Understanding Structure−Stability Relationships of Candida antartica

Biomacromolecules 2005, 6, 1457-1464

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Understanding Structure-Stability Relationships of Candida antartica Lipase B in Ionic Liquids Teresa De Diego,† Pedro Lozano,† Said Gmouh,‡ Michel Vaultier,‡ and Jose´ L. Iborra*,† Departamento de Bioquı´mica y Biologı´a Molecular B e Inmunologı´a, Facultad de Quı´mica, Universidad de Murcia, P.O. Box 4021, E-30100 Murcia, Spain, and Institut de Chimie, UMR-CNRS 6510, Universite´ de Rennes1, Campus de Beaulieu, Av. Ge´ ne´ ral Leclerc, 35042 Rennes, France Received November 22, 2004; Revised Manuscript Received January 26, 2005

Two different water-immiscible ionic liquids (ILs), 1-ethyl-3-methylimidizolium bis(trifluoromethylsulfonyl)imide and butyltrimethylammonium bis(trifluoromethylsulfonyl)imide, were used for butyl butyrate synthesis from vinyl butyrate catalyzed by Candida antarctica lipase B (CALB) at 2% (v/v) water content and 50 °C. Both the synthetic activity and stability of the enzyme in these ILs were enhanced as compared to those in hexane. Circular dichroism and intrinsic fluorescence spectroscopic techniques have been used over a period of 4 days to determine structural changes in the enzyme associated with differences in its stability for each assayed medium. CALB showed a loss in residual activity higher than 75% after 4 days of incubation in both water and hexane media at 50 °C, being related to great changes in both R-helix and β-strand secondary structures. The stabilization of CALB, which was observed in the two ILs studied, was associated with both the maintenance of the 50% of initial R-helix content and the enhancement of β-strands. Furthermore, intrinsic fluorescence studies clearly showed how a classical enzyme unfolding was occurring with time in both water and hexane media. However, the structural changes associated with the incubation of the enzyme in both ILs might be attributed to a compact and active enzyme conformation, resulting in an enhancement of the stability in these nonaqueous environments. Introduction Ionic liquids (ILs) have emerged as exceptionally interesting nonaqueous reaction media for enzymatic transformations, and research interest in this area has increased widely during recent years.1 They are salts and therefore are entirely composed of ions which are liquids below 100 °C or typically close to room temperature. Their interest as green chemicals resides in their high thermal stability and very low vapor pressure, which can be used to mitigate the problem of volatile organic solvent emission in the atmosphere. Moreover, the physical properties of ILs (density, viscosity, melting points, polarity, etc.) can be finely tuned by the appropriate selection of anions and (or) cations.2 ILs can be designed to be miscible or immiscible with water or organic solvents (e.g., hexane, toluene, ether, 1-propanol, etc.), making them more useful for easy recovery of products from the reaction mixture. Spectroscopic studies of ILs suggest that these solvents have a polarity comparable with that of the lower alcohols (e.g., methanol, ethanol, etc.).2c All of these properties, including the fact that they are recyclable, make ILs potentially ideal solvents for green chemistry. Biphasic systems based on ILs and supercritical fluids for enzyme catalysis have been put forward as the first approach of integral green bioprocesses in nonaqueous media.3 However, several authors described that, although ILs are * To whom correspondence should be addressed. Phone: (+34) 968367398. Fax: (+34) 968364148. E-mail: [email protected]. † Universidad de Murcia. ‡ Universite ´ de Rennes1.

chemicals that can be applied as solvents and/or catalysts in green chemistry processes, they cannot necessarily be considered or described as green solvents, because of their low biodegradability and high (eco)toxicological properties.4 Water-immiscible ILs have been shown to be excellent nonaqueous media for enzyme-catalyzed reactions (especially for lipases), not only because of the high level of activity and stereoselectivity displayed by enzymes in chemical transformations,5 such as ester synthesis, kinetic resolution of sec-alcohols, etc., but also because of their now well identified stabilization effect on the biocatalysts,6 even under extremely harsh conditions (e.g., supercritical carbon dioxide at 100 bar and 150 °C). However, very low enzyme activity and/or fast enzyme deactivation has been observed in the case of pure water-miscible ILs.7 The structure/stability relationship during enzyme deactivation in nonaqueous media and the enhancement of stability must be understood before enzyme technology can be applied in chemical processes. Spectroscopic (i.e., fluorescence, CD, or FTIR) measurements have been widely applied to analyzing changes in enzyme structures in an attempt to explain the stabilization or denaturation phenomena associated with enzyme environments,8 e.g., temperature, organic solvents, etc. The use of spectroscopic methods has recently been described to correlate changes in the secondary structure of monellin,9 Candida antarctica lipase B (CALB),10 or R-chymotrypsin11 with stability in ILs. CALB has been widely used for biotransformations in nonaqueous environments because of its exceptional ability to adapt to nonaqueous media, such as organic solvents12 or ILs.5,6

10.1021/bm049259q CCC: $30.25 © 2005 American Chemical Society Published on Web 03/02/2005

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This paper describes our efforts to understand why waterimmiscible ILs stabilize CALB by looking at structure/ function relationships. This work describes a comparative analysis of the synthetic activity (using the butyl butyrate synthesis from butyl vinyl ester and 1-butanol, as the reaction model) and stability of CALB in hexane and two waterimmiscible ionic liquids, i.e., 1-ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide, [emim][NTf2], and butyltrimethylammonium bis(trifluoromethylsulfonyl)imide, [btma][NTf2], at 2% v/v water content and 50 °C. The ability of these ILs to protect CALB (at the same water content) against enzyme deactivation has previously been demonstrated, even under extremely harsh conditions (e.g., supercritical carbon dioxide at 150 °C and 10 MPa).3 In this paper, it is assumed that the decay in enzyme activity during a deactivation process is directly correlated with simultaneous structural changes in the protein. Thus, the structural changes produced in CALB by incubation in ILs or hexane over 4 days were analyzed by using circular dichroism (CD) and fluorescence techniques. Experimental Section Materials. CALB (EC 3.1.1.3, 10.9 U/mg of solid) was purchased from Fluka. The purity of the enzyme was tested by the SDS-PAGE electrophoresis method described by Palomo et al,13 which showed only one protein band. Substrates, solvents, and other chemical were also purchased from Sigma-Aldrich-Fluka Chemical Co., and were of the highest purity available. Synthesis of Ionic Liquids. Specific procedures to prepare both [btma][NTf2] and [emim][NTf2] were previously described in detail.3d,6a,14 The resulting ILs were colorless, as a waterlike oil, containing 75 and 65 ppm water, respectively, as determined by Karl Fischer measurements. The chloride content of ILs was undetectable by both mass spectrometry and the AgNO3 test. Synthetic Activity of CALB. Vinyl butyrate (30 µL, 236 µmol) and 1-butanol (110 µL, 1.21 mmol) were added to screw-capped vials (1.5 mL total capacity) containing 300 µL of each ionic liquid ([emim][NTf2] and [btma][NTf2]) or hexane. The reaction was started by adding 10 µL of the aqueous solution of CALB (10 mg/mL) and run at 50 °C in an oil bath with shaking. At regular time intervals, 20 µL aliquots were taken and suspended in 1 mL of hexane. The biphasic mixture was strongly shaken for 3 min to extract all the substrates and product into the hexane phase, and then centrifuged at 3500 rpm for 10 min to separate the phases. Then, 400 µL of hexane extract was added to 100 µL of a 30 mM propyl acetate (internal standard) solution in hexane, and 1 µL of the resulting solution was analyzed by GC. In the case of hexane, aliquots dissolved in this solvent were centrifuged to give a clear solution, which was used for GC analysis. A Shimadzu (GC-17A) gas chromatograph equipped with a capillary Nukol column (30m × 0.25 mm × 0.25 µm, Supelco) and a flame ionization detector was used. The conditions were as follows: carrier gas (He) at 64 kPa (83 mL/min total flow); temperature program, 45 °C, 5 °C/min, 105 °C, 20 °C/min, 180 °C; split ratio 115:1; detector 230

De Diego et al.

°C. The retention times of the peaks were as follows: propyl acetate, 5.74 min; vinyl butyrate, 6.79 min; 1-butanol, 9.38 min; butyl butyrate, 11.23 min; butyric acid, 18.88 min. All experiments were carried out in duplicate, providing an SD of less than 0.5%. One unit of synthetic activity was defined as the amount of enzyme that produces 1 µmol of butyl butyrate/min under our assay conditions. Hydrolytic Activity of CALB. The assay was performed by measuring the increase in absorbance at 348 nm produced by the release of p-nitrophenol during the hydrolysis of 0.4 mM p-nitrophenylpropionate (pNPP) in 25 mM sodium phosphate buffer at pH 7 and 25 °C.13 To start the reaction, 20 µL of CALB (0.2 mg/mL) was added to 980 µL of the substrate solution. Experiments were carried out in duplicate, providing an SD of lower than 0.5%. One unit of hydrolytic activity was defined as the amount of enzyme that is necessary to hydrolyze 1 µmol of pNPP/min under the assay conditions. Stability of CALB. To 300 µL of each nonaqueous medium (hexane, [emim][NTf2], or [btma][NTf2]) was added 10 µL of an aqueous solution of CALB (10 mg/mL) in screwcapped vials (1.5 mL total capacity). The resulting solutions were mixed for 2 min and then incubated at 50 °C. At selected times, substrates (236 mmol of vinyl butyrate and 1.21 mmol of 1-butanol) were added to each vial, and the synthetic reaction was followed as described above. In the case of water, 10 µL of an aqueous solution of CALB (10 mg/mL) was added to 440 µL of water, and then the resulting solution was incubated at 50 °C in an oil bath. At regular intervals, 20 µL aliquots were used to measure the hydrolytic activity, as described above. Circular Dichroism Spectroscopy. CD spectra were obtained using a PiStar-180 spectrophotometer (Applied Photophysics, U.K.) equipped with a N2 purge and a Peltier system for temperature control. CD calibration was performed using (1S)-(+)-10-camphorsulfonic acid, which exhibits a 34.5 M/cm molar extinction coefficient at 285 nm, and a 2.36 M/cm molar ellipticity at 295 nm. The assayed protein concentration for both ILs was 0.22 mg/mL, because of the resulting fully clear and homogeneous solutions. Before the measurements, samples were preequilibrated at the desired temperature for 5 min. Spectra were carried out at a 20 nm/min scan speed with a response time of 1 s, and 2 nm bandwidth. For far-UV (190-240 nm) spectra, 0.1 cm cells were used, while in the case of near-UV (250-300 nm) spectra, measurements were made with 1.0 cm cells. Four spectra were acquired and averaged for each sample to eliminate signal noise. For each solvent, it was necessary to subtract a blank medium without enzyme to discard its influence on the enzyme CD spectrum. The mean molar residue ellipticity [θ]MRW was expressed in deg cm2 dmol-1, using 33000 as the molecular weight and 317 residues for CALB, and was calculated as follows: [θ]MRW ) ([θ]obs × 104.1 × 0.1)/(lc) where [θ]obs is the observed ellipticity (deg), 104.1 the mean residue molecular weight of CALB, l the optical path length (cm), and c the protein concentration (g/mL).

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Lipase in Ionic Liquids Table 1. Synthetic Activity and Selectivity of CALB for Butyl Butyrate Synthesis in Different Mediaa medium

synthetic activity (U/mg of protein)

selectivity (%)

hexane [emim][NTf2] [btma][NTf2]

89.7 ( 0.4 333.6 ( 1.2 182.6 ( 0.8

95 97 96

a Reaction conditions: 0.52 M vinyl butyrate, 2.68 M 1-butanol, 2% by volume water content and 50 °C.

The percentages of secondary structures were calculated by using an online CD, DICHROWEB (http:// www.cryst.bbk.ac.uk/cdweb/html/), which provides several curve-analyzing algorithms (e.g., CONTIN/LL, SELCON3, CDSSTR, etc.).15 Analysis of CD spectra is characterized by mean square root deviation errors (δ) and correlation coefficients (r) between the X-ray and CD estimates of secondary structure fractions. The best predicted secondary structures are given by the smallest δ and largest r; e.g., for the CONTIN/LL method, the accepted overall performance is provided by δ ) 0.075-0.069 and r ) 0.784-0.817.16 The CDSSTR method was selected to determine the secondary structure (R-helix and β-sheet) because it provided the best agreement (δ < 0.046 and r ) 0.836) between experimental and X-ray17 (obtained from the Protein Data Bank (PDB), http://www.rcsb.org/pdb/) spectra for the native structure of CALB (R/β globular protein) in water at 30 °C. Fluorescence Spectroscopy. Intrinsic fluorescence emission spectra of CALB were monitored using a PiStar-180 spectrophotometer (Applied Photophysics, U.K.), and a scanning emission monochromator (SK.2E) controlled from the PiStar software. Enzyme samples were incubated in the cell for 5 min at 50 °C before being excited at 295 nm, and emission was registered from 300 to 400 nm, using a 5 nm bandwidth in both the excitation and emission paths. The final CALB concentration in all the media was 0.22 mg/ mL. The spectrofluorometer automatically provided corrected spectra by comparison with a 1 nM standard solution of rhodamine B in glycerol, to avoid changes in lamp output and instrument geometry. The maximum emission wavelength of the sample was determined as the wavelength for which dI/dλ ) 0. In all cases, it was necessary to subtract a blank medium without enzyme at different times, to discount the influence of the solvent fluorescence on the enzyme fluorescence spectrum. Results and Discussion Activity and Stability of CALB in ILs. Table 1 shows the initial synthetic activity and selectivity exhibited by CALB for butyl butyrate synthesis from butyl vinyl ester and 1-butanol in two ILs, [emim][NTf2] and [btma][NTf2], at 2% by volume water content and 50 °C. The synthetic activity was also carried out in a classical molecular organic solvent, hexane, for comparison. The initial synthetic rate in both ionic liquids was higher than that observed in hexane, demonstrating the suitability of both ILs for the proposed biotransformation in nonaqueous environments. In all cases, the selectivity parameter, defined as the ratio between the synthetic and the acyl-donor consumption rates, was close

Figure 1. Deactivation profiles of CALB in [btma][NTf2] (b), [emim][NTf2] (9), hexane (2), and water ([) at 50 °C.

to 100% as a consequence of the low water content in the reaction media. These results are in agreement with our previous reports,3c,5f,6b where the suitability of ILs based on bistriflimide anion to synthesize esters or to kinetically resolve sec-alcohols (both catalyzed by CALB) was demonstrated. In this work, the synthetic activity exhibited by this purified enzyme preparation was slightly lower than that previously observed for the industrial raw enzyme preparation (i.e., Novozym 525L).5c Furthermore, the stability of CALB was studied by incubation of an aqueous solution of the enzyme in either hexane or ILs at 50 °C, measuring the residual synthetic activity (see Figure 1). These results were also compared with those obtained in water medium, using the hydrolysis of p-nitrophenyl propionate as an enzyme activity test. As can be seen, CALB exhibits a classical continuous loss of activity in molecular solvents, the decrease being faster in water (t1/2 ) 5.3 h) than in hexane (t1/2 ) 10.2 h). Along these lines, Salis et al. described CALB as an atypical lipase better adapted to organic media than typical lipases (e.g.. Thermomyces lanuginose lipase) with large lid regions protecting the active center, which must be opened to become active.12b The obtained results might be explained by the conformational changes of the enzyme which occur during the deactivation process as a consequence of thermal unfolding and/or enzyme dehydration by the organic solvent.8 Nevertheless, the stability of the CALB in both [emim][NTf2] and [btma][NTf2] was much greater than in either molecular solvent (t1/2 )170 and 207 days, respectively), being in agreement with previous results where the residual activity was assayed for 50 days.4c,6b The half-life of the enzyme for each IL was determined by using a two-step kinetic model previously described in detail.8a,11 In fact, after the initial activity decay observed during the first 5 h, the synthetic activity of the enzyme remained constant for one week. These results show the excellent ability of these ILs to maintain an active conformation of the enzyme, and to provide suitable microenvironments.8-12 The catalytic behavior of enzymes in ILs has usually been explained by the specific solvent properties of these molten salts and their interactions with proteins.1,5-7 Several physical and chemical characteristics (e.g., thermal properties, conductivity, electrochemical windows, density, viscosity, polarity, etc.) of ILs have been described as a function of ion type.2,14,18 However, for lipase-catalyzed synthetic reactions in nonaqueous environments, the polarity of the medium,

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together with the miscibility of the medium with water, could be considered as the key parameter which may most influence the active conformation of the enzyme. The miscibility of ILs with water is particularly interesting. Although all ILs are hygroscopic, some of them are totally miscible with water, whereas others only saturate at low water content (e.g., the water content at saturation of [emim][NTf2] is 1.8% by weight at 20 °C14,19). On the other hand, the polarity of different ILs has been widely studied by using solvatochromic empirical scales, such as the Reichardt ET(30) parameter, or by measurements of 1-octanol-water partition coefficients (log P parameter).18 In all cases, the ILs, including water-immiscible ILs, have been described as solvents with polarities comparable to those of the lower alcohols (i.e., methanol, ethanol, etc.). Indeed, the use of the [NTf2] anion yields particularly water immiscible, low melting point, and hydrophobic ILs.18d In this context, it has been described how water-immiscible ILs form a strong ionic matrix, where “wet” ILs should not be regarded as solvents with homogeneous structures, but as nanostructures with polar and nonpolar regions.2a So, an aqueous solution of free-enzyme molecules added to the IL phase could be considered as being included but not dissolved in the medium, providing a suitable microenvironment for the catalytic action and enhanced enzyme stability.6,11 In agreement with this view, the activity of free CALB-IL systems was not reduced by a liquid-liquid extraction process (e.g., with water, phosphate buffers, or hexane), and it was necessary to ultrafilter the solution through 5000 Da cutoff membranes to separate the enzyme from the IL phase.3c All these results might be explained as a function of changes in the structure of the native enzyme produced by a specific enzyme-solvent (hexane or ILs) interaction or interactions between solvent and essential water molecules surrounding the enzyme macromolecule. CD Studies of CALB Deactivation. CD spectroscopy is an important technique in structural biochemistry for determining the structure of proteins. The technique makes it possible to analyze conformational modifications and especially to quantify secondary structures of proteins from the changes observed in the CD spectrum.8 In the far-UV region (190-240 nm), which corresponds to the peptide bond absorption, the CD spectrum can be analyzed to give the content of regular secondary structural features, such as R-helix and β-sheet. Additionally, the CD spectrum in the near region (260-320 nm) reflects the environments of the aromatic amino acid side chains and thus gives information about the tertiary structure of the protein.15 Figure 2 depicts the far-UV spectra of CALB in all the assayed media (water, hexane, [emim][NTf2], and [btma] [NTf2]) after different incubation times (0, 2, and 4 days) at 50 °C. To compare the results, Figure 2A also includes the spectrum of CALB in water at 30 °C, which could be considered as the native form of the enzyme, as well as the spectrum for the unfolded protein conformation obtained by incubation of the enzyme in 8 M urea at 50 °C. As can be seen from Figure 2, the far-UV CD spectra of the enzyme differ according to the assayed medium and the incubation time, indicating that the protein has a particular distribution of the secondary structure

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Figure 2. Far-UV CD spectra of CALB in water (A), hexane (B), [emim][NTf2] (C), and [btma][NTf2] (D) at different incubation times and 50 °C (t ) 0, bold line; t ) 2 days, dotted line; t ) 4 days, dashed line). (A) Native at 30 °C, thin line; unfolded in 8 M urea at 50 °C, dotted-dashed line.

in each condition, except in the case of water, where the initial spectra at 30 and 50 °C were similar. In both cases, the CD spectrum of CALB in water has a characteristic minimum at 208 nm and a positive band at a wavelength lower than 203 nm. For initial incubation times, this minimum remained constant for [emim][NTf2], but redshifted to 212 nm for [btma][NTf2]. Several methods can be used for determining the secondary structural elements of a protein from its CD spectrum in the amide regions. We selected the CDSSTR method because it provided the best agreement between experimental and theoretical spectra (see the Experimental Section).15,17 This method is a modification of the original variable selection of proteins version, named VARSELEC,17b but can be applied with no restriction on the number of calculations, and using a reference set of 43 proteins. Table 2 shows the percentages of different secondary structure elements of CALB obtained by CDSSTR for each assayed condition. As can be seen, the secondary structure distribution of native CALB (determined from an aqueous solution at 30 °C) was maintained at 50 °C, the estimated R-helix content (36%) agreeing with the reported X-ray crystallographic data.16 CALB was reported to be an R/β-hydrolase-like fold enzyme (Mw ) 33000) in an open conformation with an accessible active site. It does not have a typical lid domain, but a short helix with high mobility which might act as a lid.16a On the other hand, the analysis of the CD spectrum of CALB in the presence of 8 M urea at 50 °C (see Figure 2A) shows a fall in both R-helix and β-strand secondary structures, which would agree with an unfolded state of the protein, which was also inactive. In this context, it is reasonable to expect that more than one type of interaction must be broken before full enzyme deactivation occurs, followed by different conformational changes of the native structure. In the case of water medium (see Figure 2A), the changes in the farUV CD spectra of CALB at 50 °C were related to the continuous decrease in both R-helix and β-strand secondary structures and the concomitant increase in the unordered fraction, which was proportional to the incubation time (see Table 2). Furthermore, this loss of native enzyme conforma-

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Lipase in Ionic Liquids Table 2. Secondary Structure Percentages of CALB in Different Media and at Different Times at 50 °C Calculated by the CDSSTR Method time

R-helix β-strand turns unordered

total

medium

(days)

(%)

(%)

(%)

(%)

(%)

δa

water, 30 °C 8 M urea water

0 0 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4

36 1.7 36 13.7 9.8 9.3 9.0 11 10 6 1.2 0.8 32 21 21 14 6 24 23 12 11 10

22 7.3 22 27.6 32.1 10.9 3.8 35 33 35 32 36 26 27 27 31 41 33 33 40 42 41

17 22 17 27.0 20.4 31.6 41.0 28 15 23 17 20 23 25 25 28 25 16 16 17 17 15

25 69 25 31.6 37.6 48.2 46.2 26 43 36 38 35 20 28 28 26 29 27 28 31 30 33

100 100 100 99.9 99.9 100 100 100 99 100 99 99 101 101 101 99 101 99 99 100 100 99

0.005 0.021 0.013 0.013 0.012 0.019 0.019 0.015 0.015 0.018 0.016 0.015 0.003 0.019 0.019 0.046 0.028 0.006 0.005 0.006 0.004 0.004

hexane

[emim][NTf2]

[btma][NTf2]

a

Mean square root deviation error.

tion is clearly correlated with the decay in activity depicted in Figure 1. However, in the case of hexane, where CALB showed a deactivation profile similar to that observed in water, the perturbations on the secondary structure of the enzyme resulted in an initial increase in β-strand and turns with respect to the native state, and a progressive decrease of R-helix. These results could be explained as a consequence of the loss of essential water molecules from the protein microenvironment to the bulk solvent, enhancing the enzyme rigidity.12a Madeira-Lau et al. also described the poor activity of CALB in several water-miscible ILs (i.e., 1-butyl-3methylimidazolium nitrate, 1-butyl-3-methylimidazolium lactate, etc.), the observed deactivation being related to a loss of R-helix and β-strand from its native conformation, as determined by FTIR.10 The ability of both water-immiscible ILs (i.e., [emim][NTf2] and [btma][NTf2]) to preserve the catalytic activity of the enzyme would reflect the maintenance of the secondary structure elements of native CALB. It is noticeable how the initial distribution of the secondary structure of the enzyme in both ILs is very similar to that of the native structure. In the same way, the loss of R-helix content with incubation time is clearly less in ILs than in both water and hexane. However, a marked increase in the β-strand content of CALB with time was also observed for both ILs. These results show how the native/active enzyme conformation is protected by ILs against deactivation, as can be seen from comparison with Figure 1. The observed increase in β-sheet could be attributed to a loss of hydrogen-bonding interactions between the water molecules and R-helices. This seems to be similar to the effect of lyophilization on the structure of lipase, where the decrease in the amount of water molecules around the protein results in an enhancement in β-strand.12a

In this way, the low solubility of water in the assayed ILs could be involved in the preservation of critical water molecules in the microenvironment of the enzyme.6 However, it is also possible that excess water molecules were partitioned between the microenvironment (around the enzyme molecule) and macroenvironment (bulk solvent), increasing the extent to which water molecules are organized around the protein. Indeed, Cammarata et al. described how water molecules dissolved in [emim][NTf2] are “free” water molecules, interacting via H-bonds with the anions in a symmetric complex.19 Water-immiscible ILs form a strong ionic matrix which retains CALB molecules in an adequate microenvironment, resulting in a supramolecular net able to keep the protein conformation active. These ILs should be regarded as being an immobilization support, rather than a reaction medium. These results are in agreement with previous stability and conformational studies of R-chymotrypsin in [emim][NTf2], where the IL was able to stabilize the enzyme through the formation of a flexible and more compact three-dimensional structure, which could be related to the preservation of the essential water shell.11 The observed evolution of R-helix to β-sheet in CALB in the assayed water-immiscible ILs to reach another active and stable enzyme conformation might be explained as a function of both the amino acid sequence and the solvent accessibility of each amino acid residue. This latter possibility was confirmed from the amino acid sequence of native CALB (obtained from the PDB) using the WHAT IF 5.0 software (http://swift.cmbi.kun.nl/WIWWWI/), based on a FORTRAN 77 computer program for macromolecular modeling.20 Figure 3 shows the primary structure of CALB obtained from the PDB, as well as the secondary structure determined for every residue and its position in the 3-D structure, which permits (or not) solvent accessibility. As can be seen, a large number of residues involved in R-helices are exposed to the outer surface (e.g., positions 12-18, 267-287, etc.), and more of them, such as V, L, A, etc., usually appear in β-strand with a higher frequency than in R-helix for globular proteins, such as CALB. The near-UV CD spectra (250-300 nm) were also monitored to obtain information about changes in the tertiary structure of the enzyme after incubation for 24 h in all the assayed media at 50 °C (Figure 4). This spectral region is dominated by the contribution of aromatic side chains (e.g., Trp, Tyr, or Phe) and disulfide bridges, while the intensity of the 250-300 nm band is affected by local conformational changes around these chromatophores. Since CALB is a monomeric protein that contains five Trp, nine Tyr, and ten Phe residues and four disulfide bridges,16b interpretation of the spectra is difficult. Nevertheless, it is known that the intensity of the near-UV band increases when the aromatic residues come into closer contact with each other.8d Then, as can be seen, the less intense CD signal in water and hexane, as compared to those measured in ILs, reflects the less compact structure of the enzyme in both molecular solvents. Furthermore, the molar mean residue ellipticity at 290 nm is characteristic of Trp residues, and Figure 4 shows the higher ellipticity observed in the assayed waterimmiscible ILs after 24 h of incubation than that observed

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Figure 3. Amino acid sequence of native C. antarctica lipase B (Protein Data Bank), listed with the secondary structure (H, R-helix; 3, helix 310; S, β-sheet, T, turn) and the solvent accessibility of each residue (A) calculated by using the software WHAT IF 5.0.20a External R-helix residues are underlined.

Figure 4. Near-UV CD spectra of CALB in water (1), hexane (2), [emim][NTf2] (3), and [btma][NTf2] (4) after 24 h of incubation at 50 °C.

in water or hexane is clearly involved with a folded and active enzyme conformation (see Figure 1). These results agree with previous studies, suggesting that the stability of enzyme in these ionic liquids was clearly improved by the evolution of the native enzyme conformation to a compact and flexible nativelike conformation able to show a high level of catalytic activity.6,11 Intrinsic Fluorescence Analysis of CALB. Intrinsic fluorescence analysis was used to study the protein unfolding process in each of the assayed media, and to understand conformational transitions that affect the tertiary structure of the protein. For proteins that contain fluorophore residues (e.g., Trp, Tyr, or Phe), such as CALB, the denaturation process can be followed by both changes in the maximal intensity of fluorescence (Imax) and the red shift of the maximal emission wavelength (λmax).8a According to the selective excitation of Trp residues at 295 nm, both fluo-

Figure 5. Fluorescence spectra of CALB in water (1), hexane (2), [emim][NTf2] (3), and [btma][NTf2] (4) obtained initially (A) and after 4 days of incubation at 50 °C (B).

rescence parameters can be related to changes in the polarity of the microenvironment of these residues in the protein globule. Figure 5A shows the initial fluorescence spectra of CALB in all the assayed media (water, hexane, [emim][NTf2], and [btma][NTf2]) at 50 °C, while Figure 5B depicts the spectra exhibited by the enzyme after 4 days in the same conditions. The maximum intensity for all the spectra has been normalized with respect to the initial spectrum obtained for the enzyme in water at 50 °C. As can be seen, the enzyme exhibits an initial λmax of 322 nm in water, which is redshifted in the other media to 337 nm for both ILs and 343 nm in the case of hexane. After incubation for 4 days, it is noticeable how the fluorescence spectra of the enzyme were clearly modified compared with the initial spectra, especially with regard to the enhancement of the Imax parameter of the enzyme in ILs, which is in contrast with the decrease of this parameter in both molecular solvents (water and hexane). Additionally, Figure 6 shows the evolution of both Imax and

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fluorescence studies, these authors also correlated this thermal stabilization with a more compact protein conformation. Also, Turner et al. described how the deactivation of the enzyme cellulase produced by water-miscible ILs (e.g., 1-butyl-3-methylimidazolium chloride) is accompanied by a fall in the Imax of the Trp parameter with respect to the native conformation in water.7b The conformational changes of CALB produced by both [emim][NTf2] and [btma][NTf2] ionic liquids, which were observed by both fluorescence and CD studies, could be attributed to a compact, flexible, and active enzyme conformation with high levels of activity and stability. Figure 6. Evolution of the fluorescence emission intensity (b) and maximal emission wavelength (9) of CALB with time in water (A), hexane (B), [emim][NTf2] (C), and [btma][NTf2] (D) at 50 °C.

λmax fluorescence parameters of CALB with incubation time in all the assayed media at 50 °C. The Imax has been normalized with respect to the initial value in each medium. As can be seen, the continuous decrease in Imax and the red shift of λmax observed in water (Figure 6A) correspond to a classical unfolding process of globular proteins and, in our case, are accompanied by an activity loss with a similar profile (see Figure 1). In fact, it is necessary to take into account that under denaturing conditions (such as 8 M urea) the absolute value of Imax was reduced 3-fold with respect to that of the native enzyme, while the λmax was increased from 322 to 347 nm, as a result of the enhancing exposure of Trp residues to the bulk solvent (e.g., the λmax of free Trp in aqueous solution is 350 nm). In the case of hexane, the unfolding process of CALB occurs in the same way, but to a lower extent than that in water, because the initial enzyme conformation in hexane was greatly modified with respect to the native conformation in water (see Table 2). Thus, the evolution of both the Imax and λmax fluorescence parameters of CALB with incubation time in hexane might also be related to the profile of enzyme deactivation (see Figure 1). However, as can be seen for Figure 6C,D, the fluorescence parameters of CALB in both [btma][NTf2] and [emim][NTf2] ionic liquids were similar because of the continuous enhancement of the Imax, which involves the reduced exposure of Trp residues to the bulk solvent, resulting in a refolded protein conformation with a high level of activity and stability (see Table 1 and Figure 1). The slight red shift observed in λmax could be related to the different positions of the Trp residues in the 3-D structure of the native enzyme. CALB has three Trp residues (positions 52, 113, and 155) located in three different R-helices, while the other two residues (positions 65 and 104) are placed in two different β-sheets.16 Thus, the evolution of R-helix to a more open secondary structure, such as β-sheet, in ILs could involve an increase in exposure of these three Trp residues, which could explain the red shift. In the same context, Baker et al. described how the incubation of a protein, i.e., monellin, in the water-immiscible IL 1-butyl-3-methylpyrrolidinium bis(trifluoromethanesulfonyl)imide at 2% by volume water content provided the protein with a considerable thermodynamic stability, the melting temperature shifting upward to over 100 °C in IL compared with 40 °C in water.9 In

Conclusions The stability of CALB in two water-immiscible ionic liquids ([btma][NTf2] and [emim][NTf2]) at 2% by volume water content and 50 °C was compared with the stability observed in water and hexane. The synthetic activity and stability exhibited by CALB in ILs was much higher than that observed in hexane, and was related to the associated conformational changes that take place in the native structure of CALB as demonstrated by fluorescence and CD spectroscopic techniques. The stabilization of CALB by ILs seems to be related to the observed evolution of R-helix to β-sheet secondary structures of the enzyme, resulting in a more compact enzyme conformation able to exhibit catalytic activity. These results improve knowledge of the excellent properties of water-immiscible ionic liquids as green alternatives to organic solvents, since both are able to stabilize enzymes and are suitable as reaction media for enzymatic biotransformations of industrial interest. Acknowledgment. This work was partially supported by CICYT (PPQ2002-03549) and SENECA Foundation (PB/ 75/FS/02) grants. We thank Prof B. A. Wallace, Department of Crystallography, Birkbeck College, University of London, for his kind assistance in the analysis of CD spectra. References and Notes (1) (a) Kragl, U.; Eckstein, M.; Kaftzik, N.Curr. Opin. Biotechnol. 2002, 13, 565. (b) Park, S.; Kazlauskas, R. J. Curr. Opin. Biotechnol. 2003, 14, 432. (c) van Rantwijk, F.; Madeira-Lau, R.; Sheldon, R. A. Trends Biotechnol. 2003, 21, 131. (d) Song, C. E. Chem. Commun. 2004, 1033. (2) (a) Dupont, J.; de Souza, R. F.; Suarez, P. A. Z. Chem. ReV. 2002, 102, 3667. (b) Wassercheid, P., Welton, T., Eds. Ionic Liquids in Synthesis; Wiley-VCH: Weinheim, Germany, 2003. (c) Poole, C. F. J. Chromatrogr., A 2004, 1037, 49. (3) (a) Lozano, P.; de Diego, T.; Carrie´, D.; Vaultier, M.; Iborra, J. L. Chem. Commum. 2002, 692. (b) Dzyuba, S. V.; Bartsch, R. A. Angew. Chem., Int. Ed. 2003, 42, 148. (c) Lozano, P.; de Diego, T.; Carrie´, D.; Vaultier, M.; Iborra, J. L. Biotechnol. Prog. 2003, 19, 380. (d) Lozano, P.; de Diego, T.; Gmouh, S.; Vaultier, M.; Iborra, J. L. Biotechnol. Prog. 2004, 20, 661. (4) (a) Jastorff, B.; Sto¨rmann, R.; Ranke, J.; Mo¨lter, K.; Stock, F.; Oberheitmann, B.; Hoffmann, W.; Hoffmann, J.; Nu¨chter, M.; Ondruschka, B.; Filser, J. Green Chem. 2003, 5, 136. (b) Swatloski, R. P.; Holbrey, J. D.; Rogers, R. D. Green Chem. 2003, 5, 361. (c) Gathergood, N.; Garcia, M. T.; Scammells, P. J. Green Chem. 2004, 6, 166. (d) Garcia, M. T.; Gathergood, N.; Scammells, P. J. Green Chem. 2005, 7, 9. (5) (a) Sho¨fer, S. H.; Kaftzik, N.; Wasserscheid, P.; Kragl, U. Chem. Commun. 2001, 425. (b) Kim, K. W.; Song, B.; Choi, M. Y.; Kim, M. J. Org. Lett. 2001, 3, 1507. (c) Lozano, P.; de Diego, T., Carrie´,

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