Tailoring of Horseradish Peroxidase Activity in Cationic Water-in-Oil

Apr 15, 2006 - Sangita Roy, Antara Dasgupta, and Prasanta Kumar Das*. Department of Biological Chemistry, Indian Association for the CultiVation of ...
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Langmuir 2006, 22, 4567-4573

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Tailoring of Horseradish Peroxidase Activity in Cationic Water-in-Oil Microemulsions Sangita Roy, Antara Dasgupta, and Prasanta Kumar Das* Department of Biological Chemistry, Indian Association for the CultiVation of Science, JadaVpur, Kolkata 700 032, India ReceiVed January 30, 2006. In Final Form: March 10, 2006 Horseradish peroxidase (HRP) in cationic water-in-oil (W/O) microemulsions has always been ignored in reverse micellar enzymology, mainly because cationic surfactants are inhibitors of enzyme peroxidase. In the present study, for the first time, we have successfully introduced the cationic W/O microemulsion as an attractive host for efficient HRP activity. To this notion, much improved activity of HRP was observed in the W/O microemulsion of cetyltrimethylammonium bromide (CTAB) with an increase in n-hexanol concentration and W0 ([water]/[surfactant]), presumably due to the increased interfacial area of the microemulsions. In support of our above observation, six surfactants were synthesized with an increased headgroup size where the methyl groups of CTAB were subsequently replaced by the n-propyl and 2-hydroxyethyl groups, respectively, to prepare mono-, di-, and tripropylated/ hydroxyethylated n-hexadecylammonium bromide. The peroxidase activity enhanced with headgroup size and also followed an overall trend similar to that found in the case of CTAB. Possibly, the reduced positive charge density at the augmented interfacial area by means of increase, either in headgroup size, cosurfactant concentration, and/or W0, is not capable of inactivating HRP. Also, the larger space at the interface may facilitate easier solubilization of the enzyme and increase the local concentration of enzyme and substrate, leading to the higher activity of HRP. The best activity was obtained with surfactant N-hexadecyl-N,N,N-tripropylammonium bromide, the highest ever found in any cationic W/O microemulsions, being almost 3 times higher than that found in water. Strikingly, this observed highest activity is comparable with that observed in an anionic bis(2-ethylhexyl)sulfosuccinate sodium salt (AOT)based system, the best W/O microemulsions used for HRP.

Introduction Water-in-oil (W/O) microemulsions (also known as reverse micelles) are optically transparent, thermodynamically stable, nanometer-scale aggregates of water and surfactants dispersed in bulk apolar solvent in which the surfactant molecules pose themselves with their polar head toward the aqueous core, most commonly known as the water pool, and the hydrophobic tail is in contact with the bulk apolar solvent.1 Enzymology in W/O microemulsions has been an area of increasing interest during the past two decades because of their wide-ranging technological as well as biotechnological applications in different branches of science.2,3 Enzymes, the active biocatalyst for all kinds of biochemical transformations in living cells, most commonly remain active * To whom correspondence should be addressed. E-mail: bcpkd@ iacs.res.in. (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) Stenius, P. ReVerse Micelles; Luisi, P. L., Straub, B. E., Eds.; Plenum: New York, 1984; p 1. (d) Fendler, J. H. Acc. Chem. Res. 1976, 9, 153. (e) Silber, J. J.; Biasutti, M. A.; Abuin, E. B.; Lissi, E. AdV. Colloid Interface Sci. 1999, 82, 189. (f) Fendler, J. H. Membrane Mimetic Chemistry; Wiley & Sons: New York, 1982. (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) Menger, F. M. Angew. Chem., Int. Ed. Engl. 1991, 30, 1086. (3) (a) Luisi, P. L. Angew. Chem, Int. Ed. Engl. 1985, 24, 439. (b) Eicke, H. F.; Rehak, J. HelV. Chim. Acta 1976, 59, 2883. (c) Bommarius, A. S.; Hatton, T. A.; Wang, D. I. C. J. Am. Chem. Soc. 1995, 117, 4515. (d) Walde, P.; Han, D.; Luisi, P. L. Biochemistry 1993, 32, 4029. (e) Martinek, K.; Levashov, A. V.; Khmelnitsky, Y. L.; Klyachko, N. L.; Berezin, I. V. Science 1982, 218, 889. (f) Martinek, K.; Klyachko, N. L.; Kabanov, A. V.; Khmelnitsky, Y. L.; Levashov, A. V. Biochim. Biophys. Acta 1989, 981, 161. (g) Menger, F. M.; Yamada, K. J. Am. Chem. Soc. 1981, 101, 6731. (h) Lissi, E. A.; Abuin, E. B. Langmuir 2000, 16, 10084. (i) Luthi, P.; Luisi, P. L. J. Am. Chem. Soc. 1984, 106, 7285. (j) Abuin, E.; Lissi, E.; Duarte, R. Langmuir 2003, 19, 5374. (k) Komives, C. F.; Osborne, D. E.; Russell, A. J. J. Phys. Chem. 1994, 98, 369. (l) Yang, F. X.; Russell, A. J. Biotechnol. Bioeng. 1995, 47, 60.

either at the surface of the biological membranes or inside them. However, the water around the enzyme at the interface, which is considered to be a crucial parameter in regulating enzyme activity, differs enormously in all aspects from “bulk” water.4 Thus, to have a better understanding of the enzyme action, reverse micellar enzymology has been evolved to provide a more realistic biomimetic model for living cells.5,6 To date, hydrolases such as lipase, chymotrypsin, and trypsin are the most extensively studied enzymes in W/O microemulsions compared to oxidoreductase, primarily because of the restored activity of the former. Horseradish peroxidase (HRP), the most popular member of the heme protein family in the plant kingdom, has already proved its importance,7 owing to its well-established property as an oxidoreductase.8 It is its stability and hence its activity over a broad pH and temperature range that have made HRP more versatile to be used in various media other than the aqueous solution.9 Nevertheless, the number of investigations on oxidoreductase in W/O microemulsions is not huge, except for a few reports on (4) Franks, F. WatersA ComprehensiVe Treatise; Plenum: New York, 1975. (5) Brockerhoff, H.; Jensen, R. G. Lipolytic Enzymes; Academic Press: New York, 1974. (6) (a) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Pantin, V. I.; Berezin, I. V. Biochim. Biophys. Acta 1981, 657, 277. (b) Luisi, P. L.; Magid, L. J. CRC Crit. ReV. Biochem. 1986, 20, 409. (c) Carvalho, C. M. L.; Cabral, J. M. S. Biochimie 2000, 82, 1063. (7) (a) Das, P. K.; Caaveiro, J. M. M.; Luque, S.; Klibanov, A. M. J. Am. Chem. Soc. 2002, 124, 782. (b) Xie, Y.; Das, P. K.; Caaveiro, J. M. M.; Klibanov, A. M. Biotechnol. Bioeng. 2002, 79, 105. (c) Xie, Y.; Das, P. K.; Klibanov, A. M. Biotechnol. Lett. 2001, 23, 1451. (d) Henriksen, A.; Schuller, D. J.; Meno, K.; Welinder, K. G.; Smith, A. T.; Gajhede, M. Biochemistry 1998, 37, 8054. (e) Perez, U.; Dunford, H. B. Biochim. Biophys. Acta 1990, 1038, 98. (8) (a) Yamazaki, I. In Molecular Mechanisms of Oxygen ActiVation; Hayaishi, O., Ed.; Academic Press: New York, 1974; Chapter 13, p 535. (b) Dunford, H. B. In Peroxidases in Chemistry and Biology; Everse, J., Everse, K. E., Grishham, M. B., Eds.; CRC Press: Boca Raton, FL, 1991; Vol 2, Chapter 1, p 1. (c) Dunford, H. B.; Stillman, J. S. Coord. Chem. ReV. 1976, 19, 187. (d) Zhu, M.; Huang, X.; Shen, H. Talanta 2001, 53, 927.

10.1021/la0602867 CCC: $33.50 © 2006 American Chemical Society Published on Web 04/15/2006

4568 Langmuir, Vol. 22, No. 10, 2006 Chart 1

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is still an overlooked area in reverse micellar enzymology. To this end, for the first time, we have been able to dramatically enhance the catalytic efficiency of the peroxidase in cationic W/O microemulsions. The catalytic activity of HRP was estimated in W/O microemulsions of cationic surfactants (CTAB and compounds 1-6, Chart 1) with varying headgroup size as a function of surfactant concentration, pH, buffer strength, W0, as well as cosurfactant concentration. Interfacial water may not have an exclusive effect on enzyme efficiency, but the space at the interface plays a crucial role in improving the enzyme efficiency. In the W/O microemulsion of surfactant 3, HRP showed the best activity, which is the highest ever found in any cationic W/O microemulsions and almost 3 times higher than that found in water. Experimental Section

understanding the structure-function relationship of HRP within the anionic W/O microemulsions.10 Peroxidase showed “super activity” in pyrogallol oxidation within the reverse micelles of bis(2-ethylhexyl)sulfosuccinate (Aerosol OT or AOT) in octane, where the catalytic activity was found to be almost 20 times higher than that in water.10a But, to the best of our knowledge, literature on peroxidase’s activity in cationic W/O microemulsions is scarce, and the activity is reported to be very poor whenever investigated.11 Cationic surfactants are known to be inhibitors of the enzyme peroxidase, except when using it in combination with an anionic surfactant to develop a catanionic microreacting system.12 This was realized by considering the Fourier transform infrared (FTIR) study of the enzyme entrapped in both AOTand cetyltrimethylammonium bromide (CTAB)-based microemulsions.13 Peroxidase within AOT reverse micelles showed much resemblance to the native structure observed in aqueous solution, whereas the enzyme structure was partly disturbed in the microemulsion derived from CTAB. However, hitherto, no such attempt has been made toward improving the efficiency of peroxidase in cationic microemulsions, while the versatility in the headgroup of cationic surfactants is becoming notably abundant.14,15 The present investigation deals with an attempt to improve the HRP’s proficiency within cationic W/O microemulsions, which (9) (a) Laszlo, J. A.; Compton, D. L. J. Mol. Catal. B 2002, 18, 109. (b) Smith, K.; Silvermail, N. J.; Rodger, K. R.; Elgren, T. E.; Castro, M.; Parker, R. M. J. Am. Chem. Soc. 2002, 124, 4247. (c) Klibanov, A. M. Nature 2001, 409, 241. (d) Kalra, B.; Gross, R. A. Green Chem. 2002, 4, 174. (e) Kamiya, N.; Inoue, M.; Goto, M.; Nakamura, N.; Naruta, Y. Biotechnol. Prog. 2000, 16, 52. (10) (a) Klyachko, N. L.; Levashov, A. V.; Martinek, K. Mol. Biol. 1984, 18, 830. (b) Gebicka, L.; Pawlak, J. J. Mol. Catal. B: Enzym. 1997, 2, 185. (c) Ma, C. S.; Li, G. Z.; Shen, R. N.; Li, S. B.; Wang, H. Q. J. Dispersion Sci. Technol. 1999, 20, 425. (d) Azevedo, A. M.; Fonseca, L. P.; Graham, D.; Cabral, J. M. S.; Prazeres, D. M. F. Biocatal. Biotransform. 2001, 19, 213. (e) Motlekar, N. A.; Bhagwat, S. S. J. Chem. Technol. Biotechnol. 2001, 76, 643. (f) Parida, S.; Parida, G. R.; Maitra, A. Colloids Surf. 1991, 55, 223. (11) Davletshin, A. I.; Kalabina, N. A.; Zaitsev, S.; Egorov, V. V. Bioorg. Khim. 1998, 6, 430. (12) Mahiuddin, S.; Renoncourt, A.; Bauduin, P.; Touraud, D.; Kunz, W. Langmuir 2005, 21, 5259. (13) Chen, J.; Xia, C.; Niu, J.; Li, S. B. Biochem. Biophys. Res. Commun. 2001, 282, 1220. (14) (a) Menger, F. M.; Catlin, K. K.; Chen, X. Y. Langmuir 1996, 12, 1471. (b) Biresaw, G.; Bunton, C. A.; Quan, C.; Yang, Z. Y. J. Am. Chem. Soc. 1984, 106, 7178. (15) (a) Das, D.; Roy, S.; Mitra, R. N.; Dasgupta, A.; Das, P. K. Chem.sEur. J. 2005, 11, 4881. (b) Mitra, R. N.; Dasgupta, D.; Das, D.; Roy, S.; Debnath, S.; Das, P. K. Langmuir 2005, 21, 12115.

Materials. HRP (EC 1.11.1.7, Type II, RZ: 2.0) was purchased from Sigma and was used as received. AOT was procured from Aldrich Chemical Co. Analytical-grade CTAB 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. Pyrogallol, the substrate used for monitoring HRP activity, was obtained from Qualigens Fine Chemical Company, India. Hydrogen peroxide (30% w/v solution), also used for measuring the catalytic activity of HRP, was from Ranbaxy, India. All these reagents were used without any further purification. 1H NMR spectra were recorded on an AVANCE 300 MHz (Bruker) spectrometer. Chemical shifts are reported in ppm, using trimethylsilane (TMS) for 1H NMR as the internal standard. Mass spectrometric (MS) data were acquired by electrospray ionization (ESI) techniques in a Q-TOF micro-quadruple mass spectrometer (Micromass, UK). The synthetic procedures for the different surfactants (Chart 1) along with their 1H NMR, elemental analysis, and mass spectroscopic data are listed below. All of the amphiphiles were prepared following the procedure mentioned in a couple of recently published protocols.15,16 Synthesis of N-Hexadecyl-N,N-dimethyl-N-propylammonium Bromide (HDMPAB; 1) and N-Hexadecyl-N-methyl-N,N-dipropylammonium Bromide (HMDPAB; 2). Hexadecylamine (1 equiv) and n-propyl bromide (1.2 equiv) were refluxed in a 30% MeOH/ MeCN solvent mixture for 36 h, and then the white solid was crystallized from MeOH/ethyl acetate three times. The desired compound with Rf ) 0.5 (in 10% MeOH/CHCl3) was isolated by column chromatography on a 60-120 mesh silica gel column using MeOH/CHCl3 as the mobile phase. The ammonium salt thus obtained was basified with ammonia and then extracted with ether, and the ether layer was washed with brine to neutrality and concentrated. The secondary amine (N-propyl-N-hexadecylamine) thus obtained was divided into two parts, and the first part was quaternized with excess methyl iodide in the presence of 2.2 equiv of K2CO3 and a pinch of 18-crown-6-ether in dry dimethylformamide (DMF) for 2 h. The reaction mixture was extracted with ethyl acetate and washed with 5% aqueous sodium thiosulfate solution and brine. It was then concentrated to get the iodide form of surfactant 1. Another part of the secondary amine was again refluxed with 1.2 equiv of n-propyl bromide. The corresponding tertiary amine was isolated following the same procedure mentioned above. The tertiary amine thus obtained was then quaternized with methyl iodide in methanol for 2 h, then methanol was evaporated on a rotary evaporator, and the material was taken in ethyl acetate and washed with sodium thiosulfate solution followed by a brine wash. The organic part was concentrated to (16) (a) Das, D.; Das, P. K. Langmuir 2003, 19, 9114. (b) Chatterjee, A.; Maiti, S.; Sanyal, S. K.; Moulik, S. P. Langmuir 2002, 18, 2998. (c) Dasgupta, A.; Das, D.; Das, P. K. Biochimie 2005, 87, 1111.

Tailoring HRP ActiVity in W/O Microemulsions produce the iodide form of surfactant 2. The iodides were then passed through an Amberlyst A-26 bromide ion-exchange column to get the corresponding bromides 1 and 2. These salts were crystallized from MeOH/ether three times to get the pure compounds. HDMPAB (1). 1H NMR (300 MHz, CDCl3): δ 0.79-0.83 (t, J ) 6.82 Hz, 3H), 0.97-1.03 (t, J ) 7.2 Hz, 3H), 1.11-1.18 (br, 26H), 1.56-1.76 (br, 4H), 3.32 (s, 6H), 3.40-3.48 (br, 4H). Anal. calcd for C21H46BrN: C, 64.26; H, 11.81; N, 3.57. Found: C, 64.35; H, 11.60; N, 3.49. MS (ESI) m/z: calcd, 312.36; found, 312.4034 (M+). HMDPAB (2). 1H NMR (300 MHz, CDCl3): δ 0.85-0.90 (t, J ) 6.81 Hz, 3H), 1.04-1.09 (t, J ) 7.23 Hz, 6H), 1.25 (br, 26H), 1.69-1.81 (br, 6H), 3.34 (s, 3H), 3.41-3.47 (br, 6H). Anal. calcd for C23H50BrN: C, 65.69; H, 11.98; N, 3.33. Found: C, 65.85; H, 11.80; N, 3.42. MS (ESI) m/z: calcd, 340.9; found, 340.4234 (M+). Synthesis of N-Hexadecyl-N,N,N-tripropylammonium Bromide (HTPAB; 3). n-Hexadecyl bromide (1.2 equiv) and tripropylamine (1 equiv) was refluxed in a 30% methanol/acetonitrile solvent mixture for 48 h. Then the solvents were removed on a rotary evaporator, and the mixture was crystallized from methanol/ diethyl ether three times to get the pure product 3 with 85% yield. HTPAB (3). 1H NMR (300 MHz, CDCl3); δ 0.77-0.79 (t, J ) 6.6 Hz, 3H), 0.96-1.01 (t, J ) 7.11 Hz, 9H), 1.17 (br, 26H), 1.601.74 (br, 8H), 3.29-3.31 (br, 8H). Anal. calcd for C25H54BrN: C, 66.93; H, 12.13; N, 3.12. Found: C, 66.59; H, 11.93; N, 3.03. MS (ESI) m/z: calcd, 368.43; found, 368.4215 (M+). Synthesis of N-Hexadecyl-N-(2-hydroxyethyl)-N,N-dimethylammonium Bromide (HHEDMAB; 4) and N-Hexadecyl-N,Nbis(2-hydroxyethyl)-N-methylammonium Bromide (HBHEMAB; 5). Briefly, 1-bromohexadecane and the corresponding amines (N,N-dimethylethanolamine for 4 and N-methyldiethanolamine for 5) were taken in the molar ratio 1.2:1 in 30% methanol/acetonitrile and refluxed. After 24 h, the solvent was evaporated on a rotary evaporator, and pure products were obtained by crystallization of the reaction mixture from methanol/ethyl acetate. The yields were 87% and 80% for 4 and 5, respectively. HHEDMAB (4). 1H NMR (300 MHz, CDCl3): δ 0.86 (t, J ) 6.9 Hz, 3H), 1.18-1.33 (m, 26H), 1.77 (br, 2H), 3.41 (s, 6H), 3.453.48 (br, 2H), 3.69 (br, 2H), 4.14 (br, 2H). Anal. calcd for C20H44BrNO: C, 60.89; H, 11.24; N, 3.55. Found: C, 60.85; H, 11.16; N, 3.39. MS (ESI) m/z: calcd, 314.34; found, 314.2605 (M+). HBHEMAB (5). 1H NMR (300 MHz, CDCl3): δ 0.88 (t, 3H), 1.18-1.36 (br, m, 26H), 1.75 (br, 2H), 3.31 (s, 3H), 3.49 (br, 2H), 3.71 (br, 4H), 4.13 (br, 4H). Anal. calcd for C21H46BrNO2: C, 59.42; H, 10.92; N, 3.30. Found: C, 59.45; H, 10.86; N, 3.08. MS (ESI) m/z: calcd, 344.60; found, 344.4506 (M+). Synthesis of N-Hexadecyl-N,N,N-tris(2-hydroxyethyl)ammonium Bromide (HTHEAB; 6). An aqueous solution of NaOH (2.72 g, 0.068 mol, in 25 mL of doubly distilled water) was added dropwise to a mixture of 2-bromoethanol (6.5 g, 0.081 mol) and hexadecylamine (5 g, 0.027 mol) under refluxing conditions. After 24 h of refluxing, the reaction mixture was extracted with chloroform (3 × 50 mL). Chloroform was removed on a rotary evaporator, followed by drying under vacuum. The residue was then crystallized from methanol/ethyl acetate and filtered. The resulting mixture showed three spots (with Rf ) 0.55, 0.4, and 0) on thin-layer chromatography (TLC) using 25:75 (v/v) methanol/chloroform as the TLC developing solvents. The dried product (with Rf ) 0.55) was purified from the white solid obtained from crystallization by column chromatography in a 230-400 mesh silica gel column with 7% methanol/chloroform. The yield was 40%. HTHEAB (6). 1H NMR (300 MHz, CDCl3): δ 0.88 (t, J ) 6.9 Hz, 3H), 1.25-1.34 (m, 26H), 1.83 (br, 2H), 3.18-3.23 (br, 2H), 3.35-3.49 (br, 6H), 4.08 (br, 6H). Anal. calcd for C22H48BrNO3: C, 58.15; H, 10.57; N, 3.08. Found: C, 58.13; H, 10.64; N, 3.08. MS (ESI) m/z: calcd, 374.62; found, 374.2458. Preparation of Microemulsions. A requisite quantity of surfactant was dispersed in isooctane in a 2 mL volumetric flask, to which the calculated amount of n-hexanol was added to attain the corresponding z ([n-hexanol]/[surfactant]) value and shaken vigorously. Finally, aqueous buffer (phosphate) solution was added (to attain the

Langmuir, Vol. 22, No. 10, 2006 4569 corresponding W0), and the whole suspension was vortexed to obtain a clear homogeneous solution. Measurement of Peroxidase Activity. The kinetics of pyrogallol oxidation, catalyzed by peroxidase, was monitored spectrophotometrically using a Shimadzu 1700 spectrophotometer. In a typical experiment, 2.25 µL of the substrate stock solution (from 0.2 M stock in acetone) and 3.0 µL of the aqueous enzyme stock solution (0.5 mg/mL) were added to the 1.5 mL of W/O microemulsion previously prepared with the desired surfactant concentration and pH (pH refers to the pH of the aqueous phosphate buffer solutions used for preparing the W/O microemulsions; the pH within the water pool of W/O microemulsions does not vary significantly, 15 mM), such a minute change in local pH was possibly diminished and stabilized both the vicinity condition of the enzyme and it’s activity. However, to obtain the most enzyme activity, both the buffer strength and the surfactant will be used at a concentration of 25 mM throughout the investigation to prepare the W/O microemulsions for HRP study. Interestingly, we observed in both preceding paragraphs that HRP showed improved activity at higher n-hexanol content while (24) Jiang, T.; Ji, X.; Yuan, Z. Acta Biochim. Biophys. Sin. 1998, 30, 132.

Tailoring HRP ActiVity in W/O Microemulsions

Figure 3. Variation in the relative enzyme activity for the HRPcatalyzed oxidation of pyrogallol at pH ) 7.0 (25 mM phosphate) in CTAB (25 mM) microemulsions with varying W0 at different z values.

optimizing the concentration of surfactant and buffer strength (Figures 1 and 2) for the best composition of microemulsions. At first glance, this was very surprising to us, since we are aware of the inhibiting action of alcohols over the catalytic activity of enzymes such as lipase through the competitive inhibition mechanism.25 Considering the fact that alcohols are generally inhibitors of enzymes, here too it was expected that HRP activity should decrease with an increase in the total n-hexanol concentration, as this would increase the interfacial concentration of the n-hexanol near the enzyme. In this context, the inhibiting ability of n-hexanol over the efficiency of peroxidase is evidenced in anionic and catanionic W/O microemulsions,12,23b but the inhibition mechanism is yet to be established. Importantly, to our knowledge, no thorough investigation has been reported to date to determine the effect of alcohol on HRP entrapped in cationic W/O microemulsions, as they were considered to be poor hosts for HRP. To understand the role of alcohol, first we checked the HRP activity in an aqueous buffer solution (25 mM phosphate; pH 7.0) with 70 mM of n-hexanol (the maximum solubility of n-hexanol in pure water is ∼70 mM). The peroxidase activity was found to be almost the same as that found in the absence of n-hexanol. Although the activity did not change, it is difficult to conclude the role of n-hexanol from this observation, as the solubility of n-hexanol is too low in water. Meanwhile, the interfacial concentration of n-hexanol in cationic W/O microemulsions was found to be in the range of 6-10 M, depending on the actual n-hexanol content in the system.19b However, to check the influence of n-hexanol on HRP activity in wide-ranging solution compositions, enzymatic oxidation of pyrogallol was investigated across a range of W0 (12-28) in a CTAB/water/isooctane/n-hexanol microemulsion with varying z from 25.6 to 51.2 (Figure 3). Regardless of W0, HRP efficiency improved markedly with increasing n-hexanol content in microemulsions. Interestingly, the activity also enhanced steadily with an increase in water content, irrespective of the solution compositions. Even though, with an increasing interfacial (25) (a) Jenta, T. R. J.; Batts, G.; Rees, G. D.; Robinson, B. H. Biotechnol. Bioeng. 1997, 54, 416. (b) Goldberg, C. S.; Tall, A. R.; Krumholz, S. J. Lipid Res. 1984, 25, 714. (c) Zhou, G. W.; Li, G. Z.; Xu, J.; Sheng, Q. Colloids Surf., A 2001, 194, 41. (d) Bousquet, D. M. P.; Graber, M.; Sousa, N. Biochim. Biophys. Acta 2001, 1550, 90. (e) Garcia, A. L. F.; Gotor, V. Biotechnol. Bioeng. 1998, 59, 163.

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Figure 4. Relative enzyme activity change with W0 for the HRPcatalyzed oxidation of pyrogallol at pH ) 7.0 (25 mM phosphate buffer) in cationic W/O microemulsions of CTAB and 1 (25 mM) at z ) 32.

concentration of n-hexanol, the possibility of more n-hexanol interacting with the enzyme increases, the HRP activity improved significantly. Literature reports to date reveal that the concentration of interfacial water is also expected to play a major part in modulating the surface-active enzyme efficiency in the W/O microemulsion. However, according to Das and Chaudhuri, the interfacial water concentration of CTAB W/O microemulsions across a W0 range of 12-44, estimated following the phenyl cation trapping protocol,26,27 is grossly unaltered.28 Yet, it was evidenced in earlier literature that the chemical reactivity of water near the enzyme was influenced by the specific ion effect with varying headgroups.23a However, besides the participation of interfacial water in regulating HRP activity, there must be some other crucial factor, presumably the structure of the W/O microemulsion, which influences the overall effect significantly. It is quite well-known that short-chain alcohols such as n-hexanol, acting as a cosurfactant for the cationic W/O microemulsions, play a very important role in controlling the microstructures. It actually lowers the interfacial tension and rigidity by preferentially adsorbing into a curved interfacial region and thus stabilizing the cationic reverse micelles by increasing the interfacial area.29 We also know that, with gradual increments in W0, the hydrodynamic diameter of the reverse micelles increases,30 which, in turn, definitely increases the area at the anisotropic interface of the corresponding W/O microemulsion. Now the question of how such microstructural changes influence the activity of the biocatalyst arises. However, it was shown by Pinna et al. in a recent paper that the positively charged organic cation (tetramethylammonium, choline) induces a significant decrease in the peroxidase activity in aqueous buffer solution.31 Such inactivation of HRP actually originated from the chaotropic nature of the organic cations due to their lower hydration ability, which makes the surface of the corresponding microaggregates (26) Chaudhuri, A.; Romsted, L. S. J. Am. Chem. Soc. 1991, 113, 5052. (27) Chaudhuri, A.; Loughlin, J. A.; Romsted, L. S.; Yao, J. J. Am. Chem. Soc. 1993, 115, 8351. (28) Das, P. K.; Chaudhuri, A. Langmuir 2000, 16, 76. (29) (a) Mitchell, D. J.; Ninham, B. W.; J. Chem. Soc., Faraday Trans. 2 1981, 77, 601. (b) de Gennes, P.; Taupin, C. J. Phys. Chem. 1982, 86, 2294. (30) (a) Maitra, A. N. J. Phys. Chem. 1984, 88, 5122. (b) Atik, S. S.; Thomas, J. K. J. Am. Chem. Soc. 1981, 103, 4367. (31) Pinna, M. C.; Bauduin, P.; Touraud, D.; Monduzzi, M.; Ninham, B. W.; Kunz, W. J. Phys. Chem. B 2005, 109, 16511.

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Roy et al.

Figure 5. Variation in the relative enzyme activity with W0 for the HRP-catalyzed oxidation of pyrogallol at pH ) 7.0 (25 mM phosphate buffer) in 25 mM cationic W/O microemulsions of 1 (a) and 4-6 (b-d, respectively) at different z values.

sufficiently hydrophobic compared to the anionic ones.32 Therefore, it is quite obvious that, as the interfacial area increased, the ammonium cation with the long hydrophobic chain may have altered its orientation over the broader interfacial space. Consequently, it will expectedly reduce the overall charge density per unit area, as evident from the earlier studies by Mahiuddin et al. who showed that the specific conductivity of W/O microemulsions decreases with simultaneous increments in n-hexanol content in the bulk composition.12 Hence, the reduced charged density arising out of the increased interfacial area is presumably not capable of inactivating HRP. Furthermore, in consistence with our previous studies with lipase,15a it is probable here that the larger space at the interface facilitates easier solubilization of the enzyme and increases the local concentration of enzyme and substrate, leading to the higher activity of HRP. To further ascertain the influence of interfacial space on an enzyme’s efficiency, HRP activity was measured in W/O microemulsions of 1 in comparison with CTAB at fixed z ) 32 (Figure 4). Surfactant 1 comprises a larger headgroup area than does CTAB.15a The n-hexanol content was kept constant to check the exclusive role of the headgroup area on HRP activity. The enhanced headgroup area of 1 dramatically improves the

Figure 6. Variation in the relative enzyme activity for the HRPcatalyzed oxidation of pyrogallol at pH ) 7.0 (25 mM phosphate buffer) in 25 mM microemulsions of 3 and AOT with varying W0 at z ) 9.6.

(32) Buchner, R.; Baar, C.; Fernandez, P.; Schro¨dle, S.; Kunz, W. J. Mol. Liq. 2005, 118, 179.

efficiency of peroxidase with varying W0. This observation distinctly describes the fact that increasing interfacial area, either

Tailoring HRP ActiVity in W/O Microemulsions

Figure 7. Variation in the relative enzyme activity for the HRPcatalyzed oxidation of pyrogallol with varying pH in 25 mM CTAB, 1, and 4/water/isooctane/n-hexanol at z ) 16 and W0 ) 24. The buffers used for different pH solutions were hydrochloric acid/KCl (pH 2 and 3), citric acid/trisodium citrate (pH 4 and 5), sodium dihydrogen phosphate/sodium monohydrogen phosphate (pH 6 and 7), tris buffer (pH 8 and 9), and bicarbonate/carbonate (pH 10).

by headgroup size, W0, or by increasing the concentration of n-hexanol, improves the HRP activity because of the possible reason discussed in the preceding paragraph. To examine the generality of the effect of n-hexanol in terms of increasing the interfacial area as well as decreasing the charged density, HRP activity was estimated in W/O microemulsions of different cationic surfactants, varying the headgroup size as well as the hydrophilicity (Figure 5). Surfactant 1 has a higher headgroup area and is more hydrophobic, as discussed earlier, and 4, 5, and 6, having one, two, and three hydroxyethyl substitutions, respectively, have increasing headgroup area and hydrophilicity. The peroxidase activity profile follows an overall trend similar to that found in the case of CTAB, that is, HRP activity increases with the increase in both n-hexanol content and W0. The activity of HRP cannot be measured in the wider solution composition of 2, as enzyme solubilization was difficult. However, at z ) 9.6, surfactant 2 exhibited relative enzyme activity from 1.6 to 2.0 in the W0 range of 8-20. In the present investigation, the best activity was obtained in the microemulsion of 3, with a z value of 9.6 (Figure 6), the highest ever found in

Langmuir, Vol. 22, No. 10, 2006 4573

any cationic W/O microemulsion. Noticeably, the observed HRP activity is 3 times higher than that found in water. Also the HRP activity in the W/O microemulsion of 3 was compared with a similar solution composition of AOT (25 mM)/isooctane/water. Strikingly, the observed highest activities in both cationic and anionic W/O microemulsions are comparable. This happened for the first time in the literature reports of HRP in cationic W/O microemulsions. Furthermore, we investigated the pH dependence of the peroxidase activity entrapped in a cationic microemulsion of CTAB and surfactant 1 and 4 at a common z ) 16 and fixed W0 ) 24, keeping all other parameters constant across a wide pH range of 2-10, using different buffer solutions (Figure 7). Interestingly, the peroxidase activity was markedly altered across the pH range of 2-10 for the monohydroxylated surfactant, 4, while no considerable change was seen with nonhydroxylated surfactants CTAB and monopropylated 1 (Figure 7) in the same pH range. This is in accordance with our previous observation for another surface-active enzyme, lipase.15b Thus, the catalytic activity of peroxidase is pH sensitive in cationic reverse micelles of hydroxylated surfactant, in contrast to those of nonhydroxylated surfactants. Most importantly, it was quite distinct from this study (Figure 7) that, although the activity in reverse micelles of CTAB and 1 is independent of pH, the enzyme efficiency is much higher in monopropylated surfactant (1) compared to CTAB. Thus, again, it is the space at the interface that is playing the crucial role in improving the enzyme efficiency.

Conclusion In closing, herein we have been able to establish cationic reverse micelles as an efficient microreactor for hosting peroxidase catalysis. Hereafter, cationic microemulsions are expected to receive more attention as attractive hosts for peroxidase instead of as inhibitors of the enzyme. Rational architectural design of cationic surfactants would enable one to improve the enzyme in a proficient manner within the microdomain. Thus, our investigation opens up a new direction in reverse micellar enzymology, which is still in its infancy and needs to be explored further. Acknowledgment. P.K.D is thankful to the Council of Scientific and Industrial Research, India for financial assistance. S.R and A.D.G acknowledge the Council of Scientific and Industrial Research, India for their Research Fellowships. We are thankful to the reviewers for their helpful comments. LA0602867