Biotechnol. Prog. 1990, 6, 465-47 1
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Modification of Enzyme Activity in Reversed Micelles through Clathrate Hydrate Formation A. Madhusudhan Rao, Huyen Nguyen, and Vijay T. John* Department of Chemical Engineering, Tulane University, New Orleans, Louisiana 70118
The physical phenomenon of clathrate hydrate formation in protein-containing reversed micelles is described. Hydrate formation in reversed micelles is a method of adjusting the water t o surfactant molar ratio, wo,which influences micellar size. Lipase and a-chymotrypsin encapsulated in large reversed micelles of high wo show significant enhancements in activity when the micelle size is reduced through hydrate formation. Alternate methods of micelle size adjustments also show enhancements in activity. The implications for improving the activity of such encapsulated enzymes recovered from fermentation media through phase transfer into reversed micelles are discussed.
Introduction In recent years, reversed micellar systems have been increasingly applied to enzymology (Leser et al., 1987; Martinek et al., 1987; Luisi et al., 1988), protein purification technology (Goklen and Hatton, 1985; Leser et al., 1986; Rahaman et al., 1988),and biomembrane mimetic studies (Wirz and Rosenbusch, 1984). These water-in-oil microemulsions, shown schematically in Figure 1, are capable of solubilizing a variety of proteins through encapsulation in the microaqueous phase. The retention of enzyme catalytic activity in reversed micelles opens up the possibility of conducting a number of novel reactions. A specific example is that of ester synthesis, where the requirement of a minimal water environment is a necessity to shift the equilibrium of the reaction toward formation of the ester bond rather than hydrolysis. In this paper we show how clathrate hydrate formation in an enzyme-containing reversed micellar system can be used to modify catalytic activity. Clathrate hydrates are crystallineinclusions of water and gas, and their formation from bulk water has been traditionally studied with reference to natural gas recovery and processing (Berecz and Balla-Achs, 1983). The phenomenon occurs when a hydrate-forming gas species is contacted with water; at appropriate thermodynamic conditions of temperature and pressure (depending on the gas species), the water crystallizes to the cagelike hydrate structure. Figure 2 illustrates the unit cell of gas hydrate of structure I; dispersion interactions between gas molecules (the guest species) located within the cavities and the host water molecules that form the cavity help stabilize the crystal structure. The formation of clathrate hydrates in reversed micellar solutions is a rather new and interesting phenomenon. In a recent paper (Nguyen et al., 1989), we have shown that hydrates of methane can form in reversed micellar solutions, wherein the water in the microaqueous phase is converted to crystalline hydrate form. Studies involving the applications of the phenomenon to protein-containing reversed micelles are in progress; here we report the use of hydrate formation to enhance catalytic activity in enzyme-containing reversed micelles. Two specific enzymes have been considered, microbial lipase and a-chymotrypsin. Lipase, being an interfacial enzyme (Sarda and
* Author to whom correspondence should be addressed. 8756-7938/90/3006-0465$02.50/0
Figure 1. Schematic of a reversed micelle.
Figure 2. Unit cell of hydrate structure I. The circles represent the host water molecules, and the filled circles illustrate the two types of cavities.
Desnuelle, 1958), would be expected to reside at the micelle wall; a-chymotrypsin, on the other hand, has been reported to reside in the core of the micelle (Pileni et al., 1985). The model reaction considered for lipase catalysis was the synthesis of the octyl oleate ester from oleic acid
0 1990 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1990, Vol. 6, No. 6
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and octanol; that for a-chymotrypsin was the hydrolysis of N-glutaryl-L-phenylalanine-p-nitroanilide (GPNA).
Materials and Methods Lipase from Candida cylindracea, a-chymotrypsin, and the substrates oleic acid and GPNA were obtained from Sigma Chemical Co.; octanol was from Aldrich Chemical Co. Reversed micelle constituents included the anionic surfactant bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT) and isooctane, both obtained from Aldrich (99% purity). Methane (Matheson, >99.9% purity) was used as the hydrate-forming gas. Double-distilled water was used in buffer preparations. Enzyme-encapsulated reversed micellar solutions were prepared by the so-called injection method, wherein the enzyme is first dissolved in buffer a t the appropriate pH: 7.5 for lipase (0.01 M phosphate buffer) and 10.5 for a-chymotrypsin (0.1 M glycine/NaOH buffer). The enzymecontaining aqueous phase is then contacted with isooctane containing AOT at the given concentration. The mixture is stirred with a magnetic stirrer till an optically transparent reversed micellar solution is obtained. The ratio of water t o surfactant, W O ,is the characteristic quantity that influences the size and aggregation number of the micelles (Zulauf and Eicke, 1979). The experimental apparatus used to conduct hydrate formation in reversed micelles is shown schematically in Figure 3 and is similar to that used by Holder and Hand (1982) for studies on hydrate thermodynamics in aqueous solutions. The essence of the setup is a glass-windowed, high-pressure cell suspended in a temperature-controlled methanol/water bath and rocked to maintain agitation of the cell contents. Sampling tubes inserted into the cell enable removal of the liquid phase a t high pressure. Activity measurements for lipase catalysis in reversed micelles were carried out a t 313 K. The reaction was conducted in a shake flask set in a water bath, and samples were analyzed as a function of time through gas chromatography (flame ionization detection, 15-m column, methylsilicone stationary phase, 1.5-pm film thickness). Since extremely good resolutions and peak reproducibilities were obtained for alcohol (octanol) and solvent (isooctane),the ratio of the octanol to isooctane peak areas was simply used as the measure of alcohol conversion and reaction progress.
We used a 1:l molar ratio of acid and alcohol in all experiments; thus the alcohol conversion is directly related to the acid conversion and the ester yield. For a-chymotrypsin, we essentially followed the procedure described by Barbaric and Luisi (1981). This involves introducing into the sample cell of a UV spectrophotometer 0.3 mL of enzyme-containing reversed micelles of a given W Qand 1.2 mL of substrate (GPNA) containing micelles of the same W O ,to give 1.5 mL of solution with final substrate and enzyme concentrations of 0.2 mM and 1.3 pM, respectively. The same procedure is followed for the reference cell with the exception that the 0.3 mL of reversed micellar solution does not contain any enzyme; the difference between the two cells is just the enzyme content. The reaction was followed by monitoring the absorbance at 366 nm due to the nitroaniline released in the process (Barbaric and Luisi, 1981; Luisi, 1985). The slope of the absorbance vs time plot, linear over the first 15 min, was used to determine initial rates. Further specifics of the experiments are described during our discussion of results.
Results and Discussion Prior to the actual description of the results, we briefly discuss the rationale for hydrate formation in proteincontaining reversed micelles. Figure 4 summarizes the essence of hydrate thermodynamics in reversed micelles, with the actual data listed in our earlier paper (Nguyen et al., 1989). Hydrate formation is dependent on the water to surfactant ratio, W O ,and is independent of the amount of surfactant. At each W O ,the system is univariant, as represented by the P-T line for a given micellar size (or W O )in Figure 4; i.e., the pressure a t which hydrates form is determined once the temperature is specified. This equilibrium pressure, or dissociation pressure as it is termed, increases as w o decreases, Le., as the micelles decrease in size. The reasoning is rather intuitive; the smaller the micelles, the more bound the water molecules and the greater the chemical potential induced driving force, manifested as the pressure (van der Waals and Platteeuw, 1959;Nguyen et al., 1989) required to reorient the water molecules to the hydrate crystalline form. The situation in the hydrate cell is represented schematically in Figure 5. As hydrates form and nucleate,
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P
T Figure 4. Characteristics of hydrate formation in reversed micelles. Each line represents the univariant P-T correlation for micelles of a specified WO. The actual data for methane hydrates is listed in Nguyen et al. (1989).
. I Protein
Hydrate
Figure 5. Schematic of the contents of the hydrate cell.
they settle to the bottom of the cell (hydrates being more dense than the bulk organic), allowing sampling of the protein-containing micellar supernatant. Examining the concept in Figure 4 from another angle, if large reversed micelles are introduced into the hydrate cell and the cell is progressively pressurized at a constant temperature, then hydrates progressively form reducing the micellar size (Nguyen et al., 1990). The system is completelyreversible; by decreasing the pressure, hydrates dissociate, and the water released is spontaneously backincorporated into the micelles, increasing their size again. Knowledge of the P-T data and size-wo correlations (Nguyen et al., 1989;Pileni et al., 1985;van der Waals and Platteeuw, 1959) thus allows alteration of micellar size to a desired value simply by manipulating the pressure at a given temperature. The potential relevance to protein extraction and biocatalysis is seen here. Protein extraction from fermentation broth into reversed micelles through phase transfer involves contacting the broth with a reversed micellar solutions constituting a separate phase (Rahamanet al., 1988;Goklen and Hatton, 1985). A maximum of water is therefore solubilized into the micelles, which thus exist at the maximum wo dictated by the ionic strength and pH of the aqueous solution. wo values of reversed micelles in contact with a bulk aqueous phase are typically above 20. For example, we have found through Karl-Fischer titration (Mettler DL18) that the wo of reversed micelles in contact with an aqueous phase containing 0.1 M KC1 and pH 7 is about 24. A t the same time, it has been reported in the literature
(Barbaric and Luisi, 1981; Shield et al., 1985; Han and Rhee, 1981; Luisi, 1985) that many enzymes exhibit a maximum in catalytic activity at an optimal wo that is usually between 7 and 15. While there is no clear rationale for the existence of an optimal W O , the explanation given by Kabanov et al. (1988) may have some merit. These authors attribute the optimal wo to a situation wherein the enzyme is locked into its optimal conformation. At higher wo values, increased conformational fluctuations may prevent acceptance of substrate at the instant of interfacial contact, while at lower wo values, the enzyme is perhaps forced into a nonoptimal conformation. Regardless of the true reasons, the phenomenon has been observed for almost all enzymes, including lipase (Han and Rhee, 1987) and a-chymotrypsin (Barbaric and Luisi, 1987). Thus, we posed the question as to whether modification of wo through hydrate formation can affect enzyme activity, using lipase and a-chymotrypsin as model enzymes. The experiment was to introduce an enzyme-containing reversed micellar solution at high wo into the hydrate cell, pressurize the cell to form hydrates, remove supernatant samples a t progressively higher pressures and thus at progressively smaller W O values, and analyze the samples for catalytic activity. The actual experiments are a little more involved. In order to form hydrates at a reasonable pressure, the system has to be cooled to temperatures approaching 273 K. For example, hydrate dissociation pressures for methane as the hydrate former at 273.15 K, the experimental temperature used, are 2.76 MPa for reversed micelles with wo 15,3.10 MPa for micelles with wo 10, and 4.83 MPa for micelles with wo 5. There is thus the need to maintain enzyme activity at low temperatures, which as we show later was not a difficulty. A second consideration is that sampling has to be done carefully to avoid deactivation through shearing during the process. We found that the process of pressurizing a sampling cell to 0.1-0.3 MPa below the temperature-controlled hydrate cell and recovering the supernatant across this small pressure gradient results in a sample with little enzyme deactivation. Once the sample is recovered, the pressure in the sampling cell can be slowly reduced to atmospheric pressure without destroying activity. Considerably lower pressures can be used with other gases; for example, xenon forms hydrates in reversed micelles of wo 15 at 0.19 MPa and 273.15 K; the corresponding dissociation pressure for ethylene hydrates is 0.62 MPa. The experiments described in this paper were done with methane as the hydrateforming gas species simply because we have studied the thermodynamicsof methane hydrate formation in reversed micelles more thoroughly than other gas species. Furthermore, protein solubility is completely retained during methane hydrate formation at the experimental temperature (273.15 K) and pressures (up to 6 MPa). Results on the two systems are next discussed. Lipasecatalyzed ester synthesis is a bimolecular reaction involving the substrates oleic acid and octanol. Since both substrates are amphiphilic, reaction proceeds at the interface of the micelle. In studying this reaction, we have found that the enzyme rapidly loses its activity in reversed micelles unless contacted with the acyl substrate (oleic acid). Figure 6 illustrates the concept. The fresh reaction mixture (filled circles) denotes the case where the reaction is started by adding both substrates immediately following encapsulation of the enzyme. Incubating the encapsulated enzyme at 313 K for 24 h prior to starting the reaction by adding the substrates results in a significantly less active enzyme; the loss of activity is somewhat mitigated by incubation at 273 K. On the other hand, incubation with
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the acyl substrate for 24 h prior to reaction initiation by adding the alcohol results in activity maintenance. Furthermore, we find that the activity is well maintained when incubation with the acyl substrate is carried out at 273 K rather than a t 313 K. Indeed, a %day incubation run at 273 K also showed good maintenance of activity. The use of the acyl substrate in stabilizing the enzyme appears reasonable when one considers that the mechanism for lipase catalysis involves initial binding to the acyl substrate (Deleuze et al., 1987): Y
AcX+E=AcE+AcY+E X
where AcX and AcY are the acyl donors (the acid and ester, respectively), ACEis the acyl-enzyme complex, and X and Y are the nucleophilic acyl acceptors (water and alcohol, respectively). Thus, the acyl-enzyme binding may optimally position the enzyme a t the interface allowing activity maintenance. The acyl-enzyme coupling is important in experiments involving hydrates, since the process of hydrate formation and sampling at progressively higher pressures takes place over the course of a day or two. Accordingly, the initial sample introduced to the hydrate cell is a lipase-containing reversed micellar solution of wo 24 and oleic acid content 0.05 M. The presence of the amphiphilic substrate then changes the nature of the micelle, and the P-T-wo diagram generated for single-surfactant micelles is no longer applicable for the added cosurfactant case. That is, it is not possible to determine simply from the pressure (at a given temperature) what the wo of the micellar supernatant is, unless a new P-T-wo diagram is generated for the dual surfactant case. We have not done this due to the long time involved in generating such extensive thermodynamic data that is incidental to the concepts of this paper; rather, we simply took out supernatant samples at progressively higher pressures and measured the water content through Karl-Fischer titration to obtain wo values. Once the samples were removed from the hydrate cell, they were contacted with 0.05 M octanol to initiate reaction, which was carried out in a shake flask maintained at 313 K. Figure 7 illustrates lipase activity obtained from reversed micelle samples recovered from the hydrate cell at different wo values (over a pressure range of 2.7-7 MPa at about 273.7 K), starting with an initial solution of w o 24. The activities are compared to those obtained from lipasecontaining micellar solutions, individually prepared a t different w o values. As observed, both sets of experiments imply an optimal wo for maximum activity. The maximum activity for the hydrate-modified case is very slightly lower than that for the individual preparation case, and the optimal wo appears to be shifted to a lower value. The
fact that lipases in reversed micelles can exhibit a significant enhancement of activity when the w ois modified by hydrate formation is indicative of the fact that hydrate formation in the micelles does not lead to enzyme deactivation. We are not able to explain the apparent shift in optimal W O ,but we speculate that it is the result of changes in the microaqueous environment brought about when part of the micellar water is removed through hydrate formation. With a-chymotrypsin, the model reaction is quite different. Although the hydrolysis of GPNA is also bimolecular, one of the substrates (water) is a constituent of the micelles. At the GPNA concentrations used (0.2 mM), less than 0.1% of the micellar water is required for full conversion; the large excess of micellar water implies that there is no effect of the reaction on the wo of the solution. The hydrate formation experiment was simply carried out by introducing an enzyme-containing reversed micellar solution at wo 17, forming hydrates at progressively higher pressures and sampling the supernatant at different pressures. The P-T-wo data developed for empty micelles (Nguyen et al., 1989) hold well for this system, and the W O of the supernatant can be determined simply by noting the equilibrated pressure, although we did verify it by Karl-Fischer titration. As soon as a supernatant sample was taken from the hydrate cell and its w o measured, the enzyme activity was assayed. Figure 8 is similar to Figure 7 and illustrates a-chymotrypsin activities in micelles of different wo values, where wo was adjusted either through individual sample preparation or through hydrate formation. Again, it is seen that the hydrate formation technique of adjusting wo leads to enzyme activity modifications; the comparable activity in both cases implies that hydrate formation within the micelles does not adversely affect enzyme activity. The small shift to a lower optimal wo is observed, which we again attribute to changes in the microaqueous phase. Luisi and co-workers (Luisi, 1985; Barbaric and Luisi, 1981) have shown that the optimal wo for chymotrypsin is a function of the pH of the buffer from which the micellar solution is prepared. In continuing work on hydrate formation in reversed micelles, we are examining the acidity of the modified microaqueous phase by using the method of 31P NMR analysis (Smith and Luisi, 1980). Thus, hydrate formation in enzyme-containing reversed micelles leads to a change in micelle size and a concomitant modification of enzyme activity. The natural question arises as to whether other methods of changing wo may just as well be used to modify enzyme activity. To examine the alternatives, we considered the three cases described next, with the results illustrated in Figures 9 and 10.
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Biotechnol. Prog., 1990, Vol. 6, No. 6
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Case I results from the addition of empty reversed micelles of much smaller w o (small micelles) t o enzymecontaining micelles of higher w oto yield a micellar solution of the optimal WO. For example, mixing micellar solutions of wo 5 and wo 25 in the volume ratio 311 leads to a solution with w o 10 (Figure 9). If the enzyme was originally contained in micelles of w o 25, the procedure leads to a %fold reduction of the enzyme concentration and hence a reduction in total reaction rate. In our comparisons of the different cases, we adjusted (increased) the enzyme concentration in making up the solutions of high W O ,so that the final enzyme concentrations were the same in all cases. The notation 25e + 5 denotes the mixing of mi-
celles of w o 25 (containing enzyme) with micelles of w o 5. Thus, 25e + 5 is identical with 10e in terms of final enzyme: surfactant:water ratios; the only difference is the method of preparation. Case I1 is the situation where AOT (in solid form) is just added to enzyme-containing reversed micelles of large w o to bring down the w o to the optimum value. Here the AOT (and water) concentrations of the final mixture are higher than in Case I, and we use this case to indirectly examine AOT concentration effects on activity. The notation 25e + A in Figure 9, for example, applies to case 11. Case I11 is a variant of cases I and 11, where dissolved AOT in isooctane (i.e., micelles of w o = 0) is added to enzyme-containing micelles of large WO. As in case I, if the AOT concentration is to be constant, the enzyme concentration decreases upon mixing the two solutions. Hence, as in case I, we adjusted the enzyme concentration in the solution of w o 25 such that the final concentration at w o 10 is 0.1 mg/mL. The notation for this case in Figure 9 is 25e A/I. Figure 11 schematically illustrates the phenomenon behind the different methods of adjusting WO. The hydrate formation method reduces w o by reversibly freezing out a part of the microaqueous phase; cases I, 11, and I11 reduce wo by mixing small surfactant aggregates with the larger protein-containing microemulsion droplets. We return to the histograms of Figures 9 and 10 to discuss the results of adjusting w othrough cases I, 11, and 111. In all cases, the final enzyme and substrate concentrations were identical; the AOT concentration for case I1 was higher than the other cases, as noted. It is observed that all methods of w o reduction to a more optimal level lead to enhancement in activity. To an extent, this is a reflection of the fact that reversed micelles are dynamic entities. During the process of collision, constituent exchange, and reformation of micelles, the encapsulated enzyme is perhaps able to alter conformation to a state dictated by the micelle size. However, activity is not enhanced to the levels obtained by encapsulating enzyme at the appropriate w o (10e in Figure 9 and 9e in Figure lo), implying that simultaneous encapsulation and reversed micelle formation leads to the most active conformation. At these wo values, the interpolated activities of the systems modified by hydrate formation are lower (Figures 7 and 8);however, we do get comparable activities at the optimal W O ,which is about 7.5 with both enzymes. We also observe in Figures 9 and 10 that at increased surfactant concentrations (case I1 in Figures 9 and 10) the
+
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Biotechnol. Prog., 1990, Vol. 6,No. 6 100
Support from the National Science Foundation (Grants CBT-8802564 and CBT-8721829) is gratefully acknowledged.
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enhancement in activity is the least. The inhibitory effect of surfactant concentration is clearly shown in Figure 12, where w o is maintained constant. This inhibitory effect of surfactant concentration on enzyme activity has been stated before (Han et al., 1987). However, without specific data on conformation as correlated to activity and as affected by surfactant content, it is hard to give a definitive rationale, besides citing adverse protein-surfactant interactions (Han et al., 1987). It is interesting to note that the inhibition occurs both for the core-resident a-chymotrypsin and for the surface-occupying lipase. As an additional consideration, increased surfactant concentrations ( a t constant W O ,substrate, and enzyme concentrations) imply a larger number of micelles, a smaller fractional occupancy of enzyme, and decreased localized substrate concentrations in the water pool or at the micelle surface. Perhaps some of the inhibition can be simply attributed to reduced enzyme-substrate contact. Finally, one might ask if just freezing the micellar solution could also affect water removal and w oadjustment. For micelles of w o 15 and lower, it has been shown that there is a significant depression of freezing point and that the micellar water crystallizes at around 233 K (Quist and Halle, 1988; Boned et al., 1986). Even more importantly, we feel that the adjustment of micellar size is much better accomplished by the pressurization/depressurization involved in hydrate formation/dissociation. Hydrate formation, while certainly just another way of freezing out some of the intramicellar water, is a controllable method.
Conclusions We have shown that hydrate formation in the water droplets of reversed micelles can alter the size of the micelles, which results in activity modification of enzymes encapsulated in the micelles. Hydrate formation leads to nucleation of intramicellar water to crystalline form and subsequent precipitation of the hydrate crystals. The enzyme remains encapsulated in the resulting micelles of reduced size. Enzyme deactivation is minimal during the process of hydrate formation. Three other methods of reducing the water to surfactant ratio (WO)of enzymecontaining reversed micelles indicate that activity can be enhanced by reduction to a more optimal w o value; full enhancement to the maximum activity is not obtained, however. The hydrate method appears to be able to bring about maximum activity, albeit at a slightly lower w ovalue. These concepts could possibly be applied to improving the activity of enzymes extracted from fermentation broths through contact with a reversed micellar phase. They may be particularly relevant to those enzymes that are to be directly used in minimal water environments.
Literature Cited Barbaric, S.; Luisi, P. L. Micellar Solubilization of Biopolymers in Organic Solvents. 5. Activity and Conformation of a-Chymotrypsin in Isooctane-AOT Reverse Micelles. J. Am. Chem. SOC.1981,103,4239-4244. Berecz, E.; Balla-Achs, M. In Studies in Inorganic Chemistry4: Gas Hydrates; Elsevier Science Publishers: Amsterdam, The Netherlands, 1983. Boned, C.; Peyrelasse, J.; Moha-Ouchane, M. Characterization of Water Dispersion in Water/Sodium Ethylhexyl Sulfosuccinate Microemulsions Using Differential Scanning Calorimetry. J. Phys. Chem. 1986,90,634-637. Deleuze, H.; Langrand, G.; Millet, H.; Baratti, J.; Buono, G.; Triantaphylides, C. Lipase-Catalyzed Reactions in Organic Media: Competition and Applications. Biochim. Biophys. Acta 1987, 91 I, 117-120. GBklen, K. E.; Hatton, T. A. Protein Extraction Using Reverse Micelles. Biotechnol. Prog. 1985, 1, 69-74. Han, D.; Rhee, J. S.; Lee, S.B. Lipase Reaction in AOTIsooctane Reversed Micelles: Effect of Water on Equilibria. Biotechnol. Bioeng. 1987, 30, 381-388. Holder, G. D.; Hand, J. H. Multiple-Phase Equilibria in Hydrates from Methane, Ethane, Propane and Water Mixtures. AIChE J . 1982, 28, 353. Kabanov, A. V.; Levashov, A. V.; Klyachko, N. L.; Pshezhetesky, A. V.; Martinek, K. Enzymes Entrapped in Reversed Micelles of Surfactants in Organic Solvents: A Theoretical Treatment of Catalytic Activity Regulation. J. Theor. Biol. 1988,133, 327-343. Leser, M. E.; Wei, G.; Luisi, P. L.; Maestro, M. Application of Reverse Micelles for the Extraction of Proteins. Biochem. Biophys. Res. Commun. 1986, 135,629-635. Leser, M. E.; Wei, G.; Luthi, P.; Haering, G.; Hochkoppler, A.; Blochliger, E.; Luisi, P. Applications of Enzyme-Containing Reversed Micelles. J . Chim. Phys. 1987, 84, 1113-1118. Luisi, P. L. Enzymes Hosted in Reverse Micelles in Hydrocarbon Solution. Angew. Chem., Int. Ed. Engl. 1985, 24, 439-450. Luisi, P. L.; Giomini, P.; Pileni, M. P.; Robinson, B. H. Reverse Micelles as Hosts for Proteins and Small Molecules. Biochim. Biophys. Acta 1988, 947, 209-246. Martinek, K.; Berezin, I. V.; Khmelnitski, Yu. L.; Klyachko, N. L.; Levashov, A. V. Enzymes Entrapped Into Reversed Micelles of Surfactants in Organic Solvents: Key Trends in Applied Enzymology (Biotechnology). Biocatalysis 1987, 1, 9-15. Nguyen, H.; Phillips, J. B.; John, V. T. Clathrate Hydrate Formation in Reversed Micellar Solutions. J. Phys. Chem. 1989,93, 8123-8126. Nguyen, H.; Reed, W.; John, V. T. Characteristics of ProteinContaining Reversed Micelles Subjected to Clathrate Hydrate Formation Conditions. J. Phys. Chem. 1990, in press. Pileni, M.-P.; Zemb, T.; Petit, C. Solubilization by Reversed Micelles: Solute Localization and Structure Perturbation. Chem. Phys. Lett. 1985, 118, 414-420. Quist, P.; Halle, B. Water Dynamics and Aggregate Structure in Reversed Micelles a t Sub-Zero Temperatures. A Deuteron Spin Relaxation Study. J . Chem. Soc., Faraday Trans. 1 1988,84, 1033-1046. Rahaman, R. S.; Chee, J. Y.; Cabral, J. M. S.; Hatton, T. A. Recovery of an Extracellular Alkaline Protease from Whole Fermentation Broth Using Reversed Micelles. Biotechnol. Prog. 1988, 4, 218-224. Sarda, L.; Desnuelle, P. Action of Pancreatic Lipase upon Esters in Emulsion. Biochim. Biophys. Acta 1958, 30, 513-521. Shield, J. W.; Ferguson, H. D.; Bommarius, A. S.; Hatton, T. A. Enzymes in Reversed Micelles as Catalysts for OrganicPhase Synthesis Reactions. Ind. Eng. Chem. Fundam. 1985, 25,603-612.
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Smith, R. E.; Luisi, P. L. Micellar Solubilization of Biopolymers in Hydrocarbon Solvents. 111. Empirical Definition of an Acidity Scale in Reverse Micelles. Helu. Chim. Acta 1980, 63,2302-2311. van der Waals, J. H.; Platteeuw, J. C. Clathrate Solutions. Adu. Chem. Phys. 1959,2, 1-57. Wirz, J.; Rosenbusch, J. P. The Formation of Reverse Mixed Micelles Consisting of Membrane Proteins and AOT in Isooctane. In Reversed Micelles; Luisi, P. L., Straub, B. E., Eds.; Plenum Press: New York, 1984; pp 231-238.
Zulauf, M.; Eicke, H.-F. Inverted Micelles and Microemulsions in the Temary System HzO/Aerosol-OT/Isooctane As Studied by Photon Correlation Spectroscopy. J. Phys. Chem. 1979, 83,480-486. Accepted August 27, 1990. Registry No. GPNA, 130013-48-8;lipase, 9001-62-1;octyl oleate, 32953-65-4; oleic acid, 112-80-1; octanol, 111-87-5; chymotrypsin, 9004-07-3.
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Modification of Enzyme Activity in Reversed Micelles through Clathrate Hydrate Formation A. Madhusudhan Rao, Huyen Nguyen, and Vijay T. John* Department of Chemical Engineering, Tulane University, New Orleans, Louisiana 70118
The physical phenomenon of clathrate hydrate formation in protein-containing reversed micelles is described. Hydrate formation in reversed micelles is a method of adjusting the water t o surfactant molar ratio, wo,which influences micellar size. Lipase and a-chymotrypsin encapsulated in large reversed micelles of high wo show significant enhancements in activity when the micelle size is reduced through hydrate formation. Alternate methods of micelle size adjustments also show enhancements in activity. The implications for improving the activity of such encapsulated enzymes recovered from fermentation media through phase transfer into reversed micelles are discussed.
Introduction In recent years, reversed micellar systems have been increasingly applied to enzymology (Leser et al., 1987; Martinek et al., 1987; Luisi et al., 1988), protein purification technology (Goklen and Hatton, 1985; Leser et al., 1986; Rahaman et al., 1988),and biomembrane mimetic studies (Wirz and Rosenbusch, 1984). These water-in-oil microemulsions, shown schematically in Figure 1, are capable of solubilizing a variety of proteins through encapsulation in the microaqueous phase. The retention of enzyme catalytic activity in reversed micelles opens up the possibility of conducting a number of novel reactions. A specific example is that of ester synthesis, where the requirement of a minimal water environment is a necessity to shift the equilibrium of the reaction toward formation of the ester bond rather than hydrolysis. In this paper we show how clathrate hydrate formation in an enzyme-containing reversed micellar system can be used to modify catalytic activity. Clathrate hydrates are crystallineinclusions of water and gas, and their formation from bulk water has been traditionally studied with reference to natural gas recovery and processing (Berecz and Balla-Achs, 1983). The phenomenon occurs when a hydrate-forming gas species is contacted with water; at appropriate thermodynamic conditions of temperature and pressure (depending on the gas species), the water crystallizes to the cagelike hydrate structure. Figure 2 illustrates the unit cell of gas hydrate of structure I; dispersion interactions between gas molecules (the guest species) located within the cavities and the host water molecules that form the cavity help stabilize the crystal structure. The formation of clathrate hydrates in reversed micellar solutions is a rather new and interesting phenomenon. In a recent paper (Nguyen et al., 1989), we have shown that hydrates of methane can form in reversed micellar solutions, wherein the water in the microaqueous phase is converted to crystalline hydrate form. Studies involving the applications of the phenomenon to protein-containing reversed micelles are in progress; here we report the use of hydrate formation to enhance catalytic activity in enzyme-containing reversed micelles. Two specific enzymes have been considered, microbial lipase and a-chymotrypsin. Lipase, being an interfacial enzyme (Sarda and
* Author to whom correspondence should be addressed. 8756-7938/90/3006-0465$02.50/0
Figure 1. Schematic of a reversed micelle.
Figure 2. Unit cell of hydrate structure I. The circles represent the host water molecules, and the filled circles illustrate the two types of cavities.
Desnuelle, 1958), would be expected to reside at the micelle wall; a-chymotrypsin, on the other hand, has been reported to reside in the core of the micelle (Pileni et al., 1985). The model reaction considered for lipase catalysis was the synthesis of the octyl oleate ester from oleic acid
0 1990 American Chemical Society and American Institute of Chemical Engineers
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and octanol; that for a-chymotrypsin was the hydrolysis of N-glutaryl-L-phenylalanine-p-nitroanilide (GPNA).
Materials and Methods Lipase from Candida cylindracea, a-chymotrypsin, and the substrates oleic acid and GPNA were obtained from Sigma Chemical Co.; octanol was from Aldrich Chemical Co. Reversed micelle constituents included the anionic surfactant bis(2-ethylhexyl) sulfosuccinate sodium salt (AOT) and isooctane, both obtained from Aldrich (99% purity). Methane (Matheson, >99.9% purity) was used as the hydrate-forming gas. Double-distilled water was used in buffer preparations. Enzyme-encapsulated reversed micellar solutions were prepared by the so-called injection method, wherein the enzyme is first dissolved in buffer a t the appropriate pH: 7.5 for lipase (0.01 M phosphate buffer) and 10.5 for a-chymotrypsin (0.1 M glycine/NaOH buffer). The enzymecontaining aqueous phase is then contacted with isooctane containing AOT at the given concentration. The mixture is stirred with a magnetic stirrer till an optically transparent reversed micellar solution is obtained. The ratio of water t o surfactant, W O ,is the characteristic quantity that influences the size and aggregation number of the micelles (Zulauf and Eicke, 1979). The experimental apparatus used to conduct hydrate formation in reversed micelles is shown schematically in Figure 3 and is similar to that used by Holder and Hand (1982) for studies on hydrate thermodynamics in aqueous solutions. The essence of the setup is a glass-windowed, high-pressure cell suspended in a temperature-controlled methanol/water bath and rocked to maintain agitation of the cell contents. Sampling tubes inserted into the cell enable removal of the liquid phase a t high pressure. Activity measurements for lipase catalysis in reversed micelles were carried out a t 313 K. The reaction was conducted in a shake flask set in a water bath, and samples were analyzed as a function of time through gas chromatography (flame ionization detection, 15-m column, methylsilicone stationary phase, 1.5-pm film thickness). Since extremely good resolutions and peak reproducibilities were obtained for alcohol (octanol) and solvent (isooctane),the ratio of the octanol to isooctane peak areas was simply used as the measure of alcohol conversion and reaction progress.
We used a 1:l molar ratio of acid and alcohol in all experiments; thus the alcohol conversion is directly related to the acid conversion and the ester yield. For a-chymotrypsin, we essentially followed the procedure described by Barbaric and Luisi (1981). This involves introducing into the sample cell of a UV spectrophotometer 0.3 mL of enzyme-containing reversed micelles of a given W Qand 1.2 mL of substrate (GPNA) containing micelles of the same W O ,to give 1.5 mL of solution with final substrate and enzyme concentrations of 0.2 mM and 1.3 pM, respectively. The same procedure is followed for the reference cell with the exception that the 0.3 mL of reversed micellar solution does not contain any enzyme; the difference between the two cells is just the enzyme content. The reaction was followed by monitoring the absorbance at 366 nm due to the nitroaniline released in the process (Barbaric and Luisi, 1981; Luisi, 1985). The slope of the absorbance vs time plot, linear over the first 15 min, was used to determine initial rates. Further specifics of the experiments are described during our discussion of results.
Results and Discussion Prior to the actual description of the results, we briefly discuss the rationale for hydrate formation in proteincontaining reversed micelles. Figure 4 summarizes the essence of hydrate thermodynamics in reversed micelles, with the actual data listed in our earlier paper (Nguyen et al., 1989). Hydrate formation is dependent on the water to surfactant ratio, W O ,and is independent of the amount of surfactant. At each W O ,the system is univariant, as represented by the P-T line for a given micellar size (or W O )in Figure 4; i.e., the pressure a t which hydrates form is determined once the temperature is specified. This equilibrium pressure, or dissociation pressure as it is termed, increases as w o decreases, Le., as the micelles decrease in size. The reasoning is rather intuitive; the smaller the micelles, the more bound the water molecules and the greater the chemical potential induced driving force, manifested as the pressure (van der Waals and Platteeuw, 1959;Nguyen et al., 1989) required to reorient the water molecules to the hydrate crystalline form. The situation in the hydrate cell is represented schematically in Figure 5. As hydrates form and nucleate,
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P
T Figure 4. Characteristics of hydrate formation in reversed micelles. Each line represents the univariant P-T correlation for micelles of a specified WO. The actual data for methane hydrates is listed in Nguyen et al. (1989).
. I Protein
Hydrate
Figure 5. Schematic of the contents of the hydrate cell.
they settle to the bottom of the cell (hydrates being more dense than the bulk organic), allowing sampling of the protein-containing micellar supernatant. Examining the concept in Figure 4 from another angle, if large reversed micelles are introduced into the hydrate cell and the cell is progressively pressurized at a constant temperature, then hydrates progressively form reducing the micellar size (Nguyen et al., 1990). The system is completelyreversible; by decreasing the pressure, hydrates dissociate, and the water released is spontaneously backincorporated into the micelles, increasing their size again. Knowledge of the P-T data and size-wo correlations (Nguyen et al., 1989;Pileni et al., 1985;van der Waals and Platteeuw, 1959) thus allows alteration of micellar size to a desired value simply by manipulating the pressure at a given temperature. The potential relevance to protein extraction and biocatalysis is seen here. Protein extraction from fermentation broth into reversed micelles through phase transfer involves contacting the broth with a reversed micellar solutions constituting a separate phase (Rahamanet al., 1988;Goklen and Hatton, 1985). A maximum of water is therefore solubilized into the micelles, which thus exist at the maximum wo dictated by the ionic strength and pH of the aqueous solution. wo values of reversed micelles in contact with a bulk aqueous phase are typically above 20. For example, we have found through Karl-Fischer titration (Mettler DL18) that the wo of reversed micelles in contact with an aqueous phase containing 0.1 M KC1 and pH 7 is about 24. A t the same time, it has been reported in the literature
(Barbaric and Luisi, 1981; Shield et al., 1985; Han and Rhee, 1981; Luisi, 1985) that many enzymes exhibit a maximum in catalytic activity at an optimal wo that is usually between 7 and 15. While there is no clear rationale for the existence of an optimal W O , the explanation given by Kabanov et al. (1988) may have some merit. These authors attribute the optimal wo to a situation wherein the enzyme is locked into its optimal conformation. At higher wo values, increased conformational fluctuations may prevent acceptance of substrate at the instant of interfacial contact, while at lower wo values, the enzyme is perhaps forced into a nonoptimal conformation. Regardless of the true reasons, the phenomenon has been observed for almost all enzymes, including lipase (Han and Rhee, 1987) and a-chymotrypsin (Barbaric and Luisi, 1987). Thus, we posed the question as to whether modification of wo through hydrate formation can affect enzyme activity, using lipase and a-chymotrypsin as model enzymes. The experiment was to introduce an enzyme-containing reversed micellar solution at high wo into the hydrate cell, pressurize the cell to form hydrates, remove supernatant samples a t progressively higher pressures and thus at progressively smaller W O values, and analyze the samples for catalytic activity. The actual experiments are a little more involved. In order to form hydrates at a reasonable pressure, the system has to be cooled to temperatures approaching 273 K. For example, hydrate dissociation pressures for methane as the hydrate former at 273.15 K, the experimental temperature used, are 2.76 MPa for reversed micelles with wo 15,3.10 MPa for micelles with wo 10, and 4.83 MPa for micelles with wo 5. There is thus the need to maintain enzyme activity at low temperatures, which as we show later was not a difficulty. A second consideration is that sampling has to be done carefully to avoid deactivation through shearing during the process. We found that the process of pressurizing a sampling cell to 0.1-0.3 MPa below the temperature-controlled hydrate cell and recovering the supernatant across this small pressure gradient results in a sample with little enzyme deactivation. Once the sample is recovered, the pressure in the sampling cell can be slowly reduced to atmospheric pressure without destroying activity. Considerably lower pressures can be used with other gases; for example, xenon forms hydrates in reversed micelles of wo 15 at 0.19 MPa and 273.15 K; the corresponding dissociation pressure for ethylene hydrates is 0.62 MPa. The experiments described in this paper were done with methane as the hydrateforming gas species simply because we have studied the thermodynamicsof methane hydrate formation in reversed micelles more thoroughly than other gas species. Furthermore, protein solubility is completely retained during methane hydrate formation at the experimental temperature (273.15 K) and pressures (up to 6 MPa). Results on the two systems are next discussed. Lipasecatalyzed ester synthesis is a bimolecular reaction involving the substrates oleic acid and octanol. Since both substrates are amphiphilic, reaction proceeds at the interface of the micelle. In studying this reaction, we have found that the enzyme rapidly loses its activity in reversed micelles unless contacted with the acyl substrate (oleic acid). Figure 6 illustrates the concept. The fresh reaction mixture (filled circles) denotes the case where the reaction is started by adding both substrates immediately following encapsulation of the enzyme. Incubating the encapsulated enzyme at 313 K for 24 h prior to starting the reaction by adding the substrates results in a significantly less active enzyme; the loss of activity is somewhat mitigated by incubation at 273 K. On the other hand, incubation with
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the acyl substrate for 24 h prior to reaction initiation by adding the alcohol results in activity maintenance. Furthermore, we find that the activity is well maintained when incubation with the acyl substrate is carried out at 273 K rather than a t 313 K. Indeed, a %day incubation run at 273 K also showed good maintenance of activity. The use of the acyl substrate in stabilizing the enzyme appears reasonable when one considers that the mechanism for lipase catalysis involves initial binding to the acyl substrate (Deleuze et al., 1987): Y
AcX+E=AcE+AcY+E X
where AcX and AcY are the acyl donors (the acid and ester, respectively), ACEis the acyl-enzyme complex, and X and Y are the nucleophilic acyl acceptors (water and alcohol, respectively). Thus, the acyl-enzyme binding may optimally position the enzyme a t the interface allowing activity maintenance. The acyl-enzyme coupling is important in experiments involving hydrates, since the process of hydrate formation and sampling at progressively higher pressures takes place over the course of a day or two. Accordingly, the initial sample introduced to the hydrate cell is a lipase-containing reversed micellar solution of wo 24 and oleic acid content 0.05 M. The presence of the amphiphilic substrate then changes the nature of the micelle, and the P-T-wo diagram generated for single-surfactant micelles is no longer applicable for the added cosurfactant case. That is, it is not possible to determine simply from the pressure (at a given temperature) what the wo of the micellar supernatant is, unless a new P-T-wo diagram is generated for the dual surfactant case. We have not done this due to the long time involved in generating such extensive thermodynamic data that is incidental to the concepts of this paper; rather, we simply took out supernatant samples at progressively higher pressures and measured the water content through Karl-Fischer titration to obtain wo values. Once the samples were removed from the hydrate cell, they were contacted with 0.05 M octanol to initiate reaction, which was carried out in a shake flask maintained at 313 K. Figure 7 illustrates lipase activity obtained from reversed micelle samples recovered from the hydrate cell at different wo values (over a pressure range of 2.7-7 MPa at about 273.7 K), starting with an initial solution of w o 24. The activities are compared to those obtained from lipasecontaining micellar solutions, individually prepared a t different w o values. As observed, both sets of experiments imply an optimal wo for maximum activity. The maximum activity for the hydrate-modified case is very slightly lower than that for the individual preparation case, and the optimal wo appears to be shifted to a lower value. The
fact that lipases in reversed micelles can exhibit a significant enhancement of activity when the w ois modified by hydrate formation is indicative of the fact that hydrate formation in the micelles does not lead to enzyme deactivation. We are not able to explain the apparent shift in optimal W O ,but we speculate that it is the result of changes in the microaqueous environment brought about when part of the micellar water is removed through hydrate formation. With a-chymotrypsin, the model reaction is quite different. Although the hydrolysis of GPNA is also bimolecular, one of the substrates (water) is a constituent of the micelles. At the GPNA concentrations used (0.2 mM), less than 0.1% of the micellar water is required for full conversion; the large excess of micellar water implies that there is no effect of the reaction on the wo of the solution. The hydrate formation experiment was simply carried out by introducing an enzyme-containing reversed micellar solution at wo 17, forming hydrates at progressively higher pressures and sampling the supernatant at different pressures. The P-T-wo data developed for empty micelles (Nguyen et al., 1989) hold well for this system, and the W O of the supernatant can be determined simply by noting the equilibrated pressure, although we did verify it by Karl-Fischer titration. As soon as a supernatant sample was taken from the hydrate cell and its w o measured, the enzyme activity was assayed. Figure 8 is similar to Figure 7 and illustrates a-chymotrypsin activities in micelles of different wo values, where wo was adjusted either through individual sample preparation or through hydrate formation. Again, it is seen that the hydrate formation technique of adjusting wo leads to enzyme activity modifications; the comparable activity in both cases implies that hydrate formation within the micelles does not adversely affect enzyme activity. The small shift to a lower optimal wo is observed, which we again attribute to changes in the microaqueous phase. Luisi and co-workers (Luisi, 1985; Barbaric and Luisi, 1981) have shown that the optimal wo for chymotrypsin is a function of the pH of the buffer from which the micellar solution is prepared. In continuing work on hydrate formation in reversed micelles, we are examining the acidity of the modified microaqueous phase by using the method of 31P NMR analysis (Smith and Luisi, 1980). Thus, hydrate formation in enzyme-containing reversed micelles leads to a change in micelle size and a concomitant modification of enzyme activity. The natural question arises as to whether other methods of changing wo may just as well be used to modify enzyme activity. To examine the alternatives, we considered the three cases described next, with the results illustrated in Figures 9 and 10.
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Biotechnol. Prog., 1990, Vol. 6, No. 6
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Case I results from the addition of empty reversed micelles of much smaller w o (small micelles) t o enzymecontaining micelles of higher w oto yield a micellar solution of the optimal WO. For example, mixing micellar solutions of wo 5 and wo 25 in the volume ratio 311 leads to a solution with w o 10 (Figure 9). If the enzyme was originally contained in micelles of w o 25, the procedure leads to a %fold reduction of the enzyme concentration and hence a reduction in total reaction rate. In our comparisons of the different cases, we adjusted (increased) the enzyme concentration in making up the solutions of high W O ,so that the final enzyme concentrations were the same in all cases. The notation 25e + 5 denotes the mixing of mi-
celles of w o 25 (containing enzyme) with micelles of w o 5. Thus, 25e + 5 is identical with 10e in terms of final enzyme: surfactant:water ratios; the only difference is the method of preparation. Case I1 is the situation where AOT (in solid form) is just added to enzyme-containing reversed micelles of large w o to bring down the w o to the optimum value. Here the AOT (and water) concentrations of the final mixture are higher than in Case I, and we use this case to indirectly examine AOT concentration effects on activity. The notation 25e + A in Figure 9, for example, applies to case 11. Case I11 is a variant of cases I and 11, where dissolved AOT in isooctane (i.e., micelles of w o = 0) is added to enzyme-containing micelles of large WO. As in case I, if the AOT concentration is to be constant, the enzyme concentration decreases upon mixing the two solutions. Hence, as in case I, we adjusted the enzyme concentration in the solution of w o 25 such that the final concentration at w o 10 is 0.1 mg/mL. The notation for this case in Figure 9 is 25e A/I. Figure 11 schematically illustrates the phenomenon behind the different methods of adjusting WO. The hydrate formation method reduces w o by reversibly freezing out a part of the microaqueous phase; cases I, 11, and I11 reduce wo by mixing small surfactant aggregates with the larger protein-containing microemulsion droplets. We return to the histograms of Figures 9 and 10 to discuss the results of adjusting w othrough cases I, 11, and 111. In all cases, the final enzyme and substrate concentrations were identical; the AOT concentration for case I1 was higher than the other cases, as noted. It is observed that all methods of w o reduction to a more optimal level lead to enhancement in activity. To an extent, this is a reflection of the fact that reversed micelles are dynamic entities. During the process of collision, constituent exchange, and reformation of micelles, the encapsulated enzyme is perhaps able to alter conformation to a state dictated by the micelle size. However, activity is not enhanced to the levels obtained by encapsulating enzyme at the appropriate w o (10e in Figure 9 and 9e in Figure lo), implying that simultaneous encapsulation and reversed micelle formation leads to the most active conformation. At these wo values, the interpolated activities of the systems modified by hydrate formation are lower (Figures 7 and 8);however, we do get comparable activities at the optimal W O ,which is about 7.5 with both enzymes. We also observe in Figures 9 and 10 that at increased surfactant concentrations (case I1 in Figures 9 and 10) the
+
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Biotechnol. Prog., 1990, Vol. 6,No. 6 100
Support from the National Science Foundation (Grants CBT-8802564 and CBT-8721829) is gratefully acknowledged.
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enhancement in activity is the least. The inhibitory effect of surfactant concentration is clearly shown in Figure 12, where w o is maintained constant. This inhibitory effect of surfactant concentration on enzyme activity has been stated before (Han et al., 1987). However, without specific data on conformation as correlated to activity and as affected by surfactant content, it is hard to give a definitive rationale, besides citing adverse protein-surfactant interactions (Han et al., 1987). It is interesting to note that the inhibition occurs both for the core-resident a-chymotrypsin and for the surface-occupying lipase. As an additional consideration, increased surfactant concentrations ( a t constant W O ,substrate, and enzyme concentrations) imply a larger number of micelles, a smaller fractional occupancy of enzyme, and decreased localized substrate concentrations in the water pool or at the micelle surface. Perhaps some of the inhibition can be simply attributed to reduced enzyme-substrate contact. Finally, one might ask if just freezing the micellar solution could also affect water removal and w oadjustment. For micelles of w o 15 and lower, it has been shown that there is a significant depression of freezing point and that the micellar water crystallizes at around 233 K (Quist and Halle, 1988; Boned et al., 1986). Even more importantly, we feel that the adjustment of micellar size is much better accomplished by the pressurization/depressurization involved in hydrate formation/dissociation. Hydrate formation, while certainly just another way of freezing out some of the intramicellar water, is a controllable method.
Conclusions We have shown that hydrate formation in the water droplets of reversed micelles can alter the size of the micelles, which results in activity modification of enzymes encapsulated in the micelles. Hydrate formation leads to nucleation of intramicellar water to crystalline form and subsequent precipitation of the hydrate crystals. The enzyme remains encapsulated in the resulting micelles of reduced size. Enzyme deactivation is minimal during the process of hydrate formation. Three other methods of reducing the water to surfactant ratio (WO)of enzymecontaining reversed micelles indicate that activity can be enhanced by reduction to a more optimal w o value; full enhancement to the maximum activity is not obtained, however. The hydrate method appears to be able to bring about maximum activity, albeit at a slightly lower w ovalue. These concepts could possibly be applied to improving the activity of enzymes extracted from fermentation broths through contact with a reversed micellar phase. They may be particularly relevant to those enzymes that are to be directly used in minimal water environments.
Literature Cited Barbaric, S.; Luisi, P. L. Micellar Solubilization of Biopolymers in Organic Solvents. 5. Activity and Conformation of a-Chymotrypsin in Isooctane-AOT Reverse Micelles. J. Am. Chem. SOC.1981,103,4239-4244. Berecz, E.; Balla-Achs, M. In Studies in Inorganic Chemistry4: Gas Hydrates; Elsevier Science Publishers: Amsterdam, The Netherlands, 1983. Boned, C.; Peyrelasse, J.; Moha-Ouchane, M. Characterization of Water Dispersion in Water/Sodium Ethylhexyl Sulfosuccinate Microemulsions Using Differential Scanning Calorimetry. J. Phys. Chem. 1986,90,634-637. Deleuze, H.; Langrand, G.; Millet, H.; Baratti, J.; Buono, G.; Triantaphylides, C. Lipase-Catalyzed Reactions in Organic Media: Competition and Applications. Biochim. Biophys. Acta 1987, 91 I, 117-120. GBklen, K. E.; Hatton, T. A. Protein Extraction Using Reverse Micelles. Biotechnol. Prog. 1985, 1, 69-74. Han, D.; Rhee, J. S.; Lee, S.B. Lipase Reaction in AOTIsooctane Reversed Micelles: Effect of Water on Equilibria. Biotechnol. Bioeng. 1987, 30, 381-388. Holder, G. D.; Hand, J. H. Multiple-Phase Equilibria in Hydrates from Methane, Ethane, Propane and Water Mixtures. AIChE J . 1982, 28, 353. Kabanov, A. V.; Levashov, A. V.; Klyachko, N. L.; Pshezhetesky, A. V.; Martinek, K. Enzymes Entrapped in Reversed Micelles of Surfactants in Organic Solvents: A Theoretical Treatment of Catalytic Activity Regulation. J. Theor. Biol. 1988,133, 327-343. Leser, M. E.; Wei, G.; Luisi, P. L.; Maestro, M. Application of Reverse Micelles for the Extraction of Proteins. Biochem. Biophys. Res. Commun. 1986, 135,629-635. Leser, M. E.; Wei, G.; Luthi, P.; Haering, G.; Hochkoppler, A.; Blochliger, E.; Luisi, P. Applications of Enzyme-Containing Reversed Micelles. J . Chim. Phys. 1987, 84, 1113-1118. Luisi, P. L. Enzymes Hosted in Reverse Micelles in Hydrocarbon Solution. Angew. Chem., Int. Ed. Engl. 1985, 24, 439-450. Luisi, P. L.; Giomini, P.; Pileni, M. P.; Robinson, B. H. Reverse Micelles as Hosts for Proteins and Small Molecules. Biochim. Biophys. Acta 1988, 947, 209-246. Martinek, K.; Berezin, I. V.; Khmelnitski, Yu. L.; Klyachko, N. L.; Levashov, A. V. Enzymes Entrapped Into Reversed Micelles of Surfactants in Organic Solvents: Key Trends in Applied Enzymology (Biotechnology). Biocatalysis 1987, 1, 9-15. Nguyen, H.; Phillips, J. B.; John, V. T. Clathrate Hydrate Formation in Reversed Micellar Solutions. J. Phys. Chem. 1989,93, 8123-8126. Nguyen, H.; Reed, W.; John, V. T. Characteristics of ProteinContaining Reversed Micelles Subjected to Clathrate Hydrate Formation Conditions. J. Phys. Chem. 1990, in press. Pileni, M.-P.; Zemb, T.; Petit, C. Solubilization by Reversed Micelles: Solute Localization and Structure Perturbation. Chem. Phys. Lett. 1985, 118, 414-420. Quist, P.; Halle, B. Water Dynamics and Aggregate Structure in Reversed Micelles a t Sub-Zero Temperatures. A Deuteron Spin Relaxation Study. J . Chem. Soc., Faraday Trans. 1 1988,84, 1033-1046. Rahaman, R. S.; Chee, J. Y.; Cabral, J. M. S.; Hatton, T. A. Recovery of an Extracellular Alkaline Protease from Whole Fermentation Broth Using Reversed Micelles. Biotechnol. Prog. 1988, 4, 218-224. Sarda, L.; Desnuelle, P. Action of Pancreatic Lipase upon Esters in Emulsion. Biochim. Biophys. Acta 1958, 30, 513-521. Shield, J. W.; Ferguson, H. D.; Bommarius, A. S.; Hatton, T. A. Enzymes in Reversed Micelles as Catalysts for OrganicPhase Synthesis Reactions. Ind. Eng. Chem. Fundam. 1985, 25,603-612.
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Smith, R. E.; Luisi, P. L. Micellar Solubilization of Biopolymers in Hydrocarbon Solvents. 111. Empirical Definition of an Acidity Scale in Reverse Micelles. Helu. Chim. Acta 1980, 63,2302-2311. van der Waals, J. H.; Platteeuw, J. C. Clathrate Solutions. Adu. Chem. Phys. 1959,2, 1-57. Wirz, J.; Rosenbusch, J. P. The Formation of Reverse Mixed Micelles Consisting of Membrane Proteins and AOT in Isooctane. In Reversed Micelles; Luisi, P. L., Straub, B. E., Eds.; Plenum Press: New York, 1984; pp 231-238.
Zulauf, M.; Eicke, H.-F. Inverted Micelles and Microemulsions in the Temary System HzO/Aerosol-OT/Isooctane As Studied by Photon Correlation Spectroscopy. J. Phys. Chem. 1979, 83,480-486. Accepted August 27, 1990. Registry No. GPNA, 130013-48-8;lipase, 9001-62-1;octyl oleate, 32953-65-4; oleic acid, 112-80-1; octanol, 111-87-5; chymotrypsin, 9004-07-3.
