‐situ aldol condensation reaction to give biaryl(hetero)chalcones in good to excellent yields. When the protocol was applied to highly lipophilic or less reactive reagents, micellar catalysis was required for good results. To achieve this, we successfully used a new surfactant obtained from renewable resources that we recently designed. Furthermore, using this additive, the catalytic system can be repeatedly recycled without significant loss of activity.

A novel aqueous micellar two-phase system composed of surfactant and mannitol for purification of polygalacturonase enzyme from Durio zibethinus Murray and recycling phase components

ABSTRACTA novel aqueous micellar two-phase system composed of surfactant and mannitol was employed for the first time to purify polygalacturonase from Durio zibethinus Murray. The influences of different parameters such as type of surfactants, composition of surfactant/mannitol, pH and crude load on polygalacturonase partitioning were investigated. The results indicated that the polygalacturonase was recovered in 25% Tween 80 and 22% mannitol, at 68.2% of Tie line length, crude load of 30% at pH 5.0. The enzyme was successfully recovered with a high purification factor of 16.1 and yield of 97.3% while phase components were also recovered and recycled above 95%.

Recovery of bromelain from pineapple stem residues using aqueous micellar two-phase systems with ionic liquids as co-surfactants

Pineapple processing industries produce a large amount of residues; for example, the pineapple stem, which is rich in bromelain. Bromelain is a group of cysteine proteinases that have major importance in the pharmaceutical field due to, among others, their anti-inflammatory, antithrombotic, and fibrinolytic activities. Since Brazil is one of the largest producers of pineapples in the world, the development of an ecofriendly and cost-effective process to extract and purify bromelain from food/fruit residues would be significant. In this study, aqueous micellar two-phase systems composed of ionic liquids as co-surfactants were evaluated in the extraction of bromelain from the pineapple stem crude extract. The main results showed that bromelain partitions preferentially toward the micelle-poor phase, with enzyme recoveries above 90% for the studied systems. Moreover, a stabilizing effect of the ionic liquids toward the enzyme was observed.