Improving ascorbyl oleate synthesis catalyzed by Candida antarctica lipase B in ionic liquids and water activity control by salt hydrates

Process Biochemistry 44 (2009) 257–261

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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio

Improving ascorbyl oleate synthesis catalyzed by Candida antarctica lipase B in ionic liquids and water activity control by salt hydrates Marek Adamczak a,*, Uwe T. Bornscheuer b a b

Department of Food Biotechnology, University of Warmia and Mazury in Olsztyn, 10-718, Olsztyn, J. Heweliusz St. 1, Poland Institute of Biochemistry, Department of Biotechnology & Enzyme Catalysis, Greifswald University, Felix-Hausdorff-St. 4, Greifswald, Germany

A R T I C L E I N F O

A B S T R A C T

Article history: Received 20 August 2008 Received in revised form 9 October 2008 Accepted 19 October 2008

Lipase catalyzed-synthesis of ascorbyl oleate was performed in ionic liquids with addition of salt hydrate pairs for water activity control. The highest yield in the synthesis of ascorbyl oleate (72%) was obtained in [BMIM][BF4] in the presence of the salt pair NaI 2/0, which corresponds to a water activity of 0.3. Purity and chemical identity of 6-O-ascorbyl oleate was confirmed by 1H and 13C NMR analysis. ß 2008 Elsevier Ltd. All rights reserved.

Keywords: Lipase 6-O-ascorbyl oleate Ionic liquid Water activity

1. Introduction Enzymatic methods for the synthesis of polyunsaturated fatty acids (PUFA) concentrates or structured triacylglycerols (sTAG) rich in PUFA or conjugated fatty acids have been described using different methods, substrates and enzymes [1]. However, functional food products containing PUFA are susceptible to oxidation that causes off-flavour [2]. To prevent unfavourable changes in lipids, they should be stored under proper conditions, e.g. at low temperature in an inert atmosphere. Ascorbyl esters of fatty acids are very popular antioxidants, e.g. E304 ascorbyl palmitate, but the most active (probably because of its higher solubility) ascorbyl ester was found to be ascorbyl oleate [3] that is not used on commercial scale in food industry, and just on smaller scale in cosmetics. Recently, also esters of fatty acids and ascorbic acid or benzoic acid were synthesized in reactions catalyzed by lipase [4], that can also act similar to ascorbyl esters as antioxidant and amphiphilic compound. Ascorbyl esters are also used in combination with the food colors carotene and xanthophylls to prevent their oxidation, as crumb-softening agents in bread and as antimicrobial agents [5]. A method of cancer treatment has been described, i.e. inhibition of proliferation of human cancer cells by interfering with cell cycle progression, induction of apoptosis by modulation of signal transduction pathways using a lipophilic

* Corresponding author. Tel.: +48 89 5233838; fax: +48 89 5233838. E-mail address: [email protected] (M. Adamczak). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.10.014

derivative of ascorbic acid [6,7] as well as the treatment of pigmentation disorders [8]. Modification of ascorbic acid by acylation with fatty acids results in oil-soluble compounds. Lipophilization reactions including synthesis of ascorbyl esters were recently reviewed by Villeneuve [9] and can be used in reactions with, e.g. vitamin A [10,11], B6 [12] or flavonoids [13–16]. For the synthesis of ascorbyl ester critical parameters are choice of substrates, solvent and water activity of the reaction medium. Medium engineering can be used to improve enzyme performance [17,18]. Beside organic solvents, also less commonly used solvent systems have been described such as supercritical fluids and more recently ionic liquids (IL). IL are alternative to organic solvents because of their low vapor pressure, chemical and thermal stability and are therefore considered as green solvents [19,20]. It was also discovered that lipases in IL exhibit greater stability compared to organic solvents [21], even up to 2300 times higher half-life [22] and enhanced enantioselectivity [23]. Properties of ionic liquids can be modified by changing either the anion and/or the cation. The ions are influencing enzyme properties based on the Hofmeister series in aqueous solutions of hydrophilic IL and in a more complicated manner in low-water activity media [24]. Water activity has also been described as important parameter to optimize enzymatic reactions and this can be adjusted by a range of methods. The most popular is pre-equilibration with saturated salt solutions or dehydration with molecular sieves [25]. A control of water activity during the reaction can be achieved by applying vacuum [26] and at best using salt hydrate pairs [27]. These were

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used as an easy way to control and regulate water activity value in organic solvents and in ionic liquids [28]. The aim of this study was to determine the parameters influencing the synthesis of ascorbyl oleate in ionic liquids, with addition of salt hydrates pairs for water activity control, using commercially available immobilized lipase B from Candida antarctica (CAL-B). 2. Materials and methods

silica gel 60 plates using a solvent mixture of chloroform/methanol/acetic acid (96/ 3/1, v/v/v) [31] or chloroform/methanol/acetic acid/water (80/10/8/2, v/v/v/v) [32]. Compounds were visualized by spraying the plates with 5% (v/v) ethanolic solution of H2SO4 and heating for about 10 min at 150 8C. Quantitative analysis was done by HPLC using an Agilent system HP1100, on a C18 Supelcosil column (5 mm, 250 mm  4.6 mm) with methanol/water (90/10, v/v) as mobile phase at 1 ml/min flow rate [33]. Detection was achieved using an evaporative light scattering detector (PL-ELS 1000, Polymer Laboratories, Church Stretton, England). Samples were filtered to remove the enzyme and molecular sieves and 100 ml of the solution was diluted with methanol/water (90/10, v/v) [33]. Conversion was calculated from the decrease in L-ascorbic acid concentration.

2.1. Enzyme and chemicals Commercially available immobilized C. antarctica lipase B, i.e. Chirazyme1 L-2, carriers: C1, C2, and C3), and free lipase C. antarctica B, Chirazyme1 L-2 (all from Roche Diagnostics, Penzberg, Germany) were used as biocatalysts. Organic solvents were purchased from POCh (Gliwice, Poland) and were HPLC grade purity. Other chemicals were purchased from Sigma–Aldrich (Poznan, Poland) and were of the highest available purity. All solvents, fatty acids or their methyl esters were dehydrated before use with activated 4 A˚ molecular sieves (Sigma–Aldrich). Ascorbyl oleate synthesis was performed in a range of ionic liquids purchased from Solvent Innovation, Germany (BioTech kit) containing: 1-butyl-3-methylimidazolium tetrafluoroborate ([BMIM][BF4]), 1-ethyl-3-methylimidazolium tosylate ([EMIM][TOS]), 1-butyl-3-methylimidazolium n-octylsulfate (ECOENGTM 418; [BMIM][OcSO4]), 1-butyl-3-methyl-imidazolium diethyleneglycolmonomethylethersulfate (ECOENGTM 41 M; [BMIM] [MDEGSO4]), trioctylmethylammoniumbis(trifluoromethylsulfonyl)imide ([OMA][BTA]), PEG-5-cocomonium methylsulfate (ECOENGTM 500; [CABHEM][MeSO4]). 2.2. Lipase-catalyzed synthesis of ascorbyl oleate Water activity (defined as the vapor pressure of water above/in a sample divided by that of pure water at the same condition) in the reaction mixture was controlled either by the addition of molecular sieves (4 A˚) or by pre-equilibration over a saturated salt solution or by the addition of salt hydrate pairs. Water activity of the reaction mixtures were adjusted by pre-equilibration in containers with saturated salt solutions (LiCl, MgCl2, K2CO3, NaCl, equivalent to water activities of 0.11, 0.33, 0.43, and 0.75, respectively) (Table 1) at room temperature for at least 2 weeks [29]. The abbreviated notation of salt hydrate pairs was used as proposed by Berberich et al. [28], e.g. NaI 2/0 refers to a mixture of NaI2H2O and anhydrous NaI. Salt hydrate pairs: MgCl2 6/4, NaI 2/0, CuSO4 5/3, and Na4P2O7 10/0 were used to maintain water activity at the values given in Table 1. Water activities of the reagents and enzymes were confirmed by measurement using a hygrometer (BTRS1, Rotronic AG, Bassersdorf, Switzerland). Typically, the condensation reaction was conducted in screw-cap glass vials protected from light and containing oleic acid (0.12 mmol), ascorbic acid (0.1 mmol), 20 mg lipase, 0.5 ml of ionic liquid and 0.2 g of the higher salt hydrate (the lower salt hydrate is formed when water is released into the medium) [28]. Reactions were performed (based on preliminary studies) at 60 8C for 24 h, with mixing by a magnetic stirrer at 300 rpm. The headspace of the vials was filled with nitrogen and refilled after each sampling. If the reactions were carried out in the presence of molecular sieves, then 150 mg were added. Control experiments were conducted without enzyme. 2.3. Semi-continuous synthesis of ascorbyl oleate Synthesis was performed for 24 h and lipase was re-used after separation for the next reaction. The enzyme was separated by Whatman filter paper, washed with n-hexane, water and then freeze-dried for 12 h (Christ alpha 1-2 LD). Stability of lipases was studied by re-using immobilized Chirazyme L-2, C2 and determining the yield after 24 h. Conversion was calculated as given in Section 2.4. 2.4. Analysis The product of the reaction was isolated by addition of 20 ml of water to the reaction mixture. Qualitative analysis of reaction mixtures was made by TLC on Table 1 Water activity of the reaction medium using solid salts addition. Water activitya

This work Ref. [28] Ref. [30] Ref. [27] a

Salt hydrates MgCl2 6/4

NaI 2/0

CuSO4 5/3

Na4P2O7 10/0

0.10 – – 0.056

0.30 0.17 0.07 0.198

0.55 0.42 0.42 0.55

0.65 0.47 0.47 0.75

Determined at 60 8C.

2.5. Surface properties determination Ascorbyl oleate (purified by semi-preparative HPLC) was dissolved in distilled water at different concentration and surface tension and critical micelle concentration (CMC) were determined at 25 8C by the ring method using tensiometer K-9 (Kru˝ ss, Hamburg, Germany). All experiments were performed in at least triplicate (except for the assay of antimicrobial activity, which were done in duplicate) and standard deviations were calculated. 2.6. NMR spectra analysis NMR spectra were recorded on a Bruker Avance II 300, at 300,1812007 MHz for H spectroscopy and at 75,4803238 MHz for 13C spectroscopy. Analysis were performed at 300 K and the samples were diluted in DMSO-d6. The chemical shift (ppm) for solvent was d = 2.50 and d = 39.51 for 1H and 13C, respectively. 1 H NMR (DMSO-d6): 11.07 (1H, br, C20 –OH); 8.36 (1H, br, C30 –OH); 5.31 (2H, m, H–9 and H–10); 4.65 (1H, d, 3JH40 –H50 = 1.58, H-40 ); 4.08 (1H, dd, 2JH60 –H60 = 10,44, 3 JH50 –H60 = 8.20, H-60 ); 4.04 (1H, dd, 2JH60 –H60 = 10.44, 3JH50 –H60 = 5.07, H-60 ); 3.96 (1H, br, H-50 ); 3.37 (1H, br, C50 –OH); 2.30 (2H, t, 3JH2–H3 = 7.40, H-2); 1.97 (4H, m, H-8 i H11); 1.53 (2H, m, H-3); 1.40–1.14 (20H, H-4, H-5, H-6, H-7, H-12, H-13, H-14, H-15, H-16, H-17); 0.85 (3H, t, 3JH17–H18 = 6.62, H-18). 13 C NMR (DMSO-d6): 172.62 (C-1); 170.33 (C-10 ); 152.10 (C-30 ); 129.58 (C-10 or C-9); 129.55 (C-9 or C-10); 118.24 (C-20 ); 75.01 (C-40 ); 65.53 (C-50 ); 64.42 (C-60 ); 33.40 (C-2); 31.32 (C-16); 29.14 (2); 28.88; 28.64; 28.53 (2); 28.50 (2); 26.60 (2, C-8 and C-11); 24.38 (C-3); 22.12 (C-17); 13.89 (C-18). 1

3. Results Ionic liquids (IL) are attractive solvents for enzymatic reaction, and especially for reactions of substrates of different polarity, as they can solubilize polar as well as non-polar compounds. Additionally, by changing cations and anions, the properties of IL can be simply changed [20]. IL are considered to be non-toxic and biocatalysis in a medium containing IL can be regarded as a green process [19]. On the other hand, the problem of a reliable estimation of IL toxicity and even the methods of testing IL (eco)toxicology should be solved [34,35]. Usually, IL have to be purified for applications in biocatalysis, e.g. washing 3-alkyl-1methylimidazolium tetrafluoroborate with aqueous sodium carbonate was needed to get active Pseudomonas cepacia lipase in the acetylation of 1-phenylethanol [36]. However, a tremendous progress was made in the last few years in the preparation and application of IL as reaction medium [37]. IL are still expensive and in some cases special treatments for obtaining enzymes active in IL are needed, e.g. the immobilization of C. antarctica lipase B with poly(ethylene glycol) was presented as the method to obtain enzyme active in glucose fatty acid ester synthesis in pure [BMIM][BF4] [38]. The most extensive research concerning synthesis of ascorbyl oleate in IL was performed by Viklund et al. [3,39,40]. However, the use of salts pairs for in situ control of water activity in lipasecatalyzed reaction for the modification of ascorbic acid has not been investigated yet. The first reports about the use of salts pairs for water activity control were published by Kuhl and Halling [41] and Halling [27]. Water plays a crucial role in enzyme structure and function of proteins and thus determines activity and stability of enzymes. Water dynamics and mechanism of salt-activation of enzymes in

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Table 2 Initial rate of ascorbyl oleate synthesis in ionic liquids. Ionic liquid

[BMIM][BF4] [EMIM][TOS] [EMIM][OcSO4] [BMIM][MDEGSO4] [Oc3MeN][BTA] [CABHEM][MeSO4] Acetone

Initial rate (mmol h1 mg1) CAL-Ba, C1

CAL-B, C2

CAL-B, C3

CAL-B

0.25 0.22 0.20 0.08 0.14 0.12 0.05

0.31 0.18 0.22 0.12 0.08 0.11 0.07

0.22 0.12 0.08 0.08 0.10 0.06 0.04

0.18 0.08 0.12 0.10 0.06 0.04 0.02

Reaction conditions: 20 mg lipase, 60 8C, 0.1 mmol ascorbic acid, 0.12 mmol oleic acid, 0.5 cm3 ionic liquid. a Candida antarctica lipase B – immobilized on carriers C1, C2, C3 or free lipase.

organic media were recently analyzed by Yang et al. [42] and Eppler et al. [43]. Park and Kazlauskas [36] correlated the high activity of P. cepacia lipase in the acetylation of 1-phenylethanol with the IL polarity, i.e. higher enzyme activity (conversion) was obtained in more polar IL. Also in our experiments lipases were more active in more polar IL (e.g. for [BMIM][BF3] polarity according to Reichardt’s scale, ETN = 0.68) than in acetone (ETN = 0.36) [36,44] (Table 2). The highest initial rate of ascorbyl oleate synthesis was obtained in reactions catalyzed by Chirazyme L-2, C2 (0.31 mmol h1 mg1) in [BMIM][BF4] (Table 2). The productivity of the lipases was in almost all cases higher in IL compared to acetone as described earlier [33]. It is well known, that salt hydrates can directly influence enzyme activity in a positive or negative manner by changing the water activity (aw) in the reaction system [45]. Additionally, it was demonstrated that salt hydrates and also zeolite molecular sieves can have acidic or basic effects on enzymes in organic media [46]. In the IL with a water activity value initially adjusted by preequilibration in saturated salt solution atmosphere, about 60% yield of ascorbyl oleate was obtained in [BMIM][BF4] at an initial aw = 0.11 (Fig. 1). However, the highest conversion and yield of ascorbyl oleate, 85% and 72%, respectively, were obtained in [BMIM][BF4] in the presence of the salt pair NaI 2/0, which corresponds to an aw of 0.3 (Fig. 1). In [2PentMIM][BF4] (3-methyl1-(1-methyl-butyl)-3-imidazolium tetrafluoroborate) the highest yield of the reaction, i.e. 61%, was obtained by the reaction controlled by using vacuum-mediated water removal [40]. The yield of the reaction of ascorbyl oleate synthesis performed in tamyl alcohol ranged from 38% [47] to 86% [3]. The most popular solvent used for ascorbyl derivatives synthesis is acetone in that Yan et al. [48] obtained 91% yield of 6-O-palmitoyl-L-ascorbic acid after 48 h, but Song and Wei [49] reported here only 19.3% ascorbyl oleate. In these experiments the most popular catalyst was lipase B from C. antarctica (used in different forms), also in continuous stirred tank reactors [50]. In addition, we observed, that the use of salt hydrates lead to about 20% higher conversion compared to the more commonly used pre-equilibration of reaction mixtures in saturated salt solutions (Fig. 2). The salt hydrates absorb the water formed during the reaction of condensation and enable to keep the water activity value at the level that stimulated ascorbyl oleate synthesis [28]. The initial adjustment of water activity enable to keep the water activity value at desired level for as little as 8 h, then the reaction of ascorbyl oleate synthesis was inhibited because of the high water activity value of the reaction mixture (Fig. 2). A similar, but just temporal, effect of water activity control was obtained when molecular sieves for water absorption were added to the reaction medium [3,33]. As expected, the increase of the amount of lipase from 10 to 40 or 60 mg increased the conversion (Fig. 3); larger

Fig. 1. Effect of water activity control on conversion and yield in the synthesis of ascorbyl oleate in [BMIM][BF4]. (A) Pre-equilibration with saturated salt solution; (B) Salt hydrates. Reaction conditions: see Table 2. MS-addition of molecular sieves; 0.11, 0.33 water activity after pre-equilibration over saturated salt solutions, LiCl and MgCl26H2O, respectively; NaI 2/0, CuSO4 5/3–salt hydrate, aw = 0.30 and aw = 0.53, respectively. Error bars represent standard deviation.

amounts of enzyme had not additional effect (data not shown). Fig. 4 shows the residual activity of lipase versus the number of batch reactions. The insert in Fig. 4 indicates (logarithmic scale) that the inactivation could be expressed a by first-order kinetic.

Fig. 2. Influence of method of water activity control on kinetic of ascorbyl oleate synthesis in [BMIM][BF4]. Reaction conditions: the same as described in Table 2.

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References

Fig. 3. Influence of enzyme amount on ascorbyl oleate synthesis in [BMIM][BF4] with addition of NaI 2/0. Reaction conditions: the same as described in Table 1. Error bars represent standard deviation.

Fig. 4. Kinetic of semi-continuous synthesis of ascorbyl oleate in [BMIM][BF4] with addition of NaI 2/0.

The critical micellar concentration (CMC) of ascorbyl oleate was determined to be 2.2  104 M, and the surface tension was reduced to 32 mN/m at 25 8C. The surface activity of the product obtained in our study were comparable to that described by Watanabe et al. [5] indicating good surface activity of the product obtained. 4. Conclusions It could be shown, that the enzymatic synthesis of ascorbyl oleate in ionic liquids is affected by water activity and especially be the method used to control it. The highest conversion (85%) and isolated yield (72%) of ascorbyl oleate was obtained after 14 h using CAL-B, C-2 in [BMIM][BF4] with NaI 2/0. Ascorbyl oleate indicated high interfacial activity and properties of an emulsifier that supports its application in water–fat phases. Acknowledgements The gift of lipases from Roche Diagnostics (Penzberg, Germany) is gratefully appreciated. This work was supported by the Committee of Scientific Research Poland (Grant KBN P0 6T 01327).

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