Feruloyl esterase-catalysed synthesis of glycerol sinapate using ionic liquids mixtures

Journal of Biotechnology 139 (2009) 124–129

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Feruloyl esterase-catalysed synthesis of glycerol sinapate using ionic liquids mixtures Christina Vafiadi a , Evangelos Topakas a , Victoria R. Nahmias a , Craig B. Faulds b , Paul Christakopoulos a,∗ a BIOtechMASS Unit, Biotechnology Laboratory, School of Chemical Engineering, National Technical University of Athens, 5 Iroon Polytechniou Street, Zografou Campus, 15700 Athens, Greece b Sustainability of the Food Chain Exploitation Platform, Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, UK

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Article history: Received 6 May 2008 Received in revised form 22 August 2008 Accepted 29 August 2008 Keywords: Feruloyl esterase Ionic liquids Glycerol sinapate LDL oxidative modification

a b s t r a c t The ability of a feruloyl esterase (AnFaeA), either in free or immobilised (cross-linked enzyme aggregates) form, to catalyse the esterification of glycerol, a major by-product of the biodiesel industry, with sinapic acid was studied in four hexafluorophosphate anion-containing ionic liquids: ([Bmim][PF6 ], [Omim][PF6 ], [C2 OHmim][PF6 ] and [C5 O2 mim][PF6 ]). Such ionic liquids are considered ‘green’ reaction systems. The synthetic reaction was optimised in [C2 OHmim][PF6 ] and the highest conversion yield was 72.5 ± 2.1%, while, at the same reaction conditions in [C5 O2 mim] [PF6 ], a similar conversion yield was obtained (76.7 ± 1.5%). AnFaeA was active in its free and immobilised form, with the latter retaining a part of its synthetic activity after 5 consecutive 24 h-period reaction cycles. Sinapic acid was esterified to one of the primary hydroxyl groups of glycerol and retained, after esterification, 63.1 ± 0.3% and 89.5 ± 1.1% of its antioxidant activity against low-density lipoprotein oxidation, when added at concentrations of 10 and 60 ␮M, respectively, in the assay mixture. © 2008 Elsevier B.V. All rights reserved.

1. Introduction The use of ionic liquids (ILs) as reaction systems for biocatalytic transformations has received growing attention during the last two decades. Unlike conventional organic solvents, ILs possess no vapour pressure, are able to dissolve many compounds and can be used to form two-phase systems with many solvents in the presence of water. According to a recent review work on biocatalysis in ILs, factors such as polarity and nucleophilicity of the anion, pH, purity of the IL and water content, have a major effect on the activity, the stability and the solubility of enzymes in these nonconventional media (van Rantwijk and Sheldon, 2007). Applying enzymes in ILs has a very short history and the types of ionic liquids and enzymes used so far are quite restricted (Yang and Pan, 2005). van Rantwijk and Sheldon (2007), in their recent review, summarised the different types of enzymes used so far in biocat-

Abbreviations: FAE, feruloyl esterase; HCA, hydroxycinnamic acid; MHCA, methyl ester of hydroxycinnamic acid; FA, ferulic acid; SA, sinapic acid; pCA, pcoumaric acid; CA, caffeic acid; MSA, methyl sinapic acid; CLEAs, cross-linked enzyme aggregates; LDL, low-density lipoprotein; MOPS, 3-[N Morpholino]propane sulphonic acid. ∗ Corresponding author. Tel.: +30 210 7723231; fax: +30 210 7723163. E-mail address: [email protected] (P. Christakopoulos). 0168-1656/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jbiotec.2008.08.008

alytic transformations in ILs (lipases, pig liver esterase, proteases, glucosidases, lyases) and claim that it is to be expected that ionic liquid-based solvent systems will have enormous potential in multicatalyst transformations. More recently, an alkaline phosphatase (Lopez-Pastor et al., 2007), three alcohol dehydrogenases (Dreyer and Kragl, 2007; Hussain et al., 2008) and a serine cutinase protease (Micaelo and Soares, 2008) were studied and found to be active in certain types of ILs. Therefore, the screening of novel enzymatic activities in ILs is critical and up to date. Recently, feruloyl esterases (FAEs) [E.C. 3.1.1.73], a subclass of the carboxyl-esterases, have been used as synthetic tools for the esterification of HCAs to aliphatic alcohols (Giuliani et al., 2001; Topakas et al., 2003a,b; Hatzakis and Smonou, 2004, 2005) and mono- and oligosaccharides (Topakas et al., 2005; Vafiadi et al., 2006, 2007) in organic solvent mixtures. It has been reported in several cases, that immobilisation of enzymes favorises their use in biocatalytic reactions in ILs (Toral et al., 2007; van Rantwijk et al., 2006; Shah and Gupta, 2007). The preparation of cross-linked enzyme aggregates (CLEAs), a new carrier-free immobilisation technique, was first introduced by Cao et al. (2000). This approach consists of the covalent cross-linking of a precipitated enzyme and has been successfully applied in the carrier-free immobilisation of several pure enzymes (Cao et al., 2000; Lopez-Serrano et al., 2002; Bode et al., 2003; Chmura et al., 2006; Yu et al., 2006).

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Hydroxycinnamic acids (HCAs) such as ferulic (FA), sinapic (SA), p-coumaric (pCA), and caffeic (CA), display proven antioxidant activities and therefore a wide range of industrial applications. HCAs are high-added value products that can be isolated from natural sources and as by-products of industrial processes. SA, for example, has been isolated from a waste stream in the processing of yellow mustard protein isolate (Prapakornwiriya and Diosady, 2008), from rice bran pitch (Kikuzaki et al., 2002) and from rice husk (Kuriakose and Rajendran, 2000). The antioxidant activities of these simple phenolic acids and their derivatives have been studied during the past few years using many different model systems, such as the low-density lipoprotein (LDL) oxidation system in vitro (Vafiadi et al., 2008; Katapodis et al., 2003; Andreasen et al., 2001; Chalas et al., 2001) and free radical scavengers tests against the 1,1-diphenyl-2-picryl hydrazyl (DPPH) (Tsuchiyama et al., 2007; Katapodis et al., 2003; Chalas et al., 2001) and 2,2 -azobis(2-amidinopropane)dihydrochloride (AAPH) (Chalas et al., 2001). These studies focus on the correlation between antioxidant activity and chemical structure, which still remains unclear. However, factors such as the number and the position of hydroxyl groups on the phenolic ring and the degree of the lipophilicity of the compound seem to play an important role (Chalas et al., 2001). The modification of this class of natural phenolics via esterification can lead to compounds with novel characteristics, expanding their use in a variety of food and non-food applications. Glycerol has been a well-known renewable chemical for centuries, but its commercial relevance has increased considerably in the last few years, because of its rising inevitable formation as a by-product of biodiesel production (Behr et al., 2008). Several reports refer to the chemistry of glycerol, starting from the classic esters and oligomers to new products like glycerol carbonate, telomers, branched alkyl ethers, propanediols and epoxides via heterogeneous, homogeneous and biocatalytic reactions (Behr et al., 2008). The enzymatic esterification of glycerol to HCAs using FAEs, has been previously reported (Tsuchiyama et al., 2006, 2007). In the present study, we investigated the esterification of glycerol to SA in PF6 − anion-containing ILs, using a FAE from Aspergillus niger (AnFaeA) as biocatalyst. Studies on the crystal structure of AnFaeA showed many similarities to those found in fungal lipases and different from that reported for other types of FAEs (Hermoso et al., 2004; Levasseur et al., 2006). Since lipases are extensively used in ILs, we estimate that this particular enzyme is a good starting point for the initial studies of FAEs synthetic activity in ILs. Furthermore, apart from its technological significance, reported already previously, esterification of glycerol was also chosen due to the presence of two primary hydroxyl groups, in which FAEs display synthetic specificity in esterification reactions (Topakas et al., 2005; Vafiadi et al., 2006, in press). Several factors affecting the synthetic reaction were optimised and the esterification product, after purification and structural identification, was tested for its antioxidant activity against the LDL oxidation system in vitro.

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2.2. Enzymes Recombinant A. niger type-A FAE (AnFaeA), expressed in Pichia pastoris, was produced as described previously (Juge et al., 2001). 2.3. Immobilisation of AnFaeA AnFaeA was immobilised according to the CLEAs methodology, using ethanol as precipitant and 100 mM glutaraldehyde (Vafiadi et al., 2008). 2.4. Esterification of glycerol Reactions were performed in the ILs [Bmim][PF6 ], [Omim][PF6 ], [C2 OHmim][PF6 ] and [C5 O2 mim][PF6 ] using different concentrations of glycerol, MSA (in the case of transesterification reactions) and SA (in the case of esterification reactions). AnFaeA was introduced in the form of concentrated stock solution in 100 mM buffer MOPS-NaOH pH 6.0. In the reactions catalysed by CLEAs, the enzyme solution was replaced by buffer solution in the required volume. All reactions were performed in duplicate in an Eppendorf thermomixer at 50 ◦ C with mixing at 1400 rpm. Quantitative analysis of samples was made by HPLC on a C18 Nucleosil column (250 mm × 4.6 mm) (Macherey Nagel, Dren, Germany) and detection of the product by a Jasco UV-975 detector set at 320 nm based on a calibration curve prepared using standard solutions of the isolated glycerol sinapate (see Section 2.5) in water/methanol 1:1 (v/v). The reaction mixtures were diluted with methanol before analysis. Elution was conducted with methanol:water:acetic acid (4:6:0.1) as the mobile phase at a flow rate of 1.0 mL/min at ambient temperature. Yields for the synthesis of glycerol ester were calculated from the amount of MSA or SA having reacted compared to their initial quantity. All reactions were performed in duplicate with <10% standard deviation for each set of results. No glycerol consumption was observed in the absence of the enzyme. 2.5. Isolation of glycerol sinapate In a preparative-scale reaction, esterification of glycerol with SA was carried out in [C5 O2 mim][PF6 ] under the optimum conditions found for the synthesis of glycerol sinapate by AnFaeA. The reaction mixture was extracted with ethyl acetate and the extract, containing SA and the esterification product, was evaporated under vacuo. After diluting the mixture with methanol:water 1:1, isolation of the esterification product was carried out by preparative HPLC on a C18 Luna 5 ␮m column (250 mm × 21.2 mm) (Phenomenex, France). Detection was achieved by a Jasco UV-975 detector set at 320 nm. Elution was conducted with methanol:water:acetic acid (4:6:0.1) as the mobile phase at a flow rate of 8.0 mL/min at ambient temperature. Fractions containing the esterification product were pooled and evaporated under diminished pressure. 2.6. Structural characterisation of glycerol sinapate

2. Experimental 2.1. Materials ILs were purchased by Solchemar (Portugal). Methyl sinapic acid (MSA) was purchased from Apin Chemicals Ltd. (Abingdon, UK) and SA from Sigma Chemical Co. (MO, USA). All other reagents were purchased from LabScan (Dublin, Ireland). The EDTA-containing stock solution of human LDL (2.0–3.5 gLDL /L) was purchased from Sigma.

Proton NMR spectroscopy was performed in CD3 OD with a Bruker AC 300 spectrometer, equipped with broad band probe at 300 MHz. 1 H NMR, CD OD, ı: 7,6 (1H, d, J = 15.9 Hz CHCHCOO), 3 6,9 (2H, s, aromatic), 6.4 (1H, d, J = 15.8, CHCHCOO), 4.25 (1H, dd, J = 4.5 Hz, COOCHHCH(OH)CH2 OH), 4.19 (1H, dd, J = 6.2 Hz, COOCHHCH(OH)CH2 OH) 3.9 (6H, s, OCH3 , OCH3 ), 3.4–3.6 (1H, m, COOCH2 CH(OH)CH2 OH), m), 3.4–3.6 (2H, dd, COOCH2 CH(OH)CH2 OH).

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Mass spectrometry was performed using an Agilent 1100 MSD ion trap. The mass spectrum of the esterification product was obtained in positive and negative mode. 2.7. Antioxidant activity against LDL oxidation The LDL solution was prepared as described previously (Vafiadi et al., 2008). Lipid oxidation of human LDL was assessed by spectrophotometric monitoring of conjugated diene lipid hydroperoxide formation at 234 nm during copper-induced oxidation (5 ␮M copper, 37 ◦ C, pH 7.4), as described previously (Vafiadi et al., 2008). To evaluate the antioxidant activities of SA and glycerol sinapate, pure compounds were tested in two different concentrations (10 and 60 ␮M) in the assay mixture. All assays were performed in duplicate with <10% standard deviation for each set of results. 3. Results 3.1. Enzymatic esterification of glycerol In order to investigate the synthetic activity of FAEs in ILs, the enzymatic esterification of glycerol with SA by AnFaeA was studied in four PF6 − anion-containing ILs, using free SA and its methyl ester as acyl donors. SA is the second strongest antioxidant, after CA, among the HCAs that exist in plants (Cheng et al., 2007; Andreasen et al., 2001). 3.1.1. Influence of the IL cation Transesterification and esterification reactions were performed in four PF6 − anion-containing ILs: [Bmim][PF6 ], [Omim][PF6 ], [C2 OHmim][PF6 ] and [C5 O2 mim][PF6 ]. The reactions were performed at 50 ◦ C and 1400 rpm, with 6% 100 mM buffer MOPS-NaOH pH 6.0 in each IL, using 200 mM glycerol, 50 mM SA or MSA and 24.5 mU AnFaeA. The esterase was able to catalyse both transesterification and esterification of glycerol only in [C2 OHmim][PF6 ] and [C5 O2 mim][PF6 ]. Esterification yields were slightly higher compared to those obtained in the cases of transesterification (Fig. 1). Conversion reached a plateau after 24 h in all cases. The synthetic activity

Fig. 2. Effect of temperature on the initial rate (䊉) and % conversion yield () of the esterification of glycerol with SA. Reactions were performed using 50 mM SA, 200 mM glycerol, with 24.5 mU AnFaeA (in 100 mM buffer MOPS-NaOH pH 6.0, 6% in the IL [C2 OHmim][PF6 ]) at 1400 rpm for 24 h.

of AnFaeA was slightly higher in [C5 O2 mim][PF6 ] (9.2 ± 0.1%) than in [C2 OHmim][PF6 ] (6.7 ± 0.1%). With regard to the initial rates, esterification (28.5 ± 1.0 and 16.9 ± 1.7 mM/h/mgprotein for [C5 O2 mim][PF6 ] and [C2 OHmim][PF6 ], respectively) was faster than transesterification (22.6 ± 0.4 and 11.4 ± 0.1 mM/h/mgprotein ) for [C5 O2 mim][PF6 ] and [C2 OHmim][PF6 ], respectively). Between the two ILs, higher initial rates were observed in the case of [C5 O2 mim][PF6 ]. Given that the obtained reaction yields are comparable in all cases, we chose to proceed to the optimisation of the enzymatic esterification of glycerol using only the [C2 OHmim][PF6 ] and SA as acyl donor (direct esterification), because the costs of [C2 OHmim][PF6 ] and SA are lower compared to those of [C5 O2 mim][PF6 ] and MSA, respectively. 3.1.2. Effect of temperature Temperature is an important factor in equilibrium synthetic reactions. As it can be seen from Fig. 2, maximum yield (5.7 ± 0.3%) is observed in the range of 50–55 ◦ C while the maximum initial rate (18.0 ± 0.8 mM/h/mgprotein ) in the range of 55–60 ◦ C. The temperature of 50 ◦ C was retained in the subsequent optimisation experiments. 3.1.3. Effect of % water content Results presented in Fig. 3, show the effect of different % water content added in [C2 OHmim][PF6 ] on the % yield and the initial rate of the esterification reaction. Both measures increased with the increase of % water content up to 15%, where the optimum values are obtained (8.3 ± 0.1% and 22.0 ± 1.7 mM/h/mgprotein , respectively).

Fig. 1. Reaction progress of the transesterification (() in [C2 OHmim][PF6 ]; () in [C5 O2 mim][PF6 ]) and esterification ((䊉) in [C2 OHmim][PF6 ]; () in [C5 O2 mim][PF6 ]) reaction between glycerol and MSA or SA, respectively, catalysed by AnFaeA. Reactions were performed using 50 mM MSA or SA, 200 mM glycerol, with 24.5 mU AnFaeA (in 100 mM buffer MOPS-NaOH pH 6.0, 6% in the IL), at 50 ◦ C with stirring at 1400 rpm for 6 days.

3.1.4. Effect of SA and glycerol concentration Under the optimum conditions obtained above (50 ◦ C, 15% content of 100 mM buffer MOPS-NaOH pH 6.0 in [C2 OHmim][PF6 ]) and maintaining the same reaction conditions (200 mM glycerol, 24.5 mU AnFaeA and stirring at 1400 rpm), the effect of different SA concentrations on the esterification reaction was examined. Maximum yield (20.9 ± 0.6%) was observed with 20 mM SA, while maximum initial rate (34.9 ± 0.6 mM/h/mgprotein ) was obtained with 15 mM SA (Fig. 4A). SA was not totally soluble in the reaction medium at concentrations higher than 150 mM. Using 20 mM SA and maintaining the rest of the optimum reaction conditions as determined above, different concentrations of

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Fig. 3. Effect of % water (buffer solution) content added in [C2 OHmim][PF6 ] on the initial rate (䊉) and % conversion yield () of the esterification of glycerol with SA. Reactions were performed using 50 mM SA, 200 mM glycerol, with 24.5 mU AnFaeA, at 50 ◦ C, with stirring at 1400 rpm for 24 h.

glycerol were tested. Maximum yield (72.5 ± 2.1%) was obtained when 2.5 M of glycerol was added and maximum initial rate (74.5 ± 2.8 mM/h/mgprotein ) was observed in the case of 3 M glycerol (Fig. 4B). When [C2 OHmim][PF6 ] was replaced by [C5 O2 mim][PF6 ], under the optimum conditions, a slight increase on the conversion yield was observed (76.7 ± 1.8%), in contrast with the initial rate, which increased dramatically (178.0 ± 12.1 mmol L−1 h−1 mg−1 ), showing that [C5 O2 mim][PF6 ] favorises the reaction kinetics (data not shown). 3.1.5. Synthetic activity and recovery of immobilised enzyme Under optimum reaction conditions, when CLEAs of AnFaeA were used in [C5 O2 mim][PF6 ], using the same amount of enzyme (mU), the reaction yield was not significantly affected (72.6 ± 1.5%) (data not shown). CLEAs were removed from the reaction mixture by brief centrifugation after 24 h and washed several times with ethyl acetate and buffer. The immobilised AnFaeA was found to retain still a part of its synthetic activity (13.4 ± 0.7%) after 5 consecutive 24 h-period reaction cycles, while no synthetic activity was found after the 6th cycle (Fig. 5).

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Fig. 5. % Retained synthetic activity of CLEAs of AnFaeA after 6 consecutive 24 hperiod reaction cycles. Each reaction was performed using 20 mM SA, 2.5 M glycerol, 15% water in [C5 O2 mim][PF6 ], at 50 ◦ C, with stirring at 1400 rpm.

3.1.6. Structural characterisation of glycerol sinapate For the structural determination of the esterification product, a preparative-scale reaction and purification was performed as described in Section 2.5. The structure of the esterification product was first analysed by mass spectrometry. The first order mass spectrum in the positive mode showed mainly the singly charged ion at m/z 619, which corresponds to the sodium adduct of two molecules of mono-esterified glycerol with SA, while in the negative mode showed mainly the singly charged ion at m/z 297, which corresponds to the deprotonated ion of monoesterified glycerol with SA (data not shown). Both results suggest that esterification took place in only one of hydroxyl groups of glycerol. No further structural information was extracted from the second order mass spectra in the positive and negative mode. In order to confirm the position of the sinapic group on the glycerol molecule and thereupon the specificity of AnFaeA, NMR spectrometry was used. The 1 H NMR spectra of the product showed that the esterification took place in one of the two primary hydroxyl groups of glycerol, as shown in Fig. 6.

Fig. 4. Effect of SA (A) and glycerol (B) concentration on the initial rate (䊉) and % conversion yield () of the esterification of glycerol with SA. Reactions were performed using 200 mM glycerol (A), 20 mM SA (B), with 24.5 mU AnFaeA (in 100 mM buffer MOPS-NaOH pH 6.0, 15% (v/v) in [C2 OHmim][PF6 ]), at 50 ◦ C, with stirring at 1400 rpm for 24 h.

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Fig. 6. Structure of glycerol sinapate.

Fig. 7. Antioxidant activity of SA and glycerol sinapate against the LDL oxidation. (䊉) Control, () SA 10 ␮M, () SA 60 ␮M, () glycerol sinapate 10 ␮M and () glycerol sinapate 60 ␮M.

3.2. Antioxidative effect against human LDL With respect to percentage inhibition values, SA, after esterification with glycerol, retained 63 ± 0% and 89 ± 1% of its antioxidant activity against LDL oxidation when added at concentrations of 10 and 60 ␮M in the assay mixture, respectively (Fig. 7). Maximal rates of conjugated lipid hydroperoxide formation where calculated on the base of the molar absorptivity of conjugated lipid hydroperoxides (ε234 = 29.500 M−1 cm−1 ). In the case of SA, rate values where 0.285 ± 0.004 and 0.125 ± 0.009 nmoldienes /mgLDL /min for 10 ␮M and 60 ␮M, respectively, and in the case of glycerol sinapate 1.622 ± 0.002 and 0.534 ± 0.001 nmoldienes /mgLDL /min for 10 ␮M and 60 ␮M, respectively. 4. Discussion Hydrophobic anion (PF6 −1 )-containing ILs were found to be appropriate reaction media for the enzymatic esterification of glycerol to SA, especially when they possess hydrophilic cations (C5 O2 mim+1 , C2 OHmim+1 ). These two ILs, in contrary with [Bmim][PF6 ] and [Omim][PF6 ], can be considered as amphiphilic, because they possess both a hydrophilic cation and a hydrophobic anion. Another issue to consider is the lower viscosity of these two ILs compared to [Bmim][PF6 ] and [Omim][PF6 ], which reduces mass-transfer limitations (Yang and Pan, 2005). Since similar yields and initial rates were obtained in the cases of esterification and transesterification reactions, we chose the first as it is more economical (SA is cheaper than MSA) and more environmental friendly (to avoid the formation of methanol as a side product in the case of the transesterification reaction) than the second one.

When the esterification reaction was performed in different temperatures, different optima were obtained for the % conversion and the reaction rate, showing that the thermodynamic optimum of the reaction differs from the kinetic one. Similar results were obtained in previous studies on the synthetic activity of AnFaeA in organic solvent–water mixtures (Vafiadi et al., 2008). The presence of water is essential for AnFaeA to act in ILs, in contrast with lipases, which require almost anhydrous conditions for effective esterification. The % conversion of the esterification reaction of glycerol with SA in [C5 O2 mim][PF6 ] reached 76.7 ± 1.8%. The yield obtained is comparable to that reported previously for the same reaction (70%) performed in a solvent free system catalysed by a purified type-A FAE from an A. niger commercial enzyme preparation (Tsuchiyama et al., 2007). According to the structural characterisation of the esterification product, SA was esterified in one of the primary hydroxyl groups of glycerol. This result is in agreement with the specificity of FAEs for the esterification of primary hydroxyl groups described previously (Topakas et al., 2005; Vafiadi et al., 2006, 2007, 2008). With regards to the antioxidant activity, glycerol sinapate acts slower as an antioxidant in the LDL oxidation model compared to the free SA, where the partition coefficient between the aqueous and the lipophilic phase influences the accessibility of antioxidants to free radicals (Rice-Evans et al., 1996). Previous studies on the antioxidant activities of HCAs and their derivatives include their antioxidant activity against the oxidation of LDL in vitro (Vafiadi et al., 2008; Katapodis et al., 2003; Andreasen et al., 2001; Chalas et al., 2001) and their activity as free radical scavengers against the 1,1-diphenyl-2-picryl hydrazyl (DPPH) (Tsuchiyama et al., 2007; Katapodis et al., 2003; Chalas et al., 2001) and 2,2 azobis(2-amidinopropane)dihydrochloride (AAPH) (Chalas et al., 2001). Ethyl and butyl esterification of HCAs increased their antioxidant activity against LDL oxidation, probably due to a better incorporation in the lipid layer of the LDL particle and the exertion of their antioxidant effect in the true site of lipoperoxidation (Vafiadi et al., 2008; Chalas et al., 2001). Despite the increased hydrophilicity, the feruloylated trisaccharide (FAX3 ) showed higher antioxidant activity against LDL oxidation than free FA (Katapodis et al., 2003). When it comes to the DPPH oxidation system, except for pCA, whose ethyl ester activity rose to 48%, the other acids had no significative change in their reducing capacity once esterified (Chalas et al., 2001). FAX3 (Katapodis et al., 2003) and glycerol esters of HCAs (Tsuchiyama et al., 2006, 2007) showed less antiradical activity than the corresponding free HCAs, which agrees with the results obtained in the present study. In the case of AAPH experiment, ethyl esters were equally or more effective than the corresponding acids in protecting erythrocytes from haemolysis (Chalas et al., 2001). In conclusion, the enzymatic esterification of glycerol with SA catalysed by AnFaeA is the first example of activity of FAEs in ILs. Esterified glycerol has a satisfactory antioxidant activity against LDL oxidation in vitro and therefore expands the use of SA as an antioxidant in adequate processes. Furthermore, the use of glycerol showed that this waste can be converted into health-improving functional compounds. The findings of the present study could be the basis for further exploitation of FAEs in ILs, for the modification of compounds insoluble in conventional reaction media.

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