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Bioconversions in a Water-in-CO2 Microemulsion J. D. Holmes, D. C. Steytler,* G. D. Rees, and B. H. Robinson School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, U.K. Received June 15, 1998. In Final Form: July 27, 1998 In a previous communication (Langmuir 1997, 13, 6980-6984) we reported stabilization of water-inCO2 (w/c) microemulsions by fluorinated dichained sulfosuccinate surfactant ‘di-HCF4’. In this paper we present a study of enzyme-catalyzed reactions affected in this w/c microemulsion in which the enzyme is located within the dispersed water droplets. Two reactions, a lipase-catalyzed hydrolysis of p-nitrophenol butyrate and lipoxygenase-catalyzed peroxidation of linoleic acid were examined. The activity of both the enzymes in the w/c microemulsion environment were essentially equivalent to that in a water-in-heptane microemulsion stabilized by Aerosol OT, a surfactant with the same headgroup as di-HCF4. The inherent condition of low pH of the water droplets in the w/c microemulsion was improved using the buffer MES [2-(N-morpholino)ethanesulfonic acid] which was shown to fix the pH in the range 5-6 depending on buffer concentration, pressure, and temperature.
1. Introduction The discovery that enzymes can remain active in highly apolar organic solvents, in the presence of small amounts of water, has led to the development of novel applications of such ‘low water’ systems in chemical synthesis.1 Not only can enzyme-catalyzed ‘biotransformations’ be performed on apolar substrates but also the low levels of water present in these systems can shift the reaction equilibrium to favor the formation of products that would otherwise be rapidly hydrolyzed in aqueous solution. The formation of peptides, glycosides, and esters, for example, are commonly produced in this way.2 Enzymes have also been solubilized in water-in-oil (w/o) microemulsions, which are thermodynamically stable dispersions of nanometer-sized water droplets in an oilcontinuous medium. The droplets are stabilized by a adsorbed monolayer of surfactant.3 For many microemulsion systems, spherical droplets are formed with a small size distribution for which the mean radius is easily controlled by the water-to-surfactant molar concentration ratio, wo, where wo ) [H2O]/[surfactant]. Enzymes compartmentalized within the droplets experience an aqueous environment and are protected from the potentially deleterious effects of the organic solvent, thereby exhibiting good stability and activity. Moreover, w/o microemulsions have the ability to function as a ‘universal solvent’ medium for solubilizing high concentrations of both polar and apolar molecules within the dispersed aqueous and continuous oil phases, respectively. Solutes of amphiphilic nature are also solubilized and preferentially adsorb at the oil-water surface of the droplets. These systems can therefore be used to exploit both the selectivity of the biocatalyst and the solvent properties of the microemulsion itself to provide a unique reaction medium for biomimetic chemistry.4 The benefits of near-critical carbon dioxide (nc-CO2) as a reaction, extraction, or treatment medium have been * To whom correspondence should be addressed. Telephone: 0044 1603 592033. Fax: 0044 1603 259855. E-mail:
[email protected]. (1) Dordick, J. S. Enzyme Microbiol. Technol. 1990, 11, 194. (2) Volkin, D. B.; Staubli, A.; Langer, R.; Klibanov, A. M. Biotechnol. Bioeng. 1991, 37, 843. (3) Robinson, B. H. Chem. Brit. 1990, 26, 342. (4) Oldfield, C. Biotechnol. Genetic Engin. Rev. 1994, 12, 255.
widely reviewed.5,6 Near-critical CO2 is nontoxic and nonflammable and therefore offers many environmental and safety advantages over conventional petrochemical solvents. A feature of being in a near-critical condition is that the density and, hence, solvating properties of the medium can be readily manipulated. Therefore, under the control of pressure and temperature, selective separation of dissolved components (e.g., reaction products) can be facilitated. However, nc-CO2 is a poor solvent for high molecular weight or highly polar materials.7 The problem associated with the low solubility of polar materials in the CO2 medium has been overcome using molecular selfassembly systems of specially designed surfactants, incorporating a ‘CO2-philic’ fluorocarbon moiety.8,9 For example, fluorocarbon-hydrocarbon block copolymers have been observed to aggregate under appropriate conditions in CO2, forming micelles with a hydrocarbon core capable of solubilizing high molecular weight polymers.10 A range of fluorocarbon surfactants have also been specifically designed to stabilize nanometer-sized water droplets in CO2, such as the hybrid fluorocarbonhydrocarbon surfactant11 (C7H15)(C7F15)CHSO4Na. In common with many w/o microemulsions, water-in-CO2 (w/c) microemulsions show a spherical droplet structure for which the droplet radius is directly proportional to wo () [H2O]/[surfactant]).12 They also behave as universal solvents for solubilizing hydrophilic, hydrophobic, and amphiphilic solutes.11,13 (5) McHugh, M. A.; Krukonis, V. J. In Supercritical Fluids Extraction: Principles and Practice, 2nd ed.; Butterworth-Heinman: Woburn, MA, 1993. (6) Supercritical Fluid Science and Technology; Johnston, K. P.; Penninger, J. M. L., Eds., ACS Symposium Series No. 406: Washington, D.C., 1989. (7) Consani, K. A.; Smith R. D. J. Supercrit. Fluids 1990, 3, 51. (8) Hoefling, T. A.; Enick, R. M.; Beckman, E. J. J. Phys. Chem. 1991, 95, 7127. (9) McFann, G. J.; Johnston, K. P.; Howdle, S. M. AlChE J. 1994, 40, 543. (10) McClain, J. B.; Betts, D. E.; Canelas, D. A.; Samulski, E. T.; DeSimone, J. M.; Londono, J. D.; Cochran, H. D.; Wignall, G. D.; ChilluraMartino, D.; Triolo, R. Science 1996, 274, 2049. (11) Harrison, K.; Goveas, J.; Johnston, K. P.; O’Rear III, E. A. Langmuir 1994, 10, 3536. (12) Eastoe, J.; Bayazit, Z.; Martel, S.; Steytler, D. C.; Heenan, R. K. Langmuir 1996, 12, 1423. (13) Heitz, M. P.; Carlier, C.; deGrazia, J.; Harrison, K. L.; Johnston, K. P.; Randolph, T. W.; Bright, F. V. J. Phys. Chem. 1997, 101, 6707.
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(2-ethylhexyl) sulfosuccinate (AOT), which is an effective surfactant for stabilizing w/o microemulsions that provide good enzyme compatibility. 2. Experimental Section
Figure 1. Scheme 1: Hydrolysis of p-nitrophenyl butyrate catalyzed by C. viscosum lipase. The formation of p-nitrophenol can be monitored by UV-vis spectrophotometry at λ ) 326 nm. Scheme 2: Peroxidation of the 1,4-cis,cis-pentadiene group of linoleic acid by soybean lipoxygenase, to form 13-hydroperoxyoctadecadienoic acid. The formation of the fatty acid hydroperoxide can be monitored by UV-vis spectrophotometry at λ ) 234 nm.
Recently, Clarke et al.14 have solubilized ionic species in the water core of a w/c microemulsion and exploited the high miscibility of both SO2 and H2S in supercritical CO2 to perform aqueous inorganic reactions. Thus aqueous potassium dichromate was reacted with gaseous SO2 in the w/c microemulsion, leading to the formation of chromium (III) ions in the aqueous phase. Similarly, aqueous sodium nitroprusside was observed to change color immediately upon the addition of H2S into the w/c microemulsion. These reactions were monitored by ultraviolet-visible (UV-vis) and infrared (IR) spectroscopy, respectively. In addition, Johnston et al.15 have demonstrated that the protein bovine serum albumin (Mw ) 67 000 Da) can be solubilized in a w/c microemulsion, but to date there have been no reports of enzyme-catalyzed reactions in w/c microemulsions. In this communication we report two enzyme-catalyzed reactions (Figure 1) in a w/c microemulsion and reveal how the potential problem of low pH of the dispersed aqueous phase can be overcome. Relatively few surfactants have been shown to be capable of stabilizing microemulsions in CO2. However, the dispersion of water in nc-CO2 using the surfactant di(1H, 1H, 5H-octafluoro-n-pentyl) sodium sulfosuccinate (di-HCF4) has recently been investigated by small-angle neutron scattering (SANS).16 The data obtained showed that the dichained fluorinated surfactant was effective at forming w/c microemulsions giving a maximum solubilization, wmax, of 40 at 20 °C and 500 bar. For the work described in this paper we have used w/c microemulsions formed by the di-HCF4 surfactant at w0 ) 10 for which a water droplet radius Rc ∼ 20 Å was determined from SANS measurements. The surfactant is related to bis(14) Clarke, M. J.; Harrison, K. L.; Johnston, K. P.; Howdle, S. M. J. Am. Chem. Soc. 1997, 119, 6399. (15) Johnston, K. P.; Harrison, K. L.; Clarke, M. J.; Howdle, S. M.; Heitz, M. P.; Bright, F. V.; Carlier, C.; Randolph, T. W., Science 1996, 271, 624. (16) Eastoe, J.; Cazelles, B. M. H.; Steyler, D. C.; Holmes, J. D.; Pitt, A. R.; Wear, T. J.; Heenan, R. K. Langmuir 1997, 13, 6980.
2.1. Chemicals. The surfactant di(1H,1H,5H, octafluoron-pentyl) sodium sulfosuccinate (di-HCF4) was prepared by the method described by Yoshino et al.17 Elemental microanalysis as well as 1H, 13C, and 19F nuclear magnetic resonance (NMR) spectra (JEOL GX400) were consistent with the desired product at purity 98-99%. The aqueous phase critical micelle concentration (cmc ) 5.0 × 10-3 M) was measured at 25 °C using a duNouy tensiometer (Kruss K10). Distilled water was used in all experiments and carbon dioxide (99%) was used as received (BOC gases). The buffers examined in this study included 2-(Nmorpholino)ethanesulfonic acid, sodium salt (MES), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), N,Nbis[2-hydroxyethyl]-2-aminoethanesulfonic acid (BES), 3-[cyclohexylamino]-1-propanesulfonic acid (CAPS), N-tris[hydroxymethyl]methylglycine, and N-[2-hydroxy1,1-bis(hydroxymethyl)ethylglycine (Tricine) were obtained from Sigma Chemicals. The hydrophilic pH indicators methyl orange (pKa ) 3.5) and methyl red (pKa ) 5.1) were BDH chemicals and p-nitrophenolsulfonate (PNPS) (pKa ) 6.5) was obtained from Aldrich. 2.2. Phase Diagram Measurements. The pressuretemperature phase stability profiles for the w/c microemulsions were determined in a high-pressure optical cell by direct observation of turbidity accompanying phase separation at the lower pressure phase boundary (LPPB). Such transitions, measured at constant temperature with decreasing pressure, are clearly visible and highly reproducible. The high-pressure cell used throughout this work has been described previously.18 The maximum operating pressure of the cell is 500 bar, and accurate thermostating is possible (°C) using a conventional bath-circulator. The cell was configured with an optical path length of 3 mm, giving a nominal volume of 12 mL on which quoted concentrations are based. Two poly(tetrafluoroethylene) (PTFE)-coated magnetic stirrer bars, one above and one below the sapphire windows, were driven by an external magnetic stirrer. 2.3. pH Measurements. For two-phase water-CO2 mixtures, an aqueous solution of the indicator and buffer was first adjusted to the pKa of the buffer and then introduced into the cell to a height covering the windows. CO2 was then added to the cell and the pressure adjusted using a small hand pump that acts on the piston within the cell via a hydraulic line. The contents of the cell were stirred vigorously to ensure equilibrium between the two phases. For pH studies in w/c microemulsions, the diHCF4 surfactant (30 mM) together with H2O (300 mM) (or buffered indicator) were added to the cell, and liquid CO2 was introduced at 20 °C. The pressure was then raised, with the contents of the cell being continuously stirred, until a singlephase microemulsion was formed. All UV-vis spectra were recorded using a Hewlett-Packard 8452A Diode array spectrophotometer fitted with a sliding cradle to accommodate the pressure cell. 2.4. Enzyme Reactions in Water-in-CO2 Microemulsions. To measure the rates of enzymatic hydrolysis, the surfactant (30 mM) and aqueous solution of Chromobacterium viscosum lipase, in MES buffer (100 mM, pH ) 5.0), were placed at the bottom of the pressure cell. The p-nitrophenyl butyrate (PNPC4) substrate was then added to the upper part of the cell to avoid contact with the enzyme. The cell was filled with CO2 at 20 °C and the pressure adjusted to 450 bar. The microemulsion (w0 ) 10) formed spontaneously on stirring, and the ensuing formation of p-nitrophenol was monitored by UV-vis spectroscopy. To avoid complications arising from the different adsorption spectra of the acid and basic forms of the product, nitrophenol, the reaction was monitored at their isosbestic point (λ ) 326 nm). (17) Yoshino, N.; Komine; N., Suzuki, J.-I.; Arima, Y.; Hirai, H. Bull. Chem. Soc. Jpn. 1991, 64, 3262. (18) Eastoe, J.; Robinson, B. H.; Steytler, D. C. J. Chem. Soc., Faraday Trans. 1990, 86, 511.
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Scheme 1
Kapp m )
Kw m Ps
(4)
and when the substrate is significantly partitioned to the droplets (Ps . 1), w Kapp m ) Kmθ
The temperature and pressure conditions and experimental procedure employed for the lipoxygenase reaction were essentially the same as just described for the lipase reaction. However, after filling the cell with CO2 (20 °C, 58 bar), gaseous O2 was introduced to a pressure of ≈110 bar ([O2] (0.5 M ≡ 1.6% w/v). A single-phase transparent microemulsion (w0 ) 10) was formed upon further increasing the pressure, and the ensuing reaction was monitored at a wavelength of 234 nm. For both reactions, control experiments were also performed with no enzyme present. 2.5. Enzyme Reactions in Water-in-Oil Microemulsions. Solutions of AOT (30 mM) and PNPC4 were prepared in n-heptane in volumetric flasks (10 mL). C. viscosum lipase in MES (100 mM aqueous, pH ) 5.0) was then added to the AOT/ heptane/PNPC4 solution, in the pressure cell, and the solution was vigorously stirred until a microemulsion formed (w0 ) 10). The reaction was monitored by UV-vis spectroscopy at a wavelength of 326 nm. The soybean lipoxygenase reaction was executed in an identical fashion with the solution exposed to atmospheric O2. The reaction was monitored by UV-vis spectroscopy at a wavelength of 234 nm.
3. Enzyme Kinetics The kinetics of enzyme-catalyzed reactions in w/o microemulsions have been treated in terms of a MichaelisMenten type model allowing for substrate partitioning between the oil and water pseudophases (Scheme 1).19 Where Ps represents the partition coefficient of the substrate S between the oil (o) and droplet (w) pseudophases of the microemulsion:
Ps )
[S]w [S]o
(1)
For this model, which assumes rapid pre-equilibrium, the initial rate, V, is given by,
V)
d[P] kcat[E]w[S]w ) w dt Km + [S]w
(2)
(5)
where θ is the droplet volume fraction in the microemulsion. When comparing enzyme reactions it is common practice to quote the maximum initial rate of enzyme turnover under condition of saturation of the enzyme with substrate (Vmax). A convenient method for simultaneous determination of Vmax and Kapp m is to use an inverted form of eq 3 app
1 1 Km 1 ) + V Vmax [S]T Vmax
(6)
A ‘Lineweaver-Burk’ plot of 1/V against 1/[S]T yields a straight line with slope Kapp m /Vmax and intercepts 1/Vmax app and - Kapp m . Hence, values can be realized for both Km , which provides information about the binding of the substrate to the enzyme and Vmax, which serves as a measure of substrate turnover by the enzyme. 4. Results and Discussion 4.1. Phase Behavior and pH Control. Many enzymes exhibit an optimum catalytic ‘turnover’ of substrate at higher pH values and would be expected to perform with a much reduced activity in this acidic environment. Previous studies have shown that the enzymes C. viscosum lipase (Mw ) 33 000 Da) and soybean lipoxygenase (Mw ) 108 000 Da) display their greatest activities at pH ∼ 6.5 and pH ∼ 9.0, respectively, in an AOT/heptane microemulsion.19,20 Consequently, before proceeding with enzyme reactions in the w/c microemulsion, the performance of a variety of buffers were tested both in ‘bulk’ water in contact with CO2 and in the microdispersed water droplets of w/c microemulsions. In view of the current interest in supercritical fluids, it is surprising that there is no published work concerning the effective buffering of water exposed to CO2. As a result of the formation of carbonic acid21 aqueous solutions in contact with CO2 at elevated pressures are acidic with pH ∼3. The equilibria concerned are: K1
CO2 + H2O y\z H2CO3 K2
where the enzyme [E]w and substrate [S]w concentrations represent substrate concentration expressed as moles per unit volume of the water (w) pseudophase. Because [S]w is not easily quantifiable, the kinetics are often expressed in terms of the concentrations per unit volume of total microemulsion (T),
V)
kcat[E]T[S]T Kapp m + [S]T
(3)
w The relationship of Kapp m to Km depends on the magnitude of Ps. For an oil-soluble substrate that partitions preferentially to the oil phase (Ps , 1),
(19) Fletcher, P. D. I.; Robinson, B. H.; Freedman, R. B.; Oldfield, C. J. Chem. Soc. Faraday Trans. 1985, 81, 2667.
H2CO3 + H2O y\z H3O+ + HCO3K3
HCO3- + H2O y\z H3O+ + CO32Because K2 . K3, the hydrogen ion concentration in water is primarily determined by the dissociation of carbonic acid, which also produces the bicarbonate anion. A simple method for pH control is to suppress this dissociation by addition of sodium bicarbonate. Our initial experiments examined the effectiveness of sodium bicarbonate and various ‘biological’ buffer solutions in contact with ncCO2 using the hydrophilic pH indicators methyl orange (20) Kurganov, B. I.; Shkarina, T. N.; Malakhova, E. A.; Davydov, D. R.; Chebotareva, N. A. Biochimie 1989, 71, 573. (21) Kamat, S. V.; Beckman, E. J.; Russell, A. J. Crit. Rev. Biotech. 1995, 15, 41.
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Figure 2. Effect of sodium bicarbonate concentration on the measured pH of water in contact with CO2. (T ) 20 °C, P ) 450 bar).
(pKa ) 3.5), methyl red (pKa ) 5.1), or p-nitrophenolsulfonate (PNPS; pKa ) 6.5). The pH of the coexisting water phase was monitored by UV-vis spectroscopy as a function of pressure and temperature, in the presence and absence of a variety of buffers. Sodium bicarbonate was found to be an effective buffer (Figure 2) particularly at concentrations >1 M (∼7 wt %) for which pH ) 6-7 could be achieved. Of the biologically compatible buffers tested, MES and HEPES were the most effective, giving an equilibrium pH of the aqueous phase in contact with liquid CO2 that increased linearly with buffer concentration from 5.2 ([buffer] ) 100 mM) to 6.0 ([buffer] ) 500 mM). As also observed in the absence of buffer, the acidity of the aqueous phase mirrored the density of the CO2 phase; that is, increasing with temperature (at constant pressure) and decreasing with pressure (at constant temperature). These effects were, however, less dramatic than the breadth of pH obtained across the range of buffers examined. The pH of the aqueous phase in the w/c microemulsion was also measured in the absence and presence of MES buffer using the same indicator probes. The pKa of the indicators in the aqueous phase of the w/c microemulsion was taken to be equivalent to their value in bulk water. Previous studies have shown that they are effective pH probes for w/o microemulsion systems.22 The pH inside both the MES-buffered and unbuffered water droplets was also observed to decrease with increasing pressure and decreasing temperature (Figure 3). The pH of the MES-buffered water droplets in the diHCF4-stabilized w/c microemulsion, at 25 °C and 450 bar, was comparable with the pH determined for bulk water under the equivalent conditions. However, the pH (∼5) obtained is significantly lower than the pKa of the buffer (6.1) and therefore the buffer is not functioning in its usual role close to the pKa. Interestingly, the increase in the ionic strength of the dispersed water phase resulting from addition of buffer shifts the lower-pressure phase boundary to higher temperatures.23 Thus, addition of MES in the w/c system provided the added advantage of lowering the pressure required to form the microemulsion (Figure 4). 4.2. Lipase-Catalyzed Bioconversions. Lipasecatalyzed bioconversions have been extensively studied in conventional w/o microemulsions in a variety of
esterification and trans-esterification processes.24,25 For our tests with the di-HCF4 w/c microemulsion, we chose the hydrolysis of PNPC4 by C. viscosum lipase, as shown in Scheme 1 in Figure 1. This reaction has previously been examined in a microemulsion stabilized by AOT in
(22) Oldfield, C.; Robinson, B. H.; Freedman, R. B. J. Chem. Soc. Faraday Trans. 1990, 86, 833. (23) Winsor, P. A. In Solvent Properties of Amphiphilic Compounds; Butterworth: London, 1954.
(24) Crooks, G. Stephenson, G. R. (25) Crooks, G. Stephenson, G. R.
Figure 3. Effect of temperature (P ) 450 bar) and pressure (T ) 20 °C,) on the measured pH of dispersed water droplets in a wo ) 10, di-HCF4-stabilized w/c microemulsion, in the absence (O) and presence (b) of MES (100 mM).
Figure 4. Pressure-temperature phase diagram showing the phase boundary of a wo ) 10 di-HCF4-stabilized w/c microemulsion with MES (100 mM) in the dispersed aqueous phase. 1θ and 2θ represent the single- and two-phase regions of microemulsion stability ([di-HCF4] ) 30 mM).
E.; Rees, G. D.; Robinson, B. H.; Svensson, M.; Biotechnol. Bioeng. 1995, 48, 78. E.; Rees, G. D.; Robinson, B. H.; Svensson, M.; Biotechnol. Bioeng. 1995, 48, 190.
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Figure 5. Reaction profiles (monitored at λ ) 326 nm) for the hydrolysis of PNPC4 by C. viscosum lipase in a di-HCF4stabilized w/c microemulsion with [PNPC4]T ) 2 mM: (A) [E]T ) 1.08 µg mL-1; (B [E]T ) 0.54 µg mL-1; (C) control (no lipase added). [MES]w ) 100 mM, [di-HCF4] ) 30 mM, w0 ) 10, T ) 20 °C, P ) 450 bar.
n-heptane.19 The hydrolysis is particularly amenable to study in a w/c microemulsion because the differences between the UV-vis spectra of the PNPC4 substrate and p-nitrophenol product allow direct and continuous monitoring of product formation by spectrophotometry. This technique avoids the experimental complication of removing samples at high pressure. To confirm that the formation of p-nitrophenol resulted from biocatalysis by C. viscosum lipase in the system, the reaction was repeated at two different enzyme concentrations, and a control incubation in which the enzyme was absent was also monitored. To ascertain the importance of the microemulsion in dispersing the enzyme, another control experiment was undertaken in the absence of surfactant. Figure 5 shows the results illustrating what we believe to be the first enzyme-catalyzed reaction monitored in a w/c microemulsion. The data clearly show that no significant reaction takes place in the absence of enzyme, but the rate of PNPC4 hydrolysis increases proportionally with increasing enzyme concentration. Similarly, no increase in absorbance was observed in the absence of surfactant which confirms the requirement of a high level of enzyme dispersion facilitated by the microemulsion. For the lipase-catalyzed reaction in w/o microemulsions, it has been established19 that the apolar substrate PNPC4 is partitioned preferentially to the oil phase surrounding the droplets (i.e., Ps , 1). This partitioning reduces the concentration of substrate in the water droplet such that Kapp m . [S]T. A zero intercept is then always obtained on a Lineweaver-Burk plot so that the parameters Kapp m and Vmax cannot be independently resolved. To overcome this limitation, the kinetics have been represented in terms of a second-order rate constant, k2, with the initial rate given by
V ) k2[E]T[S]T
(7)
where concentrations are again defined as moles per unit volume of the total microemulsion as opposed to the volume of the oil or water pseudophases. It can be shown that k2 is directly related to kcat by eq 8
k2 ) const
kcat Kapp m
(8)
Figure 6. Reaction profiles (monitored at λ ) 234 nm) for the peroxidation of linoleic acid by soybean lipoxygenase in a diHCF4-stabilized w/c microemulsion containing dissolved O2 gas with [linoleic acid]T ) 2 mM: (A) [E]T ) 0.54 µg mL-1; (B) [E]T ) 0.27 µg mL-1; (C) control (no lipoxygenase added). [MES]w ) 100 mM, [di-HCF4] ) 30 mM, w0 ) 10, T ) 20 °C, P ) 450 bar.
The rate constant k2 therefore normalizes the initial rate for both enzyme and substrate concentration and provides a convenient measure of enzyme performance for this reaction in microemulsions. The value of k2 obtained from the data in Figure 5 for the w/c microemulsion was 12.8 mL g-1 s-1, which compares favorably with 11.8 mL g-1 s-1 obtained for the equivalent AOTstabilized w/o microemulsion in heptane at the same pH. 4.3. Lipoxygenase-Catalyzed Bioconversions. A frequently cited advantage of near-critical fluids as reaction media is their ability to dissolve large concentrations of gaseous reactants, a feature which has recently been exploited to affect inorganic reactions in w/c microemulsions.14 To examine this feature in the context of enzyme-catalyzed reactions, we studied the peroxidation of linoleic acid by soybean lipoxygenase, which requires gaseous O2 as a substrate (see Scheme 2 in Figure 1). Lipoxygenases catalyze the peroxidation of polyunsaturated fatty acids possessing the 1,4-cis,cis-pentadiene group, producing a hydroperoxide. Formation of the product 13-hydroperoxyoctadecadienoic acid can be conveniently monitored by UV-vis spectrophotometry at a wavelength of 234 nm. The resulting initial rate profiles for the ‘bioconversions’ of linoleic acid are shown in Figure 6. In common with the data obtained using C. viscosum lipase, the results clearly show a progressive increase in the rate of reaction, in this case oxidation, with increasing enzyme concentration. Control experiments again confirmed that no significant reaction occurred in the absence of enzyme or surfactant. The interplay between oxygen and linoleic acid concentration on the reaction has recently been examined and it was established that both the rate and equilibrium position can be lowered when [O2] < 1 mM.26 The oxygen concentration in the both the air-saturated heptane and oxygen-enriched CO2 continuous phases of the w/o and w/c microemulsions will ensure that the level in the droplets does not fall below this limit. The reaction in these microemulsions should therefore be directly comparable with respect to the effects of the linoleic acid substrate concentration. We have therefore analyzed (26) Berry, H., Debat, H.; Larreta-Garde, V. FEBS Lett. 1997, 408, 324.
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5. Conclusions
Figure 7. Reciprocal initial rate, V -1, versus reciprocal substrate concentration, [S]T-1, (Lineweaver-Burk plot), for the peroxidation of linoleic acid by soybean lipoxygenase ([E]T ) 0.54 µg mL-1) in a di-HCF4-stabilized w/c microemulsion ([di-HCF4] ) 30 mM, w0 ) 10), T ) 20 °C, P ) 450 bar).
initial rate measurements for this reaction in the framework of a single substrate Michaelis-Menten model. Because linoleic acid shows significant partitioning to the droplets27 we were in this case able to determine the kinetic parameters using a Lineweaver-Burk representation (Figure 7). The initial rates have been expressed as concentration of product (M) per unit time (s) per unit concentration of enzyme (g mL-1 of microemulsion). A value of Kapp m ) 1.4 mM was obtained from the intercept on the x-axis, giving a value of Vmax ) 8.0 M s-1 g-1 mL from the slope. Values for these parameters of similar magnitude were obtained for an AOT-stabilized w/o microemulsion in heptane under equivalent conditions of -1 g-1 mL). pH (Kapp m ) 1.0 mM; Vmax ) 5.9 M s (27) Kurganov, B. I.; Tsetlin, L. G.; Malakhova, E. A.; Chebotareva, N. A.; Lankin, V. Z.; Tglebova, G. A.; Berezovsky, V. M.; Levahov, A. V.; Martinek, K. J. Biochem. Biophys. Meth. 1985, 11, 177.
The preliminary results we present here are extremely encouraging and demonstrate the viability of cell-free enzyme biotransformations in w/c microemulsions. The stereosynthetic potential of lipases is well-known; however, lipoxygenases are highly site specific and also stereoselective in their action. Many of the products that they generate are biologically important and include leukotrienes, lipoxins, jasmonates, and related plant growth regulators.28 In summary, our results demonstrate the viability of enzyme-catalyzed reactions within a pH-controlled w/c microemulsion and show the turnover to be comparable with that obtained in w/o systems. It is anticipated that these findings will be of value for the development of CO2 as a medium for biotransformations. With increased interest and commercialization of surfactants in CO2 as ‘environmentally acceptable’ cleaning fluids, it is conceivable that applications may soon exist in this area for a combined enzymatic biodegradation as in conventional cleaning processes.28,29 We also believe there are exciting opportunities for selective protein separations using this new medium, which will result in recovery of the proteins in an active state and with simplified isolation procedures. Ongoing studies are in progress in our laboratory involving more detailed examination of the kinetics and mechanisms of the reactions reported in this communication. In parallel with this activity, we are also examining the potential of various surfactants in CO2 as media for a wider range of enzyme reactions, and for novel bioseparations.30 Acknowledgment. The authors gratefully acknowledge financial support from the Engineering and Physical Sciences Research Council (EPSRC). LA9806956 (28) Boyington, J. C.; Gaffney, B. J.; Amzel, L. M. Science 1993, 20, 1482. (29) Wu, C. Science News 1997, 152, 108. (30) Adams, A. New Scientist 1997, 155, 12.
