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J. of Supercritical Fluids 40 (2007) 93–100

On the importance of the supporting material for activity of immobilizedCandida antarctica lipase B in ionic liquid/hexane and ionic

liquid/supercritical carbon dioxide biphasic media

Pedro Lozano a, Teresa De Diego a, Tanja Sauer a, Michel Vaultier b,Said Gmouh b, Jose L. Iborra a,∗

a Departamento de Bioquımica y Biologıa Molecular B e Inmunologıa, Facultad de Quımica, Universidad de Murcia,P.O. Box 4021, E-30100 Murcia, Spain

b Universite de Rennes-1, UMR-CNRS 6510, Campus de Beaulieu, Av. General Leclerc, F-35042 Rennes, France

Received 4 October 2005; received in revised form 23 March 2006; accepted 29 March 2006

bstract

A commercial solution of free Candida antarctica lipase B (Novozyme 525L) has been immobilized by adsorption onto 12 different silicaupports modified with specific side chains (e.g. alkyl, amino, carboxylic, nitrile, etc.). The immobilized derivatives were assayed for the kineticesolution of rac-1-phenylethanol in both ionic liquid/hexane and ionic liquid/supercritical carbon dioxide biphasic media. The best results werebtained for the supports modified with non-functionalized alkyl chains and when the in water activity increased from 0.33 to 0.90 (e.g. theALB/butyl-silica activity was enhanced up to five times). Coating immobilized enzyme particles with ionic liquids (butyltrimethylammoniumistriflimide or trioctylmethylammoniun bistriflimide) resulted in a decrease in activity (10 times), although half-life times were enhanced (up

o six times) in hexane media at 95 ◦C. However, immobilized derivatives coated with ionic liquids clearly improved their synthetic activity inupercritical CO2 by up to six times with respect to the hexane medium, which agrees with the “philicity” between alkyl chain lengths of both theilica support and the cation of ionic liquid.

2006 Elsevier B.V. All rights reserved.

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eywords: Lipase; Ionic liquids; Supercritical fluids; Enzyme immobilization;

. Introduction

The use of supercritical fluids as green non-aqueous mediaor enzyme-catalyzed synthetic reactions has been widely stud-ed during the last decade, due to their excellent recognizedroperties [1–3]. The greenness of enzymatic biotransforma-ions in scCO2 begins with the enzyme, because enzymes areatalytic proteins of living systems, and so, acts as environ-entally benign catalysts, with having high chemo-, regio- and

nantioselectivity [4]. However, enzymes have been designedo work in aqueous solutions within a narrow range of environ-

ental conditions (pH, temperature, pressure, etc.), and theirse in non-aqueous media is seriously limited by the denatura-ive action of these media on the proteins. In this way, scCO2

∗ Corresponding author. Tel.: +34 968367398; fax: +34 968364148.E-mail address: jliborra@um.es (J.L. Iborra).

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896-8446/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.supflu.2006.03.025

ansformation; Green chemistry

as been described as a solvent with a strong deactivating effectn enzymes [1,5,6], making it necessary to develop enzyme sta-ilization strategies, e.g. covalent immobilization on supportsoated with hydrophilic polymers [7].

On the other hand, ionic liquids (ILs) have recently emergeds exceptionally interesting green non-aqueous reaction mediaor enzymatic transformations [8]. They are salts and thereforentirely composed of ions that are liquids below 100 ◦C or closeo room temperature. Their interest as green solvents resides inheir high thermal stability and very low vapor pressure, whichitigates the problem of releasing volatile organic solvents into

he atmosphere. Moreover, the physical properties of ILs (den-ity, viscosity, melting points, polarity, etc.) can be finely tunedy the appropriate selection of anions and/or cations. ILs can

e designed to be miscible or immiscible with water or organicolvents (e.g. hexane, toluene, ether, 1-propanol, etc.), makingt easy to recover products from the reaction mixture [9]. Watermmiscible ILs have been shown to be excellent non-aqueous

94 P. Lozano et al. / J. of Supercritical Fluids 40 (2007) 93–100

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edia for enzyme-catalyzed reactions (especially for lipases),ecause of the high level of activity [10–12], stereoselectivity13] and stability [14–16] displayed by enzymes in chemicalransformations, even under extremely harsh conditions (e.g.cCO2 at 100 bar and 150 ◦C) [17]. Furthermore, it has beenescribed how the water activity (Aw) parameter greatly modifieshe efficiency of biotransformation in ILs, because the hydrationevel for the enzyme must be optimal, while the minimum freeater-content in the reaction media must be minimal to avoidndesired side reactions [4,8,10,13].

Biphasic systems based on ILs and supercritical fluids fornzyme catalysis have been put forward as the first approacho integral green bioprocesses in non-aqueous media, whereoth the biotransformations and extraction steps are coupled inn environmental benign and efficient reaction/separation pro-ess [18,19]. ILs can absorb large quantities of CO2 at lowressure (0.6 mol fraction at 10 MPa), although the amount ofL dissolved in CO2 is negligible. This fact not only showshe exceptional ability of scCO2 to extract a wide variety ofydrophophic compounds from ILs, but also decreases the vis-osity of ILs, thus, improving mass-transfer phenomena [20].

The aim of this paper is to push forward in our efforts inhe design of green enzymatic processes in IL/scCO2 biphasicystems, since such systems may help to meet the increas-ng demand for clean technologies in industrial chemical pro-esses [21]. For free enzyme suspended in ILs, criteria to selecthese green solvents according to certain parameters, such asctivity and stability of enzyme, as well as, the mass-transferhenomena between both IL and scCO2 non-miscible phases,ere previously established [22]. In this work and for the first

ime in the literature, the influence of the support materialpplied to immobilize Candida antarctica lipase B (CALB)s studied for the kinetic resolution of rac-1-phenylethanoly transesterification with vinyl propionate (see Fig. 1) bysing both ILs/hexane and ILs/scCO2 biphasic media at con-rolled Aw. Two ionic liquids (ILs), based on quaternary ammo-ium cations bearing different alkyl side chains, butyl-trimethylbtma], and trioctyl-methyl [toma], associated with the samenion (bis(trifluoromethane)sulfonyl imide, [NTf2] were usedo analyse CALB activity and stability for the proposed bio-ransformation.

. Materials and methods

.1. Materials

C. antarctica lipase B (Novozym 525L, EC 3.1.1.3) wasrom Novozymes S.A. (Copenhagen, DK). The enzyme solu-

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d kinetic resolution of rac-1-phenylethanol.

ion was purified by ultrafiltration, resulting a CALB solu-ion of 14.9 mg/mL [22]. The purity of the enzyme wasested by SDS-PAGE electrophoresis, which showed onlyne protein band [16]. Silica gel (63–200 m particle size,nm pore diameter) were obtained from Sigma–Aldrich–Flukao. (Madrid, Spain). Modified silica gel (40–120 �m parti-le size, 6 nm pore diameter), containing methyl (C1), ethylC2), butyl (C4), octyl (C8), octadecyl (C18), cyanopropyl,arboxylic, aminopropyl, diethylaminoethyl, quaternary ammo-ium or benzenesulfonic groups, respectively, were obtainedrom Applied Separations Inc. (Allentown, PA, USA). Molec-lar sieve UOP Type 4 (pore diameter 4 A), substrates, sol-ents and other chemicals were purchased from Sigma–Aldrich–luka Co. (Madrid, Spain), and were of the highest purityvailable.

.2. Synthesis of ionic liquids (ILs)

Specific procedures to prepare both ILs [btma][NTf2] andtoma][NTf2] were previously described in detail [12,14,22].he resulting ILs were colourless, as a water-like oil, contain-

ng 75 and 65 ppm of water, respectively, as determined byarl-Fischer measurements using a Metrohm 684 KF coulome-

er. Dynamic viscosities were 0.058 and 0.571 Pa s, respec-ively, as determined using a AR 1000 microviscometer (TAnstruments, New Castle, DE, USA) with a stainless conelate geometry (diameter 40 mm, angle 1◦1′). A flow pro-edure was applied from 0.06 to 200 s−1 in 3 min with 20oints measured at 23 ◦C. The chloride content of the ILsas undetectable by both mass spectrometry and AgNO3 test

23].NMR spectra were recorded on a Bruker AC 300 P

1H: 300.13 MHz; 13C: 75 MHz). 1H NMR of [btma][NTf2]acetone-d6, δ/ppm relative to TMS): 0.95 (t, J = 7.4 Hz,H); 1.40–1.59 (m, 2H); 1.88–2.11 (m, 2H); 3.42 (s, 9H);.60–3.75 (m, 2H). 13C NMR of [btma][NTf2] (acetone-6, δ/ppm relative to TMS): 14.14; 20.56; 25.81; 53.95 (t,C–N = 4.1 Hz); 67.71; 121.85 (q, JC–F = 321.2 Hz). HRMSES) calcd. for C16H36F6N3O4S2, [2C+, NTf2

−]+ 512.2051,ound 512.2069. 1H NMR of [toma][NTf2] (acetone-d6,00 MHz, δ ppm/TMS): 0.88 (t, 9H, J = 6.6 Hz). 1.29–148m, 30H); 1.90–1.92 (m, 6H); 3.21 (s, 3H); 3.49–3.51 (m,H). 13C NMR of [toma][NTf2] (acetone-d6, δ/ppm rela-

ive to TMS): 14.05; 22.24; 22.73; 25.57; 29.02; 29.65;1.93; 48.27; 63.26; 119.68 (q, JC–F = 321.2 Hz). HRMS (ES)alcd. for C52H108F6N3O4S2, [2C+, NTf2

−]+ 1016.7674, found016.7670.

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P. Lozano et al. / J. of Super

.3. Enzyme immobilization

Three milliliters of purified CALB solution and 2 mL of 0.1 Mhosphate buffer pH 7.8 were added to screw-capped tubes con-aining 1 g of silica gel, or modified silica gel. The mixtures werehaken for 2 h at room temperature to adsorb the enzyme, andere then centrifuged at 2800 rpm for 10 min. In each case, the

upernatant was recovered, and the support washed twice with.1 M phosphate buffer pH 7.8 (5 mL) to remove non-adsorbednzyme molecules. The liquid fractions were recovered. Theilica gel phase was frozen at −60 ◦C and lyophilized for 72 h,ielding a dry powder containing the immobilized CALB. Theupernatant and washing fractions were collected and used touantify the amount of immobilized protein by using a modifi-ation of the Lowry’s method [24].

.4. Water activity (Aw) equilibration

Both ILs (2 mL samples) and immobilized enzyme prepa-ations (100 mg samples of each one) were separately equili-rated to fixed Aw over saturated salt solutions in closed con-ainers at 25 ◦C for 1 week. The following salts were used:

gCl2 (Aw = 0.33), Mg(NO3)2 (Aw = 0.54), NaCl (Aw = 0.75)nd BaCl2 (Aw = 0.90) [11].

.5. Drying of chemicals

Water was removed from the hexane, vinyl propionate andac-1-phenylethanol by adding molecular sieves (0.1 g/mL),haking the resulting mixture for 24 h at room temperature, andnally storing them in the presence of the adsorbent.

.6. Kinetic resolution of rac-1-phenylethanol in hexaneedia

Reactions were carried out in 1-mL screw-capped vials witheflon-lined septa, containing 600 �L of substrates solution450 mM rac-1-phenylethanol and 950 mM vinyl propionate)n dry hexane. The reaction was started by adding the Aw-quilibrated immobilized enzyme preparation (25 mg), free orixed with 50 �L of either [btma][NTf2] or [toma][NTf2] ionic

iquids, and run at 50 or 95 ◦C (oil bath) for 1 h. At regular timentervals, 20 �L aliquots were taken and suspended in 380 �Lexane, and then cooled in ice bath. The biphasic mixture wastrongly shaken for 3 min to extract all the substrates and prod-cts into the hexane phase. Then, 200 �L of the hexane extractere added to 300 �L, 100 mM butyl butyrate (internal stan-ard, IS) solution in hexane, and 1 �L of the resulting solutionas analysed by GC. Profiles of both R-1-phenylethyl propi-nate and propionic acid concentrations with time were used touantify both the synthetic and hydrolytic reaction rates of theystem, respectively. Synthetic activity was determined by theatio between the synthetic reaction rate and the assayed amount

f protein. Selectivity was determined by the ratio between theynthetic reaction rate with respect to the addition of both syn-hetic and hydrolytic reaction rates (equivalent to the acyl-donoronsumption reaction rate). Enantiomeric excess of the synthetic

bpms

l Fluids 40 (2007) 93–100 95

roduct was determined by the following equation:

.e. =

[R-1-phenylethyl propionate]

−[S-1-phenylethyl propionate]

[R-1-phenylethyl propionate]

+[S-1-phenylethyl propionate]

× 100

One unit of synthetic activity was defined as the amountf enzyme that produces 1 �mol of (R)-1-phenylethyl pro-ionate per min. All the experiments were carried out inuplicate.

.7. Enzyme stability by recycling operation in hexaneedia

After a reaction cycle has been carried out as describedbove, the liquid medium was collected and the immobi-ized biocatalyst (coated or not with IL) was recovered foreuse. First, it was washed three times with dry hexane (3 mL)o ensure that all the reaction products have been extracted.hen another reaction was started by adding 600 �L of freshubstrate solution (450 mM rac-1-phenylethanol and 950 mMinyl propionate solution hexane) to the immobilized enzymeerivative. This process was repeated for 10 times, the hexanehase being carefully collected every time to avoid the loss ofiocatalyst.

.8. Continuous kinetic resolution of rac-1-phenylethanoln scCO2

Immobilized enzyme preparation (25 mg) were coated with0 �L of either [btma][NTf2] or [toma][NTf2] ionic liquids in aest tube and strongly shaken for 10 min, to ensure proper coat-ng of all silica particles. The final mixture was placed in theartridge of an ISCO 220SX (Teledyne Isco Inc., Lincoln, NE,SA) high-pressure extraction apparatus of 10 mL total capac-

ty, containing glass wool (3 g). The apparatus is equipped withsyringe pump (ISCO model 100DX, 100 mL overall volume),eedle valves and devices for pressure, temperature and flow rateontrol. The ISCO system was started by continuous pumpingf scCO2 at 10 MPa and 50 ◦C, which automatically opens thexit valve, bubbling continuously the CO2 through a calibratedeated restrictor (1 mL/min, 60 ◦C) in a controlled amount ofexane placed on ice-bath. The synthetic process was carried outy continuous pumping of an equimolar solution of pure sub-trates (4.24 M rac-1-phenylethanol and vinyl propionate) intohe scCO2 inlet flow at a 5 �L/min flow rate (21.2 �mol/min),y using a HPLC pump (model LC-10AT, Shimadzu Europe,uisburg, Germany) (see Fig. 2). The reactor was continuouslyperated for 4 h. Substrates and products were transported by thecCO2 flow through the catalytic cartridge, being then recovered

y depressurizing through the restrictor for 30 min steps. Sam-les were analysed by GC. In all cases, substrates and productsass-balances from the outlet were consistent with the sub-

trates mass-flow inlet.

96 P. Lozano et al. / J. of Supercritical Fluids 40 (2007) 93–100

kinet

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Fig. 2. Experimental set up of the enzymatic reactor for continuous

.9. GC analysis

Analysis were performed with a Shimadzu GC-17A (Shi-adzu Europe, Duisburg, Germany) equipped with FID detec-

or. Samples were analysed on a Beta DEX-120 column30 m × 0.25 mm × 0.25 �m, Supelco), using He as carrier gasnd a FID detector, as described previously [18,22]. Retentionimes of compounds are as follows: vinyl propionate (3.1 min),ropionic acid (5.6 min), butyl butyrate (internal standard,.1 min), R-1-phenhylethanol (14.8 min), S-1-phenhylethanol15.5 min), S-1-phenylethyl propionate (18.2 min) and R-1-henylethyl propionate (18.6 min).

. Results and discussion

.1. Influence of support and water activity on enzymectivity in hexane

Fig. 1 depicts the mechanism for kinetic resolution of rac-1-henylethanol catalyzed by serine hydrolases, such as lipases.n these reactions, a covalently linked acyl-enzyme intermediate

st

o

able 1mmobilization and catalytic parameters of lyophilized Candida antarctica lipase Bifferent side chains

upport Immobilized protein(mg Prot./g supp.)

Immobilization yi(%)

ilica (Si) 26.0 58.2ethyl-Si (C1) 22.6 50.6

thyl-Si (C2) 17.8 39.8utyl-Si (C4) 11.9 26.6ctyl-Si (C8) 11.8 26.4ctadecyl-Si (C18) 16.2 36.2yanopropyl-Si 17.3 38.7arboxylic-Si 21.0 47.0minopropyl-Si 15.8 35.3iethylaminoethyl-Si 17.5 39.1uaternary ammonium-Si 17.4 38.9enzenesulphonic-Si 31.8 71.2

a Product between synthetic activity and immobilized protein.b See Section 2.6.

ic resolution of rac-1-phenylethanol in IL/scCO2 biphasic system.

s formed, and the nucleophilic attack by water results in esterydrolysis, although the presence of another nucleophile (e.g.oth isomers of 1-phenylethanol) might involve the formation ofhe transesterification product. This latter synthetic pathway cane regarded as a kinetically controlled process, where the rapidccumulation of the acyl-enzyme intermediate and the preferen-ial (and stereoselective) nucleophilic attack by the alcohol aressential. The first condition is enhanced by the use of activatedcid acyl-donors such as vinyl esters, because the vinyl alcoholeleased in the degradation of the vinyl ester tautomerizes tocetaldehyde, which cannot act as a substrate for the enzyme10,13]. The second condition may arise from using reactionedia with very low water-content and a high nucleophile (e.g.

-phenylethanol) concentration. In this context, the efficiencyn these lipase-catalyzed reactions can mainly be expressed byhree parameters: the synthetic activity, the selectivity for theynthetic reaction, determined by the ratio between both the

ynthetic and the acyl-donor consumption reaction rates, andhe enantiomeric excess (e.e.) of the synthetic product.

Table 1 shows the influence of the nature of the supportn both efficiency of the immobilization and catalytic param-

derivatives resulting from adsorption of the enzyme on silica modified with

eld Immobilized activitya

(U/g supp.)Selectivityb

(%)e.e.b (%)

52.0 100.0 >99.9282.5 94.0 >99.9388.0 94.0 >99.9606.9 95.7 >99.9392.9 95.0 >99.9468.2 94.4 >99.9138.4 98.6 >99.9

2.1 100.0 >99.944.2 100.0 >99.985.7 70.0 >99.936.5 68.8 >99.90.0 – –

critical Fluids 40 (2007) 93–100 97

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P. Lozano et al. / J. of Super

ters of the assayed immobilized derivatives. As can be seen,ll the assayed supports were able to adsorb protein molecules,ith no clear dependence on the nature of the support surface,

esulting in immobilized derivatives with a protein loading of6–71% of the offered amount. On the contrary, the catalyticfficiency of these immobilized derivatives was clearly depen-ent on the chemical characteristic of support alkyl chains. Thus,oth free silica and modified silica with ionizable (or polar)roups provide the worst catalytic parameters for immobilizedALB derivatives. However, the use of silica modified withydrophobic alkyl chains resulted in good catalytic parame-ers for immobilized derivatives, which were dependent on theength of side chain. The best immobilized activity was obtainedor the CALB-C4-silica derivative (606.9 U/g supp.), which per-aps provided a more appropriate environment for the enzymedsorption, and led to an increase in the driving forces towardshe partitioning of the hydrophobic substrates. It should also beointed out how, in all cases, the selectivity for the syntheticeaction was higher than 94% (except for the anionic exchangerroups), and the e.e. of the R-1-phenylethyl propionate prod-ct was higher than 99.9%. The nature of the support surfacen both its interaction with the enzyme and its ability to retainater molecules after lyophilization was probably involved in

he observed differences in activity and selectivity.Garcia et al. [25] describes how the protonation state of the

rotein is another factor with a marked effect on catalytic activityn organic media, which can be determined by the pH of the lastqueous solution of the enzyme prior to lyophilization. In ourase, enzyme adsorption on supports with ion-exchanger groupsay produce changes in the protonation state of the protein

fter lyophilization, involving a drop in the catalytic activity ofhe dried immobilized derivative (e.g. benzenesulphonic silica).

oreover, Vecchio et al. [26] described how the lyophilizationrocess of CALB produced an increase in �-sheet secondarytructure, which results in a decrease in activity because of theoss of native structure by decreasing hydrogen-bonding inter-ctions between water molecules and �-helices. Based on theseesults, only CALB derivatives from an alkyl-silica support weresed for further studies.

Water activity (Aw) has been described as the most suit-ble parameter for analysing enzyme activity in non-aqueousedia, because of the importance of the level of hydration of

he enzyme [11,13,23–25]. Fig. 3 shows the evolution of theynthetic activity of immobilized CALB as a function of theength of the alkyl-side chain of the support, pre-equilibrated atifferent Aw values. As can be seen, all activity profiles showedell-shaped curves, the maximum level of activity being attainedhen the C4-silica support was used. Furthermore, an increase

n Aw from 0.33 to 0.90 had a positive impact on the rates ofransesterification for all immobilized CALB derivatives, whichas, in agreement with other authors [13,23,24], attributed to

n increase in enzyme dynamics. As expected, the increasen Aw slightly favored the hydrolytic pathway of the catalytic

echanism. Thus, for all derivatives, the selectivity parame-

er exhibited at both Aw = 0.33 and 0.54 was 100%, falling to8 and 96% when the assayed Aw was 0.75 and 0.90, respec-ively (results not depicted). A pre-equilibration of immobilized

ch[r

ig. 3. Influence of the alkyl side-chain length of the support on the syntheticctivity of CALB-immobilized derivatives for the kinetic resolution of rac-1-henylethanol in hexane at controlled Aw and 50 ◦C.

erivatives at 0.90 of Aw was used to analyse the thermal stabilitynd mass-transfer phenomena in both IL/hexane and IL/scCO2iphasic systems.

.2. Activity and stability of immobilized derivatives inL/hexane media at 95 ◦C

A key criterion for selecting solid support for the immobi-ization of an enzyme for a given chemical transformation is thetability of the catalyst within the reaction medium. ILs haveeen shown to be excellent media for stabilizing both free andmmobilized enzymes towards temperature and reuse in eitherne ionic liquid phase or ionic liquid/scCO2 biphasic condi-ions [10,14–18,22]. However, there are no reported studiesoncerning the catalytic behavior of immobilized enzymes iniquid/liquid biphasic systems. The influence of two differentLs (e.g. [btma][NTf2] and [toma][NTf2]) on both the activitynd the stability of immobilized CALB derivatives (from C2 to18-silica) were studied by previously coating enzyme-supportarticles with these neoteric solvents. Each type of biocatalystas incubated into a hexane solution of substrates at 95 ◦C and.9 of Aw. Fig. 4 shows the synthetic activity exhibited by immo-ilized derivatives as a function of the alkyl chain length ofhe modified silica. All the assayed biocatalysts were able toatalyze the kinetic resolution of rac-1-phenylethanol in thesextreme thermal conditions, the best results being obtained forhe CALB-C4-silica derivative (10.7 U/mg protein). For eacherivative, a loss in synthetic activity was observed in IL/hexaneedia, as compared to that obtained in hexane, the lowest activ-

ty being exhibited by the [toma][NTf2]. To explain these results,t is necessary to take into account that assayed ILs are insol-ble in hexane, and so, biotransformation occurs within a liq-id/liquid biphasic system, which clearly involves an increasen mass-transfer limitations in the reaction media. Thus, the vis-

osity of these ILs increase with the number and length of theydrophobic alkyl chain of the cation (e.g. 58 and 571 mPa s forbtma][NTf2] and [toma][NTf2], respectively), and is mainlyesponsible for this decrease in activity [9,12,18]. Furthermore,

98 P. Lozano et al. / J. of Supercritical Fluids 40 (2007) 93–100

FTp

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ig. 4. Effect of ILs-coating different CALB immobilized derivatives (seeable 1) in the synthetic activity showed for the kinetic resolution of rac-1-henylethanol in hexane at 0.90 of Aw and 95 ◦C.

hese activity results are three-fold higher than those obtainedy using an aqueous solution of CALB “dissolved” in hexaner [btma][NTf2] at 2% (v/v) [22]. The resulting homogeneousistribution of the enzyme onto a support with preferentialnzyme-surface interactions and at an optimal hydration levelre clearly involve, and can be considered key criteria in theesign of an immobilized enzyme for a given process.

Fig. 5(A) shows the deactivation profiles of the CALB-4-silica immobilized derivative, free and coated with either

btma][NTf2] and [toma][NTf2], at 95 ◦C. As can be seen, thectivity loss of the immobilized biocatalyst was greatly reducedy the presence of ionic liquids, in agreement with their nowell described stabilization effect on the biocatalysts underon-conventional conditions (e.g. temperature, solvents, etc.)14,15]. Using both circular dichroism and fluorescence spec-

roscopic techniques, the ability of water-immiscible ILs to pre-erve the catalytic activity of the CALB has been demonstratedrom the maintenance of the secondary structure elements (�-elix and �-strand) in its native conformation [16]. The low

ig. 5. (A) Deactivation profiles of the CALB-C4-silica immobilized derivative,ithout and with coating by either [btma][NTf2] or [toma][NTf2] ionic liquids,

t 0.90 of Aw and 95 ◦C in hexane. (B) Half-lives times of immobilized CALB onilica modified by different alkyl side chains at 0.90 of Aw and 95 ◦C in hexane.

hoei[brs

3i

rbspC[a4c

ig. 6. Profiles of synthetic product yield (�) and selectivity (�) for CALB-C4-ilica derivative coated with [btma][NTf2]-catalyzed continuous kinetic resolu-ion of rac-1-phenylethanol in scCO2 at 50 ◦C and 10 MPa.

olubility of water in the assayed IL could be involved in thereservation of critical water molecules in the microenviron-ent of the enzyme at the extreme temperature assayed [17].or all immobilized derivatives, deactivation profiles followedfirst-order kinetic, allowing the determination of the half-life

ime (t1/2) as a quantitative criterion of the extent to which aiocatalyst is stabilized by ILs. As can be seen in Fig. 5(B),oth ILs increase the half-life time of all immobilized CALBerivatives with respect to the biocatalysts assayed directly in theexane phase. The [btma][NTf2] ionic liquid was shown to be anxcellent protective agent for all the immobilized derivatives, theest results being obtained for the CALB-C4-silica, where thealf-life time was increased six-fold. The best activity and sta-ility levels of immobilized CALB were obtained when C4 alkylhains were present in the microenvironment of the enzyme (seeable 1 and Fig. 5(B)). To understand these results, it is neces-ary to point out that CALB consists of a polypeptide backbone317 residues) with a globular tertiary structure, of which 36.6%re accessible to the solvent [17]. The relatively high content inydrophobic (58 residues) and aromatic (9 residues) amino acidf CALB at the protein surface could be involved in the prefer-ntial catalytic behavior observed when enzyme/C4 alkyl chainnteractions occurred. Furthermore, the high hydrophobicity oftoma][NTf2], produced by C8 alkyl chains in the cation, coulde involved in the low stabilization power, and could also beelated with the decrease in catalytic activity for C8- and C18-ilica derivatives (see Table 1).

.3. Continuous kinetic resolution of 1-phenylethanol inonic liquid/scCO2 systems

The ability of CALB to catalyze ester synthesis or the kineticesolution of sec-alcohol in an IL/scCO2 biphasic system haseen demonstrated [18,19]. Fig. 6 depicts the profile of both theynthetic yield of the enantiomeric product R-1-phenylethyl pro-ionate and the selectivity in the synthetic reaction for CALB-4-silica catalyzed kinetic resolution of rac-1-phenylethanol

btma][NTf2]/scCO2 system in continuous operation at 50 ◦Cnd 10 MPa. As can be seen, the yield profile increased up to8% with reaction time, which correspond to the practical fullonversion of the R enantiomeric substrate, and the steady state

P. Lozano et al. / J. of Supercritica

Fig. 7. Influence of the alkyl side-chain of the silica supports on the synthetica[p

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atstas(AIdrcsCwtI

A

PSsaf

R

ctivity of immobilized CALB derivatives, without and with coating by eitherbtma][NTf2] or [toma][NTf2] ionic liquids, for continuous (R)-1-phenylethylropionate synthesis in scCO2 at 50 ◦C and 10 MPa.

as reached after 120 min of reaction. The synthetic productas obtained at e.e. > 99.9, while the S-1-phenylethyl propi-nate was never detected. Furthermore, the selectivity profileor the synthetic reaction remained constant at 100% throughouthe reaction time, which means that the undesired hydrolysis ofhe acyl-donor never occurred (see Fig. 1). This fact can clearlye related to the continuous flow of dry scCO2 drawing out anyree water molecules from immobilized enzyme particles (ableo act as nucleophile acceptor); hence, the maximum selectiv-ty parameter is obtained. Two additional observations can be

ade, firstly, (R)-1-phenylethyl propionate was not synthesizedn the absence of enzyme, and secondly, no enzyme activity wasetected at the exit of the reactor. This later was related withhe stability of the chromatographic profile of each sample withime. As result, the synthetic activity displayed by the CALB-4-silica/[btma][NTf2] in the scCO2 system was enhanced up5.1 U/mg protein (six-fold higher than the obtained in hex-ne/IL media). These results can be explained by an improve-ent in the transfer rate of substrates to the enzyme microenvi-

onment compared with the liquid systems, due to the excellentbility of scCO2 to transport dissolved solutes through the ILhase [3,18–20,25]. In this way, it was demonstrated how theppropriate selection of IL as a function of the molecular char-cteristic of substrates and products can greatly improved theass-transfer rate in IL/scCO2 biphasic systems [22].Fig. 7 depicts the synthetic activity level of the different

ALB-silica immobilized derivatives, both free and coated withither [btma][NTf2] or [toma][NTf2] ionic liquids, as a functionf the alkyl chain length of the modified support. Once again, therotective effect of ILs towards enzyme deactivation by directontact with scCO2 was demonstrated, because of the poor activ-ty level exhibited by all the free immobilized enzyme derivativesompared with that obtained in the presence of ILs [1,5,6,18,25].s can be seen in Fig. 7, [btma][NTf2] appears as the best IL

n all cases, while CALB-C4-silica derivative again exhibits theighest synthetic activity. These results should be explained asfunction of both the specific enzyme-IL and enzyme-support

nteractions. Both [btma][NTf2] and C4-silica provide an ade-

l Fluids 40 (2007) 93–100 99

uate microenvironment for the catalytic action of the enzyme.n this way, water-immiscible ILs have been described as liquidmmobilization support because multipoint enzyme-IL interac-ions (ionic, hydrogen bonds, van der Waals, etc.) may occur,esulting in a supramolecular net able to maintain active therotein conformation [9,16]. Therefore, we see for the first timeow the “philicity” between the alkyl side chain of both IL andilica, which provides an adequate microenvironment for lipasection, could be regarded as a key parameter in optimizing anymmobilized enzyme/IL system for operation in supercritical

edia.

. Conclusions

The catalytic activity of immobilized CALB in both hexanend scCO2 media is markedly dependent on important fac-ors, such as the nature of the surface of the immobilizationupport and water activity. Coating immobilized enzyme par-icles with ILs, based on quaternary ammonium cations withlkyl side chain and triflimide anion, is shown as an excellenttrategy for stabilizing catalytic activity in adverse conditionse.g. hexane at 95 ◦C), with enhanced mass-transfer limitations.

continuous biphasic reactor based on immobilized CALB-L-scCO2 systems was tested for two different ILs and fiveifferent CALB immobilized derivatives to kinetically resolveac-1-phenylethanol, with excellent results. The catalytic effi-iency of the system was dependent on both the immobilizationupport and the IL, and was greatly improved when the CALB-4-silica derivative coated with the [btma][NTf2] ionic liquidas used. The importance of the enzyme microenvironment in

he catalyzed reaction and the mass-transfer phenomena betweenLs and scCO2 immiscible phases is clearly demonstrated.

cknowledgements

This work was partially supported by the CICYT Ref.:PQ2002-03549 and Ref.: CTQ2005-01571/PPQ grants. Tanjaauer is an Erasmus student from the University of Kaiser-lautern (Germany). We thank Ms. C. Saez for her technicalssistance and Mr. R. Martınez from Novozymes Espana, S.A.or the gift of enzymes.

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