Nonionic Surfactants: A Key to Enhance the Enzyme Activity at

4130

Langmuir 2007, 23, 4130-4136

Articles Nonionic Surfactants: A Key to Enhance the Enzyme Activity at Cationic Reverse Micellar Interface Anshupriya Shome, Sangita Roy, and Prasanta Kumar Das* Department of Biological Chemistry and Centre for AdVanced Materials, Indian Association for the CultiVation of Science, JadaVpur, Kolkata - 700 032, India ReceiVed September 25, 2006. In Final Form: January 22, 2007 The primary objective of the present study is to understand how the different nonionic surfactants modify the anisotropic interface of cationic water-in-oil (W/O) microemulsions and thus influences the catalytic efficiency of surface-active enzymes. Activity of Chromobacterium Viscosum lipase (CV-lipase) was estimated in several mixed reverse micelles prepared from CTAB and four different nonionic surfactants, Brij-30, Brij-92, Tween-20, and Tween80/water/isooctane/n-hexanol at different z ([cosurfactant]/[surfactants]) values, pH 6 (20 mM phosphate), 25 °C across a varying range of W0 ([water]/[surfactants]) using p-nitrophenyl-n-octanoate as the substrate. Lipase activity in mixed reverse micelles improved maximum up to ∼200% with increasing content of non-ionic surfactants compared to that in CTAB probably due to the reduced positive charge density as well as plummeted n-hexanol (competitive inhibitor of lipase) content at the interfacial region of cationic W/O microemulsions. The highest activity of lipase was observed in CTAB (10 mM) + Brij-30 (40 mM)/isooctane/n-hexanol)/water system, k2 ) 913 ( 5 cm3 g-1 s-1. Interestingly, this observed activity is even higher than that obtained in sodium bis (2-ethyl-1-hexyl) sulfosuccinate (AOT)/n-heptane reverse micelles, the most popular W/O microemulsion in micellar enzymology. To ascertain the influence of non-ionic surfactants in improving the activity of surface-active enzymes is not limited to lipase only, we have also investigated the catalytic activity of Horseradish peroxidase (HRP) in different mixed W/O microemulsions. Here also following the similar trend as observed for lipase, HRP activity enhanced up to 2.5 fold with increasing concentration of nonionic surfactants. Finally, the enzyme activity was correlated with the change in the microenvironment of mixed reverse micelles by steady-state fluorescence study using 8-anilino-1-napthalenesulphonic acid (ANS) as probe.

Introduction Enzymology in self-organized aggregates has gained exponential importance for several decades due to their potential technological as well as biotechnological applications in different branches of science.1-5 When dissolved in water-in-oil (W/O) * To whom correspondence should be addressed. Fax: +(91)-3324732805. 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) Fendler, J. H. Membrane Mimetic Chemistry; Wiley & Sons: New York, 1982. (f) Stenius, P. In ReVerse Micelles; Luisi, P. L., Straub, B. E., Eds.; Plenum: New York, 1984; p 1. (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) Eicke, H. F.; Rehak, J. HelV. Chim. Acta. 1976, 59, 2883. (d) Bommarius, A. S.; Hatton, T. A.; Wang, D. I. C. J. Am. Chem. Soc. 1995, 117, 4515. (3) (a) Luisi, P. L.; Magid, L. J. CRC Crit. ReV. Biochem. 1986, 20, 409. (b) Luisi, P. L. Angew. Chem., Int. Ed. Engl. 1985, 24, 439. (c) Kelley, B. D.; Wang, D. I. C.; Hatton, T. A. Biotechnol. Bioeng. 1993, 42, 1199. (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) Bru, R.; Walde, P. Eur. J. Biochem. 1991, 199, 95. (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. (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.

microemulsions (commonly known as reverse micelles), hydrophilic enzymes like trypsin remain active inside the waterpool, whereas interfacially solubilized enzymes such as lipase and horseradish peroxidase (HRP) locate themselves at the anisotropic interfacial region of the aggregates.5 Enzymes entrapped in reverse micelles can be treated as an excellent biomimetic model for intracellular enzymes.3a,6,7 In living systems, surface-active enzymes usually operate in the cell membrane, being stabilized by lipid mono- or bilayers whose intrinsic components are cationic, zwitterionic, and anionic surfactants.8 To date, the anionic surfactant Aerosol OT (AOT) has been particularly at the center of focus in the field of reverse micellar enzymology in which surface active enzymes like lipase, HRP show superactivities.9,10 Cationic surfactants, owing to its positive charge, are basically known to cause attenuation in efficiency of surface-active enzymes3m,5d,9a,11 through inhibition at the active (5) (a) Stamatis, H.; Xenakis, A.; Kolisis, F. N. Biotechnol. AdV. 1999, 17, 293. (b) Verger, R. In Methods inEnzymology; Colowick, S. P., Kaplan, N. O., Eds.; Academic Press: New York, 1986; 340. (c) Verger.R.; De Haas, G. H. Annu. ReV. Biophys. Bioeng. 1976, 5, 77. (d) Ying, L.; Ganzuo, L.; Chengsong, M. J. Dispersion Sci. Technol. 2000, 21, 409. (d) Mitra, D.; Chakraborty, I.; Bhattacharya, S. C.; Moulik, S. P.; Roy, S.; Das, D.; Das, P. K. J. Phys. Chem. B 2006, 110, 11314. (e) Carvalho, C. M. L.; Aires-Barros, M. R.; Cabral, J. M. S. Langmuir 2000, 16, 3082. (6) Brockerhoff, H.; Jensen, R. G. Lipolytic Enzymes; Academic Press: New York, 1974. (7) (a) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Pantin, V. I.; Berezin, I. V. Biochim. Biophys. Acta 1981, 657, 277. (b) Carvalho, C. M. L.; Cabral, J. M. S. Biochimie 2000, 82, 1063. (8) Larsson, K. Moleular Organization, Physical Functions and Technical Applications; The Oily Press: Dundee, Scotland, 1994; Chapter 1.

10.1021/la062804j CCC: $37.00 © 2007 American Chemical Society Published on Web 03/10/2007

Enzymology in Organized Aggregates

site of solubilized enzymes. However, in recent dates we have been successful in converting the cationic W/O microemulsions as the proficient host for enzymology by changing the headgroup hydrophilicity, size, geometry, nature of counterion, and hydrophobic tail length of the basic building blockssurfactant.12 In every instance, the activities of biocatalysts were improved dramatically, even sometimes significantly better compared to that observed in the AOT-based microemulsion presumably due to the enhanced surface area at the reverse micellar interface or the size of water-pool and thus increasing the local molar concentration of the enzyme and substrate. Interestingly, in our previous works, we have achieved our goal by rational designing of cationic surfactants, which are not very easy to synthesize. A simpler approach for utilizing the cationic reverse micelles as an attractive host for enzyme catalysis is to modify the anisotropic interface only by adding anionic or nonionic surfactants, which are highly abundant.13a-b,14 This added surfactant is expected to reduce the inhibitory power of the cationic surfactant on enzyme by diluting the surface charge density at the interfacial region. To this end, improvement of lipase activity by reducing the charge density of AOT micellar surface in presence of Tween surfactant, along with hydrophobic modification of enzyme using amphiphilic chains of polyethylene glycols, has been reported earlier.13c This unique property of nonionic surfactants has motivated research using mixed reversed micelles, though the basic work until recently has mostly been restricted to their physicochemical characterization.14 A few reports are available on the enzymatic studies in the mixed systems with little understanding on interaction between the enzyme and nonionic surfactant.9d,13a,15 Our present investigation deals with an attempt to understand how the presence of nonionic surfactant modulates the proficiency of surface-active enzymes simply by the alteration of the interfacial properties of the cationic W/O microemulsions. To this notion, we have utilized four different nonionic surfactants Brij-30 (polyethylene glycol dodecyl ether), Brij-92 (polyoxyethylene (2) oleyl ether), Tween-20 (polyoxyethylene (20) sorbitan monolaurate), and Tween-80 (polyoxyethylene sorbitan (20) monooleate) that were coadsorbed in varying extent at the reverse micellar interface of CTAB. Catalytic activity of CVlipase in these mixed reverse micelles was found to be dependent on the nature of nonionic surfactant as well as its composition. (9) (a) Fletcher, P. D. I.; Robinson, B. H.; Freedman, R. B.; Oldfield, C.; J. Chem. Soc., Faraday Trans.1 1985, 81, 2667. (b) Skargelind, P.; Jasson, M. J. Chem. Technol. Biotechnol. 1992, 54, 277. (c) Valis, T. P.; Xenakis, A.; Kolisis, F. N. Biocatalysis 1992, 6, 267. (d) Yamada, Y.; Kuboi, R.; Komasawa, I. Biotechnol. Prog. 1993, 9, 468. (10) (a) Klyachko, N. L.; Levashov, A. V.; Martinek, K. Mol. Biol. 1984, 18, 830. (b) Gebicka, L.; Pawlak, J. J. Mol. Catal. B: Enzymatic 1997, 2, 185.(c) Azevedo, A. M.; Fonseca, L. P.; Graham, D.; Cabral, J. M. S.; Prazeres, D. M. F. Biocatal. Biotransfor. 2001, 19, 213. (11) (a) Davletshin, A. I; Kalabina, N. A.; Zaitsev, S.; Egorov, V. V. Bioorg. Khim. 1998, 6, 430. (b) Brown, E.; Yada, R.; Marangoni, A. Biochim. Biophys. Acta 1993, 66, 1161. (12) (a) Das, D.; Das, P. K. Langmuir 2003, 19, 9114. (b) Mitra, R. N.; Dasgupta, A.; Das, D.; Roy, S.; Debnath, S.; Das, P. K. Langmuir 2005, 21, 12115. (c) Dasgupta, A.; Das, D.; Mitra, R.N.; Das, P. K. J. Colloid Interface Sci. 2005, 289, 566. (d) Das, D.; Roy, S.; Mitra, R. N.; Dasgupta, A.; Das, P. K. Chem. Eur. J. 2005, 11, 4881. (e) Debnath, S.; Dasgupta, A.; Mitra, R. N.; Das, P. K. Langmuir 2006, 22, 8732. (f) Dasgupta, A.; Das, D.; Das, P. K. Biochimie 2005, 87, 1111. (g) Roy, S.; Dasgupta, A.; Das, P. K. Langmuir 2006, 22, 4567. (13) (a) Mahiuddin, S.; Renoncourt, A.; Bauduin, P.; Touraud, D.; Kunz, W. Langmuir 2005, 21, 5259. (b) Holmberg, K.; Nyden, M.; Lee, L. T.; Malmsten, M.; Jha, B. K. AdV. Colloid Interface Sci. 2000, 88, 223. (c) Otero, C.; Robledo, L.; Del Val, M. I. Prog. Colloid Polym. Sci. 1996, 100, 296. (14) (a) Chatterjee, S.; Mitra, R. K.; Paul, B. K.; Bhattacharya, S. C. J. Colloid Interface Sci. 2006, 298, 935. (b) Mitra, R. K.; Paul, B. K.; Moulik, S. P. J. Colloid Interface Sci. 2006, 300, 755. (c) Li, X.; Ueda, K.; Kunieda, H. Langmuir 1999, 15, 7973. (d) Li, X.; Kunieda, H. Curr. Opin. Colloid Interface Sci. 2003, 8, 327. (15) (a) Hossain, M. J.; Takeyama, T.; Hayasi, Y.; Kawanishi, T.: Shimizu, N.; Nakamura, R. J. Chem. Technol. Biotechnol. 1999, 74, 423. (b) Stamatis, H.; Xenakis, A.; Dimitriadis, E.; Kolisis, F. N. Biotechnol. Bioeng. 1995, 45, 33.

Langmuir, Vol. 23, No. 8, 2007 4131

In comparison to CTAB, mixed systems appear to be a better host for lipase where activity improves up to ∼200% with increasing content of nonionic surfactant. Steady-state fluorescence was studied using 8-anilino-1-napthalenesulphonic acid (ANS) in different mixed systems to establish a correlation if any between lipase activity and the hydrophobicity/microviscosity at the micellar interface.16 The generalized influence of nonionic surfactant on surface-active enzyme was also verified using HRP, whose activity was found to be improved with increasing content of nonionic surfactant up to 2.5 fold. Experimental Section Materials. CV-Lipase (EC 3.1.1.3, Type XII) and Horseradish Peroxidase (HRP) (EC 1.11.1.7, Type II, RZ: 2.0) was purchased from Sigma and were used as received. Analytical grade CTAB from Spectrochem (Mumbai, India) was recrystallized three times from methanol/ether, and the recrystallized CTAB was without minima in its surface tension plot. Nonionic surfactants Brij 30, Brij 92 were purchased from Aldrich Chemical Co., while Tween 20 and Tween 80 were from Merck, Mumbai, India. HPLC-grade isooctane, n-hexanol, solvents, and all other reagents used in the syntheses were obtained from SRL (Mumbai, India) and were of the highest analytical grade. The substrate for lipase, p-nitrophenyl-n-octanoate, was synthesized conventionally from an equimolar solution of n-octanoic acid and p-nitrophenol (discussed later). 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 catalytic activity of HRP, was from Ranbaxy, New Delhi, India. ANS, used for fluorometric analysis, was purchased from Aldrich Chemical Co. All these reagents were used without any further purification. Methods. Synthesis of p-Nitrophenyl-n-octanoate. The substrate p-nitrophenyl-n-octanoate ester was prepared conventionally by N,Ndicyclohexylcarbodiimide (DCC) coupling of n-octanoic acid with p-nitrophenol in the presence of 4-N,N-(dimethylamino) pyridine (DMAP). The acid (1 equiv) was added to p-nitrophenol (1 equiv) in presence of 1.1 equiv of DMAP and 1.1 equiv of DCC in dry dichloromethane under the nitrogen atmosphere at -20 °C. After 12-14 h stirring at room temperature, the reaction mixture was filtered and the concentrated filtrate was extracted with ether. Ether part was dried over anhydrous Na2SO4 and concentrated. The product was obtained by purification of the concentrated material through a silica gel (60-120 mesh) column with acetone/hexane solvent mixture as the eluent. The yield of the product was 75-80%. Preparation of Mixed Reverse Micelles. The required quantity of cationic and nonionic surfactant mixture was dispersed in isooctane in a 2 mL volumetric flask, to which calculated amount of n-hexanol was added to attain the corresponding z ([n-hexanol]/[surfactant]) value and shaken vigorously. Then aqueous buffer (phosphate) solution was added to reach the desired W0 ([water]/[surfactants]), and the whole suspension was vortexed to obtain a macroscopically homogeneous solution. Steady-State Fluorescence. The fluorescence study was carried out in Perkin-Elmer LS55 luminescence spectrometer using 4 × 10-6 M (5 µL from 4 mM stock solution of ANS in water was added to 5 mL reverse micellar solution) ANS in mixed reverse micelles of cationic/nonionic systems/water/isooctane/n-hexanol at varying z, W0, and 25 °C. The excitation wavelength was 360 nm, and the emission spectra were observed in the range of 400-650 nm. Fluorescence spectra of ANS were also recorded in water and isooctane. The excitation and emission slit width for the ANS fluorescence experiments were kept constant at 10 and 2.5 nm, respectively. Phase Behavior Study. The mixture of surfactants, n-hexanol, and water was titrated with isooctane to prepare the microemulsions. The surfactant mixtures (1:1, w/w) and n-hexanol taken in a constant mass ratio (1:2, w/w) were dissolved in minimum amount of water (16) Falcone, R. D.; Biasutti, M. A.; Correa, N. M.; Silber, J. J.; Lissi, E.; Abuin, E. Langmuir 2004, 20, 5732.

4132 Langmuir, Vol. 23, No. 8, 2007

Shome et al. Table 1: Isosbestic Points (λ) and Molar Extinction Coefficients (E) of p-nitrophenol/p-nitrophenolate Couple in the W/O Microemulsions of Mixed Surfactant Systems

Figure 1. Pseudoternary phase diagrams of the quaternary systems of CTAB + non-ionic surfactants in (1:1 w/w)/n-hexanol (1:2 w/w)/ water/isooctane at 25 °C. forming solutions of varying concentrations 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. The pseudoternary phase diagrams of the different surfactant mixtures are presented in Figure 1. To compare the effect owing to the difference in nature of amphiphiles, the phase diagrams of different surfactants are merged together in Figure 1a,b. The isotropy/turbidity of the solutions were checked by the 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-nitrophenyln-octanoate in cationic/nonionic reverse micelles were determined (on a Shimadzu UV-1700 spectrophotometer) at the isosbestic points as described previously.9a,12a-e,17 In a typical experiment, 4.5 µL of the aqueous enzyme stock solution (0.34 mg/mL) was added to 1.5 mL of reverse micelle 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, < 1 unit),9a,18 in a cuvette to attain the particular W0. The reverse micellar solutions get clarified within 1 min on gentle shaking. The substrate (10 µL, from 0.45 M stock solution in isooctane) was then added to the cuvette to initiate the enzymatic hydrolysis. The initial linear rate of increase in absorbance, i.e., the absorbance of the liberated p-nitrophenol, was then recorded at the isosbestic points (λiso) in the mixed reverse micelles of CTAB and nonionic surfactants. Beside the hydrolysis of p-nitrophenyl-n-octanoate, there can be a possibility of n-hexanol acting as a substrate, either via alcoholysis of p-nitrophenyl-n-octanoate or trans esterification of the free fatty (17) (a) Crooks, G. E.; Rees, G. D.; Robinson, B. H.; Svensson, M.; Stephenson, G. R. Biotechnol. Bioeng. 1995, 48, 78. (b) Carlie, K.; Rees, G. D.; Robinson, B. H.; Steer, T. D.; Svensson, M. J. Chem. Soc., Faraday Trans. 1996, 92, 4701. (18) Das, P. K.; Chaudhuri, A. Langmuir 1999, 15, 8771.

mixed reverse micelles

isosbestic point (nm)

 (M-1cm-1)

CTAB CTAB + Brij-30 CTAB + Brij-92 CTAB + Tween-20 CTAB + Tween-80 Brij-30

339.0 340.0 341.2 342.4 343.4 340.0

4370 4330 4280 4190 4130 3880

acid produced from the hydrolysis of p-nitrophenyl ester. However, gas chromatographic analysis could not detect the common product of these two side reactions, n-hexyl-n-octanoate. If n-hexanol acts as substrate for alcoholysis, that would have increased the rate of p-nitrophenol liberation at higher alcohol content instead of inhibiting the hydrolysis reaction. The overall concentrations of lipase and p-nitrophenyl-n-octanoate are 1.02 × 10-6 g cm-3 and 3 mM, respectively. The concentration of CV-lipase cannot be expressed in terms of molarity as its overall molecular weight is unknown from the source possibly due to the presence of two forms of lipase, A and B.9a Although the lipase is essentially confined to the dispersed water droplets (at oil-water interface), the concentrations of the reactants were referred to overall concentration to avoid complexity of the volume fraction of water droplets in W/O microemulsion and the partitioning of the substrate.4c,7a,9a,17 In accordance with the previous observations, here too we have measured the second-order rate constant (k2) instead of first-order Michaelis-Menten catalytic constant (kcat), since the initial rate of lipase catalyzed hydrolysis of p-nitrophenyl-n-octanoate were observed to be first order with respect to substrate concentration.9a,12a-e,17,19 Similar observations of second-order rate constant have also been found in micelle mediated organic transformations in chemical systems.20 The isosbestic points (λiso) and molar extinction coefficients () at λiso of the p-nitrophenol/ p-nitrophenolate couple in W/O microemulsions of mixed surfactants (CTAB + nonionic)/water/isooctane/n-hexanol systems were determined spectrophotometrically and the values are given in Table 1. In triplicate experiments, the activities of lipase were found to vary within ( 2-5%. Measurement of Peroxidase Activity. The kinetics of pyrogallol oxidation, catalyzed by HRP, was monitored spectrophotometrically using a Shimadzu UV-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 in a cuvette to attain the particular W0 and reactant concentrations. Gentle shaking produced clarification of the microemulsion within 1 min. Thus, the overall concentration of the substrate and the enzyme inside the cuvette was maintained at 0.3 mM and 1 µg/mL. Here also, the enzyme and substrate concentrations were referred to as the overall concentration to avoid the complexity of the volume fraction of water droplet in the W/O microemulsions and partitioning coefficient of substrates in bulk nonpolar solvent and microaggregates. Finally, 1.0 µL of the hydrogen peroxide stock solution (0.15 M in aqueous phosphate buffer) was added to attain the final concentration of H2O2, 0.1 mM inside the cuvette. The absorbance change was monitored instantaneously after immediate addition of H2O2. The progress of the reaction was monitored by the formation of purpurogallin, the oxidized product of pyrogallol at wavelength of 420 nm (λmax of purpurogallin) for initial 5 min. The initial velocity (V) of this enzymatic oxidation was determined from the slope of the absorption intensity versus time curve, which was linear for the first 5 min, using the molar absorption coefficient of purpurogallin at 420 nm (4400 M-1 cm-1).10a The enzyme activity was expressed (19) Das, P. K.; Chaudhuri, A. Langmuir 2000, 16, 76. (20) (a) Bacaloglu, R.; Bunton, C. A.; Ortega, F. J. Phys. Chem. 1989, 93, 1497. (b) Bacaloglu, R.; Blasko, A.; Bunton, C. A.; Cerichelli, G.; Ortega, F. J. Phys. Chem. 1990, 94, 5062. (c) Brinchi, L.; Profio, P. D.; Germani, R.; Savelli, G.; Bunton, C. A. Langmuir, 1997, 13, 4583.

Enzymology in Organized Aggregates

Langmuir, Vol. 23, No. 8, 2007 4133

Table 2: Mixed Surfactant Systems of Varying Compositions surfactant (mM) system

CTAB

1 2 3 4 5 6 7 8 9 10

50 40 25 10 40 25 10 40 40 -

Brij-30 Brij-92 Tween-20 Tween80 10 25 40 50

10 25 40 -

10 -

10 -

Table 3: W0 Range for the Reverse Micelle Formation by Systems 1-10 (at Different z Values) at pH 6.0 (20 mM Phosphate) and 25 °C system

z ) 3.2

z ) 4.8

z ) 6.4

z ) 8.0

1 2 3 4 5 6 7 8 9 10

4-24 4-20 4-8

40-56 4-40 4-20 20-36 4-24 4-28 4-8

28-48 20-44 4-36 4-24 20-44 4-56 4-12 4-12 4-16 4-8

16-48 4-72 4-56 4-32 4-28 4-24 4-8 4-20 4-24 4

in terms of velocity (µM/min). In triplicate experiments, the activities of HRP were found to vary within ( 2-5%. Stability of Lipase. Six stock solutions of lipase-encapsulated microemulsions were prepared with 50 mM CTAB and different mixed systems. An aliquot of these enzyme solubilized microemulsions is removed at different time interval and the lipase activity was determined using p-nitrophenyl-n-octanoate as substrate. This procedure was repeated for 7 days.

Results and Discussion The present study has been directed to understand how the presence of non-ionic surfactants modifies the anisotropic interface of the cationic W/O microemulsions and thereby influences the proficiency of surface-active enzymes. The pseudoternary phase diagrams of mixed surfactant systems (cationic + nonionic) with n-hexanol (1:2, w/w)/water/isooctane systems at 25 °C are represented in Figure 1. This was a clear systematic phase behavior study to determine the water solubilization region with different nonionic surfactants. In reverse micelles, water solubilization region is always known to be dependent upon several microstructural parameters such as the rigidity of the interfacial film which directly got influenced by the size of the polar head as well as the hydrocarbon tail of the surfactants, nature of the bulk oil system, counterions and many others.21 As a consequence, it is expected that insertion of nonionic surfactant in the W/O microemulsion of CTAB will definitely influence the water solubilization area of CTAB reverse micelles. Surprisingly, effect was not much pronounced as to our expectation except the region having high isooctane content, where the water solubilization region in the mixed system is little bit higher than that in CTAB. Among the mixed systems, CTAB + Brij-30 was found to have greater isotropic region than CTAB + Brij-92 system (Figure 1a). This could have been realized by considering the fact that Brij-30 is having a more flexible head group owing to the greater number of polyoxyethylene units22a compared to Brij-92, which allows better accommodation for water molecules at the interfacial region. No (21) Paul, B. K.; Mitra, R. K. J. Colloid Interface Sci. 2005, 288, 261.

Figure 2. Variation of the second-order rate constant (k2) for the lipase catalyzed hydrolysis of p-nitrophenyl-n-octanoate with composition of mixed reverse micellar systems at different z, 25 °C and pH 6.0 (20 mM phosphate). [Surfactant] ) 50 mM, [lipase] ) 1.02 × 10-6 gmL-1, [substrate] ) 3 mM. Error limits of k2 are within 2-5%.

marked difference in water solubilization area was observed for the mixed surfactant systems comprising Tween-20 or Tween80 and are also in comparable area range with the CTAB system (Figure 1b). Such unaltered isotropic region for both Tween surfactants might have been originated from rigidity of the sorbitan moiety at their polar head, which possibly fails to fetch more water at the interface. Thus, the pseudoternary phase diagrams generate an indication that polar head groups with flexibility posed at the interface plays a crucial role in dictating the molecular packing of the organized aggregates. Toward deciphering the role of nonionic surfactants on enzyme activity, first of all different W/O microemulsions of CTAB and nonionic surfactants were prepared (System 1-10, Table 2) with a systematic increase in the content of nonionic surfactant (Brij30, 2-4; Brij-92, 5-7; Tween-20, 8; and Tween-80, 9), keeping overall concentration of the surfactant constant, 50 mM. Brij-92, Tween-20, and Tween-80 did not form W/O microemulsions on their own in presence/absence of n-hexanol, while later two surfactants even in combination with CTAB failed to form reverse micelles in other varying compositions except the systems 8 and 9. The enzyme activity in reverse micelle formed by only Brij-30 (10) cannot be measured in wide range of W0 due to the solubilization problem of lipase as it forms W/O microemulsions only at W0 ) 4-8 (Table 3). The activity of CV-lipase was initially investigated in the mixed reverse micelles of 0.05 M systems 1-10 (Table 2)/isooctane/n-hexanol/water across a varying range of W0 at pH 6.0 (20 mM phosphate), 25 °C, and z ) 8.0. The z value was kept same for all the systems to have logistical comparison on the influence of nonionic surfactant. The second-order rate constant, k2 for the lipase catalyzed hydrolysis of p-nitrophenyl-n-octanoate was found to be independent of W0. Irrespective of nature of the nonionic surfactants, lipase entrapped in W/O microemulsions of all these mixed systems (2-9, Table 2) showed ∼13-60% improvement in its hydrolytic ability (Figure 2) compared to that was found in case of only CTAB. Even mixed systems 2-4, comprising Brij-30 (22) (a) Bakshi, M. S.; Sing, K.; Kaur, G.; Yoshimura, T.; Esumi, K. Colloids Surf. A 2006, 278, 129. (b) Bumajdad, A.; Eastoe, J.; Griffiths, P.; Steytler, D. C.; Heenan, R. K.; Lu, J. R.; Timmins, P. Langmuir 1999, 15, 5271. (c) Bumajdad, A.; Eastoe, J.; Nave, S.; Steytler, D. C.; Heenan, R. K.; Grillo, I. Langmuir 2003, 19, 2560. (d) Silas, J. A.; Kaler, E. W. J. Colloid Interface Sci. 2001, 243, 248.

4134 Langmuir, Vol. 23, No. 8, 2007

and CTAB exhibited a much improved lipase activity compared to system 10 containing only Brij-30. However, absolute activity of lipase was found to be low and also the effect of nonionic surfactant was not that remarkable possibly due to the presence of high amount of n-hexanol which is quite well-known as a competitive inhibitor for lipase.23 Thus, to improve the activity of lipase and also the modulating role of nonionic surfactant, enzyme activity was further measured lowering the n-hexanol content (z ) 6.4), at which all the systems can form W/O microemulsions. Lipase activity improved in each system compared to that found at z ) 8.0 and importantly the role of nonionic surfactant becomes more visible at z ) 6.4, as enzyme activity increased ∼26-72% compared to that was found in CTAB as well as Brij-30 with varying composition of mixed systems (Figure 2) As a continued effort to improve the lipase’s activity, we have tried to prepare the cationic W/O microemulsions of mixed systems with least amount of n-hexanol needed to form isotropic solutions. Noticeably, the n-hexanol content could be reduced maximum up to z ) 4.8 for systems 1, 5, and 6 and up to z ) 3.2 for 4, 7, and 10 (Table 3). Whereas for Tween surfactants it could not be reduced below z ) 6.4. In each system, the k2 enhances (Figure 2) on lowering the alcohol content producing the best activity for systems 4 and 7 comprising highest content of nonionic surfactant and at the same time the lowest n-hexanol content at which CTAB alone is unable to form W/O microemulsion. Highest activity was obtained with system 4, (k2 ) 913 ( 5 cm3 g-1 sec-1, z ) 3.2) is ∼ 65%, 110%, and 200% higher compared to that was observed in Brij 30 at z ) 3.2 and CTAB microemulsion at z ) 4.8 and 8.0, respectively (Figure 2). Importantly, the observed lipase activity in 4 at z ) 3.2 is even higher than the most popular AOT/n-heptane system (k2 ) 792 ( 9 cm3 g-1 sec-1) under similar experimental conditions. Thus, it is quite clear that the nonionic surfactants definitely play an important role in regulating the lipase activity. Now the question arises what is the probable reason behind this observation? The increasing amount of nonionic surfactants possibly help in reducing the positive surface charge density at the micellar interface so as to decrease the inhibiting action of cationic surfactant on the enzyme to a considerable extent. As a consequence, the lipase activity was boosted up in the mixed reverse micellar systems compared to CTAB. In addition, increasing concentration of Brij surfactants reduce the requirement of co-surfactant (Table 3) to form microemulsions with CTAB. Actually the n-hexanol stabilizes the reverse micelle by lowering the interfacial tension and rigidity by adsorbing preferentially into a curved interfacial region.24,25 Here the inherent flexibility of polyoxyethylene containing head groups of these two Brij surfactants help to stabilize the W/O interface, even at low concentration of cosurfactants. Thus, altogether the nonionic surfactant serves the purpose of a co-surfactant too. Furthermore, if we compare the lipase activity in both (CTAB + Brij-30) and (CTAB + Brij-92) mixed systems, grossly lipase shows better proficiency within the former. This could have been realized by considering the fact that the reverse micellar interface is more flexible in Brij 30 containing systems compared to the Brij-92 owing to the greater number of polyoxyethylene units in Brij-3022a, which probably enhances the interfacial area. The (23) (a) Jenta, T. R. J.; Batts, T.; Rees, G. D.; Robinson, B. H. Biotechnol. Bioeng. 1997, 54, 416. (b) Zhou, G. W.; Li, G. Z.; Xu, J.; Sheng, Q. Colliods Surf., A 2001, 194, 41. (c) Bousquet, D. M. P.; Grabber, M.; Sousa, N. Biochim. Biophys. Acta 2001, 1550, 90. (d) Garcia, A. L. F.; Gotor, V. Biotechnol. Bioeng. 1998, 59, 163. (24) Mitchell, D. J.; Ninham, B. W. J. Chem. Soc., Faraday Trans. 2 1981, 77, 601. (25) de Gennes, P.; Taupin, C. J. Phys. Chem. 1982, 86, 2294.

Shome et al.

Figure 3. Stability of CV-lipase in mixed reverse micellar systems at different z, 25 °C and pH 6.0 (20 mM phosphate). [Surfactant] ) 50 mM, [lipase] ) 1.02 × 10-6 gmL-1, [substrate] ) 3 mM. Error limits of k2 are within 2-5%.

larger numbers of polyoxyethylene units of Brij-30 leads to an enhancement in the microemulsion solubilization and phase stability.22b-d Consequently, the effective concentrations of enzyme and substrate increase in the interfacial region and thereby the lipase activity. Interestingly, mixed systems comprising Tween surfactants (8 and 9) show almost comparable activity with systems 2-7 at z ) 8.0 and even in some cases (z ) 6.4) a slightly improved activity than the CTAB + Brij systems. Probably, a larger sorbitan head group might be sometimes more effective in reducing the positive charge density at the oil-water interface and thus improving the lipase activity. Furthermore, it is not only the head group size or flexibility of the nonionic surfactants which alone dictates the enzyme activity at the interface, varying amphiphilicity of the surfactants definitely have some crucial role to play toward controlling the enzyme proficiency within the microaggregates. All the four surfactants differ in their hydrophilic-lipophilic-balance (HLB) to a great extent, which possibly has been reflected in the corresponding biocatalytic activity. Both the Tween surfactants have a very high HLB value of 15 and 16 for Tween 80 and Tween 20 respectively. Thus Tweens are too hydrophilic to locate at the interface, it will probably partition to the aqueous core mainly. But at higher n-hexanol content (z ) 6.4), they may achieve larger partitioning at the interface so that lipase shows higher activity in systems 8 and 9. Brij-92 has its HLB value 4.9, so it is very nonpolar and partitions mainly to bulk oil. Brij-30 has its HLB value (9.7) in between these two extremes having higher propensity to locate itself at the reverse micellar interface compared to Tweens or Brij-92. As a consequence the (CTAB + Brij-30) mixed reverse micellar system appears to be the most effective host for surface-active enzymes. As it is observed that presence of nonionic surfactant enhances the activity of lipase at cationic reverse micellar systems, it would be essential to determine the influence of these nonionic surfactants on the stability of the enzyme. To this notion, the stability of lipase was checked in reverse micelles of mixed systems and CTAB for 7 days (Figure 3). In pure CTAB, lipase lost ∼50% of its initial activity in 7 days, whereas in the mixed systems after the same period, 81-87% of lipase activity is retained. Thus, mixed system is even better than anionic AOT9a reverse micellar systems where lipase retains only 60% of its activity after 6 days. This result clearly indicates that nonionic

Enzymology in Organized Aggregates

Figure 4. Dependence of HRP activity on compositions of mixed reverse micellar systems at z ) 8, 25 °C, and pH 7.0 (25 mM phosphate). [HRP] ) 1µg/mL, [Pyrogallol] ) 0.3 mM.

surfactants also increase the stability of lipase in mixed reverse micellar systems. Thus, addition of nonionic surfactants can enhance both activity and the stability of surface-active enzymes. To ascertain further that the observed influence of nonionic surfactants is not only specific to lipase, we have estimated the activity of another surface-active enzyme, HRP in systems 1-7 at z ) 8 and W0 ) 20 (in case of system 7, W0 ) 8). HRP activity could not be measured in systems 8-10 owing to the problem in enzyme solubilization within the microaggregates. Following the similar trend as it was observed in case of lipase here too HRP activity enhances with increasing concentration of nonionic surfactants in mixed reverse micelles (Figure 4). At higher concentration of CTAB, for both Brij-30 (system 2) as well as Brij-92 (system 5), HRP shows similar activity (V ) 43.6 µM/ min), which is only slightly higher than that in CTAB W/O microemulsions (V ) 32.7 µM/min). This is presumably due to the fact that here concentration of CTAB is high enough to suppress the effect of nonionic surfactant. With increasing concentration of nonionic surfactants, the activity of HRP increases steadily up to 2 and 2.5 fold for Brij-92 (system 7) and Brij-30 (system 4), respectively (Figure 4), compared to that was found in CTAB only. Activity of HRP is greater in the mixed reverse micelles relative to CTAB (V ) 32.7 µM/min) as nonionic surfactants decline the inhibiting effect of cationic surfactants. Here also enzyme activity in the (CTAB+Brij-30) systems is higher compared to the (CTAB + Brij-92) systems. This again indicates that the flexibility of head group plays important role in dictating the enzyme activity. HRP shows highest activity in the reverse micellar solution of system 4 (V ) 81.8 µM/min, Figure 4). At this point, we were quite eager to know the changes in the microenvironment of cationic reverse micelle in presence of nonionic surfactants, which may have a correlation with enzyme activity. Emission spectra of ANS (4 × 10-6 M) measured in different mixed reverse micellar systems [(50 mM)/water/ isooctane/n-hexanol] are presented in Figure 5. ANS exhibited very low intensity in water at an emission wavelength (λem) 510 nm due to the small quantum yields whereas in isooctane, λem shifted drastically to 402 nm owing to the hydrophobic environment,25 and here also the intensity was found to be very low due to the poor solubility of ANS in isooctane. ANS is most likely to locate itself at the interface of mixed reverse micelles primarily due to the electrostatic attraction. The emission curves in mixed reverse micelles exhibit high intensities at ∼460-475 nm, ∼35-50 nm blue shift compared to that of water, which again proves the localization of ANS at the interface rather than

Langmuir, Vol. 23, No. 8, 2007 4135

Figure 5. Emission spectrum of ANS (4 × 10-6 M) in mixed reverse micellar systems; af ANS in water, λem ) 510 nm, bf ANS in isooctane, λem ) 402 nm, cf CTAB (50 mM), z ) 4.8, W0 ) 40, λem ) 474 nm, df CTAB (50 mM), z ) 6.4, W0 ) 40, λem ) 476 nm, ef CTAB + Brij-30 (40 + 10 mM), z ) 6.4, W0 ) 24, λem ) 472 nm, ff CTAB + Brij-30 (25 + 25 mM), z ) 4.8, W0 ) 20, λem ) 473 nm, gf CTAB + Brij-30(10 + 40 mM), z ) 4.8, W0 ) 20, λem ) 470 nm, hf CTAB + Brij-92 (40 + 10 mM), z ) 6.4, W0 ) 20, λem ) 471 nm, if CTAB + Brij-92 (25 + 25 mM), z ) 4.8, W0 ) 20, λem ) 474 nm, jf CTAB + Brij-92 (10 + 40 mM), z ) 4.8 W0 ) 20, λem ) 470 nm, kf CTAB + Tween-20 (40 + 10 mM), z ) 6.4, W0 ) 20, λem ) 465 nm, lf CTAB + Tween-80 (40 + 10 mM), z ) 6.4, W0 ) 20, λem ) 459 nm.

the water pool. This higher ANS emission intensity and the blue shift of λem was interpreted on the basis of enhanced microviscosity and decreased micropolarity at the binding site of ANS.26 While measuring emission intensity, we have tried to keep W0 constant for all the systems (except in CTAB (50 mM), at z ) 4.8, 6.4, W0 ) 40 and CTAB + Brij-30 (40 mM+10 mM), z ) 6.4, W0 ) 24, as they did not form homogeneous solution at W0 ) 20) to avoid the complexity arising out of varying W0, as it decreases with increasing W0.26b We also wanted to keep the alcohol concentration fixed throughout the experiments again to avoid the influence of n-hexanol on emission spectra. However, we had to vary the z value from 4.8 to 6.4 for systems, which do not form microemulsion at lower n-hexanol content. Thus to have sensible comparison with respect to CTAB, emission spectra were taken at both z value (4.8 and 6.4). Now, if we compare curve c (CTAB 50 mM, z ) 4.8) with curves f-j mixed reverse micelles (CTAB + Brij-30/92 at z ) 4.8), in each case, a very slight blue shift (1-4 nm) and increase in intensity are observed. Similarly, increase in intensity and blue shift (1-17 nm) in curves e, h, k, and l (CTAB + Brij-30/92/Tweens at z ) 6.4) is found compared to curve d (CTAB 50 mM, z ) 6.4). Thus, irrespective of the composition of nonionic surfactants and varying alcohol content, mixed reverse micellar interface is always more microviscous and hydrophobic than the pure CTAB. As a whole the enzyme (lipase or HRP) activity was found to improve in different mixed reverse micelles leading to the conclusion that the activity is directly related to the hydrophobicity/microviscosity at the reverse micellar interface.

Conclusion In this present work, we have been able to modify the architechture of cationic reverse micellar interface simply by addition of nonionic surfactants. They synergistically interact with the cationic surfactants to reduce the positive charge density (26) (a) Kosower, E. M.; Dodiuk, K.; Tanizawa, K.; Ottolengchi, M.; Orbach, N. J. Am. Chem. Soc. 1975, 97, 2167. (b) Manaj, K. M.; Jayakumar, R.; Rakshit, S. K. Langmuir 1996, 12, 4068. (c) Wong, M.; Thomas, J. K.; Gra¨tzel, M. J. Am. Chem. Soc. 1976, 98, 2391.

4136 Langmuir, Vol. 23, No. 8, 2007

at the interface and thereby markedly improving the enzyme activity simply by reducing the chance of cationic inhibition at the active site of the enzyme. Furthermore, nonionic surfactants like Brij, which bears a relatively flexible head group, imparts an additional effect on cationic W/O microemulsions by stabilizing it at a lower cosurfactant concentration, which surely improves enzyme activity to a significant extent. Influence of nonionic surfactants is further generalized from the improvement of another surface-active enzyme, HRP with increasing content of nonionic surfactants. Thus, our present investigation leads to the knowledge that nonionic surfactants improve the activity of surface-active

Shome et al.

enzyme in cationic W/O microemulsion by reducing the surface charge density and increasing the flexibility of interface, which has a correlation with the hydrophobicity/microviscosity at the interface. Acknowledgment. P.K.D. is thankful to Department of Science and Technology, India, for financial assistance through Ramanna Fellowship (No. SR/S1/RFPC-04/2006). A.S. and S.R. acknowledge Council of Scientific and Industrial Research, India, for their Research Fellowships. LA062804J