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Langmuir 1996, 12, 6320-6325
Structural Studies of Lecithin- and AOT-Based Water-in-Oil Microemulsions, in the Presence of Lipase S. Avramiotis,†,§ H. Stamatis,†,‡ F. N. Kolisis,‡ P. Lianos,§ and A. Xenakis*,† Industrial Enzymology Unit, Institute of Biological Research and Biotechnology, National Hellenic Research Foundation, 48 Vas. Constantinou Avenue, 11635 Athens, Greece, Chemical Engineering Department, Division IV, National Technical University of Athens, Zografou Campus, 15700 Athens, Greece, and Engineering Science Department, University of Patras, 26500 Patras, Greece Received July 12, 1996. In Final Form: October 3, 1996X Water-in-oil microemulsions based on lecithin or AOT were used to solubilize lipase from Pseudomonas cepacia. The enzyme activity was tested in the catalysis of an esterification reaction between lauric acid and various short chained aliphatic alcohols. Lipase was found to be more specific for propanol in the lecithin-based system, while in the AOT microemulsions the enzyme did not show any alcohol preference. Fluorescence energy transfer measurements were applied to detect the site of localization of the enzyme molecule within the different domains of the microemulsion droplets. In both cases the enzyme was found to be close to the surfactant interface, with the efficiency of the transfer being more pronounced in the AOT systems. Time-resolved luminescence quenching was also applied to both systems, to probe structural modifications of the microemulsions induced by the presence of lipase. Communication between the dispersed aqueous microdomains, as expressed by the observation of percolative phenomena, was found to be affected.
Introduction Water-in-oil microemulsions are fine dispersions of water in nonpolar organic solvents stabilized by surfactants molecules. These systems, also called reverse micelles, are optically isotropic, low viscous, and thermodynamically stable solutions. The dispersed water pools can solubilize various biomolecules such as enzymes, which may retain their catalytic ability.1,2 The presence of enzymes in the microemulsions results in structural changes of both the biomolecule and the reverse micelles. Most of the studies involving enzyme catalysis in microemulsions have been performed by using AOT, a well-characterized synthetic surfactant that forms spherical reverse micelles and has been considered as a model system for micellar enzymology. Nevertheless, these molecules are generally toxic and cannot be used in potential applications of enzyme-containing microemulsions in the pharmaceutical, cosmetics, or food technology domains.3 An attractive alternative could be to use as surfactants natural emulsifiers such as lecithin. Furthermore these microemulsions formed with phospholipid surfactants can be used as model systems that simulate biological membranes. Since lecithin is known to be slightly too lipophilic to spontaneous form the zero mean curvature phospholipid layers needed for the formation of reverse micelles, a forth component, such as propanol, has to be added to the system to make the polar aqueous phase less hydrophilic.4,5 In the present work lipase from Pseudomonas cepacia was solubilized in lecithin/isooctane/propanol-1/water microemulsions, in order to study the catalysis of a model * To whom correspondence should be addressed. † National Hellenic Research Foundation. ‡ National Technical University of Athens. § University of Patras. X Abstract published in Advance ACS Abstracts, December 1, 1996. (1) Luisi, P. L.; Magid, L. CRC Crit. Rev. Biochem. 1986, 20, 409. (2) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Khmelnitski, Y. L.; Berezin, Y. V. Eur. J. Biochem. 1986, 155, 453. (3) Attwood, D., Florence, A., Eds. Surfactant Systems: Their chemistry, pharmacy and biology; Chapman & Hall, London. 1983. (4) Shinoda, K.; Araki, M.; Sadaghiani, A.; Khan, A.; Lindman, B. J. Phys. Chem. 1991, 95, 989. (5) Shinoda, K.; Shibata, Y.; Lindman, B. Langmuir, 1993, 9, 1254.
S0743-7463(96)00686-5 CCC: $12.00
esterification reaction, as well as the structural modifications of the system induced by the presence of the enzyme. Lipase is a particularly attractive enzyme since apart from its physiological function to hydrolyse triglycerides it may also catalyze under specific conditions synthetic reactions involving fatty acids.6 These biotechnologically interesting reactions can be performed in low water content media such as microemulsions.7 Fluorescence energy transfer measurements were applied to detect the enzyme molecule localization, namely, whether it is in the aqueous core or anchored in the region of the water-oil interface, near the micellar membrane. The effect of the presence of enzyme on the structure of the microemulsion was investigated by time-resolved luminescence quenching spectroscopy by using Ru(bpy)32+ as luminophore and Fe(CN)63- as quencher, both well known as adequate probes of water-in-oil microemulsions.8,9 Similar studies were carried out in the model reverse micellar system of AOT in isooctane containing the same lipase. The results of the above studies are discussed, and a comparison is made between the two different microemulsion systems. Experimental Section Materials. Lipase from P. cepacia was supplied by Dr. U. Menge, GBF, Braunschweig, Germany, in a 96% pure form. The enzyme preparation had a specific activity of 8500 U/mg. The lipase activity was measured in aqueous solution according to Vorderwulbecke et al.10 using p-nitrophenyl palmitate as substrate. Bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT), 99% pure, was purchased from Sigma. Lecithin, containing 18-26% phosphatidylcholine, was purchased from Serva, Heidelberg, Germany. Lecithin was purified by column chromatography using a 35 cm × 3.2 cm column filled with basic alumina (type 5016A, Fluka, Basel, Switzerland). A chloroform/methanol (9/ (6) Sebastiao, M. J.; Cabral J. M. S.; Aires-Barros, M. R. Biotechnol. Bioeng. 1993, 42, 326. (7) Ballesteros, A.; Bornscheuer, U.; Capewell, A.; Combes, D.; Condoret, J. S.; Koenig, K.; Kolisis, F. N.; Marty A.; Menge, U.; Scheper, T.; Stamatis, H.; Xenakis, A. Biocatal. Biotransform. 1995, 13, 1. (8) Attik, S. S.; Thomas, J. K. J. Am. Chem. Soc. 1981, 103, 3453. (9) Lianos, P.; Zana, R.; Lang J.; Cazabat, A. M. In Surfactants in solution; Mittal, K. L., Bothorel, P., Eds.; Plenum Press: New York, 1986; Vol. 6, p 1365. (10) Vorderwulbecke, T.; Kieslich, K.; Erdmann, H. Enzyme Microb. Technol. 1992, 14, 631.
© 1996 American Chemical Society
Lecithin- and AOT-Based w/o Microemulsions 1, v/v) solvent system was used as eluent.11 The fractions were followed by TLC on silica plates (type 60, Merck) using a solvent mixture of chloroform/methanol/water (65/25/4, v/v). The purified phosphatidylcholine was identified by NMR,12-14 and an average molecular mass of 800 Da was determined. The fluorescent probe tris(2,2-bipyridine)ruthenium dichloride hexahydrate, Ru(bpy)3Cl2, was from GFS Chemicals, and the quencher potassium ferricyanide, K3Fe(CN)6, from Merck, Darmstadt, Germany. cis-Parinaric acid (99%) (9,11,13,15-cis,trans, trans,cis-octadecatetranoic acid) was obtained from Molecular Probes. A stock solution of cis-parinaric acid in AOT/isooctane and lecithin/isooctane was stored under argon at -20 °C in the presence of 0.1 mg/L BHT (2,6-di-tert-butyl-4-methylphenol) as antioxidant.15 All alcohols, organic solvents, fatty acids, and assay reagents used were of the highest commercially available purity. High-purity water was obtained by a Millipore Milli Q Plus water purification system. Preparation of Microemulsions. The lecithin microemulsions were prepared as follows: In 5% w/w lecithin in isooctane containing 10% v/v propanol-1, the relevant amount of the substrate lauric acid was added to give a final concentration within the range 25-500 mM. In this mixture appropriate amounts of lipase in 25 mM Tris/HCl pH 7.0 were added, and the final water content was adjusted by the addition of the required amount of buffer. Depending on the experiment wo (wo ) [H2O]/[surfactant]) ranged between 5 and 45. AOT-based microemulsions were prepared by adding a dilute solution of lipase in 25 mM Tris/HCl pH 7 to a stock solution of 0.1 M AOT in isooctane. The final water content was similarly adjusted by addition of buffer. The water content of the initial stock solutions was periodically checked by Karl-Fischer titrations. The amount of water (in general less than 1%) was taken into consideration in the calculation of the global water content. In both cases a transparent solution was obtained after gentle shaking for a few seconds. For the fluorescence experiments the microemulsions were prepared without adding lauric acid. Activity Measurements. The esterification reaction of lauric acid with various alcohols was carried out in capped vials placed in a thermostated bath. The reaction was initiated by adding the enzyme solution as described above. Aliquots of the reaction mixture were withdrawn at selected time intervals and assayed for fatty acid content by a cupric acetate spectrophotometric procedure as described elsewhere.16 For the enzyme stability measurements two microemulsions were prepared: one containing lipase in the absence of fatty acid and the second with fatty acid but with no lipase. These microemulsions were thus identical regarding all the other components. At appropriate time intervals, 0.3 mL of each microemulsion were mixed, and the reaction was followed in a thermostated bath at T ) 35 °C. The final lipase and fatty acid concentrations were 0.034 mg/mL (0.98 µM) and 56 mM, respectively. In the case of enzyme specificity measurements regarding primary aliphatic alcohols, the lecithin microemulsions were prepared using these alcohols as cosurfactants keeping the cosurfactant/surfactant molar ratio constant. The consumption of alcohol during the esterification reaction was assumed not to disturb the structure of the system, since its concentration was always in excess as compared to that of the fatty acid. Identification of Products. The produced esters were isolated by preparative TLC on silica plates (type 60, Merck) using a solvent mixture of hexane/diethylether/acetic acid (80/ 20/1). Identification of the products was carried out by IR and NMR spectroscopy. Fluorescence Energy Transfer. Fluorescence energy transfer takes place from the fluorescent lipase tryptophan residues, acting as donor molecules, to cis-parinaric acid, which acts as an (11) Singleton, W. S.; Gray, M. S.; Brown, M. L.; White, J. L. J. Am. Oil Chem. Soc. 1965, 42, 53. (12) Chapman, D.; Morrison, A. J. Biol. Chem. 1966, 241, 5044. (13) Haque, R.; Tinsley, I. J.; Schmedding, D. J. Biol. Chem. 1972, 247, 157. (14) Capitani, D.; Segre, A. L.; Sparapani, R.; Giustini, M.; Scartazzinni, R.; Luisi, P. L. Langmuir 1991, 7, 250. (15) Sclar, L.; Hudson, B. S.; Petersen, M.; Diamond, J. Biochemistry 1977, 16, 813. (16) Stamatis, H.; Xenakis, A.; Provelegiou, M.; Kolisis, F. N. Biotechnol. Bioeng. 1993, 42, 103.
Langmuir, Vol. 12, No. 26, 1996 6321 acceptor molecule.15,17 The resonance energy transfer efficiency (T) between donor and acceptor could be determined by measuring the decrease of the donor fluorescence intensity in the presence of different concentrations of acceptor, as described by eq 1:17
T ) 1 - FD/FD,o
(1)
where FD refers to the fluorescence intensity of the donor in the presence of acceptor and FD,o refers to the fluorescence intensity of the donor in the absence of acceptor. Energy transfer was examined in lecithin- and AOT-based microemulsion systems by measuring the fluorescence of P. cepacia lipase in the presence of various concentrations of cisparinaric acid ranging from 1.5 × 10-6 to 2.3 × 10-4 M. The concentration of lipase was in both cases kept constant and equal to 4.2 × 10-7 M The fluorescence emission spectra were monitored using a Perkin-Elmer 650-40 fluorospectrometer at 25 °C. The excitation and emission wavelengths were 280 and 330 nm, respectively. Luminescence Decay Measurements. Nanosecond decay profiles were recorded with the photon-counting technique using a specially constructed hydrogen flash and ORTEC electronics. A Melles-Griot interference filter was used for excitation (450 nm) and a cutoff filter (600 nm) for emission. All samples were deoxygenated by the freeze-pump-thaw method. The decay profiles were recorded in 1000 channels at 2.6 ns per channel and were analyzed by least-squares fits using the distribution of the residuals and the autocorrelation function of the residuals as fitting criteria.18 All measurements were performed in thermostated cells at 35 °C. The luminophore was Ru(bpy)32+. Its concentration was maintained at 10-5 M. The decay time of free Ru(bpy)32+ was 530 ns. The quencher was Fe(CN)63-, and its concentration was 2 × 10-4 M. The lipase concentration was varied from 5.6 × 10-6 M to 1.12 × 10-5 M in AOT/isooctane microemulsion systems and from 3.15 × 10-6 M to 7.3 × 10-6 M in lecithin/isooctane microemulsion systems. The analysis of the luminescence decay profiles was carried out with a model of stretched exponentials given by the following equation.19-22
I(t) ) I0 exp(-k0t) exp(-C1tf + C2t2f)
(2)
where 0 < f < 1, while the first order decay rate was calculated by
K(t) ) fC1tf-1 - 2fC2t2f-1
(3)
Fitting eq 2 to the experimental decay profile gives the values of the constant parameters C1, C2, and f, which can be used to find K(t) through eq 3. Equation 2 was previously shown to apply to profiles recorded by the photon-counting technique (i.e. to noisy data), as it is the present case22 and it models all quenching reactions, where the excited luminophore can in principle be quenched by any quencher present. Such a case is a diffusion-controlled quenching, like the present one. Diffusioncontrolled reactions in restricted geometries are distance dependent, which, in terms of rates, creates a time dependence. Thus K is time dependent. Since it is not practical to tabulate all K values for all time values, we usually choose to tabulate K1, i.e. the value of K(t) at the first recorded time channel, the value at the last channel KL, and an average over all values KAV. Thus K1 is the reaction probability at short times and KL at long times. In the case of compartmentalized reactants, K1 is the reaction probability between close-lying reactants and KL is the diffusionlimited rate, which is equivalent to the rate of intercompartment migration of reactants. Finally, the measuring of the noninteger exponent f can be understood in the following manner. Diffusion in organized molecular assemblies is successfully modeled by a random walk in a percolation cluster, either above or below the (17) Lakowicz, J. R. Principles of fluorescence spectroscopy; Plenum Press: New York, 1983. (18) Grinvald, A.; Steinberg, I. Z. Anal. Biochem. 1974, 59, 583. (19) Lianos, P.; Modes, S.; Staikos, G.; Brown, W. Langmuir 1992, 8, 1054. (20) Lianos, P.; Argyrakis, P. J. Phys. Chem. 1994, 98, 7278. (21) Duportail, G.; Merola, F.; Lianos, P. J. Photochem. Photobiol. A: Chem. 1995, 89, 135. (22) Lianos, P. Heterogeneous. Chem. Rev. 1996, 3, 53.
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Figure 1. Effect of the water content, wo, on the velocity of the esterification reaction of propanol-1 with 50 mM lauric acid in lecithin/isooctane/propanol-1/water microemulsions. T ) 35 °C, pH 7, [lipase] ) 0.034mg/mL.
Figure 2. Esterification rate of 100 mM lauric acid by various primary alcohols as a function of alcohol chain length in AOT/ isooctane/water microemulsions (b) and in lecithin/isooctane/ alcohol/water systens (O). T ) 35 °C, pH 7, [Lipase] ) 0.034 mg/mL.
percolation.20 Low f values correspond to communicating clusters, for example isolated, noninteracting reverse micelles. Low f values also correspond to localized (or aggregated) reactants. For example, if the number of reactants is relatively large compared to the number of micelles, then the probability of coexistence of a luminescent molecule and a quencher molecule in the same micelle is high. As a consequence, quenching is rapid. This situation is represented by low f values and relatively high K1 values.
Table 1. Kinetic Constants of the Esterification Reaction of Lauric Acid with Various Alcohols Catalyzed by P. cepacia Lipase in Lecithin Microemulsions; [Lecithin] ) 5% w/w; [Alcohol] ) 1.3 M; [Lipase] ) 0.034 mg/mL; pH ) 7; wo ) 8; T ) 35 °C
Results Enzyme Activity Studies. The P. cepacia lipase activity was studied in the catalysis of the model esterification reaction of lauric acid with propanol-1 in lecithin/ isooctane/propanol-1/water microemulsions. The effect of various parameters, such as pH and temperature, on the lipase activity in the above system, at water/surfactant molar ratio wo ) 8, was studied. In both cases optimum values for the enzyme activity were determined, namely, pH 7.0 and T ) 35 °C. A similar profile of the pH and the temperature effect on the P. cepacia lipase activity has also been observed in anionic microemulsions formulated by AOT in isooctane.23 Figure 1 shows the effect of the water content of the microemulsion, expressed in terms of the molar ratio wo on the lipase activity. As can be seen, the lipase activity follows a rather sharp bell-shaped profile with a maximum at wo ) 8, whereas at lower or higher values of wo a decrease of lipase activity is observed. It is interesting to note that the same behavior was also observed for this lipase in other microemulsion systems. Moreover, the optimum wo value determined above is similar to the one found in nonionic microemulsions formulated with tetraethylene glycol dodecyl ether in isooctane and close to the value determined in AOT systems (wo ) 9).23 This bell-shaped pattern is quite common in enzymic studies in various types of microemulsions and has been discussed in detail.24,25 Effect of Alcohol Chain Length on Enzyme Activity. The specificity of P. cepacia lipase regarding the chain length of the alcohols in both lecithin/isooctane and AOT/ isooctane microemulsions was tested in esterification reactions of lauric acid with various aliphatic alcohols with three to seven carbon atoms in their molecule. Figure 2 shows the effect of the chain length of primary alcohols on their esterification rate in both microemulsion systems (23) Stamatis, H.; Xenakis, A.; Dimitriadis, E.; Kolisis, F. N. Biotechnol. Bioeng. 1995, 45, 33. (24) Bru, R.; Sanchez-Ferrer, A.; Garcia-Carmona, F. Biochem. J. 1989, 259, 355. (25) Maestro, M. J. Mol. Liq. 1989, 42, 71.
alcohol
K m, M
Vmax, µmol min-1 mg-1
Vmax/Km
propanol butanol pentanol hexanol
0.26 0.27 0.52 0.23
102.5 77.8 68.6 16.0
394 288 132 70
for a constant concentration of lauric acid (100 mM). It can be seen that the P. cepacia lipase shows a preference for propanol in both systems, but as the alcohol chain length increases, a different catalytic behavior of the enzyme is observed in the two microemulsions. Namely, in microemulsions formed with lecithin in isooctane, a dramatic decrease of the esterification rate was observed when the alcohol chain length was increased from three to six carbon atoms. On the other hand, in the AOT microemulsions the increase in the alcohol chain length only slightly affected the esterification rate. Since the above studied esterification reaction involves two substrates, we have examined the effect of the lauric acid concentration, keeping constant the alcohol concentration, on the enzyme activity in lecithin microemulsions. The results showed that the enzyme behavior follows typical Michaelis-Menten kinetics. Table 1 presents the apparent kinetic constants of the esterification reaction as determined from Lineweaver-Burk double reciprocal plots for various alcohols. It can be noticed that increasing the alcohol chain length decreases the specificity constant expressed by the ratio Vmax/Km. This result supports the previous observation of Figure 2, confirming that in the lecithin microemulsion systems lipase shows decreasing apparent catalytic specificity as the alcohol chain length increases. In the case of AOT systems the behavior of the same lipase has also been shown to be independent of the substrate concentrations.23,26 The observed different catalytic specificity for the alcoholic substrate is probably due to structural differences between the two microemulsion types, as is examined in the following sections. Enzyme Stability in Lecithin Microemulsions. The lipase stability in lecithin/isooctane/propanol-1/water microemulsions was tested following the esterification of propanol-1 with lauric acid after preincubating the samples, in the absence of the fatty acid, under the optimal reaction conditions (pH, temperature, and wo). As can be (26) Stamatis, H.; Kolisis, F. N.; Xenakis, A.; Bornscheuer, U.; Scheper., T.; Menge, U. Biotechnol. Lett. 1993, 15, 703.
Lecithin- and AOT-Based w/o Microemulsions
Figure 3. Lipase stability in lecithin/isooctane/propanol-1/ water microemulsions as a function of preincubation time. T ) 35 °C, pH 7, [lipase] ) 0.034 mg/mL, wo ) 8, [lauric acid] ) 56 mM.
Langmuir, Vol. 12, No. 26, 1996 6323
Figure 5. Energy transfer efficiency, T, as a function of the ratio [cis-parinaric acid]/[lipase] in AOT/isooctane/water microemulsions with (b) wo ) 10 and (O) wo ) 30 and in lecithin/ isooctane/propanol-1/water microemulsions with (3) wo ) 10 and (1): wo ) 30. T ) 25 °C, pH 7, [lipase] ) 4.2 × 10-7 M, λex ) 280 nm, λem ) 330 nm. Table 2. Values of K1, KL, KAV, and f for the AOT Microemulsion System, wo ) 25, Obtained by Analyzing Time-Resolved Luminescence Quenching Profiles of 10-5 M Ru(bpy)32+ in the Presence of 2 × 10-4 M Fe(CN)63According to Eq 3, at 35 °C
Figure 4. Fluorescence emission spectra of lipase in the absence or presence of cis-parinaric acid in a 0.1 M AOT/ isooctane, wo ) 10, microemulsion. [lipase] ) 4.2 × 10-7 M, [cis-parinaric] ) 1.5 × 10-5 M, T ) 25 °C, pH 7, λex ) 280 nm.
seen in Figure 3, lipase retains about 60% of its activity after a preincubation period of 120 min. It is interesting to note that the lipase stability observed in this work was higher than that determined in AOT/isooctane microemulsions, where after 1 h a 50% loss of activity was found.26 The rapid denaturation of the lipase observed in the AOT-based microemulsions was attributed to electrostatic and hydrophobic interactions between the lipase molecule and the anionic surfactant.23 In the present case, the electric charges of the lecithin molecules are rather balanced and the enzyme behavior is, thus, less affected. A similar stabilizing effect of lipase was observed when nonionic surfactants were added in AOT microemulsion systems.27 Fluorescence Energy Transfer in Microemulsion Systems. Figure 4 shows the effect of cis-parinaric acid on P. cepacia lipase fluorescence intensity incorporated in the AOT/isooctane microemulsion system at wo ) 10. As can be seen, an increase of the cis-parinaric acid concentration decreases the fluorescence of the lipase at 330 nm. A similar effect was also observed in the lecithinbased microemulsions. It is interesting to note that in both microemulsions increasing the lipase concentration increases the cis-parinaric acid fluorescence at 420 nm (data not shown). This observation ensures that all the (27) Yamada, Y.; Kuboi, R.; Komasawa, I. Biotechnol. Progr. 1993, 9, 468.
[lipase], 106 M
K1, 106 s-1
KL, 106 s-1
KAV, 106 s-1
f
0 5.6 7.5 11.2
1.61 22.10 35.40 40.75
0.13 0.12 0.13 0.10
0.20 0.46 0.59 0.63
0.61 0.30 0.25 0.28
changes observed in the fluorescence are due to an energy transfer mechanism and not to either environmental or trivial quenching effects.28 The variation of the energy transfer efficiency, T, as a function of the ratio of the concentrations of cis-parinaric acid to lipase, for two different wo values, in both microemulsion systems is shown in Figure 5. The increase of the energy transfer efficiency is considerably higher in the anionic system than in the lecithin-based one. In the case of the anionic microemulsion, the variation of T was independent of the wo values of the system. In the case of the lecithin microemulsions increasing the wo value from 10 to 30 slightly increased the energy transfer efficiency. Time-Resolved Luminescence Quenching Data. The effect of P. cepacia lipase on the structure of AOT reverse micelles and lecithin w/o microemulsions was examined by applying the luminescence decay technique. A typical quenching reaction between the luminophore Ru(bpy)32+ and the quencher Fe(CN)63- was studied in both systems in order to clarify the similarities or the differences between them. Luminescence decays were recorded in the presence of various lipase concentrations. Table 2 shows the results of the analysis for the AOT microemulsion system. In the sense that f is a measure of the restrictions imposed on quenching by the reaction domain, its value dramatically decreases when lipase is solubilized in the reverse micelles, and within experimental error, continues decreasing while the enzyme concentration gradually increases. This fact means that the reaction environment becomes progressively more restrictive. In addition, the quenching rate at the beginning of the reaction, as reflected in the values of K1, is drastically increased upon addition of the enzyme. These large values of K1 correspond to localized interactions. As already stated, the KL value gives the reaction probability at long time, i.e. by reactant diffusion through long (28) Stryer, L. Annu. Rev. Biochem. 1978, 47, 819.
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Table 3. Values of K1, KL, KAV, and f for the Lecithin Microemulsion System, wo ) 43.5, Obtained by Analyzing Time-Resolved Luminescence Quenching Profiles of 10-5 M Ru(bpy)32+ in the Presence of 2 × 10-4 M Fe(CN)63According to Eq 3, at 35 °C [lipase] 106 M
K1, 106 s-1
KL, 106 s-1
KAV, 106 s-1
f
0 3.15 4.86 7.30
1.35 0.65 0.81 1.00
0.57 0.17 0.04 0.00
0.68 0.21 0.13 0.15
0.86 0.80 0.76 0.77
distances. In the present case KL is different from zero, pointing out that even at long times the reaction goes on with a small but constant probability. Finally, the KAV value, as already explained, gives an average estimate of the reaction rate. The latter substantially increases in the presence of the enzyme, apparently because of reactant localization. Table 3 shows similar results of the analysis for the lecithin-formulated microemulsion system. f is significantly decreased upon addition of lipase and continues decreasing with increasing enzyme concentration. The restriction of the reaction domain imposed by lipase is on the same order of magnitude as the restrictions imposed by trypsin in the same system.29 The decreased quenching efficiency is remarkable in the presence of the lipase. KAV clearly decreases when the enzyme is solubilized in the lecithin microemulsion. The KL value is strongly affected by the presence of the lipase and it is equal to zero for the highest enzyme concentration, showing that the system undergoes a nonpercolation procedure. K1 values remained relatively low, with no significant variation, if we consider that the system has incorporated a large amount of water (wo ) 43.5). Discussion In the present work we studied the behavior of the P. cepacia lipase in lecithin/isooctane and AOT/isooctane microemulsion systems, by following the catalysis of a model esterification reaction. The structural changes occurring in both systems by the enzyme presence were monitored by fluorescence energy transfer and luminescence decay measurements. The results of the enzyme catalytic behavior regarding the specificity toward the alcoholic substrate presented above in Figure 2 showed two different patterns. Namely, the specificity of the P. cepacia lipase decreased with increasing the alcohol chain length in the lecithin systems, but remained relatively unchanged in the AOT ones. This completely different behavior of the enzyme, as regards the catalysis of the very same reaction, is probably related to the particularities of the microemulsion systems. While AOT is known to form spherical reverse micelles,30 in lecithin-based systems the shape of the dispersed droplets remains unclear. In the latter case the nature and the amount of the cosurfactant used influence the extent of the monophasic area of the phase diagram31 and thus the droplet structure. It is well-known that lecithin cannot form water-in-oil microemulsions unless a cosurfactant, such as a short chain alcohol, is added.5 The presence of important quantities of alcohol, about 10%, affects most of the properties of both the dispersed aqueous phase and the surfactant membrane. A consequence of the presence of the alcoholic cosurfactant is the structural differences between AOT and lecithin microemulsions, as will also be discussed later. (29) Avramiotis, S.; Lianos, P.; Xenakis, A. Prog. Colloid Polym. Sci. 1996, 100, 286. (30) Zulauf, M.; Eicke, H. F. J. Phys. Chem. 1979, 83, 480. (31) Avramiotis, S.; Papadimitriou, V.; Cazianis, C. T.; Xenakis, A. Colloids Surf. A, 1996, in press.
The enzyme behavior is affected by the partition of alcohol between the dispersed aqueous phase and the continuous organic phase. Partition considerably varies with alcohol chain length. Short alcohols are mainly solubilized in the aqueous microphase, whereas the longer ones are found in the continuous phase or at the interface. The specificity of lipase depends on the sites of localization of both the enzyme molecule and the substrate within the different microdomains of the system.16 When the alcohol substrate is located in the continuous phase, the diffusion through the surfactant layer may slow down the catalytic process, while in the case of the hydrophilic alcohols, such as propanol, the enzyme is directly accessible. The diffusional barrier of the surfactant membrane is apparently different between the two surfactant types used. Since the AOT interface is dynamic in nature, it is quite accessible for alcohol penetration so that variations in alcohol partitioning are not reflected in the lipase activity. The fluorescence energy transfer experiments allow us to determine the precise site of the enzyme localization within the distinct microdomains of the microemulsion. The nature of the surfactant constituting the interface of the microemulsion may influence the efficiency of the energy transfer, providing thus additional information about the structure of the microemulsion droplets. The acceptor molecule cis-parinaric acid is an amphiphile that can be localized in oil-water interfaces, penetrating into the membrane, among the surfactant molecules. The donor, i.e. the enzyme, is localized in the dispersed phase either in the aqueous core or near the interfacial region, depending on the biomolecule. As the results showed (Figure 5), the water content did not influence the energy transfer efficiency in the AOT systems, while in the lecithin-based ones it was only slightly increased, with increasing wo. Thus in both systems, the size of the droplets, as defined by the water amount, did not affect the energy transfer efficiency, showing that the lipase molecule is preferentially localized near the surfactant membrane. This result is in accordance with a similar observation that has been reported for a lipase from Penicillium simplicissimum in the same AOT/isooctane system.32 In addition, Figure 5 showed that the energy transfer efficiency was almost 50% lower in the lecithin system than in the AOT one, even when cis-parinaric acid concentration was in the former case increased 10-fold for the same lipase concentration. This large difference in energy transfer efficiency between AOT and lecithin microemulsions may be attributed to the varied accessibility of the tryptophan residues to the cis-parinaric acid and to the structural differences of the microdroplets. More specifically in the AOT reverse micelles the lipase molecule is more easily accessible through the surfactant membrane than in the lecithin system, which is more rigid (see also above). The acceptor molecules being also amphiphilic can be intercalated within the hydrophobic chains of the surfactant. The flexible moiety of the parinaric acid comprises a nine carbon atom chain, the length of which is closer to the AOT hydrophobic double chain than to the 16-18 fatty acid hydrophobic part of lecithin. Furthermore, when the microemulsion droplets are spherical, the parinaric acid molecules are uniformly distributed around the enzyme and the fluorescence energy transfer mechanism proceeds independently of the orientation of the tryptophan residues. The spherical shape of the AOT reverse micelles, imposed by the double-branched tails,33 offers such a possibility, allowing an effective energy transfer. Apparently this is not the case of the lecithin(32) Stamatis, H.; Xenakis, A.; Kolisis, F. N.; Malliaris, A. Progr. Colloid Polym. Sci. 1994, 97, 253. (33) Pitre´, F. Ph.D. Thesis, University of Paris VI, 1993.
Lecithin- and AOT-Based w/o Microemulsions
Figure 6. Schematic representation of possible microdroplet structure of (a) AOT reverse micelles; (b) AOT reverse micelles with solubilized lipase; c) lecithin w/o microemulsion (adapted from ref 35); (d) lecithin w/o microemulsion with solubilized lipase.
based microemulsion droplets, where the less efficient transfer suggests a different geometry. The luminescence decay measurements were carried out in order to confirm the structural alterations of the above microemulsions induced by the presence of lipase. This technique has been previously applied in the same microemulsion systems in the presence of either R-chymotrypsin (AOT/isooctane systems)34 or trypsin (lecithinbased microemulsions).29 In the AOT microemulsion system (Table 2) the addition of lipase induced a decrease of the exponent f, corresponding to more restricted geometries, while the quenching rate constants either increased, as in the cases of K1 and KAV , or remained unchanged, as in the case of the long time reaction rate, KL. Apparently, the quenching reaction is more efficient, indicating that the simultaneous presence of an excited luminophore and a quencher is more probable in the presence of lipase. In addition, the reactant diffusion process within the dispersed phase retains a substantial value, showing that the system is percolating independently of the presence of lipase. On the contrary, when the more hydrophilic R-chymotrypsin was added in an AOT system, the quenching was less efficient.34 In view of the above findings, and considering the more lipophilic character of P. cepacia lipase,35 this enzyme seems to occupy a position close to the micellar interface. The restructure of the reverse micelles caused by the presence of lipase at the interface results in a dramatic decrease of the number of micelles, thus increasing the probability of short time interactions (high K1). This decrease of the number of micelles is most probably apparent and due to formation of clusters around lipasecontaining reverse micelles, as presented in Figure 6b. The invariability of KL in the presence of the enzyme probably reflects the result of two opposing effects: the decrease of the number of micelles decreases the probability of collision and solute exchange, i.e. decreases KL, but the modification of the interface facilitates fusion and exchange, i.e. increases KL. So finally, the overall quenching efficiency increases in the presence of enzyme, the short time quenching increases, and the long time (34) Papadimitriou, V.; Xenakis, A.; Lianos, P. Langmuir 1993, 9, 912. (35) Desnuelle, P. Adv. Enzymol. 1961, 23, 129.
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quenching efficiency remains practically unchanged. In conclusion, lipase decreases the number of individual micelles forming clusters and makes the interface less stiff and easier to penetrate. In the lecithin microemulsion, the results indicate a different behavior. First the relatively high f value in the absence of enzyme is typical of large and nonspherical structures, equivalent to large clusters in the percolation model. These large molecular assemblies may not be percolating clusters in the strict mathematical meaning of the term, but the molecular diffusion there has so many degrees of freedom that in a time period of the lifetime of the excited state it behaves as an only slightly restricted motion. The presence of the enzyme has some effect on the structure, but it is relatively small. For these reasons, the variation of K1 is also small; that is, reactant redistribution is minimal. However, two parameters do suffer a net decrease, i.e. the overall quenching efficiency and the ability of the molecular diffusion (decrease of the KAV and KL, respectively). Apparently, lipase has a net effect on the quenching by impeaching molecular diffusion. The only way that a macromolecule with a preference for the water/oil interface can induce such an effect is to block communication between already existing compartments. We thus conclude, in view of the above results, that our lecithin microemulsions consist of multicompartmental structures, for example, clusters of communicating microemulsion droplets (Figure 6c). The presence of macromolecules (i.e. the enzyme) blocks the intracluster and intercompartment communication routes by action on the surfactant/alcohol layer, i.e. the micellar interface (Figure 6d). The above observations lead to three main conclusions: (1) The solubilization site of the P. cepacia lipase is near the interfacial region of both microemulsion systems; (2) the phosphatidylcholine interface seems more rigid and impermeable than the AOT one which is more dynamic and flexible, and (3) the microstructure of the lecithin microemulsion is rather adapted to a channel-like complex network of aqueous compartments with branches than to spherical or rodlike entities, at least in the above reported composition. Figure 6c shows a possible schematic representation of such an organized microphase.36 The channels of the dispersed aqueous phase can communicate at long distances, allowing the observation of percolation phenomena. Nevertheless this communication is not due to droplet collision and fusion, as in the case of the AOT systems (Figure 6a). When an enzyme is introduced in such an organized phase, it can either be localized near the lecithin interface, as in the case of lipase, or be entirely solubilized in a water pool maintaining a water shell around its molecule, not interfering with the surfactant layer, as in the case of the hydrophilic trypsin. In the first case the incorporation of lipase blocks the communication and the diffusion of the water soluble probes, because of its intercalation in the narrow domains of the aqueous network (Figure 6d). Acknowledgment. The authors are very thankful to Dr. U. Menge, GBF, Braunschweig, Germany, for his kind gift of lipase, and Mrs. V. Bekiari for her technical assistance with the luminescence decay measurements. S.A. thanks the Greek Ministry of Education for a leave. LA9606862 (36) Cazabat, A. M.; Chatenay, D.; Guering, P.; Langevin, D.; Meunier, J.; Sorba, O.; Lang, J.; Zana, R.; Paillette, M. In Surfactants in solution; Mittal, K. L., Bothorel, P., Eds.; Plenum Press: New York, 1986; Vol. 3, p 1737.
