Insights on the laccase extraction and activity in ionic-liquid-based aqueous biphasic systems

Due to their catalytic properties, selectivity, and efficiency, enzymes are excellent biocatalysts. In particular, laccases are versatile multi-copper oxidases with great interest for a wide plethora of biotechnological and environmental applications. Even though several laccasecatalysed processes have been reported at an industrial level, the high costs of their downstream processing required to provide biocatalysts with high purity levels, stability and activity remains one of the main drawbacks when economically evaluating the overall processes. Aqueous biphasic systems based on ionic liquids (ILs) can be foreseen as a promising alternative approach for the extraction and activity maintenance/improvement of enzymes, essentially due to the designer solvents ability of ionic liquids. However, to take advantage of this feature and to use the full potential of IL-based aqueous biphasic systems, it is necessary to understand the effect of ILs as phase-forming constituents and how they affect the enzymes extraction and activity. In order to overcome the lack of information on this topic in the literature, in this work, IL-based aqueous biphasic systems were investigated to extract and enhance the laccase activity, in order to gather evidences that could be used to improve the enzymes downstream processing. To this end, a wide screening of imidazolium-, pyridinium-, pyrrolidinium-, piperidinium-, tetraalkylphosphonium-, and tetraalkylammonium-based ILs as phase-forming components of ABS was carried out. Furthermore, these ILs were used to create ABS combined with salts, polymers and used as adjuvants in polymer-based ABS. Most ABS comprising ILs revealed to be highly efficient extraction platforms, allowing the complete extraction of laccase for all the conditions tested, and with an enzyme activity enhancement by more than 50%. Overall, the obtained results demonstrate that laccase preferentially partitions to the most hydrophilic phase in ABS comprising ILs, both used as adjuvants or as phase-forming components, corresponding to the phase in which the IL is enriched. Furthermore, the IL chemical structure of the IL plays a significant role in the enzyme activity, where ILs with a higher number of hydroxyl groups seem to be relevant to improve the laccase activity.


Introduction
Enzyme biocatalysis undergone significant developments in the last decades in the production of high-value products in a variety of industries [1]. Due to their excellent catalytic properties, selectivity, efficiency, low toxicity and biodegradability, enzymes are excellent biocatalysts to be used in mild reaction conditions. Oxidative enzymes have been applied in a wide variety of processes [2], such as delignification and biobleaching [3,4], dye degradation [5][6][7], bioremediation [8], ethanol production [9], biosensors [10] and in the production of pharmaceutical drugs [11,12]. In fact, within their relevant applications, the pharmaceutical sector can be highlighted, in which the first drug synthesized by an oxidative enzyme was actinocin, an effective compound to fight cancer by blocking the transcription of the tumour cell DNA [13]. An additional example includes the coupling of katarantine and vindoline to produce vinblastine, an anti-cancer drug [14].
Laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) are versatile multi-copper oxidases [15], capable of oxidizing a large number of phenolic and non-phenolic molecules due to their low substrate specificity, using oxygen as electron acceptor and generating water as a byproduct [16]. Due to their wide range of biotechnological and environmental applications [17], laccase is one of the most used oxidative enzyme. In particular, laccase presents a great potential in different processes related with pulp and paper [18], food [19] and textile industries [20], as well as in novel fields such as bioremediation [21], biosensing [22] and lignocellulosic biorefineries [23].
Even though several laccase-catalysed processes have been reported at an industrial level over the last decades [24,25], due to its high relevance and wide plethora of applications, the high costs of the downstream processing of enzymes required to provide biocatalysts with high purity levels, stability and activity remains one of the main drawbacks when economically evaluating the overall processes [26]. On an attempt to overcome these limitations, the use of ionic liquids (ILs) has been investigated in the development of more efficient downstream processes for enzymes, as well as to provide more sustainable enzymatic reactions, particularly when the substrates have low water solubility [27]. The interest in ILs as a novel class of solvents for protein purification and biocatalytic transformations is mainly due to their designer solvents ability and excellent performance to act as extraction and/or reaction media [28][29][30][31][32][33].
Depending on the enzymes purity requirement, multiple bioprocessing steps may be required, including chromatography and other polishing steps such as ultrafiltration. Among these processes, aqueous biphasic systems (ABS) have been recognized as an economic and efficient downstream processing tool to be used in the separation and purification of biomolecules from complex biological matrices [34]. Traditional ABS consists of two immiscible aqueous-rich phases based on polymer/polymer, polymer/salt or salt/salt combinations. However, typical polymer/polymer ABS display two hydrophobic phases, while polymer/salt ABS show a highly hydrophobic polymer-rich phase and a hydrophilic salt-rich phase. Therefore, there is always a restricted polarity difference between the two phases, which may limit their performance in the selective extraction and purification of proteins. The introduction of ILs in ABS was first reported by Rogers and co-workers [35], and since then the potential of ILs as phase-forming components of ABS has been recognized in the presence of several salts, amino acids, carbohydrates or polymers [31,[36][37][38]. Essentially due to their capability to act as designer solvents, tailored polarities of the ABS coexisting phases can be obtained, thus leading to tailored affinities and improved extractions [39].
ABS based on ILs and salts or polymers have been proposed as effective separation and purification strategies for several biomolecules, such as proteins and enzymes [28,30,[40][41][42][43][44]. ILbased ABS have been also reported as effective in the extraction and in maintaining the activity of enzymes [42]. Moreover, the stability of oxidative enzymes in presence of ILs has been proved [45,46]. Although traditional polymer/polymer and polymer/salt ABS have been investigated for the extraction of oxidative enzymes, including laccase [47], to the best of our knowledge, the application of IL-based ABS for the extraction or purification of laccase, was not previously addressed.
Since ILs are designer solvents and in order to take advantage of that characteristic to reach the full potential of IL-based ABS, in this work, a wide range of imidazolium-, pyridinium-, pyrrolidinium-, piperidinium-, tetraalkylphosphonium-, and tetraalkylammonium-based ILs were evaluated in ternary and quaternary ABS, as phase-forming compounds or adjuvants, respectively, for the extraction of laccase and their effect upon the enzyme activity. Ternary ABS composed of ILs and potassium citrate buffer (C 6 H 5 K 3 O 7 /C 6 H 8 O 7 ) at pH 8, potassium citrate (C 6 H 5 K 3 O 7 ) or polypropylene glycol 400 (PPG 400) were studied; quaternary ABS composed of polymer/salt (PPG 400 + K 2 HPO 4 ) and polymer/polymer (PPG 400 + poly(ethylene)glycol 400/PEG 400) with ILs as adjuvants at three different concentrations (1, 3 and 5 wt%) were also investigated. The phase-forming abilities of the different ILs were determined through the characterization of the respective liquid-liquid phase diagrams, followed by studies on the extraction of laccase in these systems and enzyme activity. Several operational conditions, namely the type of system, the IL, salt, polymer and enzyme concentrations were investigated, allowing to address some of the main molecular mechanisms and driving forces ruling the laccase partition in IL-based ABS.  [48]. The chemical structure of all the ILs investigated on this work are presented in Table 1.

Experimental Procedure
Determination of binodal curves, tie-lines and tie-line lengths. The cloud point titration method was used to determine the new binodal curves at 25±1°C, according to a protocol previously described [49]. The binodal curves were then fitted using Equation 1, proposed by Merchuk et al. [50]: where and correspond to the weight fractions percentages of each phase-forming compound; is the salt or PPG and is the IL or PEG depending on the constituents of the ABS. , and are fitting parameters obtained by least-squares regression from the experimental data.
Tie-lines (TLs) were gravimetrically determined at 25±1°C according to the method proposed by Merchuk et al. [50], and already applied to IL-based ABS [51]. Additional details on the TLs and tie-line lengths (TLLs) determination can be found in the Supplementary Information (SI). and centrifuged at 10000 rpm for 20 min to achieve the thermodynamic equilibrium and separation between the two phases. Then, the phases were separated, their volumes were ascertained, and laccase activity was measured in both phases with the procedure described below.

Determination of the extraction efficiency and partition coefficient of active laccase in
IL-based ABS. Laccase activity measured according to the protocol described below provides a measurement of the concentration of active enzyme present in each phase of each ABS. The extraction efficiency of active laccase (EE%) was considered as the percentage ratio between the laccase activity in the IL-rich phase to that in the opposite phase, according to Equation 2: is the activity of laccase (U·L -1 ), represents the volume of each phase, and the [ ] subscripts , and represent the IL-, salt-, and PPG-rich phases, respectively.
The activity partition coefficient of laccase in each phase was obtained using Equation

3
: where the subscripts and or indicate the phase were the laccase activity is measured, respectively. or PEG 400, IL and laccase aqueous solution (7.5 µL·mL -1 ). The laccase activity measurement procedure was the same as described below, and its extraction efficiency ( ) was determined % according to Equation 4:

Screening of ILs as adjuvants in polymer
where is the activity of laccase (U·L -1 ), represents the volume of each phase, and the [ ]

subscripts
, and represent the salt-, PEG-and PPG-rich phases, respectively.
For each phase, the pH values were determined using a Mettler Toledo U402-M3-S7/200 micro electrode, and the presence/quantification of the IL was determined by 1 H NMR spectroscopy. Further details on the IL determination/quantification procedure can be found in the SI.
Laccase activity. The laccase activity was determined according to a method previously described [53]. For that purpose 100 µL of laccase solution was mixed with 0.5 mL of ABTS 0.2 mM, and 1.4 mL of citrate/phosphate buffer 0.05/0.1M at pH 4.5. The increase in absorbance was measured in kinetic model of a UV-Vis spectrophotometer (Agilent 8453) at 420 nm. The laccase activity was estimated using Equation 5: where ε is ABTS molar extinction coefficient (36000 M -1 cm -1 at 420 nm), abs·min -1 is the increase in absorbance per minute, f dil is the dilution factor of the sample, 10 6 is the conversion factor from M to μM. One unit (U) of laccase activity is defined as the amount of enzyme required to form 1 μmol of ABTS˙+ per minute, and laccase activities are expressed in U·L -1 . Blank control samples were always analysed to ascertain possible interferences of the phase-forming components on the activity assays. Blue G-250 and then distained. A molecular weight full-range marker (VWR) was used as protein standards.

Extraction/activity of laccase in IL-based ABS
In the last years there has been an increasing interest in the use of ILs in the field of biocatalysis and enzyme purification [54,55]. Previous works showed that ILs may have beneficial or deleterious effects on the stability of enzymes and their activity [56,57]. To take advantage of the ILs designer solvents aptitude and to use the full potential of IL-based ABS, it is necessary to understand the effect of ILs as a phase-forming constituents and how they affect the enzymes extraction and activity. For that purpose a wide range of ILs used as ABS phase-forming constituents were screened, namely tetraalkylammonium-, tetraalkylphosphonium-, imidazolium-,  Figure S1. The experimental weight fraction data are also given in the SI (Table S1). Initial studies were carried out with different concentrations of laccase, ranging from 3.75 to 15 µL·mL -1 , in selected ABS, to investigate the effect of increasing protein concentration on laccase partition and activity and to infer the most appropriate concentration to be used. This laccase concentration range was selected to give similar enzyme activity to those achieved during its production by fermentation, based on a previously published paper by us [58]. The results are presented in Figure S2 in the SI, showing that it is possible to extract laccase for the IL-rich phase using concentrations up to 15 µL·mL -1 , with no saturation of the phases and/or any precipitation of the enzyme at the interface. Therefore, all the investigated ABS in this work were loaded with an intermediate laccase concentration -7.5 µL·mL -1 , whose compositions of the ABS investigated are given in Table 3. All the mixture points studied were selected based on the corresponding phase diagrams in order to be close to the binodal curves, maximizing the amount of water in the systems, which is beneficial when dealing with proteins/enzymes. Also, it is important to highlight that all the ABS composed of C 6 H 5 K 3 O 7 /C 6 H 8 O 7 buffer were set at pH 8, since it consists in the optimum pH value of the laccase under study, based on a preliminary study of the laccase activity as a function of the pH (see SI, Figure S3) -being in accordance with the information provided by the supplier in the product datasheet. The comparison between the laccase activity in the top and bottom phases, as well as the extraction efficiencies (EE%) and partition coefficient of the active laccase (K act ) are given in Table 3. These ILs were then selected for further studies of laccase extraction and activity.

Effect of mixture composition on laccase partition/activity in IL-based ABS
The partition and activity of an enzyme in an ABS depends on the properties, composition  Table 4.    [40], which is the opposite to that occurring in the IL-PPG 400-systems in which the phase enriched in IL is the most hydrophilic phase. Thus, these results suggest that the hydrophobicity/hydrophilicity of the ABS phases plays a major role in the partition behaviour of active laccase, although other specific interactions occurring with the IL chemical structure may also play a role [61].
Although the complete extraction of laccase towards the IL-rich phase occurs in systems formed by cholinium-based ILs and PPG 400, a strong effect of the IL anion is however observed in laccase activity. The results given in Table 4 show that the laccase activity decreases in the  Table 4, indicate that the modification of the initial mixture composition (and consequently the systems' volume ratio) has a significant influence on the laccase activity in the IL-rich phase. In general, the activity of the enzyme decreases as the concentration of IL increases in the mixture composition. Thus, laccase activity differences obtained for the same IL at different mixture compositions, with a similar TLL and thus similar differences in the compositions between the two phases, are essentially related with the phases' volumes and saturation effects that may occur (related with the changes on the volume ratio).

Among the investigated cholinium-based ILs, [Ch]
[DHCit] is the most effective IL in the enzyme activity preservation/enhancement in the IL-rich phase, allowing to infer that also the IL chemical structure, and particularly the IL anion, plays an important role in the tailoring of the ABS polarity and subsequent partition of laccase. For these reasons, this study suggests that [Ch][DHCit]-based ABS is the best option, since it allowed the complete extraction of laccase in a single step with an enzyme activity higher than 1000 U·L -1 .

Extraction/activity of laccase in ABS comprising ILs as adjuvants
The formation of ABS using ILs requires large amounts of these chemicals. A more economical alternative is the formation of conventional polymer-polymer or polymer-salt ABS using ILs as adjuvants, at lower concentrations. This possibility was explored using the best ILs identified before. For this purpose, two conventional ABS were selected: a polymer-salt ABS (PPG 400 + K 2 HPO 4 ) and a polymer-polymer ABS (PPG 400 + PEG 400), using ILs as adjuvants at 1, 3 and 5 wt%.
The effects of the IL and its concentration upon the binodal curve of PPG 400 + K 2 HPO 4 + H 2 O were evaluated and are presented in SI, Figure S5. In general, the curves show that, for all the ILs studied, the IL concentration of 5 wt% increased the biphasic region of the systems, meaning that lower amounts of the phase-forming compounds are required to form the ABS. After performing the extraction of laccase using these ABS composed of 5 wt% of IL, other percentages of adjuvant were further tested, namely 1 and 3 wt%, and for which the binodal curves were also determined (SI, Figure S5). The binodal curves using 1 wt% of IL are very close to those without any adjuvant, meaning that at such low concentrations the IL has a negligible effect in ABS formation. Higher concentrations are required in order to obtain a more pronounced effect in the phase diagrams and respective monophasic/biphasic regimes. In all these systems, the top phase corresponds to the PPG 400-rich phase and the bottom phase corresponds to the K 2 HPO 4 -rich phase.
The influence of ILs as adjuvants at the three previously mentioned concentrations (1, 3 and 5 wt%) in IL-salt ABS on laccase partition was investigated. For all the conditions tested, including for the systems without IL, a preferential partition of laccase to the bottom salt-rich phase was found, with its complete extraction achieved in one-step for all cases (EE% = 100%).
The previous results regarding the ternary systems composed of IL + PPG 400 + H 2 O show that laccase migrates preferentially to the IL-rich phase -the most hydrophilic phase in the system.
The results for the quaternary systems composed of PPG 400 + K 2 HPO 4 + H 2 O + IL show the same behaviour, with the preferential partition of laccase to the salt-rich phase -the most hydrophilic phase in these systems, and in which the IL is retained (the complete partition of the IL was determined; the salt-rich phase is completely enriched in IL). A common mixture composition was chosen in the biphasic region of these systems: 27 wt% of PPG 400 + 5 wt% of K 2 HPO 4 + (1, 3 or 5) wt% IL. Although complete extraction was obtained for all the conditions in this study (see SI, Table S8), significant differences were found regarding the laccase activity in the salt-rich phase, as depicted in Figure 1. The respective detailed values and pH values of the phases are given in the SI, Table S8. there are no significant differences on the enzyme activity between the three concentrations tested, which means that the IL chemical structure appears to be more relevant than the IL amount present in the systems.
In the studied polymer-salt quaternary ABS, high activity of laccase after the extraction process was attained in the best conditions tested, being even higher to those obtained with the As a last approach, ILs were also tested as adjuvants in ABS composed of two different watersoluble polymers for the partition and activity of laccase. It has been shown that polymer-polymer ABS can be easily formed depending on the type of polymers combined [36]. The polymers PEG 400 and PPG 400 were chosen since both are water soluble, nontoxic (considered as safe) [63], and have been approved for human injections and oral application, being widely applied by the chemical, food and pharmaceutical industries [64].  Figure S6. As observed before with ABS formed by polymers and salts, it was found that by using ILs as adjuvants (1, 3 and 5 wt%) in polymer-polymer systems it is possible to lower the amounts of phase-forming compounds required to form the ABS. In all these systems, the top phase corresponds to the PPG-400-rich phase and the bottom phase corresponds to the PEG-400-rich phase.
After the characterization of the phase diagrams, the impact of the cholinium-based IL as adjuvants in each PPG 400 + PEG 400 ABS was studied in the laccase partition and activity.
According to the gathered results, laccase completely partitions to the bottom PEG-400-rich phase, achieving an extraction efficiency of 100% for all the conditions tested without using IL, and also with the three ILs at the three different concentrations. For this type of systems and for all the studied concentrations of IL, the complete partition of the IL to the bottom PEG-rich phase was observed. These results allow to corroborate the idea that ILs and laccase present a higher affinity for more hydrophilic phases, being the phosphate-rich phase in the systems formed by polymers and salts and the PEG-400-rich phase in the systems formed by two polymers.
Although the complete extraction was obtained for all the conditions under study (see SI, Table S9), significant differences were found regarding the laccase activity in the various systems, as depicted in Figure 2. The detailed values and pH values of the coexisting phases are presented in the SI, Table S9. For all the conditions tested, the enzyme activity increases in the bottom PEG-rich phase when compared with the control system (without IL), except for the system composed of 1 wt% Overall, the obtained results allow to conclude that laccase preferentially partitions to the most hydrophilic phase in systems formed by ILs and polymers or salts, both used as adjuvants or as phase-forming components, and particularly to the phase in which the IL is enriched. Furthermore, the IL chemical structure of the IL has a significant impact in the enzyme activity, where ILs with a higher number of hydroxyl groups appear as promising to improve the laccase activity. In this sense, evidences were gathered that could be useful to improve the extraction efficiency of enzymes downstream processing, and that may take advantage of the designer solvent ability of ILs to selectively extract laccase from complex fermentation broths.

Conclusions
In this work, we studied different types of ABS comprising ILs to extract and improve the activity of laccase aiming at gather evidences that could allow the design of effective downstream processes. Three types of IL-based ABS have been evaluated: ternary systems composed of IL + (polymer or salt) + water and quaternary systems composed of (polymer + salt + water + ILs as adjuvants) and (polymer + polymer + water + ILs as adjuvants). From the wide range of ILs evaluated, cholinium-based ILs were identified as the most promising candidates concerning the development of an effective extraction process for laccase, providing also high activity values.
Several evidences support a clear preference of laccase to the most hydrophilic phase of the ABS, independently of the type of system investigated, which is also the phase in which the IL is enriched. Both ternary and quaternary IL-based ABS showed promising results; however, ABS composed of polymers, salts and ILs as adjuvants should be highlighted as they provided the complete extraction of laccase (extraction efficiency = 100%) in a single-step, with an improvement on its activity to values higher than 150%. Furthermore, the use of ILs as adjuvants presents the advantage of requiring lower amounts of the ABS phase-forming compounds, confirmed by the shift in the respective binodal curves of the phase diagrams, as well as a small concentration of IL, thus contributing to decrease the costs associated with the process.
[Ch][DHCit], the cholinium-based IL with more hydroxyl groups in its chemical structure, was identified as the most promising IL studied, corroborating the idea that the chemical structure of the IL plays a significant role in the enzymes partitioning and activity in ABS, and as such giving insights on the best strategy to develop effective downstream processes for laccase.