Extraction of Candida antarctica lipase A from aqueous solutions using imidazolium-based ionic liquids

Extraction of Candida antarctica lipase A from aqueous solutions using imidazolium-based ionic liquids

Separation and Purification Technology 97 (2012) 205–210 Contents lists available at SciVerse ScienceDirect Separation and Purification Technology jou...

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Separation and Purification Technology 97 (2012) 205–210

Contents lists available at SciVerse ScienceDirect

Separation and Purification Technology journal homepage: www.elsevier.com/locate/seppur

Extraction of Candida antarctica lipase A from aqueous solutions using imidazolium-based ionic liquids Francisco J. Deive a,b, Ana Rodríguez a,b, Luís P.N. Rebelo a, Isabel M. Marrucho a,c,⇑ a

Instituto de Tecnologia Química e Biológica, www.itqb.unl.pt, UNL, Apartado 127, 2780-157 Oeiras, Portugal Departamento de Ingeniería Química, Universidade de Vigo, 36310 Vigo, Spain c Departamento de Química, Universidade de Aveiro, 3810-193 Aveiro, Portugal b

a r t i c l e

i n f o

Article history: Available online 23 December 2011 Keywords: Ionic liquids Aqueous biphasic system Green biotechnology Extraction Lipase

a b s t r a c t The suitability of two different imidazolium-based families of ionic liquids (ILs) involving alkylsulfate and alkylsulfonate anions to act as solvents in an aqueous biphasic system for the extraction of lipase A from Candida antarctica has been investigated. Both high charge–density salts and aminoacids have been studied in order to evaluate both their potential as two-phase forming agents and as stabilizers of the active conformation of the enzyme. Extraction with 1-ethyl-3-methylimidazolium butyl sulfate [C2MIM][C4SO4] with ammonium sulfate affords an enzyme recovery of 99%. The structural conformation of the enzyme was analyzed by Fourier transform infrared data. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Biotechnological processes usually involve many separation stages that represent a crucial part of their overall efficiency and cost due to energy and chemical consumption. The challenge is thus, to develop and scale extraction processes that are capable of improving the downstream techniques for the recovery of biologically produced compounds, making biotechnology competitive when compared with conventional chemical processes. Over the last few years, different liquid–liquid extraction schemes for biologically active compounds were approached, in order to overcome some toxic and deactivating impacts lent by conventional volatile organic solvents [1,2]. Research shaped to ILs is increasingly focused on their role as efficient separation agents [3–5] and media for biotransformations, due to their exceptional ability to maintain enzymes in active and stable conformations [6,7]. Since 2003, the concomitant effect of ILs and aqueous solutions of high charge–density inorganic salts led to the development of a specific technology based on aqueous biphasic systems (ABS), which seeks the formation of an upper IL-rich phase and a bottom inorganic salt-rich phase [8]. ABS have been recognized as an economical and efficient downstream processing method which offers many advantages such as low energy requirements, short process time, reliable scale-up and a biocompatible environment [9]. Due to the enhanced stability, activity and enantioselectivity of enzymes in aqueous solutions of ionic liquids [10,11], ILs-based ⇑ Corresponding author at: Instituto de Tecnologia Química e Biológica, www.itqb.unl.pt, UNL, Apartado 127, 2780-157 Oeiras, Portugal. Tel.: +351 214469442. E-mail address: [email protected] (I.M. Marrucho). 1383-5866/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.seppur.2011.12.013

ABS seem to be a particularly suitable technique for the extraction of biocatalysts. However, enzyme extraction by means of ILs-based ABS has been scarcely investigated, and up to date, only two articles have been published dealing with lipase extraction [12,13]. Lipases (EC 3.1.1.3) are enzymes able to catalyze a wide range of reactions such as hydrolysis, interesterification, esterification, alcoholysis, acidolysis and aminolysis [14]. Their biocatalytic potential has triggered a great interest in areas such as food, detergent, paper or oleochemical industries, making up one of the three largest groups of enzymes in terms of sales. In this study, the lipase A (CaLA) from the basidiomyceteous yeast Candida antarctica was selected as a model enzyme to perform this study. This lipase is active in a wide range of temperature, with an optimum activity above 90 °C, which is higher than the homologous lipase B from the same microorganism [15]. We have recently showed that short alkyl chain length in imidazolium cation combined with ethylsulfate anion ([C2MIM][C2SO4]) renders the preservation of high levels of enzyme activity [12]. These results encouraged us to search and test similar anions. To this end, 1-ethyl-3-methyl imidazolium paired with both alkylsulfate and alkylsulfonate anions, [C2MIM][CnSOm] with n = 2, 4 and 6 and m = 3 and 4, were selected to be studied as enzyme media in this work. In our case, ABS is formed by mixing an aqueous solution of CaLA and an IL acting as water destructuring compound, and a high charge–density inorganic salt selected on the basis of the Hofmeister series. Aminoacids were also tested as possible salting out agents. The Hofmeister series establishes the strength of ions, ordering them according to their lyotropic degree (from salting in to salting out). This nomenclature has been proposed as a

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replacement of the traditional chaotropic/kosmotropic terms, since the latter address protein stabilization effects as a result of the interaction not only with water but with water and salt. However, it has been recently stated that other interactions between the protein and the molten salt or the solute can also play a decisive role on the stability and the extraction process [16,17]. In general terms, a protein solution is normally stabilized by salting out anions and salting in cations but destabilized by salting in anions and salting out cations. Nevertheless, it would be too simplistic forgetting that other properties such as, pH, ion size, isoelectric point of the enzyme, hydrophobicity, nucleophylicity or H-bond basicity of the solvents may strongly alter the hydration shells surrounding the enzyme molecule to disrupt the protein hydration, and to modify their enzyme affinity for the substrate [18]. This information leads us to consider K3PO4, K2CO3, (NH4)2SO4 as salt models with different lyotropic degrees, to carry out the salting out effect. Additionally, two recent reports suggest the use of aminoacids as phase segregation promoters since they might have a more benign effect on biocompounds [17,19]. Therefore, based on these results, lysine, alanine and glycine were also investigated. The knowledge of the deactivation of the enzyme in the presence of the selected ILs and the salting out promoters is essential for the design of a successful IL-based ABS extraction methodology of CaLA. Therefore, the strategy followed was to analyze the kinetics of the enzyme deactivation in the selected compounds, taking into account thermodynamic and Fourier transform infrared (FTIR) data. In summary, the aim of this work is to propose a viable separation process to extract C. antarctica lipase A from aqueous solutions by means of an IL-based ABS. The reliability of this process was assessed by choosing a suitable combination of IL (1-ethyl-3-methyl imidazolium cation paired with alkylsulfate or alkylsulfonate anions) and inorganic salts rendering possible high enzymatic activity levels. 2. Experimental 2.1. Chemicals C. antarctica lipase A was acquired from Novozymes (Novozyme 735). [C2MIM][C2SO4] (Solvent Innovation, >99%), [C2MIM][C4SO4] and [C2MIM][C6SO4] (Merck, >98%) were used after purification. [C2MIM][CnSO3] (n = 2, 4 and 6) were prepared according to literature procedure (purity >99%) [20]. All ILs were submitted to vacuum at least for 4 h prior to their use, in order to remove water and other volatile solvents. When necessary, ultrapure, bidistilled water was used. K3PO4 and K2CO3 and (NH4)2(SO4) were purchased from Sigma Aldrich with purities over 99%. The amino acids: L-glycine (Riedel-de-Häen, >99.1%), L-alanine (BDH Chemicals, >99%), and DL-lysine monohydrochloride (BDH Chemicals, >98.5%), were used as purchased without further purification. 3. Experimental procedure 3.1. Partition of C. antarctica lipase A The solubility curves of the systems (water + IL + salting out compound) were determined previously in our lab at 298 K and atmospheric pressure [21,22]. The partition measurements of the enzyme started with the addition of a known amount of IL and aqueous solution of the enzyme, until the total volume reached 5 mL value. The mixture was vigorously stirred and the phase promoter agent was added until turbidity was attained. The aqueous

biphasic system assays were performed in a jacketed glass vessel containing a magnetic stirrer connected to a 25 °C temperature controlled circulating bath (controlled to ±0.01 K). The temperature was measured with a four-wire platinum resistance thermometer coupled to a Yokogawa 7561 multimeter with an accuracy of ±0.01 K. The mixture was left to settle for 1 h for complete phase separation. Samples from both phases were simultaneously taken with a syringe from the upper and lower layers, respectively. Lipolytic activity of the phases was determined by UV spectrophotometric assay described ahead. 3.2. Fourier transform infrared (FT-IR) measurements Infrared spectra of the commercial lipase and solutions in IL, and inorganic salts with different deactivation capacities were recorded by means of an IFS-66/S FTIR spectrometer from Bruker (Bruker Daltonics, MA, USA) using a nine-reflection ATR cell (DuraDisk). Acquisition was accomplished in the MCT-detection mode using an accumulation rate of 32 scans at a resolution of 2 cm1 at room temperature, in the spectral range of 4000–600 cm1, and its processing was done by the OPUS software package (Bruker) with a wavenumber accuracy of 0.1 cm1. Interference of water vapor and CO2 was avoided by purging the sample and detector compartments with N2, at a flow rate of 18 L min1. The spectra were corrected by subtraction of the background spectrum (using water, IL and salts as appropriate). 3.3. Lipolytic activity assay Lipolytic activity was determined spectrophotometrically using an aqueous solution of 2.5 mM of p-nitrophenyl laurate (Sigma, St. Louis, MO, USA) as substrate [23] at pH 7.0, 25 °C and 20 min reaction time. One of the products of the hydrolysis reaction, p-nitrophenol, was monitored by the increase in the absorbance band at 400 nm. One activity unit is defined as the amount of enzyme that produced 1 lmol of p-nitrophenol per minute under standard assay conditions. The activities were expressed in (U/L) and all the results shown in the graphs are mean values of triplicates with standard deviations less than 15%. 4. Results and discussion 4.1. Operating conditions for phase partition with maintained activity of C. antarctica lipase A 4.1.1. Selection of IL The first stage of the study comprises the investigation of the lipolytic activity of CaLA in different alkylsulfate- and alkylsulfonate-based ILs. Thus, the enzyme was settled in vials containing 75% (v/v) of [C2MIM][CnSOm], n ranging from 2 to 6, and m = 3 or 4. The results obtained are shown in Fig. 1. Important differences are detected on the basis of the increase in the alkyl chain length of the anion. In general, the presence of alkylsulfates entails higher biocatalytic potential of the CaLA. A recent work of our group focused on studying the differences between alkylsulfate and alkylsulfonate-based ILs [24]. Despite their similarities, the existence of an extra oxygen bridge-atom in the sulfate anion confers the possibility of forming more extensive polar networks, which could be the basis of their different interplays with the selected protein. This characteristic can strongly modify the bulk water structure, affecting the enzyme-water interactions, and directly interplaying with the enzyme molecules, which can drastically alter the enzyme conformation [25]. On the other hand, according to the CaLA inhibitory effectiveness, the IL anions can be ordered as follows: [C2SO4] > [C6SO4] > [C4SO4].

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[CC2MIMC6SO3 2MIM][C6SO3] [CC2MIMC4SO3 2MIM][C4SO3] [CC2MIMC2SO3 2MIM][C2SO3] [CC2MIMC6SO4 2MIM][C6SO4] [CC2MIMC4SO4 2MIM][C4SO4] [CC2MIMC2SO4 2MIM][C2SO4] Water

0

2000

4000

6000

8000

10000

Lipolytic Activity (kU/L) Fig. 1. Lipolytic activity of CaLA in different ILs.

Several aspects, such as the biospecific contributions or solvent properties can be playing a decisive role in the CaLA lipolytic activity. Also, ion size of the IL could influence enzyme activity, since sterically demanding ions would require many hydrogen bonds to be broken to create fewer new ones, which could contribute to protein disruption [26]. On the contrary, more lipophilic compounds could further the active conformation of the enzyme [27]. The final lipolytic activity is a balance between all those factors. The low solubility in water of sulfonates with alkyl chain lengths longer than six carbon atoms does not allow the full checking of this behavior. Therefore, since water is the medium to be mimicked as model solvent for dissolving proteins, the anions [C4SO4] and [C6SO3] were selected for further study.

k1

k2

E ! Ea11 ! Ea22

ð1Þ

The enzyme residual activity (a/a0) can be described by the following equation, as a function of time (t):

!1=2 ð3Þ

i

where zexp and zadjust the experimental and calculated data, respectively, and nDAT the number of data. An excellent fitting to the model was obtained, as can be observed in Fig. 2. The free energy (DG0) of the thermal deactivation process was also calculated by means of Eq. (4):

DG0 ¼ RT ln



k1 h kB T

 ð4Þ

where kB is Boltzmann’s constant (J K1), h is Planck’s constant (J min), R is the gas constant (J mol1 K1) and T is the temperature (K). All the calculated kinetic and thermodynamic parameters are shown in Table 1. A visual inspection of the results permits one to conclude that more than one type of bond is broken during the deactivation process, since the fractional rate of deactivation is not constant. This behavior was coincident with the findings reported by Deive et al. [12,29] when studying the deactivation kinetics of a lipase from other mesophilic yeast and fungus. Furthermore, the previously selected IL for this other lipase was [C2MIM] [C2SO4], confirming thus that [C2MIM][C4SO4] and [C2MIM][C6SO3] 1.2 1 0.8

a/a0

4.1.2. Deactivation kinetics of C. antarctica lipase A with the selected ILs The investigation of the kinetic and the thermodynamic parameters of enzyme deactivation can help us to unravel the conformational changes occurring in the enzyme structure. Additionally, it permits to quantitatively ascertain the standard free energy of deactivation (DG0), which licenses further insight into the stability of the CaLA in different media. When high free energy is obtained, the stability of the enzyme is increased, as a consequence of a strengthening of intramolecular forces contributing to the cohesion of the native protein molecule. The experimental data were modeled through a series-type deactivation equation involving two first-order steps with an intermediate enzyme form E1 and a final state E2, with relative specific activities a1 and a2 [28].

RMSD ¼

nDAT X ðzexp  zadjust Þ2 =nDAT

0.6 0.4 0.2 0

    a a1 k1 a2 k 2 k1 ¼ 1þ  expðk1 tÞ  ða1  a2 Þexpðk2 tÞ þ a2 a0 k2  k1 k 2  k1 k 2  k1

0

ð2Þ

The RMSD function shown in Eq. (3) was minimized, providing the first order deactivation rate constants k1 and k2, the specific relative activities a1 and a2, and the regression coefficients.

200

400

600

800

1000

1200

1400

1600

t (min) Fig. 2. Thermal deactivation profiles of C. antarctica lipase A in the presence of (4) water, (s) [C2MIM][C6SO3] and (h) [C2MIM][C4SO4]. The symbols represent the experimental data and the solid lines indicate the fittings to a two-step series type deactivation model.

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Table 1 Kinetic and thermodynamic parameters of C. antarctica lipase A deactivation according to a two-step series – type deactivation model. Compound [C2MIM][C6SO3] [C2MIM][C4SO4] Water

a1

a2

0.6256 0.7039 0.7068

0.0113 0.1353 0.1342

k1 (min1)

k2 (min1) 4

0.0059 0.0290 0.0239

1.3  10 1.7  105 2.8  105

dissolution properties are similar to those offered by water, since they entail similar degree of deactivation in both lipases. This fact is very advantageous, in terms of using ionic liquids as solvents for biocatalysis.

DG0 (kJ mol1)

RMSD

201.1 202.2 201.5

1.4  104 1.3  104 3  105

1.20

1.00

[C2MIM][C2SO3] [C2MIM][C4SO3] [C2MIM][C6SO3] [C2MIM][C2SO4] [C2MIM][C4SO4] [C2MIM][C6SO4] Ala

Lys

K3PO4 K2CO3 (NH4)2SO4

Fig. 3. Phase segregation ability of different aminoacids and high charge–density inorganic salts with alkylsulfate and sulfonate 1-ethyl-3-methyl imidazolium ILs. Gray boxes mean two phase formation, and white boxes mean no phase formation.

a /a 0

0.80

4.1.3. Selection of salting out agents licensing maintained lipolytic activity of C. antarctica lipase A The last step to achieve a successful ABS is finding a phase segregation promoter that provides an effective salting out process, at the same time that lends a protective effect on the enzyme surface. Different high charge–density inorganic salts with known salting out ability such as K3PO4, K2CO3, and (NH4)2SO4, and aminoacids like glycine (Gly), lysine (Lys) and alanine (Ala) [17] were assessed. As qualitatively depicted in Fig. 3, it can be concluded that the use of aminoacids does not permit the formation of two phases with the ILs evaluated in this work. As stated by Domínguez-Pérez et al. [19], these zwitterionic species have a less pronounced salting-out inducing behavior than the Hofmeister series-based inorganic salts. Also registered in this figure is the fact alkylsulfonate-based ILs cannot be segregated by (NH4)2SO4, as we found in our previous investigations [22]. The analysis of the enzyme lipolytic activity in the presence of the salting out promoter, chosen on the basis of the previous systematic study, seems to be crucial for obtaining an adequate extraction process. Therefore, the lipolytic activity of CaLA in K3PO4, K2CO3 and (NH4)2SO4 at different concentrations was evaluated. Both K3PO4 and K2CO3 involve drastic enzyme inhibition after just 15 min of contact time. An ion may affect the enzyme performance by playing the role as a substrate, a cofactor, or an inhibitor to the activity. Generally, the specific ion effects should be better understood by including their ability to alter the bulk water structure, to affect the protein-water interaction, and to directly interplay with the enzyme. In our case, the phosphate and the carbonate anions, both possessing strong salting out character, are destabilizing the CaLA due to their strong interaction with the enzyme moiety [30]. On the contrary, ammonium sulfate did not involve so extensive deactivation effects. This inorganic salt has been widely reported for enzyme purification, since protein precipitation with ammonium sulfate is one of the most common protein purification methods [31]. The deactivation profile at different concentrations of ammonium sulfate is presented in Fig. 4, and a lower deactivation potential with increased concentrations of inorganic salt can be observed.

0.60

0.40

0.20

0.00 0

200

400

600

800

1000

1200

1400

1600

t (min) Fig. 4. Thermal deactivation profiles of C. antarctica lipase A in the presence of (NH4)2SO4 at different concentrations: (h) 0.5 m; (4) 1 m and (s) 1.5 m.

4.1.4. Structural changes of C. antarctica lipase A Aiming at unraveling the role of the selected neoteric solvent and the high charge–density inorganic salts on the stability of CaLA, and to achieve a deeper understanding of the phenomena occurring at the molecular level, FTIR data were collected. This technique is a valuable tool to reach a better understanding of the conformational changes governing the catalytic ability of the enzyme. FTIR spectroscopy is a useful technique for the characterization of protein secondary structure and the identification of protein components involved in events such as ligand binding and electron-transfer reactions [32,33,26]. The protein spectra obtained through this technique enables the analysis of the characteristic amide bands from different vibrations of the peptide moiety. The amide I frequencies were used to determine primary structural components and to monitor unfolding reactions in the experiments described in this work. This IR region, from 1600 to 1700 cm1, is associated to [email protected] stretching vibration, and thus, it is considered the most conformational sensitive area for a detailed study of the protein structure. The conformation of lipase A from C. antarctica lipase under [C2MIM][C4SO4], K2CO3 and (NH4)2SO4 was compared with the native commercial enzyme, and the data obtained is shown in Figs. 5a–c. It can be observed that the second-derivative of the spectrum of CaLA in contact with [C2MIM][C4SO4] shows slight differences compared to that of the aqueous enzyme. Specifically, the absence of a band at 1634 cm1 and a merging of peaks at 1656 and 1659 cm1 into one peak at 1657 cm1 is noticed, which indicates a modification in the content of a-helix and b-turn. This fact was also observed by Van Rantwijk et al. for other lipase from the same yeast [26]. However, these changes do not trigger any drastic fall of CaLA biocatalytic potential, as previously mentioned for the other lipase. Contrarily to this, the presence of K2CO3 even after a short contact time with CaLA entails much more profound alterations in the enzyme conformation. The absence of one peak at 1642 cm1 and the presence of two bands at 1640 and 1644 cm1, corresponding to the region of a-helix and random coil conformations seems to be playing a decisive role in the protein unfolding leading to a total

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F.J. Deive et al. / Separation and Purification Technology 97 (2012) 205–210 Table 2 Separation parameters, extraction efficiency Wrec and the distribution ratio DCaLA, for IL-based ABS of C. antarctica lipase A. Concentration wt.% ((NH4)2SO4/[C2MIM][C4SO4])

Wrec

DCaLA

(12.4/38.8) (19.6/20.8) (32.2/9.2)

0.44 0.51 0.99

695 956 1639

4.2. IL-based ABS of C. antarctica lipase 1590

1610

1630

1650

1670

1690

1710

Wavenumber (cm-1) Fig. 5a. Comparison of the second derivative infrared spectra of aqueous C. antarctica lipase A (dashed line) and in contact with [C2MIM][C4SO4] (solid line) after 1 h of contact.

1590

1610

1630

1650 1670 Wavenumber (cm -1)

1690

1710

Fig. 5b. Comparison of the second derivative infrared spectra of aqueous C. antarctica lipase A (dashed line) and in contact with K2CO3 (solid line) after 1 h of contact.

1590

1610

1630

1650

1670

1690

1710

Wavenumber (cm-1) Fig. 5c. Comparison of the second derivative infrared spectra of aqueous C. antarctica lipase A (dashed line) and in contact with (NH4)2SO4 (solid line) after 1 h of contact.

loss of the enzyme activity. On the other hand, the enzyme in a (NH4)2SO4 environment does not show important variations in the number of peaks with regard to the spectrum obtained for the aqueous solution. The absence of a band at 1634 cm1 corresponding to b-sheets assignments, as in the case of the IL, that does not seem to exert any relevant modification, preserving the enzyme catalytic activity. Therefore, the FTIR data gives further insights into the enzyme deactivation behavior, and license to state that there are drastic changes in the enzyme secondary structure behind the loss of the catalytic potential.

The relevance of researching lipase stability in ILs is clearly demonstrated through several articles published in the last year [12,34–36]. These essential data mark the onset for the proposal of a viable, biocompatible and greener separation technique licensing a concomitant use of ILs as withdrawal solvents, and, further, as media for catalytic applications. In this work, the formation of an upper IL and lipase-rich phase was pursued after having investigated: (i) a suitable IL preserving enzyme activity, (ii) a salting out compound with phase segregation ability and without deactivation potential and (iii) the deactivation profiles in the presence of ILs and salts. Previous studies from our group [21,22] focused on the ABS behavior of the systems including 1-ethyl-3-methyl imidazolium alkylsulfate and 1-ethyl-3-methyl imidazolium alkylsulfonate with K3PO4, Na2CO3, K2CO3 and (NH4)2SO4. As expected, the later inorganic salt involves lower phase segregation capacity, and it was not able to form ABS with none of the studied alkylsulfonate-based ILs. In this particular case, based on the abovementioned experimental data concerning lipase activity, only the ABS containing [C2MIM][C4SO4] and (NH4)2SO4 was selected to perform the lipase extraction. Since the objective of this work is to effectively extract the enzyme from an aqueous solution, the extraction efficiency Wrec and the distribution ratio DCaLA were the parameters chosen to describe the enzyme partition in the separation process:

W rec ¼

W IL W0

ð5Þ

DCaLA ¼

½enzyme ðU=LÞIL ½enzyme ðU=LÞAq

ð6Þ

being WIL the lipolytic activity units in the upper-IL rich phase and W0 the total lipolytic activity units in the system. The ability of the IL as enzyme extraction agent is proved by the values of the separation parameters shown in Table 2 for different IL and salt concentrations. It is clear that the operation at high salt concentrations yields enzyme recoveries of almost 100% and partition coefficients more than two folds higher than the other cases. These results are clear improvements of the extraction efficiency reported by Cao et al. [37] and Deive et al. [12] with a horseradish peroxidase and a fungal lipase, respectively. Also, in agreement with our previous findings, the use of higher amounts of inorganic salt has been reported to be beneficial for lipolytic activity [12]. Therefore, the extraction procedure at (wsalt, wIL) % = (32.2, 9.2) allows the most successful separation of lipase A secreted by C. antarctica from aqueous solutions and confirms the hypothesis proposed that hydrophilic ILs are suitable for lipase separation. 5. Conclusions In this study, the enormous potential of ABS for lipase extraction is clearly shown. Moreover, the use of the ionic liquids’ superior solvent quality in the recovery and purification of enzymes constitute a step forward in the reduction of the environmental impact of these

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processes. The structural FTIR study, aimed at identifying the enzyme behavior in the selected compounds involved in the aqueous two-phases separation process, allowed to conclude that some changes in the secondary structure of the enzyme strongly influence its biocatalytic potential. In this sense, the addition of an imidazolium-based IL paired with an alkylsulfate anion reflects an enhancement in terms of: (i) process time (just one hour), (ii) very low energy consumption, (iii) mild operating conditions, (iv) high extraction capacity (>99%) and (v) possible use of the ionic liquid as medium for biocatalytic transformations.

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