Imidazolium Bromide-Based Ionic Liquid Assisted Improved Activity of

Feb 9, 2010 - the biocatalyst, as they tend to get localized toward the interfacial region of ... experimentation was carried out in the presence of I...
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Imidazolium Bromide-Based Ionic Liquid Assisted Improved Activity of Trypsin in Cationic Reverse Micelles Sisir Debnath, Dibyendu Das, Sounak Dutta, and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the Cultivation of Science Jadavpur, Kolkata - 700 032, India Received March 9, 2009. Revised Manuscript Received January 18, 2010 The present work reports the imidazolium-based ionic liquids (ILs) assisted enhancement in activity of water-pool solubilized enzyme trypsin in cationic reverse micelles of CTAB. A set of imidazolium ILs (1-alkyl-3-methyl imidazolium bromides) were prepared with varying lengths of their side arm which results in the differential location of these organic salts in the reverse micelles. The different ILs offered varied activating effects on the biocatalyst. The activity of trypsin improved ∼30-300% in the presence of 0.1-10 mM of different ILs in reverse micelles of CTAB. Trypsin showed ∼300% (4-fold) increment in its activity in the presence of IL 2 (1-ethyl-3-methyl imidazolium bromide, EMIMBr) compared to that observed in the absence of IL in CTAB reverse micelles. The imidazolium moiety of the IL, resembling the histidine amino acid component of the catalytic triad of hydrolases and its Br- counterion, presumably increases the nucleophilicity of water in the vicinity of the enzyme by forming a hydrogen bond that facilitates the enzyme-catalyzed hydrolysis of the ester. However, the ILs with increasing amphiphilic character had little to no effect on the activity of trypsin due to their increased distance from the biocatalyst, as they tend to get localized toward the interfacial region of the aggregates. Dynamic light scattering experimentation was carried out in the presence of ILs to find a possible correlation between the trypsin activity and the size of the aggregates. In concurrence with the observed highest activity in the presence of IL 2, the circular dichroism (CD) spectrum of trypsin in CTAB reverse micelles doped with IL 2 exhibited the lowest mean residue ellipticity (MRE), which is closest to that of the native protein in aqueous buffer.

Introduction Enzymology in organized assemblies has been an area of wide research interest for the past few decades because of its potential in biotechnological applications.1-5 Studies on structure and activity of enzymes particularly in confined media have always shown considerable significance from basic research to technological *To whom correspondence should be addressed. E-mail: [email protected]. (1) (a) Eicke, H. F.; Shepherd, T. C.; Steinmann, A. J. Colloid Interface Sci. 1976, 56, 168. (b) Zana, R.; Lang, J. In Solution Behavior of Surfactants, Mittal, K. L., Fendler, E. J., Eds.; Plenum: New York, 1982; Vol. 2. (c) Fendler, J. H. Acc. Chem. Res. 1976, 9, 153. (d) Silber, J. J.; Biasutti, M. A.; Abuin, E. B.; Lissi, E. Adv. Colloid Interface Sci. 1999, 82, 189. (e) Biasutti, M. A.; Abuin, E. B.; Silber, J. J.; Correa, N. M.; Lissi, E. A. Adv. Colloid Interface Sci. 2008, 136, 1. (f) Stenius, P. Reverse Micelles, Luisi, P. L., Straub, B. E., Eds.; Plenum: New York, 1984; p 1. (g) Shinshi, M.; Sugihara, T.; Osakai, T.; Goto, M. Langmuir 2006, 22, 5937. (h) Kimura, M.; Michizoe, J.; Oakazaki, S.; Furusaki, S.; Goto, M.; Tanaka, H.; Wariishi, H. Biotechnol. Bioeng. 2004, 88, 495. (2) (a) Holmberg, K. Adv. Colloid Interface Sci. 1994, 51, 137. (b) Paul, B. K.; Moulik, S. P. J. Dispersion Sci. Technol. 1997, 18, 301. (c) Bommarius, A. S.; Hatton, T. A.; Wang, D. I. C. J. Am. Chem. Soc. 1995, 117, 4515. (d) Maruyama, T.; Hosogi, T.; Goto, M. Chem. Commun. 2007, 4450. (e) Moniruzzaman, M.; Kamiya, N.; Nakashima, K.; Goto, M. ChemPhysChem 2008, 9, 689. (f) Biswas, R.; Das, A. R.; Pradhan, T.; Touraud, D.; Kunz, W.; Mahiuddin, S. J. Phys. Chem. B 2008, 112, 6620. (g) Stamatis, H.; Xenakis, A.; Kolisis, F. N. Biotechnol. Adv. 1999, 17, 293 and references therein. (h) Hagen, A. J.; Hatton, T. A.; Wang, D. I. C. Biotechnol. Bioeng. 2006, 95, 285. (3) (a) Azevedo, A. M.; Fonseca, L. P.; Graham, D.; Cabral, J. M. S.; Prazeres, D. M. F. Biocatal. Biotransfor. 2001, 19, 213. (b) Holmes, J. D.; Steytler, D. C.; Rees, G. D.; Robinson, B. H. Langmuir 1998, 14, 6371. (c) Oldfield, C.; Freedman, R. B.; Robinson, B. H. Faraday Discuss. 2005, 129, 247. (d) Komives, C. F.; Osborne, D. E.; Russell, A. J. J. Phys. Chem. 1994, 98, 369. (e) Menger, F. M. Angew. Chem., Int. Ed. Engl. 1991, 30, 1086. (f) Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1990, 94, 6411. (g) Carlile, K.; Rees, G. D.; Robinson, B. H.; Steer, T. D.; Svensson, M. J. Chem. Soc., Faraday Trans. 1996, 92, 4701. (h) Walde, P.; Han, D.; Luisi, P. L. Biochemistry 1993, 32, 4029. (i) Yang, F. X.; Russell, A. J. Biotechnol. Bioeng. 1995, 47, 60. (j) Martinek, K.; Levashov, A. V.; Khmelnitsky, Y. L.; Klyachko, N. L.; Berezin, I. V. Science 1982, 218, 889. (k) Menger, F. M.; Yamada, K. J. Am. Chem. Soc. 1979, 101, 6731. (l) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Khmelnitski, Y. L.; Berezin, I. V. Eur. J. Biochem. 1986, 155, 453. (m) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Kabanov, A. V.; Khmelnitsky, Y. L. Biochim. Biophys. Acta 1989, 981, 161. (n) Yamada, Y.; Kuboi, R.; Komasawa, I. Biotechnol. Prog. 1993, 9, 468.

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applications. Among these confined systems, water-in-oil (w/o) microemulsions (also known as reverse micelles) are very attractive hosts for enzyme-catalyzed reactions, because they can solubilize both hydrophobic and hydrophilic substrates/reactants.3-5 W/o microemulsions are optically transparent, macroscopically isotropic, thermodynamically stable nanometer scale aggregates of water and surfactant in apolar solvent. In our previous studies, we have shown particularly in the case of surface-active enzymes, lipase and horseradish peroxidase (HRP), how the efficiency of these enzymes was progressively improved in cationic w/o microemulsions by changing the microstructural parameters of the selforganized aggregates including headgroup hydrophilicity, size, geometry, nature of counterion, and hydrophobic tail length of the surfactant and also by addition of nonionic surfactant.6 Remarkably, the cationic w/o microemulsion also served as a (4) (a) Lissi, E. A.; Abuin, E. B. Langmuir 2000, 16, 10084. (b) Luthi, P.; Luisi, P. L. J. Am. Chem. Soc. 1984, 106, 7285. (c) Abuin, E.; Lissi, E; Duarte, R. Langmuir 2003, 19, 5374. (d) Ono, T.; Goto, M. Biochem. Eng. J. 2006, 28, 156. (e) Mahiuddin, S.; Renoncourt, A.; Bauduin, P.; Touraud, D.; Kunz, W. Langmuir 2005, 21, 5259. (f) Pinna, M. C.; Bauduin, P.; Touraud, D.; Monduzzi, M.; Ninham, B. W.; Kunz, W. J. Phys. Chem. B 2005, 109, 16511. (5) (a) Abuin, E; Lissi, E.; Calderon, C. J. Colloid Interface Sci. 2007, 308, 573. (b) Falcone, R. D.; Biasutti, M. A.; Correa, N. M.; Silber, J. J.; Lissi, E.; Abuin, E. Langmuir 2004, 20, 5732. (c) Brissos, V.; Melo, E. P.; Martinho, J. M. G.; Cabral, J. M. S. Biochim. Biophys. Acta 2008, 1784, 1326. (d) Carvalho, C. M. L.; Cabral, J. M. S. Biochimie 2000, 82, 1063. (e) Mitra, D.; Chakraborty, I.; Bhattacharya, S. C.; Moulik, S. P.; Roy, S.; Das, D.; Das, P. K. J. Phys. Chem. B 2006, 110, 11314. (f) Carvalho, C. M. L.; Aires-Barros, M. R.; Cabral, J. M. S. Langmuir 2000, 16, 3082. (g) Bauduin, P.; Touraud, D.; Kunz, W.; Savelli, M. P.; Pulvin, S.; Ninham, B. W. J. Colloid Interface Sci. 2005, 292, 244. (h) Minofar, B.; Vacha, R.; Wahab, A.; Mahiuddin, S.; Kunz, W.; Jungwirth, P. J. Phys. Chem. B 2006, 110, 15939. (6) (a) Mitra, R. N.; Dasgupta, A.; Das, D.; Roy, S.; Debnath, S.; Das, P. K. Langmuir 2005, 21, 12115. (b) Dasgupta, A.; Das, D.; Mitra, R. N.; Das, P. K. J. Colloid Interface Sci. 2005, 289, 566. (c) Das, D.; Roy, S.; Mitra, R. N.; Dasgupta, A.; Das, P. K. Chem.;Eur. J. 2005, 11, 4881. (d) Debnath, S.; Dasgupta, A.; Mitra, R. N.; Das, P. K. Langmuir 2006, 22, 8732. (e) Dasgupta, A.; Das, D.; Das, P. K. Langmuir 2007, 23, 4137. (f) Das, D.; Das, P. K. Langmuir 2003, 19, 9114. (g) Shome, A.; Roy, S.; Das, P. K. Langmuir 2007, 23, 4130. (h) Debnath, S.; Das, D.; Das, P. K. Biochem. Biophys. Res. Commun. 2007, 356, 163.

Published on Web 02/09/2010

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Chart 1. Chemical Structure of Ionic Liquids

proficient host to exhibit superior activity of structurally deprived enzyme-carbon nanotube hybrids.7 Although several measures were taken to boost the efficiencies of surface-active enzymes in reverse micelles,6 attempts to improve the activity of water-pool solubilized hydrophilic enzyme like trypsin have been really scarce. Very few attempts have been made to either correlate or enhance the activity of trypsin by increasing the local molar concentration of water and the size of the waterpool with an increase in W0 (mole ratio of water to surfactant) and by varying the headgroup size of the surfactant.8 Recently, we found that the efficiency of lipase and HRP was enhanced by almost 100-200% in the mixed reverse micelles of cetyltrimethylammonium bromide (CTAB) and imidazolium-based surfactants that are popularly known as “surfactant ionic liquids (ILs)”.9 The observed enhancement in the enzyme activity with the increasing amphiphilic character (alkyl chain length) of surfactant ILs is presumably due to the activating effect of the IL by improving the nucleophilicity of H2O and H2O2 in the vicinity of the enzyme through hydrogen bonding. It is well-known that the proteolytic enzyme trypsin with its hydrophilic character would localize itself deep inside the waterpool of reverse micelles.3k To establish the activating effect of the ILs, it is necessary to investigate the trypsin activity in the presence of imidazolium-salt-based ionic liquids (ILs) with high water solubility. The effect of such imidazolium-based ILs on the activity of trypsin has not yet been explored in the w/o microemulsion, and moreover, trypsin activity has never been tested in any IL-in-oil microemulsion. In the present work, we made an attempt to enhance the activity of trypsin in the CTAB reverse micelles using varying concentrations of water-soluble ILs (1-alkyl-3-methyl imidazolium bromides, 1-4, Chart 1) with different alkyl chain length from C-1 to C-4. Maximum activation in the efficiency of trypsin was found when concentrations of 1 and 2 were around 0.5-1 mM. Trypsin showed ∼300% (4-fold) activation in the presence of IL 2 (1-ethyl-3-methyl imidazolium bromide, EMIMBr) compared to that observed in only CTAB reverse micelle. However, the activation effect of imidazolium bromide was not prominent in the case of ILs 3 (1-propyl-3-methyl imidazolium bromide, PMIMBr) and 4 (1-butyl-3-methyl imidazolium bromide, BMIMBr). Dynamic light scattering experimentation was carried out over the entire W0 range to find out the influence of ILs having varying side chain lengths on the size of the aggregates and consequently on the trypsin activity. Circular dichroism (CD) experiments were carried out to see the change of secondary structure of the enzymes in the microemulsions in the presence of different ILs and its correlation with the trypsin activity.

Experimental Section Materials. Trypsin (EC 3.4.21.4) and N-R-benzyloxycarbonyl-L-lysine-p-nitrophenyl ester hydrochloride were purchased from Sigma, USA, and used as received. Analytical-grade CTAB (7) Das, D.; Das, P. K. Langmuir 2009, 25, 4421. (8) Dasgupta, A.; Das, D.; Das, P. K. Biochimie 2005, 87, 1111. (9) Das, D.; Das, D.; Das, P. K. Biochimie 2008, 90, 820.

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from Spectrochem (India) was recrystallized three times from methanol/ether, and the recrystallized CTAB was without minima in its surface tension plot. HPLC-grade isooctane, n-hexanol, solvents, and all other reagents used in the syntheses were obtained from SRL (India) and were of the highest analytical grade. Amberlyst A-26 bromide ion-exchange resin from Lancaster was used to convert the iodide salts to their corresponding bromide forms. 1H NMR spectra were recorded on AVANCE 300 MHz (BRUKER) spectrometer. Chemical shifts are reported in ppm, using TMS for 1H NMR as internal standard. Mass spectrometric (MS) data were acquired by electrospray ionization (ESI) techniques in Q-TOF Micro-Quadruple Mass Spectrometer, Micromass, UK. The synthetic procedures for different ionic liquids (Chart 1) along with their 1H NMR, elemental analysis, and mass spectroscopic data are listed below. The UV-visible absorption spectra were recorded on a Varian Cary-50 spectrophotometer.

Synthesis of Imidazolium ILs (1-4). Synthesis of IL 1 and 2. 1-Methylimidazole (36.5 mmol) was taken in two roundbottom flasks each, and excess methyl iodide and ethyl bromide were separately added to the flasks for the synthesis of 1 and 2, respectively. The reaction mixtures were stirred for 6 h at room temperature. After completion of the reaction, the excess alkyl halides were evaporated, and the residue was washed repeatedly with ethyl acetate, hexane, and ether to get the iodide salt of 1 and the bromide salt 2. Then, ∼1 g of the ILs were dissolved in HPLCgrade methanol and treated with activated charcoal and filtered through a Celite bed under hot conditions. The procedure was repeated three times to make the ILs colorless. Finally, the ILs were concentrated from the filtrate and dried in vacuum at 70 °C for three days. After purification, the iodide salts of IL 1 and 2 were colorless. The iodide salt of 1 was finally passed through Amberlyst A-26 bromide ion exchange column to get the IL 1 (bromide salt). 1 H NMR (300 MHz, D2O) of 1 (1,3-dimethyl imidazolium bromide) δ/ppm = 3.78 (s, 6H), 7.30 (s, 2H). Anal. Calcd for C5H9BrN2: C, 33.92; H, 5.12; N, 15.82. Found C, 33.55; H, 5.15; N, 15.47. MS (ESI) m/z. calcd (for C5H9N2þ the 4° ammonium ion, 100%) 97.0766, found 97.1400 (Mþ). 1 H NMR (300 MHz, D2O) of 2 (1-ethyl-3-methyl imidazolium bromide) δ/ppm = 1.40 (t, 3H), 3.79 (s, 3H), 4.09-4.14 (q, 2H), 7.31 (m, 1H), 7.38 (m, 1H). Anal. Calcd for C6H11BrN2: C, 37.72; H, 5.80; N, 14.66. Found C, 37.51; H, 6.01; N, 14.52. MS (ESI) m/z. calcd (for C6H11N2þ the 4° ammonium ion, 100%) 111.0922, found 111.0523 (Mþ). Synthesis of IL 3. 1-Methylimidazole and propyl bromide were taken in a round-bottom flask and stirred for 6 h at 60 °C with a reflux condenser. After the reaction, the excess propyl bromide was evaporated and the product was purified in similar ways as described for IL 1 and 2. The IL was dried in vacuum at 70 °C for three days. 1 H NMR (300 MHz, D2O) of 3 (1-propyl-3-methyl imidazolium bromide) δ/ppm = 0.8 (t, 3H), 1.74-1.79 (m, 2H), 3.79 (s, 3H), 4.05 (t, 2H), 7.33 (m, 1H), 7.37 (m, 1H). Anal. Calcd for C7H13BrN2: C, 40.99; H, 6.39; N, 13.66. Found C, 40.71; H, 6.45; N, 13.52. MS (ESI) m/z. calcd (for C7H13N2þ the 4° ammonium ion, 100%) 125.1079, found 125.0568 (Mþ). Synthesis of IL 4. IL 4 was synthesized according to a previously published protocol.10 Briefly, 36.5 mmol of 1-methylimidazole and 40.15 mmol of 1-bromobutane were taken in a roundbottom flask, and the reaction mixture was heated intermittently in a microwave oven at 320 W (20 s irradiation with 10 s mixing), until a clear single phase is obtained. The resulting IL was cooled and purified in similar ways as described for IL 1 and 2. The IL was dried in vacuum at 70 °C for three days. 1 H NMR (300 MHz, CDCl3) of 4 (1-butyl-3-methyl imidazolium bromide) δ/ppm = 0.97 (t, 3H), 1.40 (m, 2H), 1.91 (m, 2H), 4.1 (s, 3H), 4.34 (t, 2H), 7.45 (m, 1H), 7.57 (m, 1H), 10.38 (s, 1H). (10) Das, D.; Dasgupta, A.; Das, P. K. Tetrahedron Lett. 2007, 48, 5635.

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Article Anal. Calcd for C8H15BrN2: C, 43.85; H, 6.9; N, 12.78. Found C, 43.55; H, 7.15; N, 12.47. MS (ESI) m/z. calcd (for C8H15N2þ the 4° ammonium ion, 100%) 139.1235, found 139.0744 (Mþ). Preparation of Microemulsions. CTAB (910 mg) was taken in 25 mL (required to prepare 0.1 M solution) isooctane/n-hexanol solution ([n-hexanol]=0.8 M and z=8, where z=[n-hexanol]/ [surfactant]) and allowed to disperse by vortexing in a volumetric flask. During preparation of IL containing microemulsion, the required amount of IL was either added directly with CTAB or added via aqueous buffer solution when the concentration of IL was too low. To the dispersed suspension of surfactant and IL, the desired volume of aqueous phosphate buffer (pH = 6.0, 20 mM) was added to obtain a particular W0. Finally, the volume was increased up to the mark of the volumetric flask by the isooctane/ n-hexanol and vigorously vortexed to get a macroscopically homogeneous solution. The concentrations of the ILs, enzyme, substrate, the cosurfactant (n-hexanol), and CTAB are referred to as the overall concentration with respect to the total volume of the reverse micellar solution. Substrate Stock Solution in Reverse Micelles. Substrate (N-R-benzyloxycarbonyl-L-lysine-p-nitrophenyl ester hydrochloride) stock solution (5-62.5 mM) was prepared in HPLC-grade DMSO. The reaction concentration of substrate (20-250 μM) was achieved by taking 6 μL of the stock solution to 1.5 mL of the reacting solution in the cuvette. Enzyme Solution. Trypsin stock solution (1.2  10-3) M was prepared in pH = 3 buffer solution containing 1 mM CaCl2 in a 1 mL volumetric flask. This stock solution of enzyme was diluted 10 times with pH = 6 (20 mM phosphate buffer) solution just before the experiment. The final concentration of trypsin (0.32 μM, 7.62 μg/mL) was reached by the addition of 4 μL of diluted solution of enzyme to 1.5 mL of the reaction volume in the cuvette. Measurement of Trypsin Activity. The kinetic parameters (kcat) for the hydrolysis of N-R-benzyloxycarbonyl-L-lysinep-nitrophenylester hydrochloride by the compartmentalized trypsin (0.32 μM) in reverse micelles or in aqueous buffer were determined spectrophotometrically (on a Cary 50, Varian UV-visible spectrophotometer) following the formation of p-nitrophenol at the isosbestic points. The isosbestic points (λiso) and the molar extinction coefficients (∈) at λiso of the p-nitrophenol/p-nitrophenolate couple in w/o microemulsions of CTAB (0.1 M)/water/ isooctane/n-hexanol at z = 8 were determined spectrophotometrically. The λiso and ∈ were found to be 340.6 nm, 4370 M-1 cm-1 and 340 nm, 6500 M-1 cm-1, respectively, in CTAB/(water/ isooctane/n-hexanol) in the presence and absence of different imidazolium ILs and in aqueous solution of pH=6.0. In a typical experiment, 6 μL of the different substrate stock solutions prepared in HPLC dimethyl sulfoxide were added to 1.5 mL microemulsion or aqueous solution in a cuvette at pH = 6 (here, pH refers to the pH of the aqueous phosphate buffer solutions used for the preparation of w/o microemulsions, as the pH within the water-pool of w/o microemulsions does not vary significantly, 5 mM) in the reverse micelles, off-scale noise in the spectra was observed. Consequently, the spectra could not be run below 230 nm, as the imidazolium ions at these concentrations absorb strongly in this region (below 230 nm). Again, when a lower concentration of the ILs was used, no significant effect of the ILs on the CD spectra of enzyme was observed. This is the reason for using a 5 mM concentration of ILs in the CD experiments so that the spectra of the enzyme can be measured up to 220 nm without the problem of off-scale signals. The final overall concentration of protein is 20 μg/mL. This is the lowest possible concentration of the enzyme used for the CD experiments to get measurable signal intensity. However, at an even lower concentration of trypsin, the CD signal intensity was too weak to measure. Also, this concentration is only ∼2.5-fold higher than the concentrations of protein used in the activity measurement study.

Results and Discussion Enzyme and biomolecules solubilized in reverse micelles are protected from bulk organic solvent by a layer of surfactant molecule without a significant loss of its activity.3n Enzymes entrapped in such reverse micelles can be treated as an excellent biomimetic model for intracellular enzymes. Improvement in the activity of enzymes entrapped in the w/o microemulsions is always demanding because of its extensive potential in technological and biotechnological applications.1-5 In this context, we mentioned in the Introduction that the efficiency of surface-active enzymes was modulated by the alteration of microstructural parameters of the self-organized aggregates and also by using surfactant ILs.6-9 Herein, we have tried to improve the activity of water-pool solubilized hydrophilic enzyme trypsin in cationic reverse micelles Langmuir 2010, 26(6), 4080–4086

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by using highly water-soluble imidazolium bromide-based ILs as activators. It is expected that the catalytic efficiency of the encapsulated trypsin would be influenced by the change in the microstructural parameters of the water-pool in the reverse micelles. The imidazole moiety was found to influence the activity of many enzymes by helping in the respective catalytic processes.10,12,13 To investigate the prospect of the imidazolium bromide ILs as activators for water-pool solubilized enzyme, the activity of trypsin was examined in the w/o microemulsions and (water þ IL)-in-oil microemulsions of CTAB (0.1 M)/isooctane/n-hexanol/water in the presence of 0.1-10 mM ILs (1-4) at z = 8, pH 6.0 (20 mM phosphate), and room temperature across a varying range of W0 at which isotropic solutions were formed. The solubility of the ILs in the water phase of the isotropic solutions was examined by UV-vis spectroscopy (Figure S1, Supporting Information). For this investigation, the most polar IL having the methyl side chain (1) and comparatively less polar IL having the n-butyl side chain (4) were employed. These ILs (20 mM) were taken in isooctane, water, and the w/o microemulsion containing 100 mM CTAB. All the solutions were vigorously shaken to ensure the maximum solubilization of ILs in the respective systems. An indistinguishable peak of very low intensity at ∼280 nm was observed for the insoluble ILs in isooctane. However, in the macroscopically homogeneous solution of water and microemulsions, a peak of substantial intensity was observed at ∼280 nm. This confirms the localization of ILs in the water phase of w/o microemulsions. Initially, we have started the experiment with IL 1, which is highly soluble in water and expected to be solubilized deep inside the water-pool. In the absence of any IL, the activity of trypsin (kcat) in CTAB reverse micelles was found to improve from 1.7 to 9.5 s-1 (Figure 1) with increasing W0 from 8 to 40, which is consistent with the previous report, as the size of water-pool increases and the local molar concentration of water reaches the concentration of bulk water.8 In the presence of 0.05 and 0.1 mM of IL 1, trypsin showed an enhancement in the kcat from ∼3.9 to 8.9 s-1 (Figure 1) across the similar range of W0. Interestingly, the enzyme showed notable enhancement in the activity particularly in the lower W0 range, 8-24. The kcat increased from 1.7 to 5.3 s-1 and 5.8 to 8.9 s-1 for W0 = 8 and 16, respectively, in the presence of IL 1 compared to that observed in only CTAB microemulsions. Hence, the activation in the trypsin efficiency was ∼50-200% in the presence of 0.05 and 0.1 mM of 1. However, in W0 > 24, the kcat was almost comparable in the presence or absence of IL 1 in CTAB reverse micelles. At a slightly higher concentration of 1 (0.5 and 1.0 mM), the activation effect of the IL was still prominent in lower W0. However, activity of trypsin decreased a little at W0 > 24. Surprisingly, with further increase in the concentration of 1 (5 mM), significant deactivation was observed in the catalytic efficiency of trypsin in CTAB microemulsions (Figure 1). Thus, IL 1 at its lower concentration served as an activator for hydrophilic enzyme trypsin in CTAB reverse micelles. Interestingly, as the concentration of the IL increases toward 5 mM, shrinkage of the W0 range was observed. The W0 range decreased from 4 to 40 in the presence of 0.05 mM 1 to 8-24 in the presence of 5 mM 1. The decrease in the W0 range strongly indicates that the ILs reside in the water phase of the reverse micelles and participate in the formation of the hydrophilic pool (12) (a) Newmyer, S. L.; Ortiz de Montellano, P. R. J. Biol. Chem. 1996, 271, 14891. (b) Newmyer, S. L.; Ortiz de Montellano, P. R. Biochemistry 1996, 35, 12788. (13) (a) Liu, B. K.; Wang, N.; Chen, Z. C.; Lin, X. F. Bioorg. Med. Chem. Lett. 2006, 16, 3769. (b) Ganske, F.; Bornscheuer, U. T. Org. Lett. 2005, 14, 3097. (c) Itoh, T.; Han, S. H.; Matsushita, Y.; Hayase, S. Green Chem. 2004, 6, 437. (d) Liu, B. K.; Wu, Q.; Xu, J. M.; Lin, X. F. Chem. Commun. 2007, 295.

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Figure 1. Trypsin activity profile (kcat) in CTAB (0.1 M)/water/ isooctane/n-hexanol (z=8) w/o microemulsions in the presence of varying concentration of IL 1 across the W0 range at room temperature and pH = 6.0 (20 mM phosphate buffer). In duplicate experiments, observed kcat values are variable within (5%.

(IL þ water). With increasing concentration of IL, accommodation of more water in the same pool is not possible. In fact, in the presence of more than 10 mM IL there was no formation of w/o microemulsion (Table S1, Supporting Information). At this point, we were curious to know the reasons for the activation. The imidazole moiety is part of the histidine amino acid, which is an important residue of the catalytic triad of trypsin and known to participate in hydrogen bonding with water. To this end, the bromide counterion of the imidazolium IL also has strong hydrogen bond-accepting capability.14 So, Br- being the counterpart of the imidazolium cation presumably helps in the enhancement of nucleophilicity of water present in the vicinity of the enzyme through hydrogen bonding, which consequently facilitates the enzyme-catalyzed hydrolysis of ester.13d To investigate the role of the imidazolium moiety on the activation of the enzyme, we checked the trypsin activity in the presence of 1 mM and 5 mM NaBr keeping all other experimental conditions identical. No activation of the protein was observed, which consequently proved the essential presence of the imidazolium moiety along with the bromide counterion in the improvement of trypsin activity. Because of its high solubility in water, IL 1 also possibly participated in the formation of the hydrophilic pool (water þ IL) of the microemulsion at a concentration of g1 mM. The IL at such a high concentration in the hydrophilic pool can interact with the active site of the enzyme through hydrogen bonding in addition to its hydrogen bonding ability with water.13a Such interaction with the active site of the trypsin by the IL probably inhibited the catalytic process resulting in poor efficiency of the enzyme.13a,d,15 On the other hand, an activation effect of imidazolium bromide at the lower concentration was not observed in the higher W0 range possibly due to its unrestricted mobility in a bigger water-pool that reduces the hydrogen bonding between the IL and water in the vicinity of the enzyme. Hence, the nucleophilicity of water around the active site of the enzyme did not increase. (14) (a) Swatloski, R. P.; Spear, S. K.; Holbrey, J. D.; Rogers, R. D. J. Am. Chem. Soc. 2002, 124, 4974. (b) Fukaya, Y.; Hayashi, K.; Wadab, M.; Ohno, H. Green Chem. 2008, 10, 44. (15) Baker, G. A.; Heller, W. T. Chem. Eng. J. 2009, 147, 6.

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Figure 2. Trypsin activity profile (kcat) in CTAB (0.1 M)/water/ isooctane/n-hexanol (z=8) w/o microemulsions in the presence of varying concentration of IL 2 across the W0 range at room temperature and pH = 6.0 (20 mM phosphate buffer). In duplicate experiments, observed kcat values are variable within (5%.

Interestingly, trypsin showed remarkable enhancement in its activity in the CTAB reverse micelles in the presence of the next higher homologue IL, 2, under identical experimental conditions as those used for 1 (Figure 2). Here, also, the activating effect is more prominent in the lower W0 range where we have found ∼200-300% (3-4-fold) enhancement in the enzyme efficiency as the kcat increased from 1.7 to 7.2 s-1 and 5.8 to 16.3 s-1 at W0 = 8 and 16, respectively, at concentrations of 0.5 and 1.0 mM of 2. Importantly, trypsin also showed higher activity at W0 >24 in the presence of varying concentration of 2 compared to that observed for only CTAB reverse micelles. For example, at W0 = 40, in the presence of 2, the kcat varied from 10.3 to 15.7 s-1, which was 9.5 s-1 in the absence of any IL (Figure 2). Interestingly, the activation effect in higher W0 range offered by the IL 2 is sufficiently high in contrast to the observed comparable or lowered activity of trypsin in the presence of 1 (Figure 1). This activation effect increased with increasing concentration of 2 and became saturated at ∼1 mM, after which no remarkable improvement in the catalytic efficiency of trypsin was observed. Grossly, trypsin activity improved ∼30-300% in the presence of 0.1-10 mM of 2 (Figure 2). The activation effect of this IL is obviously caused by the same mechanism discussed in the preceding paragraph. However, surprisingly no deactivation in the efficiency of trypsin was noted at a higher concentration of 2. A very minute increase in the dimension of the ILs from 1,3-dimethyl imidazolium (1) to 1-ethyl-3-methyl imidazolium (2) probably hinders the hydrogen bonding of the latter with the active site of enzyme. Additionally, the water-pool localization of 2 was also evident from a similar shrinkage of the W0 range as observed for 1 (Table S1, Supporting Information). The W0 range decreased from 4 to 40 in the presence of 0.1 mM 1 to 8-20 in the presence of 20 mM 2. As the enzyme activity was found to increase from IL 1 (C-1) to IL 2 (C-2), we were very curious to see whether the higher homologue ILs with C-3 (3) and C-4 (4) chain length could further increase the activity of trypsin. However, the results were not as encouraging as observed for 1 and 2. There was ∼2-3-fold enhancement in the trypsin activity only at W0 = 8 in varying concentrations (0.1-10 mM) of 3 and 4. In other W0 range up to 40, with increasing concentration of 3, trypsin activity was either 4084 DOI: 10.1021/la9040419

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Figure 3. Trypsin activity profile (kcat) in CTAB (0.1 M)/water/ isooctane/n-hexanol (z=8) w/o microemulsions in the presence of varying concentration of IL 3 across the W0 range at room temperature and pH = 6.0 (20 mM phosphate buffer). In duplicate experiments, observed kcat values are variable within (5%.

Figure 4. Trypsin activity profile (kcat) in CTAB (0.1 M)/water/ isooctane/n-hexanol (z = 8) w/o microemulsions in the presence of varying concentration of IL 4 across the W0 range at room temperature and pH = 6.0 (20 mM phosphate buffer). In duplicate experiments, observed kcat values are variable within (5%.

comparable to or even lower than that observed in only CTAB reverse micelles (Figure 3). Similar results were also found for IL 4 having the longest chain length in our study. IL 4 also showed no activation of trypsin at W0 > 8 (Figure 4). In most of the cases with varying concentrations of 4, the activity profile of trypsin remained almost comparable to the activity profile in CTAB (Figure 4). Only in higher concentration of 4 and at higher range of W0 did activity of trypsin drop to a certain extent. However, the observed activation effect only at lowest W0 =8 was possibly due to the confinement of IL and enzyme in a very small-sized waterpool. This compelled the BMIMBr and water to come into vicinity of the enzyme resulting in enhanced nucleophilicity of water through hydrogen bonding. In general, ILs 3 and 4 did not exhibit notable activation effect on the activity of trypsin presumably due to their increasing amphiphilic character with increase in chain length. Accordingly, they prefer to locate themselves Langmuir 2010, 26(6), 4080–4086

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Figure 5. Pictorial representation of the activating effect of imidazolium bromide IL on trypsin activity at the water pool of the CTAB reverse micelles.

toward the interfacial region of the aggregates instead of deep inside the water-pool as expected for ILs 1 and 2 (Figure 5). This possible partition of ILs 3 and 4 between the water-pool and the interfacial region of w/o microemulsions might have increased the distance between the IL and the water in the vicinity of the trypsin, which reduces the hydrogen bonding among them (Figure 5). As a result, no improvement in the nucleophilicity of the water in the vicinity of the enzyme is possible. The localization of these ILs (3 and 4) toward the interfacial region of the water phase was further confirmed from their minimized shrinkage effects on the W0 range of the CTAB microemulsion. The formation of isotropic solutions was possible even when the ILs 3 and 4 are present at concentrations of 30 and 50 mM, respectively (Table S1, Supporting Information). Overall among all the imidazolium-based ILs, trypsin showed its highest activity (kcat = 16.3 s-1) in the presence of 2 at its concentration of 1 mM and W0 =16. Also in other concentrations and W0, IL 2 always had a better activation effect than others. IL 2 probably possesses the perfect balance between size and water solubility that is suitably fitted for hydrogen bonding with the water in the vicinity of enzyme, but not with the active site of trypsin. Hence, the activity of trypsin was found to be maximum in the presence of IL 2. To investigate the effects of the ILs with varying side chain lengths on the hydrophilic (water þ IL) pool size, we have calculated the hydrodynamic diameter (Dh) of the reverse micelles through dynamic light scattering (DLS) experiments. Initially, the aggregated sizes of the CTAB-based reverse micelles in the absence of ILs were investigated throughout its W0 range from 8 to 40. The Dh was found to increase with the rise in the water content of the system (Dh increased from 4.1 to 13.0 nm, Figure 6). This is in agreement with the works reported by other authors, which has shown that the increase in the water content in the reverse micellar media, keeping the concentration of the surfactants fixed, results in a concomitant increase of the size of the aggregate.5d In the presence of ILs in the reverse micelles of CTAB, there was an overall increase of the aggregate sizes for all the ILs used compared to that of only CTAB reverse micelles at comparable W0 (Figure 6). However, the increase in the aggregate size Langmuir 2010, 26(6), 4080–4086

Figure 6. Variation of the hydrodynamic diameter (Dh) with W0 in different CTAB-based w/o microemulsions in the presence of 5 mM IL 1-4.

becomes more significant as the side chain lengths of the ILs decreases. The Dh values of the systems containing the larger side chain analogues (for C-3 and C-4, Figure 6), showed a comparatively lower increase with respect to the CTAB systems alone. On the other hand, for the smaller ILs the size of the aggregate increases sharply reaching up to a maximum value of Dh = 15.5 nm at W0 = 24 for IL 1. Strikingly, this value is even higher than the dimension of the CTAB reverse micellar aggregates at the highest W0 value of 40. These results conclusively prove that size of reverse micelles as a whole increases in the presence of ILs, and particularly, the smaller-sized ILs (C-1 and C-2 analogue) have significant influence in modulating the dimension of the CTABreverse micellar aggregates. Therefore, the highest Dh observed for ILs 1 and 2 throughout their W0 range strongly implies that these ILs are the most polar and localized inside the water-pool. Consequently, the hydrophilic pool of water and IL was formed, DOI: 10.1021/la9040419

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Figure 7. CD spectra of trypsin in CTAB (0.1 M)/water/isooctane/ n-hexanol w/o microemulsions at W0 = 16, z = 8, pH = 6.0 (20 mM phosphate) in the presence of different ILs at the concentration of 5 mM.

which obviously has an impact on altering the size of the reverse micelles. As a result, their concurrent effect on the activation of tryspin, which resides in the deep of the water pool, also increases. Additionally, these results also prove that the location of the ILs is in the water phase of the reverse micelles, as there was a greater or lesser increment of the water-pool dimension in the case of all the ILs. This observation corroborates well with the literature that the size of the reverse micellar aggregates increases with the increase of the polar components.16 This was also found to be true for the different IL-in-oil microemulsions reported in the literature.17 The smaller size obtained for 3 and 4 is presumably because of the partitioning of these ILs between water-pool and interface, and consequently, the ILs move away from the enzyme (Figure 5) as the size of the arm length increases. Hence, these two ILs have little impact on the activity of trypsin in w/o microemulsion. At this point, we were interested in understanding the influence of the synergistic effect of imidazolium ILs (1-4) and the size of the hydrophilic pool on the secondary structure of trypsin, if any, and finding a correlation between conformational change and activity. The CD signal in the far-UV region and the mean residue ellipticity (MRE) indicate the secondary structure of the protein.18 The concentration of enzymes used in CD experimentation was 20 μg/mL at W0 =16 to get a reasonable CD signal, and 5 mM of ILs was used. To compare the observed secondary structural changes with the native structure of trypsin,18d the spectra of the protein was also measured in 20 mM (pH= 6) phosphate buffer. The spectrum in buffer had the most negative MRE value of all the systems (Figure 7). Interestingly, among all four ILs, the MRE value of trypsin decreased to a maximum extent in the presence of IL 2 in CTAB reverse micelles (Figure 7) and reached closest to the value observed for protein in buffer. This result strongly indicates that trypsin possesses native-like conformation in the (16) Gao, Y.; Li, N.; Zheng, L.; Zhao, X.; Zhang, J.; Cao, Q.; Zhao, M.; Li, Z.; Zhang, G. Chem.;Eur. J. 2007, 13, 2661. (17) (a) Zech, O.; Thomaier, S.; Bauduin, P.; Ruck, T.; Touraud, D.; Kunz, W. J. Phys. Chem. B 2009, 113, 465. (b) Gao, Y.; Voigt, A.; Hilfert, L.; Sundmacher, K. ChemPhysChem 2008, 9, 1603. (18) (a) Walde, P.; Peng, Q.; Fandavis, W. N.; Battistel, E.; Luisi, P. L. Eur. J. Biochem. 1988, 173, 401. (b) Peng, Q.; Luisi, P. L. Eur. J. Biochem. 1990, 188, 471. (c) Naoe, K.; Takeuchi, C.; Kawagoe, M.; Nagayama, K.; Imai, M. J. Chromatogr. B 2007, 850, 277. (d) Ghosh, S.; Banerjee, A. Biomacromolecules 2002, 3, 9.

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reverse micelles of CTAB with IL 2. Thus, in addition to its activating effect by means of hydrogen bonding with the water in the vicinity of the enzyme, the secondary structure of trypsin was also preserved in the larger reverse micelles in the presence of 2 (Figure 6). This is in complete concurrence with the observed highest activity of trypsin in the reverse micelles of CTABþIL 2 (Figure 2). However, the spectra of the protein in systems CTABþIL 3, CTABþIL 4, and only CTAB are close to each other but have less negative value compared to the native conformation of trypsin in aqueous buffer (Figure 7). This is also in nice agreement with the activity of trypsin, as the observed kcat is comparable in all three (CTAB, CTAB þ IL 3, and CTAB þ IL 4) w/o microemulsions (Figures 3 and 4) and also did not have a notable activation effect as observed in the case of 2. In the presence of 5 mM 1, the trypsin was found to be deactivating, which is reflected by its less negative MRE value in the CD spectra (Figure 7). Interestingly, in aqueous buffer solution the MRE values of trypsin were almost the same in the presence of any of the ILs 1-4 or in the absence of IL. This also corresponds to the activity of trypsin in aqueous solution that was found to be unaffected by the presence of ILs 1-4 even up to 20 mM concentration (data not shown). This result clearly indicates that the imidazolium bromide-assisted activation effect on trypsin takes place only when they are in close proximity in a confined system like reverse micelles.

Conclusion In this work, we have exploited the activating effect of the water-soluble imidazolium bromide ILs on hydrophilic enzyme trypsin in the reverse micelles of CTAB. The presence of watersoluble IL in CTAB-based w/o microemulsions makes it a better host of trypsin over the microemulsions formed by CTAB only. The side-chain lengths of the ILs were varied, and the best performance of trypsin was observed in the system having the IL with C-2 tail length (2). Optimum activation from the imidazolium bromide-assisted enzyme hydrolysis was observed in ∼0.5-1 mM concentration of the organic salts. The activity of trypsin in the presence of 2 was significantly higher (3-4-fold) compared to that observed in only CTAB w/o microemulsion. The IL 2 is the best activator of trypsin because of its perfect balance in size and water-pool solubility. 1-Ethyl-3-methyimidazolium bromide (2) can suitably offer its activating effect to the trypsin by improving the nucleophilicity of water in the vicinity of the enzyme through hydrogen bonding and also by preserving the secondary structure in the larger-sized reverse micelles. Thus, our present investigation leads to the first use of water-soluble imidazolium-bromide IL as activator for trypsin, maximizing its efficiency in CTAB reverse micelles. Acknowledgment. P.K.D. is thankful to Department of Science and Technology, India, for financial assistance through the Ramanna Fellowship (No. SR/S1/RFPC-04/2006). S.D., D.D., and S.D. acknowledge Council of Scientific and Industrial Research, India, for their Research Fellowships. Supporting Information Available: W0 range of CTAB in the presence of different ILs and the UV spectrum of the IL in the various solvents. Representative DLS data of the aggregate size distribution in different w/o microemulsions in the presence and absence of ILs. This material is available free of charge via the Internet at http://pubs.acs.org.

Langmuir 2010, 26(6), 4080–4086