Aqueous Two-phase Systems Research Articles

Aqueous biphasic systems containing sodium L-tartrate dihydrate and PEG, experimental and application of extended thermodynamic models

In the current study, liquid-liquid equilibrium data of aqueous two-phase systems (ATPS) containing polyethylene glycol at the different molecular weights (1000, 2000, 6000, and 8000) - sodium L-tartrate dihydrate - water at 298.15 K were obtained. Binodal curves of mentioned systems have been determined and the effect of the molecular weight of polymer on binodal curves, tie-lines, and two-phase region sizes was studied. The results showed that increasing the molecular weight of the polymer enhances the biphasic area of the system. Also, an extended UNIQUAC equation and an extended form of Virial expansion models as new models of activity coefficient were examined to predict the phase equilibria of mentioned systems. The fitted binary interaction parameters of the model were reported. Both investigated models were capable to correlate the VLE and LLE data of the pertinent binary and ternary systems successfully. It could be observed that the extended Virial model leads to a better presentation of the data in comparison to the extended UNIQUAC model.

Generation of polyethylene glycol-dextran aqueous two-phase system droplets using different culture media under in vitro conditions

Aqueous two-phase systems (ATPS) involve the combination of two immiscible polymers that at certain concentrations form a biphasic separation system. Due to the physicochemical properties and the low interfacial tension of the ATPS-forming polymers, the generation of ATPS droplets began to be explored. This work aims to characterize the generation of ATPS droplets evaluating four polyethylene glycol (PEG)-dextran (DEX) systems (combinations of molecular weights and concentrations) using Essential 8 (Gibco), DMEM-F12 (Gibco), StemLine II (Sigma-Aldrich), MSCs Basal (ATCC), RPMI 1640 (Gibco), and NutriStem (Sartorius) as ATPS solvents under in vitro conditions. In 96 well-plates, one DEX droplet of 1 μL was immersed at the bottom of the well filled with 50 μL of PEG bulk phase. ATPS droplets were generated in all tested PEG-DEX systems showing circular morphology with a mid-range diameter size in the six-culture media of 1.30 ± 0.18 mm, 1.30 ± 0.22 mm, 1.30 ± 0.43 mm, 1.21 ± 0.27 mm, 1.20 ± 0.16 mm, and 1.07 ± 0.21 mm, respectively. Uniformity was susceptible to the culture media composition showing a coefficient of variation range of 0.64–12.40 % among the four studied PEG-DEX systems. By controlling the geometry of ATPS droplets, specific cell patterns can be determined for the further construction of different cell microenvironments in 3D cultures using ATPS.

Aqueous Two-Phase Systems Based on Cationic and Anionic Surfactants Mixture for Rapid Extraction and Colorimetric Determination of Synthetic Food Dyes.

In this study, aqueous two-phase systems (ATPSs) containing a cationic and anionic surfactants mixture were used for the preconcentration of the synthetic food dyes Allura Red AC, Azorubine, Sunset Yellow, Tartrazine, and Fast Green FCF. A rapid, simple, low cost, affordable, and environmentally friendly methodology based on microextraction in ATPSs, followed by spectrophotometric/colorimetric determination of the dyes, is proposed. The ATPSs are formed in mixtures of benzethonium chloride (BztCl) and sodium N-lauroylsarcosinate (NaLS) or sodium dihexylsulfosuccinate (NaDHSS) under the molar ratio close to equimolar at the total surfactant concentration of 0.01-0.20 M. The density, viscosity, polarity, and water content in the surfactant-rich phases at an equimolar ratio BztCl:NaA were determined. The effects of pH, total surfactant concentration, dye concentration, and time of extraction/centrifugation were investigated, and the optimum conditions for the quantitative extraction of dyes were established. The smartphone-based colorimetric determination was employed directly in the extract without separating the aqueous phase. The analytical performance (calibration linearity, precision, limits of detection and quantification, reproducibility, and preconcentration factor) and comparison of the spectrophotometric and smartphone-based colorimetric determination of dyes were evaluated. The method was applied to the determination of dyes in food samples and food-processing industrial wastewater.

Extraction, structural characterization, and antioxidant activity of polysaccharides derived from Arctium lappa L.

Arctium lappa L. root has high nutritional and medicinal values and has been identified as a healthy food raw material by the Ministry of Health of the People's Republic of China. In the present study, an aqueous two-phase system (ATPS) of polyethylene glycol (PEG)-(NH4)2SO4 was used to extract Arctium lappa L. polysaccharides (ALPs) from the Arctium lappa L. roots, the optimal extraction conditions of crude ALPs were optimized by using the single-factor experiment and response surface methodology. The structure and composition of ALPs were determined by fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), and high-performance liquid chromatography (HPLC). At the same time, the antioxidant activity of ALPs was investigated by in vitro antioxidant experiment. The optimized extraction parameters for extraction ALPs were as follows: the PEG relative molecular weight of 6,000, a quality fraction of PEG 25%, a quality fraction of (NH4)2SO4 18%, and an extraction temperature of 80°C. Under these conditions, the extraction rate of ALPs could reach 28.83%. FTIR, SEM and HPLC results showed that ALPs were typical acidic heteropolysaccharides and had uneven particle size distribution, an irregular shape, and a rough surface. The ALPs were chiefly composed of glucose, rhamnose, arabinose, and galactose with a molar ratio of 70.19:10.95:11.16:6.90. In addition, the ALPs had intense antioxidant activity in vitro with IC50 values in the ·OH radical (1.732 mg/ml), DPPH radical (0.29 mg/ml), and superoxide anion (0.15 mg/ml) scavenging abilities. The results showed that ATPS was an efficient method to extract polysaccharides and could be used for the extraction of other polysaccharides. These results indicated that ALPs had great prospects as a functional food and could be exploited in multiple fields.

Multimodal peptide ligand extracts parvovirus from interface in affinity aqueous two-phase system.

Aqueous two-phase systems (ATPS) have found various applications in bioseparations and microencapsulation. The primary goal of this technique is to partition target biomolecules in a preferred phase, rich in one of the phase-forming components. However, there is a lack of understanding of biomolecule behavior at the interface between the two phases. Biomolecule partitioning behavior is studied using tie-lines (TL), where each TL is a group of systems at thermodynamic equilibrium. Across a TL, a system can either have a bulk PEG-rich phase with citrate-rich droplets, or the opposite can occur. We found that porcine parvovirus (PPV) was recovered at a higher amount when PEG was the bulk phase and citrate was in droplets and that the salt and PEG concentrations are high. To improve the recovery, A PEG 10kDa-peptide conjugate was formed using the multimodal WRW ligand. When WRW was present, less PPV was caught at the interface of the two-phase system, and more was recovered in the PEG-rich phase. While WRW did not significantly increase the PPV recovery in the high TL system, which was found earlier to be optimal for PPV recovery, the peptide did greatly enhance recovery at a lower TL. This lower TL has a lower viscosity and overall system PEG and citrate concentration. The results provide both a method to increase virus recovery in a lower viscosity system, as well as provide interesting thoughts into the interfacial phenomenon and how to recover virus in a phase and not at the interface.

Extraction, structural characterization, and biological activities of a new glucan from Codonopsis pilosula

In this study, a powerful and rapid aqueous two-phase system (ATPS) method was used to extract polysaccharides from Codonopsis pilosula. The ATPS process was investigated with response surface methodology (RSM). At an ammonium sulfate concentration of 17%, ethanol concentration of 30%, and extraction temperature of 40 °C at pH 6, the total extraction yield of polysaccharides reached (31.57 ± 1.28)%. After separation and purification, a homogenized polysaccharide CPP 2–4 with molecular weight of 3.9 × 104 kDa was obtained from the bottom phase. The physicochemical properties and structural features confirmed that CPP 2–4 was an α-1,6-glucan. Activity studies showed that the IC50 of CPP 2–4 for DPPH radical scavenging was 0.105 mg/mL. The FRAP and ABTS assays showed that CPP 2–4 had strong antioxidant activity in a dose-dependent manner. Furthermore, CPP 2–4 inhibited NO release in RAW264.7 cells induced by lipopolysaccharide, which indicated a certain anti-inflammatory effect. This study improved the extraction rate of polysaccharides from C. pilosula and identified a glucan for the first time, that can contribute to a better understanding of the composition and structure of polysaccharides from C. pilosula and provide data support for the medicine and food homology of C. pilosula.

Binodal Curves and Tie-Line Compositions for a Poly(ethylene glycol) 6000 + Lithium Citrate + Water Aqueous Two-Phase System and the Partitioning of Salbutamol Drug at T = 288.15–318.15 K

The partitioning of salbutamol drug in a poly(ethylene glycol) 6000 + lithium citrate + water system was investigated at T = 288.15, 298.15, 308.15, and 318.15 K. The binodal curves and tie-line compositions were determined at different temperatures, and the studied aqueous two-phase system (ATPS) was used to study the partitioning of salbutamol drug. The results showed that the ability of the system for two-phase formation increases at higher temperatures. The good performance of the empirical Merchuk equation was obtained to model the binodal curves. The tie-line compositions were also fitted using the Setschenow-type equation and electrolyte Wilson models. Furthermore, the ability of the studied ATPSs for the extraction of salbutamol is shown based on the obtained partitioning parameters, and the partitioning coefficient was increased from the value of ∼0.48 at T = 288.15 K to ∼1.7 at T = 318.15 K. Overall, the studied ATPS can be employed to extract salbutamol from aqueous medium in a range of biological temperatures.

Prediction and validation of monoclonal antibodies separation in aqueous two-phase system using molecular dynamic simulation

In order to predict how mAbs partition in 20% ethylene oxide/80% propylene oxide (v/v) random copolymer (EO20PO80)/water aqueous two-phase system (ATPS), a molecular dynamic simulation model was developed using Gromacs and then validated by experiments. The ATPS was applied with seven kinds of salt, including buffer salt and strong dissociation salt that were commonly employed in the purification of protein. Na2SO4 was shown to have the best effects on lowering EO20PO80 content in the aqueous phase and enhancing recovery. The content of EO20PO80 in the sample solution was decreased to 0.62%±0.25% and the recovery of rituximab increased to 97.88%±0.95% by adding 300 mM Na2SO4 into back extraction ATPS. The viability determined by ELISA was 95.57% at the same time. A strategy for constructing a prediction model for the distribution of mAbs in ATPS was proposed in consideration of this finding. Partition of trastuzumab in ATPS was predicted by the model created using this method and the prediction result was further validated by experiments. The recovery of trastuzumab reached 95.63%±2.86% under the ideal extraction conditions suggested by the prediction model.

Two-Step Purification and Partial Characterization of Keratinolytic Proteases from Feather Meal Bioconversion by Bacillus sp. P45

This study aimed to purify and partially characterize a keratinolytic protease produced by Bacillus sp. P45 through bioconversion of feather meal. Crude protease extract was purified using a sequence of an aqueous two-phase system (ATPS) in large volume systems (10, 50, and 500 g) to increase obtaining purified enzyme, followed by a diafiltration (DF) step. Purified protease was characterized in terms of protein profile analysis by SDS-PAGE, optimum temperature and pH, thermal deactivation kinetics at different temperatures and pH, and performance in the presence of several salts (NaCl, CaCl2, MnCl2, CaO, C8H5KO4, MgSO4, CuSO4, ZnSO4, and FeCl3) and organic solvents (acetone, ethanol, methanol, acetic acid, diethyl ether, and formaldehyde). ATPS with high capacities resulted in purer protease extract without compromising purity and yields, reaching a purification factor up to 2.6-fold and 6.7-fold in first and second ATPS, respectively, and 4.0-fold in the DF process. Recoveries were up to 79% in both ATPS and reached 84.3% after the DF step. The electrophoretic analysis demonstrated a 25–28 kDa band related to keratinolytic protease. The purified protease’s optimum temperature and pH were 55 °C and 7.5, respectively. The deactivation energy (Ed) value was 118.0 kJ/mol, while D (decimal reduction time) and z (temperature interval required to reduce the D value in one log cycle) values ranged from 6.7 to 237.3 min and from 13.6 to 18.8 °C, respectively. Salts such as CaCl2, CaO, C8H5KO4, and MgSO4 increased the protease activity, while all organic solvents caused its decrease. The results are useful for future studies about ATPS scale-up for enzyme purification and protease application in different industrial processes.

Novel Biorefinery Approach for Phycocyanin Extraction and Purification and Biocrude Production from Arthrospira platensis.

A new biorefinery from Arthrospira platensis was proposed to obtain phycocyanin (PC) and a biocrude by hydrothermal liquefaction (HTL). PC is a high-added-value phycobiliprotein widely used as a food colorant and in the nutraceutical and pharmaceutical industries. However, the use of conventional solvents in the extraction process and the purity grade of the extract are shortcomings in bioproduct production. PC was extracted using a reusable ionic liquid [EMIM][EtSO4], achieving a PC purity of the lowest commercial grade. Therefore, two downstream processes were applied: (1) dialysis + precipitation and (2) aqueous two-phase system (ATPS) + dialysis + precipitation. After the second purification process, the PC purity increased remarkably to reach the analytical grade for pharmaceutical and nutraceutical applications. The waste biomass (WB) obtained in the PC extraction was valorized by hydrothermal liquefaction (HTL) to produce a biocrude. The biocrude yield and composition remarkably enhanced using isopropanol at 350 °C as a cosolvent.

Extraction of Gallic Acid and Ferulic Acid for Application in Hair Supplements

Food supplements based on antioxidants and vitamins are often prescribed to correct inefficiencies in the human diet and delay diseases such as premature aging and alopecia (temporary or permanent hair loss), given the free radical scavenging activity of these biomolecules. By reducing the concentration of reactive oxygen species (ROS), which promote abnormal hair follicle cycling and morphology, follicle inflammation and oxidative stress are reduced, minimising the effects of these health issues. Gallic acid (GA), which is significantly present in gallnuts and in pomegranate root bark, and ferulic acid (FA), commonly found in brown rice and coffee seeds, are very important antioxidants for the preservation of hair colour, strength and growth. In this work, these two secondary phenolic metabolites were successfully extracted in the Aqueous Two-Phase Systems (ATPS) {ethyl lactate (1) + trisodium citrate (2) + water (3)} and {ethyl lactate (1) + tripotassium citrate (2) + water (3)} at 298.15 K and 0.1 MPa, moving towards the application of these ternary systems in extracting antioxidants from biowaste and their a posteriori processing as food supplements for hair fortification. The studied ATPS provided biocompatible and sustainable media for the extraction of gallic acid and ferulic acid, yielding low mass losses (<3%) and contributing to an eco-friendlier production of therapeutics. The most promising results were obtained for ferulic acid, which attained maximum partition coefficients (K) of 15 ± 5 and (3 ± 2) · 101 and maximum extraction efficiencies (E) of (92.7 ± 0.4)% and (96.7 ± 0.4)% for the longest tie-lines (TLL = 69.68 and 77.66 m%) in {ethyl lactate (1) + trisodium citrate (2) + water (3)} and {ethyl lactate (1) + tripotassium citrate (2) + water (3)}, respectively. Moreover, the effect of pH on the UV-Vis absorbance spectra was studied for all the biomolecules to minimise errors in solute quantification. Both GA and FA were found to be stable at the used extractive conditions.

Partitioning-Induced Isolation of Analyte and Analysis via Multiscaled Aqueous Two-Phase System.

Most fluorescence-based bioanalytical applications need labeling of analytes. Conventional labeling requires washing to remove the excess fluorescent labels and reduce the noise signals. These pretreatments are labor intensive and need specialized equipment, hindering portable applications in resource-limited areas. Herein, we use the aqueous two-phase system (ATPS) to realize the partitioning-induced isolation of labeled analytes from background signals without extra processing steps. ATPS is formed by mixing two polymers at sufficiently high concentrations. ATPS-based isolation is driven by intrinsic affinity differences between analytes and excess labels. To demonstrate the partitioning-induced isolation and analysis, fluorescein isothiocyanate (FITC) is selected as the interfering fluorophore, and a monoclonal antibody (IgG) is used as the analyte. To optimize ATPS compositions, different molecular weights and mass fractions of polyethylene glycol (PEG) and dextran and different phosphate-buffered saline (PBS) concentrations are investigated. Various operational scales of our approach are demonstrated, suggesting its compatibility with various bioanalytical applications. In centimeter-scale ATPS, the optimized distribution ratios of IgG and FITC are 91.682 and 0.998 using PEG 6000 Da and dextran 10,000 Da in 10 mM PBS. In millimeter-scale ATPS, the analyte is enriched to 6.067 fold using 15 wt % PEG 35,000 Da and 5 wt % dextran 500,000 Da in 10 mM PBS. In microscale ATPS, analyte dilutions are isolated into picoliter droplets, and the measured fluorescence intensities linearly correlated with the analyte concentrations (R2 = 0.982).

Aqueous Two‐Phase Enabled Low Viscosity 3D (LoV3D) Bioprinting of Living Matter (Adv. Sci. 8/2023)

Embedded Bioprinting In article number 2204609 by Jeroen Leijten and co-workers, liquid-in-liquid printing of low viscous solutions is enabled by the novel use of aqueous two-phase systems. Use of low viscous solutions allows for cytocompatible biofabrication of embedded sacrificial strands with an exceptionally large resolution range. The low viscous nature of bio-ink also allows for direct printing of intricate, interconnected, and perfusable networks within 3D structures.

Quantitative Analysis of Camellia oleifera Seed Saponins and Aqueous Two-Phase Extraction and Separation.

At present, the technology used for the extraction and purification of Camellia oleifera saponins generally has the problems of high cost and low purity, and the quantitative detection of Camellia oleifera saponins also has the problems of low sensitivity and easy interference from impurities. To solve these problems, this paper aimed to use liquid chromatography for the quantitative detection of Camellia oleifera saponins, and to adjust and optimize the related conditions. In our study, the average recovery of Camellia oleifera saponins obtained was 100.42%. The RSD of precision test was 0.41%. The RSD of the repeatability test was 0.22%. The detection limit of the liquid chromatography was 0.06 mg/L, and the quantification limit was 0.2 mg/L. In order to improve the yield and purity, the Camellia oleifera saponins were extracted from Camellia oleifera Abel. seed meal by methanol extraction. Then, the extracted Camellia oleifera saponins were extracted with an ammonium sulfate/propanol aqueous two-phase system. We optimized the purification process of formaldehyde extraction and aqueous two-phase extraction. Under the optimal purification process, the purity of Camellia oleifera saponins extracted by methanol was 36.15%, and the yield was 25.24%. The purity of Camellia oleifera saponins obtained by aqueous two-phase extraction was 83.72%. Thus, this study can provide a reference standard for rapid and efficient detection and analysis of Camellia oleifera saponins for industrial extraction and purification.

The Recent Advances in the Utility of Microbial Lipases: A Review

Lipases are versatile biocatalysts and are used in different bioconversion reactions. Microbial lipases are currently attracting a great amount of attention due to the rapid advancement of enzyme technology and its practical application in a variety of industrial processes. The current review provides updated information on the different sources of microbial lipases, such as fungi, bacteria, and yeast, their classical and modern purification techniques, including precipitation and chromatographic separation, the immunopurification technique, the reversed micellar system, aqueous two-phase system (ATPS), aqueous two-phase flotation (ATPF), and the use of microbial lipases in different industries, e.g., the food, textile, leather, cosmetics, paper, and detergent industries. Furthermore, the article provides a critical analysis of lipase-producing microbes, distinguished from the previously published reviews, and illustrates the use of lipases in biosensors, biodiesel production, and tea processing, and their role in bioremediation and racemization.

Performance of Organic Salts as Salting-Out Media on Recovering Nicotine from Water

Three degradable organic salts, sodium formate (NaCHO2), sodium succinate (Na2C4H4O4), and sodium citrate (Na3C6H5O7), were evaluated for the extraction of nicotine from aqueous solutions by forming two-phase systems. The detailed phase diagrams, including the binodal curves and tie-lines, were experimentally determined at 298.15 K and atmospheric pressure by the cloud point method. The three empirical equations including Merchuk, Pirdashti, and Hu models gave a perfect correlation of the experimental binodal data, and the reliability of liquid–liquid equilibrium data was evaluated using the Othmer–Tobias and Bancroft equations. It could be found that all adopted organic salts promoted the expansion of two-phase regions and had a high partition yield for nicotine extraction from water. The relative tendency of the anions to form aqueous two-phase systems being C6H5O73– > C4H4O42– > CHO2– was consistent with their valence order, which could be explained by the Gibbs hydration free-energy theory. Furthermore, the salting-out ability of salt anions was also in accordance with the parameter order (Na3C6H5O7 > Na2C4H4O4 > NaCHO2) of the calculated effective excluded volume. Increasing the temperature from 298.15 to 318.15 K and the pH value from 8.0 to 11.0 at 298.15 K was conducive to the two-phase formation of the ternary nicotine + Na3C6H5O7 + water system to increase the distribution coefficients and recovery percentages of nicotine. Computational thermodynamics of cloud point indicated that the more negative Gibbs free energy with temperature promoted the desolvation of nicotine to migrate preferentially to the top phase. It was concluded that degradable organic salts would be effective for nicotine extraction at an industrial scale.

Surfactant-Enhanced Extraction of Lutein from Marigold Petals using an Aqueous Two-Phase System

The extraction of lutein from marigold petals using a surfactant-based aqueous two-phase system is reported. In this work, the effectiveness of the hydrophilic-lipophilic balance of surfactants on extraction performance for the extraction of lutein from marigold petal powder was demonstrated using aqueous solutions of a wide range of non-ionic surfactants. The response surface methodology was applied to obtain optimised conditions for maximum extraction of lutein. At the optimised conditions (Temperature = 37.5 °C, S/L = 0.00375, and surfactant amount = 1.5% (v/v)), 12.12 ± 0.16 mg/g of lutein was obtained. Furthermore, the surface morphology of marigold petal powder (MPP) was analysed using SEM micrographs. Significant changes in surface morphology were observed which suggested better access of surfactant solution to the targeted biomolecule implanted in the matrix. Finally, the antioxidant activity of the obtained lutein extract was analysed using 2,2-diphenyl-1-picrylhydrazyl (DPPH). Results suggest that the antioxidant activity of the lutein extract obtained by the surfactant-based system is more than that of the lutein extract obtained by organic solvents. The aforementioned results suggest that the lutein can be extracted using a surfactant-based aqueous two-phase system (ATPS).

Autonomous Liquid–Liquid Extraction Operation in Biologics Manufacturing with Aid of a Digital Twin including Process Analytical Technology

Liquid–liquid extraction has proven to be an aid in biologics manufacturing for cell and component separation. Because distribution coefficients and separation factors can be appropriately adjusted via phase screening, especially in aqueous two-phase systems, one stage is frequently feasible. For biologics separation, aqueous two-phase systems have proven to be feasible and efficient. The simple mixer–settler equipment type is still not standard in biologics manufacturing operations. Therefore, a scalable digital twin would be of aid for operator training, process design under the regulatory demanded quality by design approach for risk analysis, design and control space definition, and predictive maintenance. Autonomous operation is achieved with the aid of process analytical technology to update the digital twin to real time events and to allow process control near any optimal operation point. Autonomous operation is first demonstrated with an experimental feasibility study based on an industrial type example of pDNA manufacturing via lysis from E. coli with and without cell separation performance.

Cleaning Phenolic Compounds Present in Water Using Salting-Out Effect with DCA-Based Ionic Liquids

Water is an essential natural resource, and its contamination is an important issue at present. This study aimed to increase the techniques that can be used to clean and reuse industrial wastewater by studying the feasibility of an aqueous two-phase system to eliminate phenolic compounds from aqueous systems. The system was prepared using two hydrophilic ionic liquids based on dicyanamide anion, 1-ethyl-3-methylimidazlium dicyanamide [EMim] [DCA], and 1-butyl-3-methylimidazolium dicyanamide [BMim][DCA], and one inorganic salt, K3PO4, at three different concentrations (20, 30, and 40%). The process was tested for the removal of phenol, o-cresol, 2-chlorophenol, and a mixture of them (PCM) at initial concentrations from 0.003 to 15 g·L−1 in water. The extraction efficiencies for all the studied systems were calculated. The influence of the structure of the cation, the concentration of salt, and the initial concentration of the extracted compounds in the extraction yields were recorded. In general, the obtained results were high for all the studied systems, with extraction efficiencies of more than 90% representing the highest removal of the 2-chlorophenol compound using [EMim] [DCA] at the highest concentration of salt.

Intensification of endo-1,4-Xylanase Extraction by Coupling Microextractors and Aqueous Two-Phase System

The extraction of xylanase was performed using an aqueous two-phase system (ATPS) based on polyethylene glycol (PEG1540) and various salts. Preliminary studies in a batch extractor showed that the highest extraction efficiency, E = 79.63 ± 5.21%, and purification factor, PF = 1.26 ± 0.25, were obtained with sodium citrate dihydrate-H2O-PEG1540-based ATPS for an extraction time of 10 min. The process was optimized using the experimental Box-Behnken design at three levels with three factors: extraction time (t), xylanase concentration (γ), and mass fraction of PEG in the ATPS (wPEG). Under optimal process conditions (γ = 0.3 mg/mL, wPEG = 0.21 w/w, and t = 15 min), E = 99.13 ± 1.20% and PF = 6.49 ± 0.05 were achieved. In order to intensify the process, the extraction was performed continuously in microextractors at optimal process conditions. The influence of residence time, different feeding strategies, and channel diameter on extraction efficiency and purification factor was further examined. Similar results were obtained in the microextractor for a residence time of τ = 1.03 min (E = 99.59 ± 1.22% and PF = 6.61 ± 0.07) as in the experiment carried out under optimal conditions in the batch extractor. In addition, a batch extractor and a continuous microextractor were used for the extraction of raw xylanase produced by Thermomyces lanuginosus on solid supports.