Laccase Activation in Deep Eutectic Solvents

The research on alternative solvents and cosolvents is relevant when envisioning the improvement of biocatalytic reactions. Among these solvents and cosolvents, deep eutectic solvents (DES) may be considered as customizable new reaction media for biocatalysis. Accordingly, in this work, 16 DES aqueous solutions, as well as the individual DES components at the same conditions, have been investigated in laccase-catalyzed reactions. Cholinium- and betaine-based DES formed with polyols at different molar ratios and concentrations were evaluated. The results reported show that in the presence of most DES the laccase activity is preserved and, with a particular DES, enhanced up to 200%. Molecular docking studies demonstrated that while most DES components establish hydrogen bonds with the enzyme amino acids, those that establish stronger interactions with the enzyme (expressed by absolute values of docking affinity energies) lead to an enhanced laccase activity. Finally, the laccase stability was evaluated in a...


INTRODUCTION
The research on alternative and greener solvents has led to advances in biocatalysis by allowing to perform enzymatic bioreactions in partially or fully nonaqueous environments. 1,2 This fact is of particular relevance when dealing with hydrophobic substrates, where the presence of organic solvents could promote enzyme deactivation or denaturation. 3 During the last decades, promising alternative solvents, such as ionic liquids (ILs), have been reported as solvents or co-solvents in enzyme-catalysed reactions with lipase, laccase, peroxidase, among others. 4 However, some ILs based on pyridinium or imidazolium cations, most of the times combined with fluorinated anions, may raise some toxicity and biodegradability concerns 5 and may lead to enzymes deactivation. 6 In addition to ILs as alternative solvents and bioreaction media, deep eutectic solvents (DES) have been attracting widespread interest from academics and industrial sector since these solvents can be produced by the simple mixing of low-cost and safe compounds, most of the times from natural sources. 7 Contrarily to ILs that are pure compounds composed solely of anions and cations, DES are a mixture of weak Lewis acids (hydrogen bond donor (HBD)) and bases (hydrogen bond acceptor (HBA)) which may contain anionic and/or cationic species (e.g. ammonium or phosphonium salts) mixed with non-ionic species. This means that although some physical properties of DES may be similar to ILs, their chemical properties are different. DES have the same 'designer solvent' characteristic of ILs, but with a much broader possibility for design since the solvent properties can be additionally fine-tuned by selecting different DES components at different proportions. 8 In biotransformations, DES can be used as solvents, cosolvents or as a second phase in water-DES mixtures. 9 4 Laccases are multicopper oxidases catalysing oxidative reactions. This type of enzymes only uses molecular oxygen and the substrate to initiate catalysis, i.e., electrons are removed from the reducing substrate molecules and transferred to oxygen to produce water. 10 Laccase has received great attention from both academia and industry due to these simple requirements and ability to degrade a diversity of substrates of industrial interest. As reviewed in the literature, 11,12 laccase oxidizes an extensive variety of organic and inorganic compounds, being applied in the bioremediation of phenolic compounds and dyes, and in pulp and paper processing, biosensors, medical care and synthesis of fine chemicals. However, some of these laccase substrates are highly hydrophobic and cannot be completely dissolved in aqueous medium, requiring therefore the use of organic solvents. 13 Thus, DES may be considered potentially safer alternatives to organic solvents in enzymatic reactions involving laccases. 14 The use of DES prevents enzymes denaturation and deactivation often detected in conventional organic solvents, as observed in some works with horseradish peroxidase 8 and hydrolases. 15 To date, only two works on DES as alternative solvents for laccasecatalyzed reactions has been reported. 16,17 Khodaverdiana et al. 16 show encouraging results for laccase activity and stability using betaine-based natural DES. Amongst the DES studied, laccase was found to be more active and stable in the glycerol:betaine DES at a molar ratio of 2:1. Additionally, the authors proved that laccase was more stable and efficient in a glycerol:betaine DES than in aqueous solutions comprising the DES components. 16 Ünlü et al., studied the polycatechin synthesis by laccase in DES composed of choline chloride:glycerol, choline chloride:glycerol and betaine:mannose at 5 and 50% (v/v). 17 The results show that the replacement of acetone by the DES was possible for the synthesis of polycatechin. 17 Nevertheless, and particularly taking into 5 account the DES designer solvents feature, the use of DES as solvent media for enzymatic reactions with laccase must be more extensively studied aiming a deeper understanding on how DES and their compositions and concentrations in aqueous solutions can affect the enzymes activity and stability.
Chemical structures, hydrophobicity, polarity, viscosity, hydrogen bond basicity/acidity, among other properties, have been described to influence the enzymes activity and stability. 18,19 However, the multitude of DES and their constituents and possible interactions with the enzymes amino acids turns the molecular-level understanding of their impact towards enzymatic activity a difficult task to be experimentally addressed.
Aiming at solving this puzzle, computational methods can be applied. Among these, molecular docking is a fast and easy approach to identify possible preferential interactions between the amino acids of a target enzyme and the DES constituents, while providing additional values on the binding affinity of macromolecules (receptor) and small molecules. 20 In this work, the effect of DES as co-solvents aqueous solution on the laccase activity was evaluated at different temperatures and DES concentrations. Sixteen cholinium-or betaine-based DES were investigated by combining four HBAs, viz. choline chloride (ChCl), choline dihydrogen phosphate (ChDHP), choline dihydrogen citrate (ChDHC) and betaine (Bet), with four HBDs compounds, namely ethylene glycol (EtG), glycerol (Gly), erytritol (Ery) and xylitol (Xyl). To better understand the molecular-level mechanisms responsible for the laccase activation or inactivation, molecular docking studies were carried out. 6
All compounds were of analytical grade and used without additional purification.

Synthesis of DES
The preparation of cholinium-and betaine-based DES was carried out by adding each hydrogen-bond acceptor (Bet, ChCl, ChDHC or ChDHP) to each hydrogen-bond donor (Ery, Gly, Xyl or EG) in glass vials at three molar ratios, namely 1:2, 1:1 and 2:1, resulting in the formation of liquid mixtures. Whenever necessary, the temperature was increased up to 80-100ºC until the formation of a homogenous and clear liquid, particularly for Ery and Xyl based DES. All mixtures were stirred for 1 h. After the synthesis of DES, three different concentrations (10, 25 and 50 wt%) in aqueous solution were prepared for each DES and each molar ratio. The chemical structures of the investigated DES are given in

Laccase stability in DES
Laccase stability was evaluated in a phosphate buffer (50 mM, pH 7.0) aqueous solution containing DES at concentrations of 10%, 25% and 50% (wt%) at 25ºC. In all tests, the final laccase concentration was 0.25 g L -1 . The mixture was incubated at room temperature for 10 min and then a sample was taken for laccase activity assay. Results presented in this work are the mean from at least three experiments.
The activity of laccase was assayed according to the method described by Ander and Messner. 21 The enzymatic reaction was carried out at 25ºC by adding 100 L of sample in 500 L of 0.4 mM 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and 1400 L of 50 mM citrate/100 mM phosphate buffer at pH 4.5. Since the ABTS oxidation is dependent on the pH, the laccase activity method was carried out in acidic conditions.
One unit (U) of laccase activity is defined as the amount of enzyme able to oxidize 1 µmol of ABTS per minute. The oxidation of ABTS was monitored by the increase in absorbance measured in kinetic model of a UV-Vis spectrophotometer (Agilent 8453) at 420 nm (ε = 36.000 M −1 cm −1 ). Laccase activities are presented in U L -1 (for more details see the Supporting Information).
Laccase thermal stability was additionally evaluated by incubating the enzyme in absence and presence of DES aqueous solutions at -80C and 60C. Samples were collected at various intervals of time, and the residual activity was measured as described.

Molecular Docking
The interaction sites of laccase with all HBDs and HBAs that compose each DES were identified using the Auto-dock vina 1.1.2 program 22 . The crystal structure of laccase PDB: 1kya was used. Auto DockTools (ADT) 20 was used to prepare the enzyme input 9 files by merging non-polar hydrogen atoms, adding partial charges and atom types.
Ligand (HBDs and HBAs) 3D atomic coordinates were computed by Gaussian 03w and ligand rigid root was generated using AutoDockTools (ADT), setting all possible rotatable bonds defined as active by torsions. The grid center at the center of mass (x-, y-, and z-axes, respectively) to cover the whole interaction surface of Laccase was 54 Å× 68 Å × 74 Å. The binding model that has the lowest binding free energy was searched out from 10 different conformers for each ligand (HBDs and HBAs).

Effect of DES nature and concentration on the laccase activity
In the literature it has been demonstrating that enzymes are able to maintain and even enhance their activity and stability on DES-based media, thus acting as improved solvents or co-solvents over conventional organic solvents. 8 10 Besides the high viscosity of pure anhydrous DES when compared to water, Leija et al. 27 proved that the viscosity of aqueous solutions of DES has no influence on enzyme activity. Other works have shown that the presence of water in DES results in a high viscosity decrease, attaining values close to the viscosity of water. 26,28,29 The laccase activity at 25ºC in presence ChCl-based DES at different concentrations and compositions, including with the two DES constituents alone, are shown in Figure 1.
Detailed results are given in the Supporting Information (Table S1 and Table S2) ion as reported in the literature, 30 and/or due to an inhibitory effect of ChCl on the laccase activity by disrupting the intramolecular hydrogen bonds. 16,31 The results obtained with polyols agree with literature, for instance, the activity of β-glucosidase was higher when 11 polyols, namely ethylene glycol and propylene glycol, were used as constituent in ChCl based DES. 32 The authors explain that a single constituent molecule may diffuse into the enzyme core causing its denaturation by disrupting intramolecular hydrogen bonds. 32 On the other hand, the formation of hydrogen bonds in DES may prevent its diffusion into the enzyme active centre. 32 (Table S1 and Table S2). In general, and only by replacing the cholinium- The last cholinium-based DES investigated comprises ChDHC, which shows to be the most appropriate HBA amongst the several salts studied. The results presented in Figure   3 show that the laccase activity depends on the DES constituents, molar ratio and concentration. Detailed results are given in the Supporting Information (Table S1 and   Table S2). Contrarily to what has been show before with ChCl and ChDHP, ChDHC induces a relevant increase in the enzyme activity, which is even higher than the It has been shown previously that betaine is able to preserve proteins against deactivation and aggregation. 33 Furthermore, Khodaverdiana et al. 16 provided encouraging results for improving laccase activity and stability using betaine-based natural DES. Therefore, the effect of betaine-based DES on the activity of laccase was investigated using the same polyols as HBD, allowing to address the effect of DES formed by cholinium-based salts versus betaine. Detailed results are given in the Supporting Information (Table S1 and   Table S2). As presented in Figure 4, while this seems to hold true for the betaine aqueous 16 solutions, the activity of laccase is similar for all solutions of betaine-based DES, with a maximum activity enhancement of 20% when compared to the buffer control results.
Furthermore, betaine, unlike some of the cholinium-based salts studied before, does not lead to synergetic effects with polyols when combined to form DES. Overall, the best results were achieved with DES based on ChDHC and Ery or Xyl at concentrations of 25 and 50 wt%, which seems to mainly derive from the increased number of hydroxyl groups in their chemical structures and their synergetic effect.  (Table S4 and Table S5; Figures    Relative activity (%) is addressed by comparison with the free-DES phosphate buffer 50 mM, pH 7.0 (control, 100%). 21

CONCLUSIONS
The catalytic activity of laccase in presence of sixteen cholinium-and betaine-based DES and of the respective constituents, and at different molar ratio, concentration and temperature was investigated. Although all polyols investigated are favorable to increase the laccase activity, ChCl and ChCl-based DES lead to a decrease in the enzyme activity.
However, the replacement of the chloride anion in the cholinium salts leads to significant improvements in the laccase activity. In particular, the DES ChDHP:Xyl and all ChDHCbased DES are improved co-solvents and significantly enhance the activity of laccase in oxidative reactions. Moreover, the enzyme activity in these DES aqueous solutions is higher than in aqueous solution of its individual components and in the buffer control, demonstrating a synergetic effect. Overall, enhancements in the relative laccase activity up to 200% at 25ºC were observed. Furthermore, it was demonstrated that aqueous solutions of DES are better storage media than the respective buffer control at -80ºC, where the relative activity of laccase is kept up to 135% for 20 days.
By molecular docking studies, it was demonstrated that all polyols and HBD components mainly establish hydrogen-bonding with the enzyme amino acids, with the exception of [Ch] + and betaine that also establish electrostatic interactions with aspartic acid amino acids. Furthermore, the higher the HBD-or HBA-laccase absolute values of docking affinity energies the higher the activity of laccase, which is dependent on the number of hydroxyl groups present in the DES components and their ability to hydrogen-bond with the enzyme amino acids. Although interactions established between the DES components and the side chain amino acids may lead to conformational rearrangements of the enzyme leaving its active site more accessible to substrates, the overall results indicate that the 22 increase in the enzymatic activity could be a direct consequence of interactions established with histidine at the catalytic cluster.
The present work demonstrates that the type of DES, molar ratio and concentration are fundamental issues to improve laccase oxidation reactions. These results suggest that by choosing DES appropriately they can act as remarkable solvents or co-solvents to improve biocatalysis performance. The results provided reveal the potential perspectives of application of these benign solvents or co-solvents to multiple oxidative biotransformations employing non-aqueous soluble laccase substrates, such as lignin (biodelignification and biobleaching of paper pulp), dyes (wastewater decolorization), phenolic compounds (bioremediation) and organic substrates used by pharmaceutical and food industries.

AUTHOR INFORMATION
Corresponding Author *E-mail address: aptavares@ua.pt