Effect of Counterions on the Activity of Lipase in Cationic Water-in-Oil

Sisir Debnath, Antara Dasgupta, Rajendra Narayan Mitra, and Prasanta Kumar Das*. Department of Biological Chemistry, Indian Association for the CultiV...
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Langmuir 2006, 22, 8732-8740

Effect of Counterions on the Activity of Lipase in Cationic Water-in-Oil Microemulsions Sisir Debnath, Antara Dasgupta, Rajendra Narayan Mitra, and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the CultiVation of Science, JadaVpur, Kolkata-700032, India ReceiVed May 25, 2006. In Final Form: July 26, 2006 This paper delineates how the different counterions affect the physicochemical properties of the aqueous aggregates and thereby the lipase activities at the interface of cationic water-in-oil microemulsions. To this end, we have synthesized a series of cetyltrimethylammonium-based surfactants, 1-14, having aliphatic, aliphatic with aromatic substitution at the R position, and aromatic carboxylate anion as the counterion. The physicochemical characterizations of these aqueous aggregates were done by conductometric, tensiometric, fluorometric techniques to determine counterion binding (β), critical micelle concentration (cmc), and micropolarity at the microenvironment. It has been found that the activity of lipase mainly increases with hydrophobicity (which is directly proportional to the counterion binding (β) of the surfactant) of the counterion and reaches a maximum when the β value is around 0.5. Increase in hydrophobicity as well as β leads to the attachment of more counterions at interface resulting in enhancement of interfacial area. Consequently, the enzyme may attain flexible secondary conformation at the augmented surface area and also allow larger population of substrates and enzyme molecules at the interface leading to the enhancement in lipase activity. After an optimum value of β, further increase probably produces a steric crowding at the interface, hindering the smooth occupancy of enzyme and the substrate in this region leading to decrease of enzyme activity, while molecular surface area of the counterion did not show any virtual influence on the lipase activity. Thus, the variation in the counterion structure and hydrophobicity plays a crucial role in modulating the lipase activity.

Introduction Water-in-Oil (W/O) microemulsion, fine dispersion of water in nonpolar organic solvents stabilized by surfactant molecules,1,2 possess the potential for technological and biotechnological applications due to their enhanced interfacial area and increased ability to solubilize otherwise immiscible substrates.3-5 Enzymology in W/O microemulsions has been an area of increasing * To whom correspondence should be addressed. Fax: +(91)-3324732805. E-mail: [email protected]. (1) (a) Eicke, H. F.; Kvita, P. ReVerse micelles; Luisi, P. L., Straub, B. E., Ed.; Plenum: New York, 1984; p 21. (b) Zana, R.; Lang, J. In Solution behaVior of surfactants; Mittal, K. L., Fendler, E. J., Eds.; Plenum: New York, 1982; Vol. 2. (c) Petit, C.; Bommarius, A. S.; Pileni, M. P.; Hatton, T. A. J. Phys. Chem. 1992, 96, 4653. (d) Fendler, J. H. Acc. Chem. Res. 1976, 9, 53. (2) (a) Fendler, J. H. Membrane mimetic chemistry; Wiley & Sons: New York, 1982. (b) Das, D.; Roy, S.; Das, P. K. Org. Lett. 2004, 6, 4133. (c) Silber, J. J.; Biasutti, M. A.; Abuin, E. B.; Lissi, E. AdV. Colloid Interface Sci. 1999, 82, 189. (d) Roy, S.; Das, D.; Dasgupta, A.; Mitra, R. N.; Das, P. K. Langmuir 2005, 21, 10398. (3) (a) Skargelind, P.; Jasson, M. J. Chem. Technol. Biotechnol. 1992, 54, 277. (b) Valis, T. P.; Xenakis, A.; Kolisis, F. N. Biocatalysis 1992, 6, 267. (c) Yamada, Y.; Kuboi, R.; Komasawa, I. Biotechnol. Prog. 1993, 9, 468. (d) Dasgupta, A.; Das, D.; Das, P. K. Biochimie 2005, 87, 1111. (e) Lissi, E. A.; Abuin, E. B. Langmuir 2000, 16, 10084. (f) Abuin, E.; Lissi, E.; Duarte, R. Langmuir 2003, 19, 5374. (g) Mitra, D.; Chakraborty, I.; Bhattacharya, S. C.; Moulik, S. P.; Roy, S.; Das, D.; Das, P. K. J. Phys. Chem. B 2006, 110, 11314. (4) (a) Stamatis, H.; Xenakis, A.; Kolisis, F. N. Biotechnol. AdV. 1999, 17, 293 and references therein. (b) Luisi, P. L.; Magid, L. J. CRC Crit. ReV. Biochem. 1986, 20, 409. (c) Luisi, P. L. Angew. Chem., Int. Ed. Engl. 1985, 24, 439. (d) Kelley, B. D.; Wang, D. I. C.; Hatton, T. A. Biotechnol. Bioeng. 1993, 42, 1199. (e) Komives, C. F.; Osborne, D. E.; Russell, A. J. J. Phys. Chem. 1994, 98, 369. (f) Menger, F. M. Angew. Chem., Int. Ed. Engl. 1991, 30, 1086. (g) Mosler, R.; Hatton, T. A. Curr. Opin. Colloid Interface Sci. 1996, 1, 540. (h) Leodidis, E. B.; Hatton, T. A. J. Phys. Chem. 1990, 94, 6411. (i) Roy, S.; Dasgupta, A.; Das, P. K. Langmuir 2006, 22, 4567. (j) Walde, P.; Han, D.; Luisi, P. L. Biochemistry 1993, 32, 4029. (k) Yang, F. X.; Russell, A. J. Biotechnol. Bioeng. 1995, 47, 60. (l) Bommarius, A. S.; Hatton, T. A.; Wang, D. I. C. J. Am. Chem. Soc. 1995, 117, 4515. (m) Yang, F. X.; Russell, A. J. Biotechnol. Bioeng. 1994, 43, 232. (n) Martinek, K.; Levashov, A. V.; Khmelnitsky, Y. L.; Klyachko, N. L.; Berezin, I. V. Science 1982, 218, 889. (o) Menger, F. M.; Yamada, K. J. Am. Chem. Soc. 1979, 101, 6731. (5) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Kabanov, A. V.; Khmelnitsky, Y. L. Biochim. Biophys. Acta 1989, 981, 161.

interest during the past two decades. Enzymes solubilized in W/O microemulsions are mainly of two classes: those within the water pool, hydrophilic enzymes, and those interacting with the micellar interface, surface-active enzymes. Lipases, a class of surface-active enzymes,4a are widely employed for the various transformations in W/O microemulsions.3,6-8 The catalytic activity of such encapsulated enzyme was considered to be primarily dependent on the local molar concentration of water and other ions present in the proximity of the enzyme.8,9 In this respect Das and Chaudhuri for the first time showed that the activity of Chromobacterium Viscosum (CV) lipase in cetyltrimethylammonium bromide (CTAB)/water/ isooctane/n-hexanol reverse micelles remains grossly unchanged across the W0 (mole ratio of water to surfactant) range apparently due to unaltered interfacial concentration of water, [H2O]i (28.131.8 M).9a However, the earlier studies from our group revealed that the correlation between the lipase activity and the [H2O]i is far from being straightforward and the architecture as well as geometric constraints at the surfactant headgroup plays a very crucial role in modulating lipase activity probably because of variation in the interfacial area.10-12 Investigations were directed (6) Fletcher, P. D. I.; Robinson, B. H.; Freedman, R. B.; Oldfield, C. J. Chem. Soc., Faraday Trans. 1985, 81, 2667. (7) (a) Stamatis, H.; Xenakis, A.; Menge, U.; Kolisis, F. N. Biotechnol. Bioeng. 1993, 42, 931. (b) Stamatis, H.; Kolisis, F. N.; Xenakis, A.; Bornscheuer, U.; Scheper, T.; Menge, U. Biotechnol. Lett. 1993, 15, 703. (c) Rees, G. D.; Robinson, B. H.; Stevenson, G. R.; Biochim. Biophys. Acta 1995, 1259, 73. (8) (a) Barbaric, S.; Luisi, P. L. J. Am. Chem. Soc. 1981, 103, 4239. (b) Damodaran, S. Colloids Surf., B 1998, 11, 231. (c) Das, P. K.; Srilakshmi, G. V.; Chaudhuri, A. Langmuir 1999, 15, 981. (d) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Pantin, V. I.; Kabanov, A. V.; Berezin, I. V. Biochim. Biophys. Acta 1981, 657, 277. (e) Carvalho, C. M. L.; Cabral, J. M. S. Biochimie 2000, 82, 1063. (9) (a) Das, P. K.; Chaudhuri, A. Langmuir 2000, 16, 76. (b) Srilakshmi, G. V.; Chaudhuri, A. Chem.sEur. J. 2000, 6, 2847. (10) (a) Das, D.; Das, P. K. Langmuir 2003, 19, 9114. (b) Dasgupta, A.; Das, D.; Mitra, R. N.; Das, P. K. J. Colloid Interface Sci. 2005, 289, 566. (11) Das, D.; Roy, S.; Mitra, R. N.; Dasgupta, A.; Das, P. K. Chem.sEur. J. 2005, 11, 8114.

10.1021/la061487d CCC: $33.50 © 2006 American Chemical Society Published on Web 09/14/2006

Enzymology in Organized Aggregates Chart 1. Cationic Surfactants with Different Counterions

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lipase activity. However, with further increase of β after an optimum value (above 0.5), the activity of lipase was found to decrease presumably due to the steric crowding of the counterions at the interface making it difficult for smooth occupancy of lipase and substrate. Experimental Section

toward understanding the effect of headgroup hydrophilicity, size, and geometric constraints on the activity of interfacially solubilized lipase. It was found that the interfacial area in the aggregated structure essentially increases with the increase in the surfactant headgroup size and its flexibility. As a consequence, the local concentrations of enzyme and substrate also expectedly increase and the lipase may be smoothly occupied at the augmented interface which leads to higher activity of CVlipase.10-12 However, until now, the studies were primarily focused on the alteration in the surfactant’s headgroup and its effect on the lipase activity, while the counterion, the other intrinsic constituent of ionic surfactants, is an overlooked parameter in micellar enzymology. How important is the influence of counterions on the activity of lipase in W/O microemulsions is still remaining to be explored. In our study, attempts are concerted to develop a possible correlation, if any, between the catalytic efficiency of lipase in the cationic W/O microemulsions with varying nature of the counterions. To this end, we have synthesized a series of cetyltrimethylammonium-based surfactants having aliphatic (series I), aliphatic with aromatic substitution at alpha position (series II), and aromatic carboxylate anion (series III) as the counterions (1-14, Chart 1). The catalytic activity of CV-lipase was then estimated in W/O microemulsions of 1-14. It has been found that the catalytic activity of CV-lipase generally enhances with the increase of hydrophobicity and the degree of counterion binding (β), reaching a maximum when β value is around 0.5. However, the dependency of lipase activity on counterion size/ surface area is less significant than β. It may be that the increase in β (up to 0.5) enhances the interfacial area of the W/O microemulsion by means of increasing concentration of counterion, which in turn might lead to increase in population of enzyme and substrate at the interface leading to enhancement in

Materials. Chromobecterium Viscosum lipase (EC 3.1.1.3, type ΧΙΙ) was purchased from Sigma and was used as received. Analytical grade CTAB from Spectrochem (Mumbai, India) was recrystallized three times from methanol/diethyl ether, and the recrystallized CTAB was without minima in its surface tension plot. 2,2-Dimethylbutyric acid and pyrene were purchased from Alfa Aesar and Fluka, respectively. Dowex 1×8-400 hydroxide ion-exchange resin was procured from Lancaster. HPLC grade isooctane, n-hexanol, n-octanol solvents, and all other reagents used in the syntheses was obtained from SRL (Mumbai, India) and were of highest analytical grade. The UV-visible absorption spectra were recorded on Shimadzu UV-1700 spectrophotometer. 1H NMR spectra were recorded on a Bruker Avance DPX-300 spectrophotometer. The fluorescence spectra were recorded with a Perkin-Elmer LS55 luminescence spectrophotometer. The calculations of molecular surface area of the counterions of surfactant have been performed using MOPAC 99 module of Chem3D Pro Program (CambridgeSoft, Cambridge, MA). After the structure of the counterion was drawn at Chem3D Pro, the energy was minimized using MOPAC 99, and finally the Connolly molecular area was analyzed by computation. Methods. Synthesis of p-Nitrophenyl n-Hexanoate. The substrate p-nitrophenyl n-hexanoate ester was prepared conventionally by N,N-dicyclohexylcarbodiimide (DCC) coupling of hexanoic acid with p-nitrophenol in the presence of 4-(N,N-dimethylamino)pyridine (DMAP). The hexanoic acid (1 equiv) was added to p-nitrophenol (1 equiv) in the presence of 1.1 equiv of 4-(N,N-dimethylamino)pyridine (DMAP) and 1.1 equiv of DCC in dry dichloromethane under the nitrogen atmosphere at -20 °C. After 12-14 h of stirring, the reaction mixture was filtered and the concentrated filtrate was extracted with ether. After concentration of this ether part, the product was obtained by purification through a silica gel (60-120 mesh) column with an acetone/hexane solvent mixture as the eluent. The yield of the product was 75-80%. Syntheses of Surfactants. The surfactants 2-14 were prepared by using the ion-exchange procedure.13 A hydroxide ion-exchange column was made with Dowex 1×8-400. This column was activated by passing 300 mL of 2 N sodium hydroxide solution and followed by washing with water (2 L) and methanol (0.5 L). Three times recrystallized CTAB (1 g) dissolved in the minimum volume of methanol was then loaded on the column, and cetyltrimethylammonium hydroxide ((CTA)OH) was collected as eluant by passing methanol slowly through this column. This methanolic solution of CTAOH was neutralized with an equivalent amount of acid to prepare the surfactant with desired counterion. The solvent was then evaporated at 40 °C using a rotary evaporator, and the product obtained was crystallized twice from methanol-ether. The yield of the product was ∼80-85%. 1H NMR (300 MHz, CDCl ) and elemental analysis of the 3 synthesized compounds are as follows. Hexadecyltrimethylammonium acetate (2): δ ) 0.88 (t, J ) 7.2 Hz, 3H), 1.18-1.34 (br, 26H), 1.72-1.74 (br, 2H), 1.93 (s, 3H), 3.38 (s, 9H), 3.41-3.47 (m, 2H). Anal. Calcd for C21H45NO2 (1 mol % crystal water): C, 69.75; H, 13.10; N, 3.87. Found: C, 69.56; H, 13.01; N, 3.75. Hexadecyltrimethylammonium butyrate (3): δ ) 0.85-0.94 (m, 6H), 1.17-1.33 (br, 26H), 1.58-1.71 (m, 4H), 2.11-2.17 (t, J ) 7.5 Hz, 2H), 3.37 (s, 9H), 3.41-3.44 (br, 2H). Anal. Calcd for C23H49NO2 (0.5 mol % crystal water): C, 72.57; H, 13.24; N, 3.68. Found: C, 72.49; H, 13.27; N, 3.64.

(12) Mitra, R. N.; Dasgupta, A.; Das, D.; Roy, S.; Debnath, S.; Das, P. K. Langmuir 2005, 21, 12115.

(13) Jiang, N.; Li, P.; Wang, Y.; Wang, J.; Yan, H.; Thomas, R. K. J. Colloid Interface Sci. 2005, 286, 755.

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Table 1. Critical Micelle Concentration (Cmc) (Tensiometric and Conductometric), Micropolarity Values (I1/I3), and Molecular Surface Area of the Counterions of the Surfactant 1-14 surfactant 1 2 3 4 5 6 7 8 9 10 11 12 13 14

104 × cmc (M)a tensiometric conductometric 9.05 5.8 5.67 4.66 3.60 2.14 4.06 3.4 1.37 0.8 2.84 3.24 2.54 0.46

9.5 17.16 12.73 6.94 7.53 3.01 6.01 7.61 2.15 1.85 6.05 6.35 4.4 1.38

counterion binding (β)b 0.68 0.36 0.42 0.46 0.51 0.78 0.47 0.54 0.54 0.56 0.48 0.69 0.59 0.84

I1/I3c

molecular surf area (Å2) of the counterions

1.35 1.34 1.27 1.27 1.16 1.28 1.19 1.28 1.01 1.03 1.23 1.35 1.09 1.14

52 70 107 142 132 111 140 144 178 205 122 127 150 185

aExperimental error within (2% and (3-5% for cmc measurements by tensiometric and conductometric methods, respectively, in duplicate experiment. b Experimental error for the calculation of β within (0.5-1%. c Intensity ratios due to first and third vibronic peak of pyrene steady-state fluorescence, I1/I3, indicate the micropolarity at the micellar interface.

Hexadecyltrimethylammonium hexanoate (4): δ ) 0.85-0.89 (m, 6H), 1.18-1.32 (br, 28H), 1.33 (br, 2H), 1.57-1.62 (m, 2H), 1.71-1.74 (br, 2H), 2.12-2.18 (t, J ) 8.1 Hz, 2H), 3.39 (s, 9H), 3.41-3.46 (br, 2H). Anal. Calcd for C25H53NO2 (1 mol % crystal water): C, 71.88; H, 13.27; N, 3.35. Found: C, 72.01; H, 13.26; N, 3.38. Hexadecyltrimethylammonium 2,2-dimethylbutyrate (5): δ ) 0.82-0.89 (m, 6H), 1.09 (s, 6H), 1.14-1.32 (br, 26H), 1.47-1.54 (q, 2H), 1.70 (br, 2H), 3.42 (s, 9H), 3.44-3.49 (m, 2H). Anal. Calcd for C25H53NO2 (0.5 mol % crystal water): C, 73.47; H, 13.32; N, 3.43. Found: C, 73.59; H, 13.24; N, 3.49. Hexadecyltrimethylammonium trichloroacetate (6): δ ) 0.88 (t, J ) 6.8 Hz, 3H), 1.18-1.32 (br, 26H), 1.71 (br, 2H), 3.32 (s, 9H), 3.37-3.43 (m, 2H). Anal. Calcd for C21H42NO2Cl3: C, 56.44; H, 9.47; N, 3.13. Found: C, 56.50; H, 9.52; N, 3.13. Hexadecyltrimethylammonium phenylacetate (7): δ ) 0.88 (t, J ) 6.9 Hz, 3H), 1.18-1.33 (br, 26H), 1.56 (br, 2H), 2.97 (s, 9H), 3.12-3.18 (m, 2H), 3.5 (s, 2H), 7.11-7.19 (m, 1H), 7.22-7.26 (m, 2H), 7.32-7.35 (m, 2H). Anal. Calcd for C27H49NO2 (1 mol % crystal water): C, 74.09; H, 11.74; N, 3.20. Found: C, 74.18; H, 11.63; N, 3.28. Hexadecyltrimethylammonium hydroxyphenylacetate (8): δ ) 0.88 (t, J ) 6.9 Hz, 3H), 1.19-1.33 (br, 26H), 1.54-1.55 (br, 2H), 2.99 (s, 9H), 3.06-3.15 (m, 2H), 4.83 (s, 1H), 7.15-7.27 (m, 3H), 7.5-7.53 (m, 2H). Anal. Calcd for C27H49NO3 (0.5 mol % crystal water): C, 72.92; H, 11.33; N, 3.15. Found: C, 72.79; H, 11.25; N, 3.19. Hexadecyltrimethylammonium naphthalene-1-ylacetate (9): δ ) 0.88 (t, J ) 6.9 Hz, 3H), 1.12-1.32 (br, 28H), 2.7 (s, 9H), 2.832.88 (br, 2H), 3.95 (s, 2H), 7.34-7.42 (br, 4H), 7.61-7.63 (br, 1H), 7.75-7.77 (br, 1H), 8.3-8.32 (br, 1H). Anal. Calcd for C31H51NO2 (1 mol % crystal water): C, 76.34; H, 10.95; N, 2.87. Found: C, 76.41; H, 11.04; N, 2.91. Hexadecyltrimethylammonium diphenylacetate (10): δ ) 0.88 (t, J ) 6.9 Hz, 3H), 1.19-1.33 (br, 26H), 1.42-1.44 (br, 2H), 2.92 (s, 9H), 2.98-3.04 (m, 2H), 4.9 (s, 1H), 7.11-7.13 (m, 2H), 7.197.26 (m, 4H), 7.39-7.42 (m, 4H). Anal. Calcd for C33H53NO2 (1 mol % crystal water): C, 77.14; H, 10.79; N, 2.73. Found: C, 77.16; H, 10.75; N, 2.63. Hexadecyltrimethylammonium benzoate (11): δ ) 0.88 (t, J ) 6.9 Hz, 3H), 1.15-1.33 (br, 26H), 1.51 (br, 2H), 3.25 (s, 9H), 3.453.47 (m, 2H), 7.26-7.3 (m, 3H), 8.01-8.04 (m, 2H). Anal. Calcd for C26H47NO2 (1 mol % crystal water): C, 73.71; H, 11.66; N, 3.31. Found: C, 73.62; H, 11.56; N, 3.42. Hexadecyltrimethylammonium 4-hydroxybenzoate (12): δ ) 0.88 (t, J ) 6.9 Hz, 3H), 1.18-1.33 (br, 28H), 2.84 (s, 9H), 2.91-2.95 (br, 2H), 6.79-6.81 (d, 2H), 7.76-7.79 (d, 2H). Anal. Calcd for C26H47NO3 (1 mol % crystal water): C, 71.03; H, 11.23; N, 3.19. Found: C, 70.97; H, 11.26; N, 3.26.

Hexadecyltrimethylammonium 4-methoxybenzoate (13): δ ) 0.88 (t, J ) 6.9 Hz, 3H), 1.16-1.33 (br, 26H), 1.52-1.53 (br, 2H), 3.193.25 (br, 2H), 3.27 (s, 9H), 3.77 (s, 3H), 6.77-6.8 (d, 2H), 7.967.99 (d, 2H). Anal. Calcd for C27H49NO3 (1 mol % crystal water): C, 71.48; H, 11.33; N, 3.09. Found: C, 71.36; H, 11.31; N, 3.06. Hexadecyltrimethylammonium 4-tert-butylbenzoate (14): δ ) 0.88 (t, J ) 6.9 Hz, 3H), 1.14 (s, 9H), 1.21-1.36 (br, 26H), 1.49-1.51 (br, 2H), 3.23 (s, 9H), 3.67 (br, 2H), 7.29-7.31 (d, 2H), 7.92-7.95 (d, 2H). Anal. Calcd for C30H55NO2 (1 mol % crystal water): C, 75.10; H, 11.97; N, 2.92. Found: C, 74.99; H, 11.85; N, 3.01. Surface Tension Measurements. The critical micelle concentration (cmc) values of the surfactants in Chart 1 were measured using a tensiometer (Jencon, Kolkata, India) by applying the Du Nou¨y ring method at 25 ( 0.1 °C in water. The cmc values were determined (Table 1, Figure 1) by plotting surface tension (γ) versus concentration of surfactant. The accuracy measurements in duplicate experiments were within (2%. Conductometry. In the conductance method, a concentrated solution of the surfactant was added in installments with a micropipet in 20 mL of water (doubly distilled having specific conductivity 2-4 µS cm-1 at 25 °C) placed in a wide-mouth test tube fitted with a dip-type conductivity cell of cell constant 1 cm-1. After each addition, the specific conductance (κ) of the solution was measured with a Toshniwal Instruments Mfg. Pvt. Ltd., Ajmer, India, conductometer at 25 ( 0.1 °C. The specific conductance values were reproducible within the limits (3-5%. The cmc values (Table 1, Figure 2) were obtained from the break point of specific conductance (κ) versus concentration of surfactant plot. Steady-State Fluorescence. The steady-state pyrene monomer fluorescence measurements were performed using a Perkin-Elmer LS55 luminescence spectrometer at 25 °C. The concentration of the pyrene used in all the measurements was ∼1 × 10-7 M. The excitation wavelength was 337 nm, and emission spectra were recorded from 357 to 600 nm. The ratio of intensities of the first vibrational peaks (I1) to those of the third vibrational peaks (I3) of the pyrene emission spectra is very sensitive to the micropolarity of the medium around the probe, and hence, this ratio is used in monitoring the medium polarity. At the same temperature the emissions due to pyrene in water and n-hexane were also measured. Preparation of Microemulsions (Phase Behavior). The mixture of surfactants, n-hexanol, and water was titrated with isooctane to prepare the microemulsions. A constant mass ratio (1:2) of the surfactant and n-hexanol was dissolved in water, forming solutions of different concentrations taken in different screw-topped test tubes and stirred until the solutions became clear. Isooctane was then added to these solutions in measured quantities at 25 °C until just turbid or phase separation. To compare the effect owing to the variation in nature of the counterion of the amphiphiles, the pseudoternary phase diagrams of series I-III surfactants are merged

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Figure 1. Plots of surface tension (γ) vs concentration of surfactants 1-14 at 25 °C.

Figure 2. Plots of specific conductance (κ) vs concentration of surfactants 1-14 at 25 °C. together in Figure 3. The isotropy/turbidity of the solutions were checked by naked eye, which means the measured phase boundaries are of fair accuracy.

Activity of Interfacially Solubilized CV-Lipase. The secondorder rate constant (k2) in lipase-catalyzed hydrolysis of p-nitrophenyl n-hexanoate in cationic reverse micelles was determined (on a

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Figure 4. Variation of the second-order rate constant (k2) for the lipase-catalyzed hydrolysis of p-nitrophenyl n-hexanoate in different cationic W/O microemulsions formed at z ) 8, 25 °C, and pH ) 6.0 (20 mM phosphate). [Surfactant] ) 50 mM, [enzyme] ) 1.02 × 10-6 g mL-1, and [substrate] ) 3 mM. The errors in measured k2 values were within the range (2-6%.

Figure 5. Variation of the second-order rate constant (k2) for the lipase-catalyzed hydrolysis of p-nitrophenyl n-hexanoate in different cationic W/O microemulsions of surfactants using the least amount of n-hexanol required at 25 °C and pH ) 6.0 (20 mM phosphate). [Surfactant] ) 50 mM, [enzyme] ) 1.02 × 10-6 g mL-1, and [substrate] ) 3 mM. The experimental errors have been mentioned in text. W0 ranges where activity was measured are 40-56, 28-36, 28-36, 36-56, 4-16, 4-32, 36-52, 40-64, 40-64, 4-24, 4064, 36-52, 48-68, and 4-12 for surfactants 1-14, respectively. The errors in measured k2 values were within the range (2-6%.

Figure 3. Pseudoternary phase diagrams of the quaternary systems of (1-14)/n-hexanol (1:2 w/w)/water/isooctane at 25 °C. Scale magnitudes are reduced by 1/100th of the plot. Shimadzu UV-1700 spectrophotometer) at the isosbestic points as described previously.6,9a,10,14 In a typical experiment, 4.5 µL of the aqueous enzyme stock solution (0.34 mg/mL) and the substrate (10 µL, from 0.45 M stock solution in isooctane) were added to 1.5 mL of reverse micelles previously prepared with the desired surfactant concentration and pH (pH refers to the pH of the aqueous buffer solutions used for preparing the W/O microemulsions; pH within the water-pool of reverse micelles does not vary significantly,