Deep Eutectic Solvents as Media for Peroxidation Reactions

Feb 24, 2016 - ABSTRACT: In the present study, the effect of various choline chloride (ChCl) and ethylammonium chloride (EAC) based deep eutectic solv...
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Deep Eutectic Solvents as Media for Peroxidation Reactions Catalyzed by Heme-Dependent Biocatalysts Athena A. Papadopoulou, Evdoxia Efstathiadou, Michaela Patila, Angeliki C. Polydera, and Haralambos Stamatis* Department of Biological Applications and Technologies, Laboratory of Biotechnology, University of Ioannina, University Campus, 45110 Ioannina, Greece ABSTRACT: In the present study, the effect of various choline chloride (ChCl) and ethylammonium chloride (EAC) based deep eutectic solvents (DES), formed with three biodegradable hydrogen bond donors (urea, glycerol, and ethylene glycol), on the catalytic behavior and structure of heme-dependent biocatalysts, such as cytochrome c and horse radish peroxidase, was investigated. The peroxidase activity of biocatalysts strongly depends on the nature of the ammonium salt and hydrogen bond donor used for the formation of DES, as well as on DES concentration in the reaction media. UV−vis and circular dichroism spectroscopic studies indicate that the effect of DES on the biocatalytic behavior of cyt c is correlated with heme microenvironment perturbations. Moreover, EACbased DES stabilize cyt c, enhance its activity for the biodegradation of an industrial dye and are successfully reused up to four times, indicating that these environmentally friendly solvents could be promising media for biocatalytic processes of industrial interest.

1. INTRODUCTION One of the main challenges that chemical and pharmaceutical industries face is the development of efficient processes, which properly set out to reduce the release waste generation and energy consumption. Biocatalysis is one of the greenest technologies that offers many advantages for the industrial production of chemicals, since it reduces synthetic steps, energy, and raw material consumption, and minimizes the yield of undesired side products, generating less waste.1 The application of enzymes for the development of such green processes can be greatly enhanced by the use of novel environmentally friendly biodegradable reaction media rather than hazardous volatile organic solvents.2 In this context, the development of bioprocesses in ionic liquids (ILs) and deep eutectic solvents (DES) is a promising approach.3,4 DES are natural mixtures of low-cost biodegradable components such as quaternary ammonium salts (e.g., choline chloride) and uncharged hydrogen-bond donors (HBD) (such as urea, carboxylic acids, or polyols) with lower melting points than those of their parental compounds.5 The use of DES as media for chemical and biochemical reactions has attracted the scientific interest, since they represent a new generation of solvents that overcome the high cost and toxicity of second generation ILs.6,7 Although DES have an uncharged component and are not entirely ionic, they possess many unique IL-like solvent properties such as negligible vapor pressure, high chemical and thermal stability, and high solubility of various compounds. The main advantages of © XXXX American Chemical Society

DES include their straightforward synthesis that does not require any purification of the solvent, their high biodegradability, and low toxicity.3 Considering these aspects, the use of DES as reaction media for enzymatic reactions alone, or as cosolvents, has been developed over the past decade. The beneficial effect of some DES used as cosolvents on various enzymes, such as lipases, epoxide hydrolases, proteases, and horseradish peroxidase, has been well demonstrated in previous studies.8−14 Recently, Parnica and Antalik have shown that cyt c can be efficiently solubilized and stored for a long time in DES such as guanidine hydrochloride−urea (GuHCl:U) and guanidine thiocyanate−urea (GuSCN:U).15 In the present study, the effect of ChCl and EAC-based DES formulated with three biodegradable HBD, such as urea (U), glycerol (Gly), and ethylene glycol (EG), on the peroxidation activity and stability of heme-dependent biocatalysts, such as cytochrome c (cyt c) and horseradish peroxidase (HRP), was investigated. The structures of the six DES used in the present work are depicted in Figure 1. Using cyt c, which is one of the most thoroughly physicochemically characterized metalloproteins,16 as a model protein, the effect of DES on the structure of Special Issue: International Conference on Chemical and Biochemical Engineering 2015 Received: December 21, 2015 Revised: February 15, 2016 Accepted: February 24, 2016

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DOI: 10.1021/acs.iecr.5b04867 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 1. Structure of DES components used in the present work.

NaOH) to the required pH before being used in the biocatalytic reactions. All experiments were performed in triplicate. Control experiments without biocatalyst were also carried out and no substrate conversion was observed in all cases studied. All the reaction rates were calculated from the slope of the linear portion of plots of absorbance versus time. The relative activity was expressed in each case as the ratio of the activity in the presence of DES to that observed in pure buffer solutions. 2.4. Stability Study. Stability studies of cyt c in the presence of aqueous solution of DES (30% v/v) were performed by incubating an amount of protein for 24 h at 40 °C. Remaining peroxidase activity was determined by the enzymatic oxidation of guaiacol, as described above. 2.5. UV−vis Spectroscopy. The absorption spectrum of cyt c (25 μg/mL) was recorded in the presence of 30% and 70% v/v of DES, in a double-beam UV−vis spectrophotometer (UV-1601 Shimadzu, Tokyo, Japan). 2.6. Circular Dichroism Study. Soret region CD spectra (350−450 nm) of cyt c (200 μg mL−1) in 0.5 mM sodium phosphate buffer pH 7.0 and in 30% (v/v) aqueous solution of ChCl:U and EAC:EG were obtained using a Jasco J-815 spectropolarimeter (Tokyo, Japan) in a 1 cm path length quartz cell. All spectra were obtained at 25 °C with a 2 nm bandwidth and a scan speed of 10 nm min−1. For each medium scanned, a baseline was recorded and subtracted from the protein spectrum. All scan measurements were performed in triplicate. 2.7. Dye Decolorization and Recycling of DES. Decolorization activity of cyt c was measured spectrophotometrically by recording the color elimination of pinacyanol chloride. In a typical oxidation reaction, pinacyanol chloride (130 μM) and cyt c (80 μg/mL) were added in aqueous solutions of DES (30% and 50% v/v). The reaction was started by adding 0.3 mM H2O2 at 27 °C. At predetermined time intervals, 30 μL aliquots were removed from the reaction mixture and the remaining concentration of the dye was monitored at 603 nm using an extinction coefficient for pinacyanol chloride equal to ε = 82 350 M−1 cm−1.19 The reusability of DES was investigated as described elsewhere.6 Briefly, 30 mg mL−1 of immobilized biocatalyst (containing 2 μg of cyt c per mg of Celite) were added to the reaction mixture (0.5 mL) containing 130 μM pinacyanol chloride. The immobilization of biocatalyst on Celite was prepared according to previous study.20 The reaction was started by adding 0.3 mM

the heme-active center was investigated using a combination of spectroscopic techniques. The effect of these DES on the ability of cyt c to catalyze the degradation of an industrial dye was also investigated.

2. EXPERIMENTAL SECTION 2.1. Materials. Cytochrome c from equine heart (>95% protein content) with a specific activity of 552 U/mg solid (1 unit corresponds to the amount of enzyme that causes an increase in absorbance at 470 nm of 0.01 per minute at pH 7.0 and 25 °C in a reaction mixture containing guaiacol and hydrogen peroxide), and horseradish peroxidase (HRP, type VI, ∼ 66% protein content) 261 U/mg solid (1 unit corresponds to the amount of enzyme which produces 1 mg of purpurogallin from pyrogallol in 20 s at pH 6.0 at 25 °C) were purchased from Sigma-Aldrich and were used without further purification. 2-Methoxyphenol (guaiacol), pinacyanol chloride, and hydrogen peroxide (30% w/v) were purchased from Sigma. H2O2 concentration was determined spectrophotometrically at 240 nm (ε240 = 43.6 M−1 cm−1).17 Choline chloride (ChCl), ethylammonium chloride (EAC), urea (U), glycerol (Gly), and ethylene glycol (EG) were of analytical grade and were obtained from Sigma-Aldrich, Merck or Applichem. 2.2. Preparation of DES. DES were prepared as described elsewhere.18 Briefly, ammonium salts (ChCl or EAC) and hydrogen bond donors (U, Gly, and EG) were added at a molar ratio of 1:2, or 1:1.5 in the case of EAC-based DES, in a beaker and incubated at 100 °C, or 80 °C (in the case of glycerolbased DES), for 1 h with intermediate stirring, until a colorless clear liquid was formed. The resulting DES were then dried over P2O5 in a desiccator at room temperature for at least 2 weeks prior to use. 2.3. Oxidation Reactions. The peroxidase activity of cyt c and HRP was determined by following the color formation of guaiacol oxidation at 470 nm with H2O2. The oxidation reactions were carried out at 30 °C in 0−90% v/v aqueous solutions of DES in 50 mM sodium phosphate buffer pH 7.0 in the case of cyt c, or pH 6.5 in the case of HRP. Reaction conditions were adjusted according to the type of biocatalyst used. In the case of cyt c, 2 mM guaiacol, 20 mM of H2O2, and 13.8 U/mL of protein were added in the reaction mixture. In the case of HRP (0.026 U mL−1), the concentration of the substrate was 20 mM and the concentration of H2O2 was 0.2 mM. All the reaction media were readjusted (with HCl or B

DOI: 10.1021/acs.iecr.5b04867 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

Figure 2. Relative peroxidase activity of cyt c for the oxidation of guaiacol in the presence of various amounts of (a) ChCl-based and (b) EAC-based DES. The peroxidase activity of cyt c in 50 mM phosphate buffer pH 7.0 is indicated as 1.0.

Figure 3. Relative peroxidase activity of HRP for the oxidation of guaiacol in the presence of various amounts of (a) ChCl-based and (b) EAC-based DES. The peroxidase activity of HRP in 50 mM phosphate buffer pH 6.8 is indicated as 1.0.

H2O2 and the mixture was incubated at 27 °C for 1 h. Upon completion of the reaction, 1 mL of water was added to the reaction mixture and the immobilized biocatalyst was filtered off. The aqueous filtrate containing the DES was washed with ethyl acetate, in order to remove any amount of the residual substrates and products, and then the water was evaporated in vacuo. The residual DES was dried under high vacuum at 40 °C until constant weight, while the purity of the recycled DES was verified by 1H NMR. All experiments were performed in triplicate.

concentration. More specifically, in DES-based media an increased peroxidase activity was observed at most concentrations tested, compared to that in pure buffer. It is noteworthy that the presence of EAC-based DES led to a significant higher cyt c peroxidase activity compared to ChClbased DES. More specifically, when ChCl:U was added in the reaction mixture at a concentration of 30% v/v, an 8-fold peroxidase activity enhancement was observed compared to that in buffer, while at the same concentration of EAC:U the activation was almost 100-fold higher indicating that the nature of ammonium salts used for the formation of DES is crucial for the peroxidase activity of cyt c. It must be noted that such activation by DES was not observed for another heme-dependent enzyme, such as HRP, which catalyzes the same reaction (Figure 3). In this case, the addition of DES led to a decrease of HRP activity compared to that observed in buffer, while this deactivation was more intensive as the concentration of DES in the reaction mixture increased. The different effect of DES on the catalytic behavior of enzymes indicates that this effect is enzyme dependent and

3. RESULTS AND DISCUSSIONS 3.1. Effect of DES on Peroxidase Activity. In the present study, the effect of various ChCl-based and EAC-based DES (ChCl:U ChCl: Gly, ChCl:EG, EAC:U, EAC:Gly, and EAC:EG) on the peroxidase activity of cyt c and HRP was investigated. As it can be seen in Figure 2, in all cases studied, the presence of DES in the reaction mixture affected the peroxidase activity of cyt c and this effect strongly depended on the nature of the ammonium salt and the hydrogen bond donor used for the formation of DES, as well as on their C

DOI: 10.1021/acs.iecr.5b04867 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Figure 4. Relative peroxidase activity of cyt c for the oxidation of guaiacol in the presence of various amounts of (a) EAC:U and (b) EAC:EG and their individual components. The peroxidase activity of cyt c in 50 mM phosphate buffer pH 7.0 is indicated as 1.0.

possible DES dissociation in water and the synergistic action of their individual components. As seen in Figure 4, the addition of equal amounts of the individual components of EAC:U and EAC:EG in the reaction mixture slightly affects the biocatalytic activity of cyt c, in contrast to the significant effect of the corresponding DES. Similar behavior was observed for the components of all the DES tested in the present work (data not shown). This observation clearly indicates that the beneficial effect of DES on the peroxidase activity of cyt c is the result of the presence of DES complex in the reaction mixture, rather than the effect of its individual components, which is in accordance to that observed for other enzymes in choliniumbased DES.14,22 The relatively high peroxidase activity of cyt c in the presence of high concentrations of U, a strong unfolding agent for proteins, could be attributed to the exposed heme prosthetic group of the unfolded cyt c, that is able, in some extent, to catalyze peroxidation reactions.27 3.2. Effect of DES on Stability of Cyt c. In order to further investigate the effect of these DES on the biocatalytic behavior of cyt c, its functional stability was investigated, by incubating the protein in various DES at 40 °C. After 24 h incubation of the protein in buffer and 30% v/v aqueous solutions of DES, the remaining peroxidase activity of cyt c was determined. As it can be seen in Figure 5, cyt c loses about 35% of its initial activity after 24 h incubation in buffer, while the remaining peroxidase activity in almost all DES-based media is significantly higher, indicating that the presence of DES increases the stability of the protein. It is interesting to note that in both ChCl-based and EAC-based DES, the stability of cyt c increased by the following HBD order: U > Gly > EG. The beneficial effect of U on protein stability, when U is used as a hydrogen-bond donor compared to Gly and EG, has also been observed in the case of HRP (data not shown), which is in accordance to that recently reported.14 It was proposed that the lower stability of the enzyme in the presence of ChCl:Gly and ChCl:EG compared to ChC:U is correlated with conformational changes of the heme prosthetic group of the enzyme.14 3.3. Structural Studies of Cyt c in DES-Based Media. In order to evaluate any conformational changes of the heme

could be attributed to the specific interactions of DES with protein molecules. The increased peroxidase activity of cyt c in EAC-based DES compared to that observed in ChCl-based DES could be attributed to different interactions of these DES with cyt c molecules, as well as to the difference in their viscosities.21 The higher viscosity of ChCl-based DES could increase the mass transfer limitations of the substrates to the cyt c microenvironment, which would be expected to reduce its peroxidase activity. Consequently, the appropriate ammonium salt selection is an important factor that significantly affects the activity of cyt c. The nature of hydrogen-bond donors used for the formation of DES also affects significantly the catalytic activity of cyt c. The use of U causes a relatively higher activation of cyt c than the other two HBD, Gly and EG, independently of the ammonium salt used for the formation of DES. This effect differs to that reported for other enzymes, such as lipase and epoxide hydrolase, since these enzymes exhibited an enhanced catalytic activity in DES containing Gly and EG as HBD rather than in those containing U.11,22,23 The beneficial effect of DES on the peroxidase activity of cyt c was more obvious at lower or moderate concentrations, while at high concentrations the peroxidase activity of cyt c was reduced, which is in accordance to that reported for other enzymes.10,11,24,25 The presence of DES, especially at high concentrations, in the reaction mixture can significantly affect the properties of the reaction medium such as polarity and viscosity and thus the biocatalytic behavior of enzymes.11 The decreased biocatalytic activity at high DES concentrations could be attributed to protein destabilization or active site perturbation,11,22,23 as well as to mass transfer limitation of the substrate to the biocatalyst active site.26 Moreover, it has been proposed that the “entrapment” of the substrate within DES structure through H-bonding, especially at very high DES concentrations, could reduce the availability of the substrate to the protein microenvironment and thus decrease its biocatalytic activity.12 Since a DES is prepared by mixing two individual components (ammonium salts and hydrogen bond donors), it is interesting to investigate if the effect of DES in the peroxidase activity of cyt c described before is attributed to a D

DOI: 10.1021/acs.iecr.5b04867 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Changes in the conformation of the heme crevice of protein in the presence of DES were further investigated through CD spectroscopic study. The Soret region of the CD spectrum (350−450 nm) of the native cyt c exhibits a negative peak at 416 nm and a positive peak at 402 nm due to a split Cotton effect.33,34 As seen in Figure 7, cyt c in buffer exhibits all of the

Figure 5. Stability of cyt c in buffer and 30% v/v aqueous solutions of various ChCl and EAC-based DES, after incubation at 40 °C for 24 h. As 100% is indicated the activity at t = 0 min.

prosthetic group of cyt c in aqueous solutions of DES, the UV− vis spectra of the protein was recorded. In its native conformation, cyt c displays characteristic absorption bands in the UV−vis region; a sharp Soret band at 409 nm, a weaker broad band at 530 nm, and a very weak charge-transfer band at 695 nm arising from the coordination of the sulfur atom of Met80 (the axial ligand) with heme iron.28−31 As it can be seen in Figure 6, the addition of DES such as ChCl:U and EAC:U induces significant changes in the UV−vis spectrum of the protein and these changes depend on the concentration of DES. More specifically, in the presence of both DES tested, the absorbance of the Soret and Q bands was increased and a small blue shift (up to 3 nm) was observed, while the 695 nm band was completely lost at high DES concentrations. These spectral changes could be correlated with changes in the microenvironment of heme of cyt c, due to the perturbation or cleavage of the coordination bond of Met80 with the heme iron.16,32

Figure 7. Soret region CD spectrum of cyt c in 50 mM phosphate buffer, pH 7.0, and in the presence of 30% v/v aqueous solution of ChCl:U and EAC:EG.

above characteristic bands in the Soret region, while in the presence of 30% v/v of EAC-based or ChCl-based DES, spectral changes were observed. These spectral changes observed in the CD Soret region also indicate that the heme plane undergoes a reorientation in the active site pocket.35,36 This reorientation probably makes the heme active center of cyt c more accessible to the substrates, thus leading to a higher peroxidase activity compared to that observed in buffer29,32−34 which is in accordance to that described in Figure 2. On the other hand, the complete loss of the CT-band at 695 nm in the UV−vis spectrum, at a high DES concentration (70% v/v) indicates that the structural changes in the heme microenvironment of cyt c are more intensive, which probably

Figure 6. UV−visible spectra (300−600 nm) of cyt c in 50 mM phosphate buffer, pH 7.0, and in the presence of 30% and 70% v/v of (a) ChCl:U and (b) EAC:U DES. (insets) Respective absorption spectra of cyt c at the charge transfer band (695 nm). E

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Figure 8. Cyt c-catalyzed decolorization of pinacyanol chloride with H2O2 in phosphate buffer 50 mM, pH 7.0, and in the presence of (a) 30% v/v and (b) 50% v/v of EAC-based DES. Decolorization rate (μM min−1) in buffer 3.4; 30% EAC:U 4.3; 30% EAC:Gly 3.2; 30% EAC:EG 5.4; 50% EAC:U 11.1; 50% EAC:Gly 4.2; 50% EAC:EG 11.3.

correlate with global protein denaturation37,38 and therefore to reduced peroxidase activity observed in this case (Figure 2). 3.4. Decolorization Study of a Dye. In order to exploit the positive effect of DES-based media on a biocatalyzed process of industrial interest, we have investigated the effect of various DES on the decolorization activity of cyt c, using pinacyanol chloride, a symmetric trimethinecyanine dye with industrial use,39 as a model substrate. As shown in Figure 8, the presence of DES enhances the decolorization activity of cyt c compared to that observed in buffer. This effect was more evident in the presence of 50% v/v of DES in the reaction mixture. More specifically, the decoloriazation rate in 50% v/v of EAC:U and EAC:EG was about 3.3 fold higher in both cases studied, compared to that in buffer. This observation correlates well with the results obtained before, regarding the activity studies of cyt c in these media (Figure 2). The decolorization of pinacyanol chloride by cyt c was further used as a model reaction, in order to investigate the reusability of DES as reaction media. For this purpose, the reusability of EAC:U and EAC:EG was studied in the same decolorization reaction using cyt c immobilized onto Celite as a biocatalyst, as described in Section 2.7. As seen in Figure 9, both EAC:EG and EAC:U were successfully reused up to three times, resulting in comparable decolorization yields with those observed for the initial reaction, indicating that these DES could be considered as promising environmentally friendly solvents for biocatalytic reactions of industrial interest.

Figure 9. Recycle of EAC:EG and EAC:U (50% v/v) in the decolorization of pinacyanol chloride using immobilized cyt c as biocatalyst after 1 h of incubation at 27 °C.

buffer. Spectroscopy studies indicated that the presence of DES induces conformational changes in the micronenvironment of heme, which are more pronounced in higher DES concentrations. The successful application of DES on the biocatalytic degradation of an industrial dye and their efficient recyclability and reuse indicate that these DES are promising environmentally friendly solvents for enzymatic processes with industrial interest.

4. CONCLUSION Summarizing the results presented here, we can conclude that ChCl and EAC-based DES could efficiently be used as cosolvents for biocatalytic oxidations catalyzed by hemedependent biocatalysts. The catalytic behavior of these hemedependent proteins is strongly affected by the nature of the ammonium salt and the hydrogen bond donor used for the formation of DES, as well as by their concentration in the reaction mixture. EAC-based DES have a considerable more beneficial effect on the peroxidase activity of cyt c compared to ChCl-based DES. Moreover, the presence of all DES studied enhances the functional stability of the proteins compared to



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DOI: 10.1021/acs.iecr.5b04867 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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ACKNOWLEDGMENTS This research project has been cofinanced by the bilateral Personnel Exchange Programme between Greece and Germany (IKYDA 2015). The authors would like to thank the Atherothrombosis Research Centre of the University of Ioannina for providing access to the facilities.



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DOI: 10.1021/acs.iecr.5b04867 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX