Performance of Cholesterol Oxidase Sequestered within Reverse

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Performance of Cholesterol Oxidase Sequestered within Reverse Micelles Formed in Supercritical Carbon Dioxide† Maureen A. Kane, Gary A. Baker, Siddharth Pandey, and Frank V. Bright* Department of Chemistry, Natural Sciences Complex, University at Buffalo, The State University of New York, Buffalo, New York 14260-3000 Received December 8, 1999. In Final Form: February 18, 2000 We report the first results on an enzyme-induced reaction within the water core of reverse micelles that have been formed in supercritical CO2 (scCO2). By using a perfluoropolyether ammonium carboxylate (PFPE) surfactant, we form reverse micelles in scCO2 with water cores and we show that the oxidation of cholesterol by cholesterol oxidase (ChOx) obeys Michaelis-Menten kinetics. The results of our experiments also show that (1) the optimum ChOx activity occurs when the molar ratio of H2O-to-PFPE (R) exceeds ∼12, (2) the rate constant describing the conversion of the ChOx-cholesterol complex to product (kcat,app) is similar to values reported using reverse micelle systems formed in liquid alkanes, (3) the equilibrium constant that describes the ChOx-cholesterol complex dissociation (Km,app) is optimal at high R values, (4) the best-case Km,app is ∼2-fold better than the value reported using reverse micelles formed in liquid alkanes, (5) there is little change in the ChOx kcat,app and Km,app as we adjust the CO2 pressure between 100 and 260 bar, and (6) the ChOx was active within the PFPE water pool for at least 5 h; however, after 8 or more hours within the PFPE water pool, ChOx became temporarily inactive.

Introduction Enzyme-catalyzed transformations, associated with high selectivity and rapid turnover under nonaggressive conditions, offer an attractive means to prepare natural products, specialty chemicals, foodstuffs, and fine pharmaceuticals.1 Enzyme-mediated reactions were originally thought to be limited for two reasons. First, some researchers believed that enzymes would only perform well within an aqueous environment. Second, certain important substrates (e.g., steroids) are not particularly soluble in aqueous media. In an effort to overcome these impediments, several research groups began to explore the potential of reverse micelles and microemulsions formed in organic solvents (e.g., isooctane) for performing enzymatic reactions.2 In this approach the micelle core offers a water-like environment that is compatible with the enzyme, and the organic continuous phase provides a way to increase substrate loadings. The concern about enzymes functioning only within an aqueous environment was erased when it was shown that enzymes could perform well in organic liquids.3 However, even though one can simultaneously increase substrate * To whom all correspondence should be directed: 716-645-6800 ext. 2162 (voice); 716-645-6963 (FAX); [email protected] (Email) † A preliminary account of this work was presented at the 6th International Conference on Supercritical Fluids, Nottingham, U.K., April 1999, poster no. 103. (1) (a) Thompson, K. N.; Johnson, R. A.; Lloyd, N. E. U.S. Patent 3 788 945, 1974. (b) Chibata, I.; Tosa, T.; Sato, T.; Mori, T. In Fermentation Technology Today; Terui, G., Ed.; Society of Fermentation Technology: Osaka, Japan, 1972; pp 383-389. (c) Wong, C. H.; Halcomb, R. L.; Ichikawa , Y.; Kajimoto, T. Angew. Chem., Int. Ed. Engl. 1995, 34, 521. (d) Takayama, S.; McGarvey, G. J.; Wong, C. H. Chem. Soc. Rev. 1997, 26, 407. (e) Jones J. B., DeSantis, G. Acc. Chem. Res. 1999, 32, 99. (2) Several of the pioneering works in this field include: (a) Martinek, K.; Levashov, A. V.; Klyachko, N. L.; Berenzin, I. V. Dokl. Akad. Nauk SSSR 1977, 236, 920. (b) Menger, F. M.; Yamada, K. J. Am. Chem. Soc. 1979, 101, 6731. (c) Douzou, P.; Keh, E.; Balny, C. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 681. (d) Luisi, P. L.; Bonner, F.; Pellegrini, A.; Wiget, P.; Wolf, R. Helv. Chim. Acta 1979, 62, 740. (3) For general reviews on this subject see: (a) Klibanov, A. M. CHEMTECH 1986, 16, 354. (b) Dordick J. S. Enzyme Microb. Technol. 1989, 11, 194. (c) Klibanov, A. M. Trends Biotechnol. 1997, 15, 97.

loadings and perform enzyme-based reactions in organic solvents (with or without micelles), there are practical limitations to this approach. For example, organic solvents can be expensive to purchase, they can be difficult to separate from reactants, products, and/or the enzyme(s), they present a difficult recycling challenge, they can be impractical to dispose of without incurring substantial costs, and they may adversely affecting the environment and/or personnel. These problems are exacerbated further because the Montreal Protocol recommends limits for and in some instances proposes bans on the use of many common organic solvents.4 As a result, despite the attractive features associated with performing an enzymatic reaction within an organic solvent, there is clear economic, environmental, energy-related, and political motivation to replace or at the very least minimize the use of certain organic solvents. Supercritical fluids exhibit liquidlike densities and gaslike mass transfer, which make them appealing solvents for extractions, chemical reactions, and separations.5 The appeal arises primarily because one can continuously tune a supercritical fluid’s physicochemical properties (e.g., density and dielectric constant) by simply adjusting the system pressure and temperature. Carbon dioxide is among the more attractive supercritical solvents because its critical points are easy to reach (Tc ) 31.1 °C, Pc ) 73.8 bar), it is inexpensive, it is nonflammable and (4) (a) Noble, D. Anal. Chem. 1993, 65, 693A. (b) Via, J.; Taylor, L. T. CHEMTECH 1993, November, 38. (c) A presidential directive published in the Federal Register (S8 FR: 65018; Dec. 10, 1993) implemented January 1, 1996. (5) (a) Supercritical Fluid Science and Technology; Johnston, K. P., Penninger, J. M. L., Eds.; ACS Symposium Series 406; American Chemical Society: Washington, DC, 1989. (b) Supercritical Fluid TechnologysReviews in Modern Theory and Applications; Bruno, T. J., Ely, J. F., Eds.; CRC Press: Boca Raton, FL, 1991. (c) Supercritical Fluid TechnologysTheoretical and Applied Approaches in Analytical Chemistry; Bright, F. V., McNally, M. E. P., Eds.; ACS Symposium Series 488; American Chemical Society: Washington, DC, 1992. (d) Supercritical Fluid Engineering SciencesFundamentals and Applications; Kiran, E., Brennecke, J. F., Eds.; ACS Symposium Series 514; American Chemical Society: Washington, DC, 1993. (e) McHugh, M. A.; Krukonis, V. J. Supercritical Fluids Extraction: Principles and Practice, 2nd ed.; Butterworth-Heineman: Newton, MA, 1993.

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nontoxic, it is effectively separated from a system by a decompression step, it can be recycled, and it is an environmentally responsible solvent. Unfortunately, hydrophiles and proteins are not particularly soluble in CO2. Despite this impediment, several research groups have shown that one can carry out enzymatic reactions directly in supercritical fluids such as CO2 by using immobilized enzymes or enzyme suspensions.6,7 Several research groups have shown that one can form thermodynamically stable reverse micelles and microemulsions in scCO2.8,9 The most widely reported of these new micelle systems with aqueous water pools are those based on PFPE (perfluoropolyether ammonium carboxylate).8a,10-12 Over the past few years several key aspects of the PFPE/scCO2/H2O system have been investigated. For example, Heitz et al. explored their phase equilibria, water pool and micelle shape, and internal dynamics.10 Niemeyer and Bright reported that the pH within an unbuffered PFPE water pool was ∼3.5.11 Holmes et al. showed how one could use concentrated aqueous buffer solutions to increase the water pool pH within PFPE micelles.12 The Johnston and Howdle groups have shown that one can use the PFPE/CO2/H2O system to perform inorganic13a and organic13b,c reactions directly within the micelle water pool. Most recently, Johnston and co-workers showed that one can prepare tailored, uniform CdS nanoparticles within the water pool of PFPE micelles formed in scCO2.13d However, despite the utility13 of the PFPE/CO2/H2O system, there have not been any enzymatic reactions performed with this system. In fact, there have not been any literature reports at all on enzyme catalysis within the water pool of any reverse micelle system formed in scCO2. The only related literature report is by Holmes et al. on the lipase-catalyzed hydrolysis of p-nitrophenolbutyrate and lipoxygenase-catalyzed peroxidation of linoleic acid within the water pool of di-HCF4 (fluorinated dichained sulfosuccinate surfactant) reverse micelles formed at 20 °C in liquid CO2.14 In this paper we report the first enzymatic catalysis within the water pool of a reverse micelle formed in neat scCO2. We also report on the quantitative behavior of the (6) The primary works on enzymatic reactions scCO2 include: (a) Randolph, T. W.; Blanch, H. W.; Prausnitz, J. M.; Wilke, C. R. Biotechnol. Lett. 1985, 7, 325. (b) Hammond, D. A.; Karel, M.; Klibanov, A. M.; Krukonis, V. J. Appl. Biochem. Biotechnol. 1985, 11, 393. (c) Nakamura, K.; Chi, Y. M.; Yamanda, Y.; Yano, T. Chem. Eng. Commun. 1985, 45, 207. (7) For authoritative reviews on enzymatic reactions in supercritical fluids see: (a) Kamat, S. V.; Beckman, E. J.; Russell, A. J. Crit. Rev. Biotechnol. 1995, 15, 41. (b) Mesiano, A. J.; Beckman, E. J.; Russell, A. J. Chem. Rev. 1999, 99, 623. (8) Pioneering work on reverse micelles and microemulsions formed in scCO2 include: (a) 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. (b) Eastoe, J.; Cazelles, B. M. H.; Steytler, D. C.; Holmes, J. D.; Pitt, A. R.; Wear, T. J.; Heenan, R. K. Langmuir 1997, 13, 6980. (c) Ghenciu, E. G.; Russell, A. J.; Beckman, E. J.; Steele, L.; Becker, N. T. Biotechnol. Bioeng. 1998, 58, 572. (d) Salaniwal, S.; Cui, S. T.; Cummings, P. T.; Cochran, H. D. Langmuir 1999, 15, 5188. (9) McClain, J. B.; Betts, D. E.; Canelas, D. A.; Samulski, E. T.; De Simone, J. M.; Londono, J. D.; Cochran, H. D.; Wignall, G. D.; ChilluraMartino, D.; Triolo, R. Science 1996, 274, 2049. (10) Heitz, M. P.; Carlier, C.; de Grazia, J.; Harrison, K. L.; Johnston, K. P.; Randolph, T. W.; Bright, F. V. J. Phys. Chem. 1997, 101, 6707. (11) Niemeyer, E. D.; Bright, F. V. J. Phys. Chem. B 1998, 102, 1474. (12) Holmes, J. D.; Ziegler, K. J.; Audriani, M.; Lee, C. T., Jr.; Bhargava, P. A.; Steytler, D. C.; Johnston, K. P. J. Phys. Chem. B 1999, 103, 5703. (13) (a) Clarke, M. J.; Harrison, K. L.; Johnston, K. P.; Howdle, S. M. J. Am. Chem. Soc. 1997, 119, 6399. (b) Jacobson, G. B.; Lee, C. T., Jr.; Johnston, K. P. J. Org. Chem. 1999, 64, 1201. (c) Jacobson, G. B.; Lee, C. T., Jr.; da Rocha, R. P.; Johnston, K. P. J. Org. Chem. 1999, 64, 1207. (d) Holmes, J. D.; Bhargava, P. A.; Krogel, B. A.; Johnston, K. P. Langmuir 1999, 15, 6613. (14) Holmes, J. D.; Steytler, D. C.; Rees, G. D.; Robinson, B. H. Langmuir 1998, 14, 6371.

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enzyme as a function of water pool “pH”, water loading, CO2 pressure, and incubation time within the water pool. Toward these ends, we have investigated the oxidation of cholesterol by cholesterol oxidase (ChOx) to produce 4-cholesten-3-one (Figure 1). A second enzyme, catalase, is used here to consume any H2O2 that might adversely affect the ChOx. We chose to study this particular reaction for several reasons. First, cholesterol exhibits limited solubility in water (∼4.7 µM), but it is far more soluble in scCO2 (Figure 2).15 Second, Gupte et al.16 have previously studied this reaction within Aerosol OT (AOT) reverse micelles formed in liquid isooctane, providing us with a convenient point of comparison. Third, Randolph et al.17 have shown that ChOx is active in neat scCO2. Fourth, 4-cholesten-3-one (cholestenone) is a commercially viable seed material for the production of androst-1,4-diene3,17-dione which is an estradiol precursor used to prepare oral contraceptives.17b Finally, cholestenone absorbs at 242 nm so we can use in situ UV absorbance spectroscopy to follow product formation. Experimental Section Chemicals and Reagents. PFPE (Figure 1) was prepared by reacting Fomblin Y(Aussimont) with an excess of NH4OH. ChOx (EC 1.1.3.6) from Pseudomonas fluorescens, catalase (EC 1.11.1.6) from Asperigillus niger, AOT, and cholesterol were products of Sigma. Isooctane (spectrophotometric grade) was purchased from Aldrich. CO2 (SFC grade) and O2 (99%) were purchased from Scott Specialty Gases. The buffers we used in this study were phosphate, citrate, acetate, 2-(N-morpholino)ethanesulfonic acid, sodium salt (MES), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), and tris(hydroxymethyl)aminomethane (TRIS). Buffer concentrations were 0.05 or 1.0 M. AOT was purified by the method of Kotlarchyk,18 and it was stored in a desiccator over CaCl2. All other reagents were used as received, and aqueous solutions were prepared using doubly distilled-deionized water. In Situ Spectroscopy. A simplified schematic of the highpressure system that was used to follow the ChOx-induced cholesterol oxidation reaction is shown in Figure 3. The system consists of an Isco 260-D syringe pump, an in-line HPLC injector with several sampling loops, a high-pressure optical cell,19 a UVVis spectrophotometer (model 1201, Spectronic Instruments), a home-built temperature controller ((0.1 °C), and a pressure monitoring ((0.2 bar) system (Heise gauge). The high-pressure cell internal volume is 3.5 mL. Enzyme Reactions in AOT/Isooctane/H2O. Benchmark ChOx-induced oxidation experiments were performed at 35 °C in 0.068 M AOT reverse micelles formed in liquid isooctane. The total solution volume was kept at 3.5 mL, and the total amount of ChOx and catalase within the cuvette was 2 and 4 µg, respectively. For AOT/isooctane experiments we prepared two solutions: (A) 2.0 mM cholesterol and 0.068 M AOT dissolved in isooctane and (B) a series of 0.05 M aqueous buffers between pH ∼ 3 and pH ) 9 that contained the appropriate amount of ChOx and catalase. An appropriate volume (3.5 mL) of solution A was mixed with a predetermined volume of solution B (adjusted to achieve a particular molar ratio of water to surfactant, R ) [H2O]/ [surfactant]) within a standard 1-cm2 quartz cuvette, and the reaction was followed spectrophotometrically. Results were similar when we used 0.05 or 1.0 M buffers to form the AOT micelle water pool. (15) Yun, S. L. J.; Liong, K. K.; Gurdial, G. S.; Foster, N. R. Ind. Eng. Chem. Res. 1991, 30, 2476. (16) Gupte, A.; Nagarajan, R.; Kilara, A. Ind. Eng. Chem. Res. 1995, 34, 2910. (17) (a) Randolph, T. W.; Blanch, H. W.; Prausnitz, J. M. AIChE J. 1988, 34, 1354. (b) Randolph, T. W.; Clark, D. S.; Blanch, H. W.; Prausnitz, J. M. Science 1988, 239, 387. (18) Kotlarchyk, M.; Chen, S. H.; Huang, J. S.; Kim, M. W. Phys. Rev. A 1984, 29, 2054. (19) (a) Betts, T. A.; Bright, F. V. Appl. Spectrosc. 1990, 44, 1196. (b) Zagrobelny, J.; Bright, F. V. J. Am. Chem. Soc. 1993, 115, 701.

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Figure 1. Simplified schematic of the enzymatic oxidation of cholesterol to cholestenone by cholesterol oxidase within the PFPE/ scCO2/H2O system. The structure of PFPE is also shown.

Figure 2. Effects of temperature and pressure on the solubility of cholesterol in scCO2 (data from ref 15).

Figure 3. Simplified schematic of the system used to follow the ChOx-induced oxidation of cholesterol in situ. ChOx activities were determined from the absorbance vs time profile and the reported cholestenone molar extinction coefficient (15 500 mol-1 dm3 cm-1 at 240 nm).16 ChOx activities are given as micromoles of cholestenone formed per minute per milligram of enzyme.16 To establish a benchmark on the ChOx activity vs AOT water pool pH, we performed a set of experiments at R ) 10. Enzyme Reactions in PFPE/scCO2/H2O. These experiments were performed at pressure by adding the following directly into the high-pressure optical cell: 1.4 wt % PFPE, a known amount of cholesterol (0-2 mg), and the appropriate volume of

a particular buffer to produce a given R value. The optical cell temperature was then adjusted to 35 °C, and the cell was carefully flushed with 4-5 bar of CO2, charged with O2 (0 or 10 bar), and then charged to ∼95% of the final CO2 pressure. The cell contents were then mixed with a magnetically coupled stir bar for 1 h to ensure that the micelles formed and that all the cholesterol was dissolved. Visual inspection of the cell contents showed only a single clear phase. To initiate the reaction, we injected the aqueous ChOx/catalase mixture (in the proper buffer)12 directly into the high-pressure cell by simultaneously activating the inline HPLC injector and raising the CO2 pump pressure to the desired final value. The injected amounts of ChOx and catalase were kept at 1 and 2 µg, respectively. ChOx activities were determined as described above. The R values reported here are based on the total volume of buffer added. In our first optimization step, we set out to determine how the ChOx activity was influenced by the PFPE water pool “pH”. This was done by performing an initial set of experiments using 0.05 or 1.0 M aqueous buffers at R ) 11 (200 bar CO2 and 10 bar O2). These experiments were followed by ones that aimed to determine the enzyme activity vs cholesterol concentration at the optimum water pool “pH” that we determined from step 1. LineweaverBurke analysis20 of these cholesterol-dependent rate data yielded an apparent Michaelis-Menten constant describing the formation of the enzyme-substrate (ES) complex (Km,app ) [ES]) and an apparent first-order rate constant (kapp,cat ) [P]/[ES]) for the reaction of ES to form the product (P):

E + S h ES f P + E

(1)

A third series of experiments were performed at the optimum water pool “pH” and buffer concentration as a function of CO2 pressure. Finally, we explored the effects of ChOx incubation time within the PFPE reverse micelle water pool on the apparent ChOx activity. Reproducibility Studies. All reported experiments were carried out in at least triplicate. All results are reported as the average of all experiments under a given set of conditions. Error bars represent plus or minus one standard deviation. (20) Bohinski, R. C. Modern Concepts in Biochemistry, 3rd ed.; Allyn and Bacon: Boston, MA, 1979; Chapter 6.

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Figure 4. Effects of buffer solution pH on the ChOx activity in AOT/buffer (0.05 M)/isooctane (b), PFPE/scCO2 using 0.05 M buffer (O), and PFPE/scCO2 using 1.0 M buffer (1) at 35 °C.

Results and Discussion Effects of Water Pool pH. Figure 4 summarizes the effects of water pool “pH” on the ChOx activity within AOT reverse micelles formed in liquid isooctane (R ) 10) and PFPE micelles formed in scCO2 (R ) 11) each at 35 °C. The AOT results (b) are in line with the previous work on ChOx (from Nocardia erythropolis),16 showing an optimum near pH ∼ 5. The activity results for ChOx sequestered within the PFPE reverse micelles using 0.05 M buffers to form the water pool (O) shows that the ChOx is not particularly active and there is no clear optimum. In contrast, when we increased the buffer concentration from 0.05 to 1.0 M, we notice (1) a substantial increase in the ChOx activity relative to the lower concentration buffer and an optimum is clearly seen near pH ) 7. These results are fully consistent with the more concentrated buffer providing enough buffer capacity against the excess CO2 to raise the water pool pH from ∼3.5 up to 5 or 6.12 A series of control experiments akin to those performed previously11 (results not shown) do not suggest that the presence of the enzymes per se affected the water pool pH significantly. The apparent shift in the pH optima when comparing the AOT and PFPE results likely comes about because a more basic aqueous buffer is needed to off set the substantially more acidic environment encountered within the PFPE/scCO2 water pool arising from the formation of carbonic acid.11,12 Estimation of ChOx kcat,app and Km,app within PFPE Reverse Micelles in scCO2. The ChOx-induced oxidation of cholesterol follows Michaelis-Menten kinetics within the PFPE/scCO2/H2O system. Figure 5 summarizes the effects of R on the ChOx kcat,app and Km,app when we form the water pool by using a pH 7 buffer (1.0 M) at 150 bar CO2 and 10 bar O2. These results show that kcat,app is optimal at R ∼ 12 and that Km,app is optimal at R g 12. Our optimized kcat,app value for ChOx sequestered within the PFPE reverse micelles is very similar to the reported value16 for ChOx in AOT reverse micelles formed in liquid isooctane. Our best-case Km,app value is ∼2-fold better when compared to the value16 for ChOx in AOT reverse micelles formed in liquid isooctane. Together these results show that, at the optimum water loading and water pool pH, the intrinsic ability of ChOx to produce cholestenone (once the enzyme-substrate complex is formed) is not particularly different in PFPE/scCO2 compared to AOT/isooctane; however, the intrinsic ability of ChOx to form its enzymesubstrate complex with cholesterol in the PFPE reverse micelles is 2-fold more favored relative to the AOT/ isooctane system. Thus, the ChOx-cholesterol complex formed in the PFPE/scCO2 system is more thermodynamically stable compared to ChOx in AOT/isooctane. This increased stability may arise from something unique

Figure 5. Effects of water loading, R, on the ChOx kcat,app (upper panel) and Km,app (lower panel) in PFPE reverse micelles formed in scCO2 using 1.0 M pH 7 buffer at 150 bar CO2 and 10 bar O2.

Figure 6. Effects of water loading, R, on the apparent overall reaction rate in PFPE reverse micelles formed in scCO2 using 1.0 M pH 7 buffer at 150 bar CO2 and 10 bar O2.

within the PFPE micelle interfacial region and/or from PFPE binding to the ChOx. Figure 6 presents the results from Figure 5 in terms of a total second-order rate constant. These results show that the overall ChOx performance is optimal in the PFPEbased reverse micelles above R ≈ 12-15. Water loadings above R ) 20 were not explored here because the system became two phases above this water loading. Figure 7 summarizes the effects of CO2 pressure on the ChOx kcat,app and Km,app within the PFPE micelles when we form the water pool with a pH 7 buffer (1.0 M) at 10 bar O2 and R ) 14. The results of these experiments show that there is little change in kcat,app or Km,app over this pressure range. CO2 pressures below 100 bar were not investigated here because the system tends to form two phases below 85-90 bar CO2 and the cholesterol solubility below 90 bar CO2 limited our ability to cover a suitable range of cholesterol concentrations for a full LineweaverBurke analysis. Figure 8 presents the results from Figure 7 in terms of a total second-order rate constant. These results show

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Figure 9. Effects of incubation time on the ChOx activity within PFPE reverse micelles formed in scCO2.

Figure 7. Effects of CO2 pressure on the ChOx kcat,app (upper panel) and Km,app (lower panel) in PFPE reverse micelles formed in scCO2 using 1.0 M pH 7 buffer at 150 bar CO2, 10 bar O2, and R ) 14.

200 bar CO2, 10 bar O2, R ) 11, 1 µg of ChOx, and 2 µg of catalase. The results of these experiments are summarized in Figure 9. ChOx sequestered within the PFPE reverse micelles remains active for at least 5 h; however, after 8 h within the PFPE reverse micelle, the ChOx is completely inactive. Interestingly, when we slowly (over 3-4 h) decompress the high-pressure optical cell and assay the ChOx, we found (results not shown) that the ChOx activity was within 20% of the value for a fresh enzyme preparation prior to being subjected to PFPE/scCO2/H2O. This result suggests that ChOx is slowly inactivated within the PFPE reverse micelles, but this inactivation is reversed following a slow decompression step. On rapid decompression (1-5 min), the ChOx remained completely inactivated. These results suggest that there is a slow reversible conformational change in the ChOx active site brought on by it being sequestered within the PFPE reverse micelle and/or a slow, reversible binding of the PFPE to the ChOx that eventually leads to ChOx inactivation. The “pH” within a buffered water pool is stable for up to several days. Conclusions

Figure 8. Effects of CO2 pressure on the apparent overall reaction rate in PFPE reverse micelles formed in scCO2 using 1.0 M pH 7 buffer at 150 bar CO2, 10 bar O2, and R ) 14.

that the overall ChOx performance in the PFPE reverse micelles is essentially constant between 100 and 260 bar CO2. ChOx Activity with PFPE Reverse Micelles Formed in scCO2. To determine how stable the ChOx is within the PFPE reverse micelles, we performed a series of experiments where a fixed amount of ChOx (0.5-3 µg) was loaded into the high-pressure optical cell along with the buffer, PFPE, and O2. We then brought the cell to 95% of its final CO2 pressure, allowed the ChOx to incubate for different lengths of time (at 35 °C), and then injected cholesterol to initiate the reaction and determine the ChOx activity. These experiments were carried out by using 1.4 wt % PFPE, a pH 7 (1.0 M) buffer to form the water pool,

We report the first results on an enzyme-induced reaction within the water pool of reverse micelles formed in scCO2. The results of our experiments show that ChOx obeys Michaelis-Menten kinetics within the PFPE reverse micelles formed in scCO2. At the optimal buffer pH and water loading, the ChOx kcat,app is comparable to the value reported in AOT/isooctane. In contrast, the corresponding Km,app is ∼2-fold better when compared to the best value for ChOx in AOT/isooctane. There is no significant change in kcat,app or Km,app when we change the CO2 pressure between 100 and 260 bar. The ChOx activity is a function of its residence time within the PFPE micelle water pool. At short contact times (e5 h), the ChOx activity is essentially constant, but after 5 or more hours within the PFPE water pool, the ChOx activity drops. The ChOx activity returns partially after a slow decompression step. Acknowledgment. We thank U.S. Department of Energy for financial support. LA991604N