Enzyme-mediated free radical polymerization of acrylamide in deep eutectic solvents

Abstract

Deep eutectic solvents (DES) have attracted considerable attention as reaction media in biocatalysis due to their particular solvent properties. It has been demonstrated that some enzymes do not denature in these solvents and that under certain conditions, enzyme-catalyzed reactions in DES can result in higher conversions and reaction rates than those obtained in conventional media. In this work we report the enzyme-mediated free radical polymerization of acrylamide in nearly non-aqueous choline chloride–urea (CCl–U) and choline chloride–glycerol (CCl–Gly) DES. The catalytic activity of the enzyme, horseradish peroxidase (HRP), was observed to be lower at high DES concentrations than in phosphate buffer solution, whereas the thermal stability was enhanced. According to fluorescence and UV-vis spectroscopy studies, the drop in the enzyme activity could be a consequence of the partial denaturation of HRP in the “hydrated” DES. Despite this significant loss in activity, HRP was still able to initiate the free radical polymerization of acrylamide with full monomer conversions. For the CCl–U system, polymers with similar average molecular weight and slightly narrower polydispersities compared to those synthesized in a totally aqueous environment were obtained. Furthermore, taking advantage of the low freezing point of CCl–Gly, it was possible to synthesize polyacrylamide at 4 °C, while no polymer was obtained using water at the same temperature. These results illustrate the potential of DES to enable biotransformations in a wide range of temperatures, leading for instance to new strategies in materials synthesis, such as enzymatic cryo-polymerization.

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Introduction

Nowadays, the concept of green chemistry has become more relevant in organic, inorganic and polymer synthesis due to the fact that many synthesis pathways involve the use of toxic and non-biodegradable compounds as precursors, reaction media and catalysts. In pursuit of green and sustainable alternatives, the use of enzymes for materials synthesis has been proposed as a replacement for inorganic catalysts aside from advantages such as their high specificity and the fact that they can readily be immobilized.¹ Enzymes have been employed successfully in the synthesis of small molecules to large polymers.² These bio-catalysts usually work in aqueous solution under mild conditions of temperature and pH.³ Although it is well known that an aqueous environment, the natural medium of enzymes, is preferred to maintain their activity, there are many enzymatic reactions that are difficult to carry out in water, either because of poor solubility of the substrate/product or because undesirable hydrolytic side-reactions can occur. Such restrictions have led to the use of alternative media capable of providing better conditions to maintain or improve the enzyme performance for those specific reactions.

Attempts of carrying out reactions with enzymes in non-conventional media range from aqueous solutions of acetone/ethanol and biphasic mixtures to nearly non-aqueous or even anhydrous organic solvents,⁴ but it still remains challenging. Interestingly, some enzymes are able to maintain their catalytic activity and show remarkable novel properties when in organic media;⁵ sometimes exhibiting faster rates and higher selectivity and stability than in water. Further, in some cases it is possible to recover or recycle them easily. Among the solvents used for this goal, ionic liquids (ILs), low melting point salts, have shown promise for application in biocatalytic processes due to their high chemical and thermal stability, negligible vapor pressure and tuneable properties.⁶

In 2000 Erbeldinger and co-workers reported the first enzymatic reaction carried out in ILs.⁷ They described the thermolysin-catalysed synthesis of Z-aspartame using [BMIM][PF₆]/H₂O (95/5%, v/v) as solvent, where the enzyme exhibited higher stability than in ethylacetate/H₂O with comparable reaction rates. Shortly after, other research groups showed that it is possible to catalyse a variety of transesterification, ammonolysis, epoxidation⁸ and enantioselective reactions^9,10 using a lipase B in anhydrous imidazolium based ILs with better reaction rates and higher yield and selectivity than those observed in conventional organic media. Since enzymes seem to better tolerate ILs than conventional organic solvents, imidazolium based ILs, mainly [BMIM][BF₄] and [BMIM][PF₆],⁶ have been employed as co-solvents with water, as a secondary phase or non-aqueous solvents for a number of enzymatic reactions.

The vast majority of research on enzymatic reactions in ILs to date has focused on hydrolases.^6,8 Nevertheless, there has also been an increasing number of studies using oxidoreductases in these media due to their wide application range in organic synthesis, e.g., synthesis of chiral alcohols from aldehydes/ketones,^11,12 polymer synthesis^13–16 and oxidative degradation of pollutants.^17,18 In particular, horseradish peroxidase (HRP) has shown good activity in [BMIM][BF₄] in the presence of small amount of water (4.53% v/v), and high stability in this IL upon immobilization in agarose hydrogels for biosensing applications.¹⁹ The activity of HRP was reported to increase 30 to 240-fold in a tailor made IL (tetrakis (2-hydroxyethyl) ammonium trifluoromethanesulfonate) compared to that in conventional ILs, and more than 10 times greater than that in methanol.²⁰ This enzyme has also been used in water-in-IL microemulsions for the oxidation of pyrogallol by H₂O₂ and a significant increase of the reaction rate was obtained in contrast with that in oil microemulsions.²¹

Despite all of these seemingly attractive scenarios, ILs' toxicity, and the high costs derived from their synthesis and post-purification limit their use in large-scale applications. The emergence of a new generation of ILs with striking properties, called deep-eutectic solvents (DESs), offers an alternative to overcome the previously mentioned drawbacks. DESs are typically obtained by mixing a quaternary ammonium salt with hydrogen-bond donors.^22,23 They exhibit a much lower freezing point than that of their individual components. In general DESs are biodegradable, inexpensive, and easy to synthesize. As a type of ILs, DESs have proved to be very good solvents for a great variety of substances and they have shown potential use in biocatalysis,^24–27 dissolution of proteins and natural polymers,^28–30 polymer synthesis^31–33 and materials preparation.³⁴ In addition to all these advantages, we propose that the use of DES as non-volatile solvents coupled with enzymes as catalysts, represents a further step towards greener alternatives in polymer synthesis by free-radicals.

Recently, the work of Wu and collaborators suggested that HRP activity is favored in highly diluted solutions of DES's constituents (ca. below 8% w/w).³⁵ Motivated by this and the fact that some oxidoreductases, such as HRP, can induce polymerization of vinyl monomers,^13–15 we studied the effect of these solvents containing small amounts of water on the HRP performance. Furthermore, we carried out an enzyme-mediated polymerization of acrylamide (AA) in DES-aqueous mixtures in order to demonstrate for the first time, that it is not only possible for HRP to retain catalytic activity at relatively high temperatures and low pressure, but also to initiate the free-radical polymerization of an acrylate in these non-conventional media with similar average molecular weights of the polymer compared to those obtained in aqueous buffered solution.

Results and discussion

HRP-mediated polymerization of AA was carried out in three different solvents, two DES-aqueous mixtures consisting of choline chloride–urea (CCl–U) and choline chloride–glycerol (CCl–Gly) (both in a 1 : 2 molar ratio, respectively) at 80% v/v DES (20% of water and 80% v/v of the corresponding DES), and a third one consisting of pure water as reference. For all these systems, initiation by free-radical species was generated by the catalytic system HRP/H₂O₂/2,4-pentanodione as reported by Emery et al. for polyacrylamide (PAA) synthesis in water (Scheme 1).¹⁴ As in their experiments, no polymer was formed in absence of enzyme, of hydrogen peroxide, and of 2,4-pentanodione, further confirming that the combination of the three components is the sole responsible of initiation. The free-radical polymerization was performed at room temperature (RT) and also at 50 °C.

Scheme 1 Reaction scheme of HRP-mediated free radical polymerization of AA in water¹⁴ (we suggest that the same reaction mechanism occurs when using DES since DES appeared to be an inert solvent during polymerization).

Prior to polymerizations experiments we determined the specific activity and thermal stability of HRP in phosphate buffer solution (0.1 M, pH 7) and DES-aqueous mixtures at different DES concentrations. Fig. 1 shows that HRP activity is higher in CCl–Gly than CCl–U aqueous mixtures, whereas in both media the activity decreases with increasing DES concentration. Given the large difference in viscosity between water and DESs (Table 1),^23,36–40 the drop on the enzyme activity may be related to some extent to diffusional and mass transfer limitations of the reagents in both reaction media. It is important to consider that although the viscosity of CCl–Gly in its pure state is almost three times lower than that of CCl–U, CCl–Gly has a higher viscosity than CCl–U when in aqueous mixtures with more than 6% w/w of water.⁴¹ If this were the only factor involved, it would be expected that the catalytic activity of HRP would be higher in CCl–U than CCl–Gly aqueous mixtures with less than 90% v/v DES concentration. This is clearly not the main factor for the drop on HRP activity.

Fig. 1 Relative specific activity of HRP in DESs-aqueous mixtures at RT and different DES concentrations (experiments were at least triplicated, see Fig. S1 in the ESI†).

Table 1 Freezing temperature and viscosity of the used DESs and DESs-aqueous mixtures

Quaternary ammonium salt	Hydrogen bond donor (HBD)	Salt : HBD (molar ratio)	Tf (°C)	Viscosity (cP)
				Pure	80 v/v% DES^a (20 °C)	80 v/v% DES^a (50 °C)
a Viscosity values were taken from the corresponding references by looking up the wt% equivalent concentrations.
		1 : 2	12, ref. 23	750 (25 °C), ref. 37	17.6, ref. 39	6.5, ref. 39
		1 : 2	−40, ref. 36	376 (20 °C), ref. 38	41.2, ref. 40	13.3, ref. 40

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Changes on the enzyme conformation, triggered either by the solvent or temperatures effects, can also significantly affect the catalytic activity of enzymes. The impact of the solvents on the enzyme conformation was investigated by fluorescence spectroscopy of the tryptophan residue of HRP in phosphate buffer solution (PBS) and DES-aqueous mixtures at a high DES concentration (Fig. 2a). Alterations of the environment surrounding the tryptophan residue, reflected on shifts and intensity variations of the amino acid fluorescence peak, can be used to infer changes on the tertiary structure of the enzyme. By comparing the fluorescence emission spectra of HRP in CCl–Gly and CCl–U to that obtained in PBS, an intensification of the tryptophan fluorescence signal can be observed. In heme proteins, this has been attributed to an increase of the distance between the tryptophan residue and the heme group upon protein denaturation.⁴²

Fig. 2 (a) Tryptophan fluorescence emission spectra of HRP (excitation wavelength 295 nm), and (b) UV-vis spectra of the Soret region of HRP in PBS (0.1 M, pH 7) and DESs-aqueous mixtures (80% v/v DES) at RT.

The spectra of HRP in CCl–Gly displayed also a slight blue shift of the maximum intensity (λ_max), which may indicate a rearrangement of the enzyme structure in this medium, where the heme group partially dissociates from the active site.^43,44 On the other hand, in the CCl–U aqueous mixture, an evident red shift of λ_max is observed as a result of the HRP unfolding and exposition of the indole ring to the polar solvent.^45,46 These results are in agreement with the absorbance measurements of the Soret region of HRP.

Fig. 2b shows that DESs induced a decrease in the Soret band absorption with respect to the same band in aqueous solution. For CCl–Gly no detectable shift in wavelength for the band is observed as it has been seen earlier for inactivation of HRP in presence of sodium azide due to partial degradation of the heme group.⁴⁷ HRP absorption spectra in CCl–U showed a red shift that coincides with that observed for heme non-covalently bonded proteins such as hemoglobin and myoglobin, where this has been attributed to the expulsion of the prosthetic group into solution.⁴⁸ As it can be seen from Fig. 1, the partial denaturation of HRP in CCl–U due to unfolding accompanied by expulsion of the heme group into solution seems to have a greater negative impact on the catalytic activity than the partial dissociation of the prosthetic group in CCl–Gly.

Regardless of this decrease on the activity at higher DES concentrations, CCl–U and CCl–Gly concentrations in the reaction media for AA polymerization were fixed to 80% v/v mainly for two reasons: (i) to preserve the halide–hydrogen bond donor (HBD) supramolecular complexes characteristic of DES, which integrate water within up to certain threshold;^{41,44,49–51} and (ii) to lower the viscosity of these solvents and thus, facilitating its handling and homogeneous stirring during the reaction,^52,53 taking also into account that the viscosity of these reaction systems increases as soon as the polymerization starts.

As for the thermal stability of the enzyme at 80% v/v DES, we found that it was notably higher in CCl–U and comparable in CCl–Gly to that in PBS at 50 °C (Fig. 3). The half-life time (t_1/2) obtained from Fig. 3 for each enzyme solution is shown in Table 2. The enhanced thermal stability of enzymes generally observed in conventional ILs has been mainly ascribed to the high viscosity of these solvents, which according to some authors slows the migration of the protein domain from the active conformation into the inactive one;⁶ and to the ability of ILs to compact the native structural conformation of the enzyme.⁵⁴

Fig. 3 Thermal stability of HRP in PBS (0.1 M, pH 7) and DES aqueous mixtures (80% v/v DES) at 50 °C (experiments were at least triplicated).

Table 2 Half-life time values (t_1/2) for HRP obtained in PBS and DESs-aqueous mixtures (80% v/v DES) at 50 °C

Reaction medium	PBS	CCl–U	CCl–Gly
t1/2 (min)	281	>360	283

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Although the underlying causes of this stabilization effect are not completely well elucidated in DES, a recent study based on molecular dynamic simulations confirmed the last assumption, and lead us to believe that in the case of CCl–U a similar phenomenon occurs. Monhemi et al. showed that the compactness of a lipase remains constant in CCl–U DES even at high temperatures (i.e. 100 °C), which means that the enzyme has a rigid structure in this solvent.⁵⁵ Moreover, it was found that urea, choline and chloride ions form hydrogen bonds with the surface residues of the enzyme and that these rigid structures formed in the enzyme surface help to promote the protein stability. This may explain the high thermal stability of HRP in CCl–U where the enzyme, in spite of exhibiting a partially unfolded structure, retains higher catalytic activity than buffered aqueous solutions and CCl–Gly for a prolonged period of time. Otherwise, in the case of CCl–Gly, the presence of glycerol as the HBD in the DES structure has a different influence on the thermal stability of HRP.

It seems that the interactions between CCl–Gly molecules with the surface residues of the enzyme are not strong enough to maintain the enzyme structure “docked” upon heating, leading to a behavior similar to that observed in the aqueous environment. Thus, further studies for a better understanding of DES composition on the thermal stability of the enzyme are underway in our laboratory.

Polymerizations of AA were successfully achieved with very high reaction yields in all cases (Table 3). PAA solutions and the corresponding polymers recovered after precipitation are shown in Fig. 4. Despite the low catalytic activity of the enzyme at high DES concentration, it proved to be enough to initiate the polymerization reaction. ATR-FTIR and ¹H NMR spectra of all samples showed the characteristic bands and signals for PAA (see Fig. S4 and S5 in the ESI†).

Table 3 Yield and M_n of PAA samples obtained by HRP-mediated initiation in H₂O and DESs-aqueous mixtures (80% v/v DES)

Reaction medium	T °C	Yield^a wt%	Mn g mol^−1	Mw g mol^−1	PDI
a The yield corresponds to the weight of PAA recovered after precipitation, filtration and drying, to the weight of AA used initially.
H2O	RT	99	1.61 × 10^5	3.92 × 10^5	2.4
	50	96	1.23 × 10^5	4.39 × 10^5	3.6
CCl–U	RT	100	1.60 × 10^5	3.28 × 10^5	2.1
	50	90	7.79 × 10^4	2.16 × 10^5	2.8
CCl–Gly	RT	100	2.27 × 10^4	9.85 × 10^4	4.3
	50	99	8.33 × 10^3	6.96 × 10^4	8.3
	4	99	3.00 × 10^4	1.47 × 10^5	5.0

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Fig. 4 Top left: PAA solutions in H₂O and DESs-aqueous mixtures (80% v/v DES). Top right: PAA precipitated with methanol. Bottom: PAA synthesized in (a) H₂O, (b) CCl–U aqueous mixture, and (c) CCl–Gly aqueous mixture at RT after precipitation and drying.

The different reaction systems yielded PAA samples with a number-average molar mass (M_n) and polydispersity index (PDI) ranging from 161 000 to 8330 g mol⁻¹ and 2.1 to 8.3, respectively. Notwithstanding the significantly reduced catalytic activity of HRP in CCl–U (almost 170 times lower to that in PBS, see Fig. S1†), this solvent allowed the synthesis of PAA with similar M_n and even slightly narrower polydispersity to the corresponding experiment in water at RT. Actually the PDI in CCl–U is close to 2, which is the theoretical limit of conventional polymerization via free radicals,⁵⁶ i.e. contribution from intra- and intermolecular chain transfer reactions during polymerization is minimized. In this regard, although AA has proven to form DES with choline chloride,⁵⁷ the ATR-FTIR spectra of the reaction mixture and the resulting PAA solution in DES (80% v/v) after polymerization do not show noticeable changes in the bands of the DES constituents, thus reflecting no significant changes in the H-bonding network of DES^58,59 at any stage of polymerization at the concentrations tested (Fig. S6 to S9 in the ESI†). In addition ¹H NMR spectra of the polymers synthesized in DES reveal small amounts of the corresponding solvent (Fig. S10†) but not taking part of the polymer structure, confirming the inertness of the DES towards free radical polymerization of AA.

Contrary to these results, replacement of water by CCl–Gly in the polymerization reaction does not offer any benefit in terms of molecular weight or PDI. For instance, PDI increases two-fold or more compared with water and CCl–U; M_n also decreases ca. one order of magnitude. A decrease in the catalytic activity of HRP in CCl–Gly coupled with a thermal stability similar to that in the buffered aqueous media at 50 °C resulted in PAA with the lowest M_n values. High polydispersity in CCl–Gly can be explained on the same basis than in CCl–U case, actually chain transfer during spontaneous polymerization of AA has been reported to occur in glycerol alone.⁶⁰

Another interesting feature resulting from the use of DES as solvent, in this case with “hydrated” DES, is the lower water activity (a_w) of these systems (Fig. 5). Derived of this, the rather narrow conditions of pH (5.4–8) for the polymerization of AA in water are circumvented,^61,62 since DES provide a unique environment where acid–base equilibrium is different in nature than in water.^63,64 DES containing up to 20% v/v of water as co-solvent demonstrated to be stable (e.g. no weight lost) under vacuum at 50 °C or RT during the course of the experiments. The possibility of applying vacuum to the systems during the first stages of polymerization was exploited in order to minimize the effect of oxygen,⁶⁵ which is a well-known factor that reduces the efficiency of propagation in free radical polymerization; therefore helped in obtaining 100% of conversion.

Fig. 5 Thermodynamic activity of water (a_w) as a function of water content in CCl–U and CCl–Gly aqueous mixtures. Note: error bars are almost all within the size of the plot symbols.

Taking advantage of the low freezing point of CCl–Gly in its pure state (−40 °C)^66,67 we carried out an additional polymerization experiment at 4 °C in this solvent and water. As it was expected, given the low temperature of the system, no polymer precipitated from the AA solution when water was used as solvent. However, in CCl–Gly, PAA with M_n of 30 000 g mol⁻¹ was obtained (Table 3) which suggests that the enzyme conformation is not drastically disrupted at this temperature. It is also worth noting that M_n of PAA synthesized at 4 °C was higher than those obtained in the same solvent at RT and 50 °C. Undoubtedly, this intriguing behaviour of HRP in CCl–Gly deserves a deeper study, but showed the potential use of these solvents to carry out biocatalytic reactions in a wide range of temperatures, useful for instance for the synthesis of polymeric materials by cryo-polymerization^68,69 mediated by enzymes.

Conclusions

In summary, we have described the use of hydrated DESs as alternative solvents for enzyme-mediated free radical polymerizations under conditions of temperature and pressure inaccessible for PAA synthesis in water. HRP retained enough catalytic activity to initiate the polymerization of AA in hydrated DES owed to its high thermal stability regardless exhibiting a partially unfolded structure. Resulting polymers showed comparable M_n values to those obtained in water for CCl–U system with complete conversion. The slightly enhancement in polydispersity obtained in the CCl–U 80% v/v system reflects the suitable environment provided for AA propagation that minimize contributions from intra- and intermolecular chain transfer reactions during polymerization.

Several properties of the polymer can be potentially tuned in by taking advantage of the solvent properties of the DES. Further, the conditions provided by DES allowed the exploration of higher and lower temperatures that in aqueous media. Finally, accessing to low temperatures for polymerization in the CCl–Gly system paves the way for unprecedented possibilities in materials synthesis through enzyme-mediated free radical cryo-polymerization in nearly non-aqueous media.

Experimental section

Materials

Choline chloride, urea, glycerol, acrylamide, phenol, 4-aminoantipyrine, 2,4-pentanodione and horseradish peroxidase were purchased from Sigma Aldrich, and H₂O₂ (30% solution) from J. T. Baker. Except for choline chloride all reagents were used without further purification.

DESs synthesis

Prior to DESs synthesis, CCl was recrystallized from absolute ethanol and dried. Choline chloride–urea (CCl–U) and choline chloride–glycerol (CCl–Gly) were prepared by heating the two corresponding components in a 1 : 2 molar ratio at 90 °C and stirring until a homogeneous liquid was formed.

Enzyme activity assays

The enzyme activity was measured using an Ocean Optics USB4000 UV-vis spectrometer following a standard colorimetric method.⁷⁰ The assay was carried out at RT and the reaction was initiated by the addition of 5 μL of HRP (type I, 52 U mg⁻¹ using pyrogallol) solution (1 mg mL⁻¹) to a cuvette with 995 μL of PBS (0.1 M, pH 7.0) containing phenol, 4-aminoantipiryne and H₂O₂ at a final concentration of 10 mM, 0.2 mM and 2.4 mM, respectively. Upon addition of the enzyme solution, the cuvette was capped and inverted three times prior to being placed in the spectrometer. The change in absorbance over time was monitored at 510 nm during the first minute. The slope of the linear increase of absorbance and a molar extinction coefficient of 7100 L mol⁻¹ cm⁻¹ were used to determine the catalytic activity. The activity of HRP in DESs-aqueous mixtures at different DES concentrations (v/v%) was determined following the same procedure. The measurements were at least triplicated and the average is reported.

Thermal stability tests

For the thermal stability tests, 100 μL of HRP solution (145.7 U mg⁻¹, 10 mg mL⁻¹) were added to 900 μL of PBS (0.1 M, pH 7.0) and to DESs-aqueous mixtures, respectively. The samples were incubated at 50 °C and the final concentration of DESs-aqueous mixtures was 80% v/v DES. During a period of 6 hours, aliquots of 5 μL were taken at different times for the activity assay as mentioned above. For each enzyme solution t_1/2 was calculated from the time-dependent loss in activity. All thermal stability experiments were at least triplicated and the average of HRP activity after incubation in the reaction media is reported.

HRP-mediated free radical polymerization of acrylamide

The HRP-mediated free-radical polymerization of AA was performed following the experimental procedure previously reported for PAA synthesis in water.¹⁴ Prior to the addition of the catalytic system to the solvents, water and DESs-aqueous mixtures containing AA were degasified by N₂ bubbling. HRP (145.7 U mg⁻¹, 37.5 μL, 10 mg mL⁻¹), H₂0₂ (10 μL, 0.82 M) and 2, 4-pentanodione (12.84 μL) were added into a flask containing 6.25 mL of 0.64 M aqueous AA solution. In the case of the DES containing systems, the AA (284.4 mg) was first dissolved in CCl–U or CCl–Gly, and the same amount of the remaining reagents was subsequently introduced into each DES–AA solution. In the final solutions, 80% of the total H₂O volume was replaced by DES. After 72 h of reaction at RT (24 h under vacuum), the solutions were added dropwise to a large excess of methanol to precipitate the PAA, filtered off and washed several times to ensure complete removal of the DES. PAA samples were dried under vacuum at 50 °C. The experimental procedure for AA polymerization at 50 and 4 °C was the same followed at RT. The reaction yield was determined gravimetrically and corresponds to the weight of PAA recovered compared to the weight of AA used initially.

Characterization

Determination of water activity in DESs-aqueous mixtures. DESs-aqueous mixtures with various water contents (20–90% v/v) were freshly prepared. The thermodynamic water activity (a_w) was determined at 25.5 °C using an AquaLab CX-2.

Fluorescence spectroscopy. The fluorescence of the tryptophan residue of HRP was measured using a steady-state FluoroMax-3 spectrofluorometer by Jobin Yvon Horiba. For the assays, 5 μM HRP solutions were prepared in PBS (0.1 M, pH 7.0) and DESs-aqueous mixtures (80% v/v DES), respectively. The spectra were acquired at RT and 90° with an excitation wavelength of 295 nm to avoid the contribution of the tyrosine residues of HRP. A quartz cuvette with a path length of 10 mm was used for the experiments. Fluorescence intensity of the corresponding solvent was subtracted from each spectrum that contained HRP to eliminate the contributions of the solvents on the fluorescence measurements and only the deconvoluted tryptophan signal is shown (see Fig. S2 in the ESI† for the raw fluorescence spectra).

UV-visible spectroscopy. Absorbance spectra of HRP were determined with 5 μM enzyme in PBS (0.1 M, pH 7.0) and DESs-aqueous mixtures (80% v/v DES), respectively. The enzymes solutions were equilibrated for 30 min at RT before collection of the spectra on an Agilent 8453 UV-vis spectrophotometer.

Fourier transmission infrared spectroscopy. ATR-FTIR spectra of PAA samples were acquired at RT using a Perkin-Elmer Spectrum GX FTIR spectrometer in the range from 4000 to 650 cm⁻¹ with a resolution of 4 cm⁻¹ and an average of 32 scans.

Nuclear magnetic resonance. ¹H spectra were conducted in a 500 MHz Bruker Avance III using a 5 mm direct broad band with Z-grad (PABBO-1H/D Z-GRAD) probe at RT and with D₂O as solvent. The ¹H spectra were recorded with the presaturation mode. The chemical shifts in the spectra were referenced to residual no deuterated solvent.

Gel permeation chromatography. Molecular weight (M_n and M_w) and molecular weight distribution (MWD) of polymers were measured using an Agilent 1260 Infinity HPLC-GPC size exclusion chromatograph (SEC) equipped with an auto sampler and a differential refractive index detector. The SEC equipment was fitted with three PL aqua-gel-OH columns; 30 (8 mm, 300 × 7.5 mm, range molecular weight 100–30 000), 40 (15 mm, 300 × 7.5 mm, range molecular weight 10 000–200 000) and 50 (15 mm, 300 × 7.5 mm, range molecular weight 50 000–1 000 000); and one aqua-gel-OH (15 mm, 50 × 7.5 mm) guard column (Polymer Laboratories). Phosphate buffer solution (pH 7) was used as eluent at 0.5 mL min⁻¹ and 40 °C. The molecular weights were determined relative to polyacrylic acid standards (100 to 1 000 000 Da).

Acknowledgements

R. J. Sánchez-Leija acknowledges CONACYT for her doctoral fellowship. Financial support by CONACYT through projects 261425 and CB/2012-181678 is greatly appreciated. The authors are grateful to Araceli Mauricio for the FTIR measurements, Carlos Regalado for the water activity measurements, Rivelino Flores for his assistance in fluorescence measurements and Pablo Zubieta and Rafael Vazquez Duhalt for their helpful discussion.

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Footnote

† Electronic supplementary information (ESI) available: Details of HNMR and FTIR analyses. See DOI: 10.1039/c5ra27468k

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