Langmuir 2009, 25, 977-982
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Biocatalysis in Water-in-Ionic Liquid Microemulsions: A Case Study with Horseradish Peroxidase M. Moniruzzaman,† N. Kamiya,†,‡ and M. Goto*,†,‡ Department of Applied Chemistry, Graduate School of Engineering, and Center for Future Chemistry, Kyushu UniVersity, 744 Moto-oka, Fukuoka 819-0395, Japan ReceiVed September 23, 2008. ReVised Manuscript ReceiVed NoVember 2, 2008 In this article we report the first results on the enzymatic activity of horseradish peroxidase (HRP) microencapsulated in water-in-ionic liquid (w/IL) microemulsions using pyrogallol as the substrate. Toward this goal, the system used in this study was composed of anionic surfactant AOT (sodium bis(2-ethyl-1-hexyl)sulfosuccinate)/hydrophobic IL [C8mim][Tf2N] (1-octyl-3-methyl imidazolium bis(trifluoromethylsulfonyl)amide)/water/1-hexanol. In this system, the catalytic activity of HRP was measured as a function of substrate concentrations, W0 (molar ratio of water to surfactant), pH, and 1-hexanol content. The curve of the activity-W0 profile was found to be hyperbolic for the new microemulsion. The apparent Michaelis-Menten kinetic parameters (kcat and Km) were estimated and compared to those obtained from a conventional microemulsion. Apparently, it was found that HRP-catalyzed oxidation of pyrogallol by hydrogen peroxide in IL microemulsuions is much more effective than in a conventional AOT/water/isooctane microemulsion. The stability of HRP solubilized in the newly developed w/IL microemulsions was examined, and it was found that HRP retained almost 70% of its initial activity after incubation at 28 °C for 30 h.
Introduction Ionic liquids (ILs) have been established as a potential alternative to highly volatile organic solvents.1 Indeed, their many unique and attractive physicochemical properties such as negligible vapor pressure, a wide liquid range, good dissolution properties, and high thermal stability make ILs great candidates for volatile organic compound (VOC) replacements. ILs are tunable solvents owing to the huge number of possible cation/ anion combinations. In the past decade, ILs have been exploited for use in a wide range of applications, including extraction,2 organic synthesis,3 separation,4 nanomaterial synthesis,5 and enzymatic reactions.6-9 In particular, the application of environmentally benign ILs as media for biocatalysis has received * To whom correspondence should be addressed. Tel: +81-92-802-2806, fax: +81-92-802-2810, e-mail:
[email protected]. † Department of Applied Chemistry, Kyushu University. ‡ Center for Future Chemistry, Kyushu University. (1) (a) Welton, T. Chem. ReV. 1999, 99, 2071. (b) Wasserscheid, P.; Keim, W. Angew. Chem., Int. Ed. 2000, 39, 3772. (c) Earle, M. J.; Esperanca, J. M. S. S.; Gilea, M. A.; Lopes, J. N. C.; Rebelo, L. P. N.; Magee, J. W.; Seddon, K. R.; Widegren, J. A. Nature 2006, 439, 831. (d) Anderson, J. L.; Ding, J.; Welton, T.; Armstrong, D. W. J. Am. Chem. Soc. 2002, 124, 14247. (e) Bonhoˆte, P.; Dias, A. P.; Papageorgiou, N.; Kalyanasundaram, K.; Gra¨tzel, M. Inorg. Chem. 1996, 35, 1168. (f) Huddleston, J. G.; Visser, A. E.; Reichert, W. M.; Willauer, H. D.; Broker, G. A.; Rogers, R. D. Green Chem. 2001, 3, 156. (2) (a) Visser, A. E.; Swatloski, R. P.; Griffin, S. T.; Hartman, D. H.; Rogers, R. D. Sep. Sci. Technol. 2001, 36, 785. (b) Chun, S.; Dzyuba, S. V.; Bartsch, R. A. Anal. Chem. 2001, 73, 3737. (3) (a) Zerth, H. M.; Leonard, N. M.; Mohan, R. S. Org. Lett. 2003, 5, 55. (b) Van Rantwijk, F.; Lau, R. M.; Sheldon, R. A. Trends Biotechnol. 2003, 21, 131. (4) Anderson, J. L.; Armstrong, D. W. Anal. Chem. 2005, 77, 6453. (5) Antonietti, M.; Kuang, D.; Smarsly, B.; Zhou, Y. Angew. Chem., Int. Ed. 2004, 43, 4988. (6) (a) Van Rantwijk, F.; Sheldon, R. A. Chem. ReV. 2007, 107, 2757. (b) Yang, Z.; Pan, W. Enzyme Microb. Technol. 2005, 37, 19. (c) Kragl, U.; Eckstein, M.; Kaftzik, N. Curr. Opin. Biotechnol. 2002, 13, 565. (d) Park, S.; Kazlauskas, R. J. Curr. Opin. Biotechnol. 2003, 14, 432. (7) (a) Erbeldinger, M.; Mesiano, A. J.; Russel, A. J. Biotechnol. Prog. 2000, 16, 1129. (b) Eckstein, M.; Sesing, M.; Kragl, U.; Adlercreutz, P. Biotechnol. Lett. 2002, 24, 867. (c) Turner, M. B.; Spear, S. K.; Huddleston, J. G.; Holbrey, J. D.; Rogers, R. D. Green Chem. 2003, 5, 443. (d) Cull, S. G.; Holbrey, J. D.; Vargas-Mora, V.; Seddon, K. R.; Lye, G. J. Biotechnol. Bioeng. 2000, 69, 227. (e) Kaar, J. L.; Jesionowski, A. M.; Berberich, J. A.; Moulton, R.; Russel, A. J. J. Am. Chem. Soc. 2003, 125, 4125. (f) Park, S.; Kazlauskas, R. J. J. Org. Chem. 2001, 66, 8395. (g) Kim, K. W.; Song, B.; Choi, M. Y.; Kim, M. J. Org. Lett. 2001, 3, 1507.
tremendous attention in the past few years. In ILs many enzymes retain their catalytic activity; examples are lipases, alcohol dehydrogenase, proteases, oxidoreductases, and so forth.6a,b Compared to those observed in molecular organic solvents, enzymes in ILs have presented enhanced activity, stability, and selectivity as well as better recoverability and recyclability.6-9 Unfortunately, the practical obstacle of using ILs for biocatalysis is that many enzymes do not dissolve readily in most ILs, which has ruled out many potential biotechnological applications. In fact, enzymes that show catalytic activities in ILs normally do not dissolve in ILs. When enzymes become active in ILs, they remain suspended as a powder. Although some ILs can dissolve enzymes through weak hydrogen bonding interactions, they often induce enzyme conformational changes resulting in inactivation.8 For example, EAN (ethylammonium nitrate) can dissolve CaLB through strongly coordinating nitrate anion resulting in denaturation.8b To improve enzyme solubility as well as activity in ILs, various attempts, including adsorption onto an acrylic resin, PEG modification, and immobilization through multipoint attachment in polyurethane foam, have been investigated.9 However, such approaches are often laborious and timeconsuming as well as tedious, and they also suffer from high costs. Another strategy for enhancing enzyme solubility in ILs involves adding a small amount of water, but the dissolved enzymes show low catalytic activity due to their conformational change.7a-c Recently, several researchers have reported enzymatic reactions in a mixture of hydrophilic ILs and water with a high concentration of water.10 However, such media are not suitable (8) (a) Sheldon, R. A.; Lau, R. M; Sorgedrager, M. J.; Van Rantwijk, F.; Seddon, K. R. Green Chem. 2002, 4, 147. (b) Lau, R. M.; Sorgedrager, M. J.; Carrea, G.; Van Rantwijk, F.; Secundo, F.; Sheldon, R. A. Green Chem. 2004, 6, 483. (9) (a) Ohno, H.; Suzuki, C.; Fukumoto, K.; Yoshizawa, M.; Fujita, K. Chem. Lett. 2003, 32, 450. (b) Itoh, T.; Matsushita, Y.; Abe, Y.; Han, S.; Wada, S.; Hayase, S.; Kawatsura, M.; Takai, S.; Morimoto, M.; Hiroshe, Y. Chem. Eur. J. 2006, 12, 9228. (c) Nakashima, K.; Maruyama, T.; Kamiya, N.; Goto, M. Chem. Commun. 2005, 4297. (d) Aorroyo, M.; Sanchez-Montero, J. M.; Sinisterra, J. V. Enzyme Microb. Technol. 1999, 24, 3. (e) LeJeune, K. E.; Russel, A. J. Biotechnol. Bioeng. 1996, 51, 450. (10) (a) Das, D.; Dasgupta, A.; Das, P. K. Tetrahedron Lett. 2007, 48, 5635. (b) Walker, A. J.; Bruce, N. C. Chem. Commun. 2004, 2570.
10.1021/la803118q CCC: $40.75 2009 American Chemical Society Published on Web 12/29/2008
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Figure 1. Oxidation of pyrogallol by horseradish peroxidase and hydrogen peroxide. The formation of an orange color purpurogallin can be monitored by UV-vis spectrophotometry at λ ) 420 nm.
for synthetic reactions. Obviously, these observations demand the development of an easy and effective way to combine enzyme solubility and activity in ILs to expand the use of them as a green solvent. One of the most promising approaches to solve this problem is to form nano/micrometer-sized water domains in an IL continuous phase (noted as w/IL microemulsions) stabilized by the suitable surfactant. It is well documented that enzymes can be solubilized in organic solvents by the use of surfactants (generally called w/o microemulsions) without the loss of their catalytic activity.11,12 In this microheterogeneous medium, enzyme molecules are entrapped in tiny water domains and thus become protected against unfavorable contact with the surrounding organic solvent by a layer of water and surfactant molecules, thereby exhibiting good stability and activity. Indeed, w/o microemulsions are highly versatile reaction media, which are currently used for many applications. The insolubility of enzymes in compressed or supercritical carbon dioxide (SC CO2) was also solved by the formation of water domains in CO2 (w/c microemulsions),13 which is now being used for various purposes including enzymatic reactions.14 Therefore, development of w/IL microemulsions to solubilize enzymes and other biomolecules is desirable and challenging. To this end, we have reported the formation of aqueous droplets in a hydrophobic IL [C8mim][Tf2N] (1-octyl-3-methylimidazolium bis(trifluromethylsulfonyl)amide) stabilized by the anionic surfactant AOT [sodium bis(2-ethyl-1-hexyl)sulfosuccinate] molecules.15 Normally, the surfactant AOT, which has been used extensively for the formation of microemulsions in nonpolar organic solvents16 and CO2,17 cannot be solubilized in IL [C8mim][Tf2N]. This limitation was overcome using 1-hexanol as a cosurfactant. The results indicated that such a water domain can dissolve various enzymes and other biomolecules. In our recent communication18 we have reported the first enzymatic reaction conducted in a w/IL microemulsion using lipase as a model enzyme. Preliminary results indicated that the catalytic activity of lipase in this new microemulsion became higher than in a w/o microemulsion. This notable finding inspires us to expand the portfolio of the new microemulsions by examining the catalytic activity of a variety of enzymes. For example, horseradish peroxidase (HRP), a interfacially active enzyme, exibits enhanced activity in w/o microemulsions.11a,19,20 We believe that HRP would behave similarily in w/IL microemulsions. In addition, the activity of enzymes entrapped in w/o microemulsions depends markedly on various physical parameters, mainly W0 (molar ratio of water to surfactant) and the pH of the system11b,d because such parameters can be readily tuned to purposefully alter the conditions in the system. Thus, it is definitely interesting to see how the catalytic activity of the enzyme in the w/IL microemulsions are influenced by changing such parameters. Here, for the first time, we investigate the activity of HRP entrapped in w/IL microemulsions.To our knowledge, this is only the second report of enzymatic catalysis within the water pool of w/IL microemulsions after our first one.18 HRP-catalyzed oxidation of pyrogallol by H2O2 was used as a model reaction
(Figure 1), which has previously been performed in w/o microemulsions.19,20a The reaction rate is optimized with respect to various physicochemical parameters of the system. We also observe the stability of HRP in the new environment.
Experimental Section Materials. IL [C8mim][Tf2N] (1-octyl-3-methylimidazolium bis(trifluromethylsulfonyl)amide) was synthesized and stored as described elsewhere.1e The purity of IL was verified by elementary analysis, showing that analytically calculated values and experimental values were the same. The surfactant AOT, sodium bis(2-ethyl-1hexyl)sulfosuccinate (>99%), was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO) and was used as received. The reagents for the synthesis of IL were obtained from Sigma-Aldrich Chemical Co., while isooctane and 1-hexanol were from Wako, Japan. The substrate pyrogallol ((1,2,3-benzentriol) and purpurogallin (2,3,4,6tetrahydroxy-5H-benzocycloheptene-5) were purchased from Tokyo Chemical Industries Co. Ltd., Japan, while the extra pure hydrogen peroxide (H2O2)(30% w/w) was from Sigma. Horseradish peroxidase (grade I) was obtained from Wako, Japan, and was used as received. All other reagents used in the experiments were analytical grade. Preparation of IL Microemulsions. The required amount of AOT was dissolved in IL containing 10% (v/v) 1-hexanol. Then, a small amount of buffer solution was added to prepare a microemulsion. Clear and stable solutions were obtained by vortex mixing and used as stock solutions. The molar ratio of water to AOT (W0, calculated by subtracting the independently known amount of water soluble in pure IL without surfactant from the total amount of water)15 of the microemulsions were manipulated by injecting an appropriate amount of buffer solution. Preparation of Reaction Solutions. Stock HRP solutions were prepared in the buffer solution. The peroxidase concentration was determined spectrophotometrically using a molar absorption of 9.1 (11) (a) Martinek, K.; Levashov, A. V.; Khmelnitskii, Y. L.; Klyachko, N. L.; Berezin, I. V. Science 1982, 218, 889. (b) Luisi, P. L.; Magid, I. J. CRC Crit. ReV. Biochem. 1986, 20, 409. (c) Rees, G. D.; Robinson, B. H.; Stephenson, G. R. Bichim. Biophys. Acta 1995, 1257, 239. (d) Han, D.; Walde, P.; Luisi, P. L. Biocatalysis 1990, 4, 153. (e) Fletcher, P. D. I.; Robinson, B. H. J. Chem. Soc., Faraday Trans. 1 1985, 81, 2667. (12) (a) Barbaric, S.; Luisi, P. L. J. Am. Chem. Soc. 1981, 103, 4239. (b) Lissi, E. A.; Abuin, E. B. Langmuir 2000, 16, 10084. (c) Das, P. K.; Srilakshmi, G. V.; Chaudhuri, A. Langmuir 1999, 15, 981. (13) (a) Johnston, K. P.; Harrison, K. L.; Clark, 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.; Steyler, D. C.; Holmes, J. D.; Pitt, A. R.; Wear, T. J.; Heenan, R. K. Langmuir 1997, 13, 6980. (c) Salaniwal, S.; Cui, S. T.; Cummings, P. T.; Cochran, H. D. Langmuir 1999, 15, 5188. (14) (a) Holmes, J. D.; Steytler, D. C.; Rees, G. D.; Robinson, B. H. Langmuir 1998, 14, 6371. (b) Kane, M. A.; Baker, G. A.; Pandey, S.; Bright, F. V. Langmuir 2000, 16, 4901. (15) Zaman, M. M.; Kamiya, N.; Nakashima, K.; Goto, M. Chem. Phys. Chem. 2008, 9, 689. (16) De, T.; Maitra, A. AdV. Colloid Interface Sci. 1995, 59, 95. (17) Liu, J.; Ikushima, Y.; Shervani, Z. J. Supercrit. Fluids 2004, 32, 97. (18) Zaman, M. M.; Kamiya, N.; Nakashima, K.; Goto, M. Green Chem. 2008, 10, 497. (19) (a) Klyachko, N. L.; Levashow, A. V.; Martinek, K. Mol. Biol. USSR 1984, 18, 830. (b) Gebicka, L.; Pawlak, J. J. Mol. Catal. B: Enzym. 1997, 2, 185. (c) Bauduin, P.; Touraud, D.; Kunz, W.; Savelli, M. P.; Pulvin, S.; Ninham, B. W. J. Colloid Interface Sci. 2005, 292, 244. (20) (a) Roy, S.; Dasgupta, A.; Das, P. K. Langmuir 2006, 22, 4567. (b) Das, D.; Roy, S.; Mitra, R. N.; Dasgupta, A.; Das, P. K. Chem. Eur. J. 2005, 11, 4881. (c) Shome, A.; Roy, S.; Das, P. K. Langmuir 2007, 23, 4130. (d) Mahiuddin, S.; Renoncourt, A.; Bauduin, P.; Touraud, D.; Kunz, W. Langmuir 2005, 21, 5259.
Biocatalysis in Water-in-Ionic Liquid Microemulsions
Figure 2. Dependence of the initial rate of the enzyme-catalyzed oxidation of pyrogallol on horseradish peroxidase (HRP) concentration at 35 °C. Conditions were: [AOT] ) 200 mM, [pyrogallol]T ) 2 mM, [H2O2]T ) 0.1 mM, W0) 4 and buffer pH (50 mM phosphate) ) 7. The data represents the average of the three experiments, and the error bars indicate the standard deviation.
× 104 M-1 cm-1 at 403 nm.21 The pyrogallol solutions were prepared in acetone while the H2O2 solutions were prepared by diluting the initial concentrated solution with buffer immediately prior to the experiment. For enzymatic reactions in the AOT/water/IL/1-hexanol systems, a typical experiment was performed as follows. To 0.97 mL of stock solution was added 2.5 µL of pyrogallol solution (0.8 M) and specific amounts of buffer solution to reach the desired W0. The mixture was vortexed to obtain a macroscopically homogeneous solution. After adding 5 µL of HRP (1.25 mg/mL), the contents were shaken mildly and kept at 35 °C so that they were combined with the reaction mixture at ambient temperature. Finally 2 µL of H2O2 buffer solution (0.4 M) was added to initiate the reaction. In our experiment, the concentrations of enzyme and substrates were defined as the overall concentration to avoid the complexity of the volume fraction of the water droplet in the w/IL microemulsions and partitioning coefficient of substrates in the bulk IL and microaggregates. Measurement of HRP Activity. The kinetics of pyrogallol oxidation catalyzed by HRP was monitored spectrophotometrically using a UV-vis spectrophotometer (Jasco V-570). The progress of the reaction was recorded at 420 nm (λmax for the product, purpurogallin) after immediate addition of hydrogen peroxide. From the slopes of the linear portions of the absorbance versus time curves (linear at least during the first few minutes), obtained by leastsquares fitting, the values of the initial rates (V) were measured in µM min-1 considering the value of the molar extinction coefficient of purpurogallin in the IL microemulsion (3450 M-1cm-1). Here, a temperature of 35 ( 1 °C was used throughout to facilitate the handling and transfer of the viscous IL [C8mim][Tf2N]. All initial rates were corrected by subtracting the non-enzymatic-catalyzed rates. Stability of HRP in Microemulsions. The microemulsion solutions containing HRP were incubated in the absence of substrates. After incubation, the samples were withdrawn at predetermined time intervals in order to measure the remaining enzyme activity by the addition of substrates (pyrogallol and H2O2). The stability of enzyme was expressed as the residual activity, which was calculated as a percentage of the original activity (considered 100%), obtained at t ) 0 min incubation.
Results and Discussion As a preliminary study, it was important to address the question of whether dissolved HRP in IL microemulsions shows catalytic activity. In order to answer this question, we have investigated the rate of pyrogallol oxidation at various HRP concentrations (Figure 2). It is seen from this figure that the rate of the reaction (liberation of purpurogallin) increased proportionally with increasing enzyme concentration. However, we found that no (21) Meathly, A. C. Methods Enzymol. 1953, 2, 801.
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Figure 3. Reaction profiles (monitored at 420 nm) for HRP-catalyzed pyrogallol oxidation at 35 °C. Total HRP concentrations is 0.1 µM, and the other experimental conditions are the same as described for Figure 2. (a) HRP in microemulsions with 0.5 mM H2O2; (b) HRP in microemulsions with 0.1 mM H2O2; (c) HRP in water saturated IL; (d) HRP in IL alone.
reaction had taken place in the absence of HRP. The results clearly demonstrate that HRP is active in the AOT-based w/IL microemulsions. With the aim of ascertaining the importance of the w/IL microemulsions in solubilizing the enzyme, a control experiment was undertaken in the absence of surfactant, where the activity of HRP was measured only in IL and in water-saturated IL. For this experiment, powder HRP was added to the IL and was shaken mildly for a few minutes. The supernatant was separated by centrifugation. The HRP in the supernatant was assayed for its ability to oxidize pyrogallol by injecting H2O2. The data showed that no significant increase in absorbance was observed in the IL or even in the water-saturated IL (Figure 3). This finding can be explained by the insolubility of enzymes in IL. Even if a small amount of HRP was dissolved in IL, the enzyme was likely inactivated because of interactions with IL. However, the rate of pyrogallol oxidation increased substantially in the presence of surfactant (Figure 3), which clearly confirms that the aqueous domain in ILs is required to facilitate the activity of many enzymes. The results obtained in the preceding paragraphs clearly demonstrate that the AOT-stabilized w/IL microemulsions can be used as a suitable reaction medium for HRP. However, under these reaction conditions, which were selected based on the experiments on HRP-catalyzed oxidation of pyrogallol conducted in w/o microemulsions, the reaction rates are modest. To enhance the activity of HRP in the new microemulsions, it is essential to optimize the various reaction conditions. Toward this goal, the influence of various experimental parameters on the reaction rate was conducted as described below. To improve HRP activity in AOT-based w/IL microemulsions, it was important to optimize the H2O2 concentration since the hydrogen peroxide is an inhibitor of HRP; particularly, a high concentration of H2O2 inactivates the enzyme easily.22 The effect of H2O2 concentration on the activity of HRP was investigated by varying its concentration while keeping the concentrations of the other parameters constant (Figure 4). We found that with an increase in hydrogen peroxide concentration, HRP activity increased to a maximum value and then began to decrease. This observation has been well documented for HRP, where at relatively high concentrations of peroxide the enzyme undergoes substrate inhibition.22 In this study, the maximum activity was found at about 0.8 mM H2O2.This observation is in contrast to that observed in w/o microemulsions, where HRP had an activity (22) Nicell, J. A.; Wright, H. Enzyme Microb. Technol. 1997, 21, 302.
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Figure 4. Effect of hydrogen peroxide on the activity of HRP encapsulated in water-in-IL microemulsions at 35 °C. Conditions were [AOT] ) 200 mM, [HRP]T ) 0.1 µM; [pyrogallol]T ) 2 mM; W0 ) 4, pH 7 (50 mM phosphate buffer). The error bars correspond to the standard deviation from three experiments.
Figure 5. The pH dependence of HRP activity in w/IL microemulsions. The concentration of hydrogen peroxide is 0.8 mM and the other experimental conditions are the same as described in Figure 4. The error bar represents the standard deviation from three experiments. The buffers used for different pH solutions were pH 5-7 (phosphate), pH 8-9 (tris-HCl), and pH 10 (borate).
maximum at about 0.1 mM hydrogen peroxide.22,23 The difference in observed optimal H2O2 concentrations in two microemulsions can be explained from the H2O2 partitioning to the continuous phases. The polar substrate H2O2 can be well distributed to the IL phase whereas it is hardly soluble in apolar organic solvents. In our study, the H2O2 partitioning to the IL continuous phase reduces the concentration in the aqueous pseudophase, and the enzyme only has access to the fraction of H2O2 solubilized in the aqueous pseudophase. To obtain a maximum of enzyme activity, 0.8 mM hydrogen peroxide was used for subsequent measurements. Another important parameter that needs to be optimized is the pH of the aqueous pseudophase in the microemulsions. Since the measurement of the pH inside the water pool is difficult, the pH mentioned refers to that of the buffer before solubilization in IL microemulsions. A range of buffer was used to ensure sufficient buffer capacity in the aqueous dispersion. Herein, we have examined the pH dependence of the HRP-catalyzed oxidation of pyrogallol in w/IL microemulsions at constant enzyme and substrate concentrations. Typical results for the pH profile obtained in w/IL microemulsions are shown in Figure 5. It was observed that HRP showed significant activity between pH 7 and pH 9, with the optimal value at pH 8. Similar behavior was found for HRP-catalyzed reactions in AOT stabilized w/o microemulsions in organic solvents.19a,24 However, the maximum rate of HRP oxidation of various substrates in a aqueous solution (23) Azevedo, M.; Fonseca, L. P.; Graham, D. L.; Cabral, J. M. S.; Prazeres, D. M. F. Biocatal. Biotransform. 2001, 19, 213. (24) (a) Parida, S.; Parida, G. R.; Maitra, A. N. Colloids Surf. 1991, 55, 223. (b) Barbaric, S.; Luisi, P. L. J. Am. Chem. Soc. 1981, 103, 4239.
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Figure 6. Effect of 1-hexanol content on the activity of HRP encapsulated in water-in-IL microemulsions at 35 °C. The pH is 8 and the other experimental conditions are the same as described in Figure 4. The error bars correspond to the standard deviation from three experiments.
was found at pH 7.25 It has been well-known that there is some decrease in pH (generally 1-2 units) when bulk water of a certain pH is transferred into the AOT-stabilized microemulsions in organic solvents because of the anionic nature of the surfactant.11b,26 It seems that such a shift in the pH profile to the alkaline direction is still valid for the HRP-catalyzed oxidation in the newly developed AOT/water/IL microemulsions. Since HRP shows maximum activity at pH 8.0, Tris-HCl buffer was used for subsequent measurements reported in this article. The influence of the total 1-hexanol content on the activity of HRP encapsulated in w/IL microemulsions was investigated because 1-alcohols have a negative effect on the enzymatic activity. It should be noted that 10% (v/v) 1-hexanol in IL is required to prepare w/IL microemulsions using AOT as the surfactant. Figure 6 shows the effect of gradual replacement of IL by an equivalent volume of 1-hexanol on the initial rate of the HRP-catalyzed pyrogallol oxidation in w/ IL microemulsions at a fixed surfactant concentration and fixed W0 value. It is evident from this figure that the catalytic activity of HRP decreased significantly with increasing 1-hexanol content. A similar behavior is observed when HRP microencapsulated in w/o microemulsions.20c,d Probable reasons for the low enzymatic activity with increasing 1-hexanol concentration are due to the followings. First, one would expect that the concentration of 1-hexanol at the interfacial region increases with increasing of total 1-hexanol concentration. Obviously, the concentration of 1-hexanol increases near the active site of HRP. As a consequence, HRP is inactivated because 1-alcohols can denature the enzyme structure.27 Second, the change in structure of the new microemulsion with increasing 1-hexanol content possibly decreased the catalytic activity of HRP. However, for all the systems prepared with different 1-hexanol content for this study (Figure 6), the microemulsions were found to be stable and transparent. In our next optimization step, we checked the activity of HRP in w/IL microemulsions as a function of water content (W0), a key parameter which plays a significant role in the enzymecatalyzed reactions in w/o microemulsions.11d,e,28 In common with many w/o microemulsions, water-in-IL microemulsions show a spherical droplet structure for which the droplet radius is directly proportional to W0 value,15 and thus the microenvi(25) Dunford, H. B.; Stillman, J. S. Coord. Chem. ReV. 1976, 19, 187. (26) Walde, P.; Mao, Q.; Bru, R.; Luisi, P. L.; Kuboi, R. Pure Appl. Chem. 1992, 64, 1771. (27) (a) Shimizu, S.; Shimizu, K. J. Am. Chem. Soc. 1999, 121, 2387. (b) Bauduin, P.; Touraud, D.; Kunz, W.; Savelli, M. P.; Ninham, B. W. J. Colloid Interface Sci. 2005, 292, 244. (28) (a) Martinek, K.; Levashov, A. V.; Klyachko, N.; Khmelnitski, Y. L.; Berezin, I. V Eur. J. Biochem. 1986, 155, 453. (b) Carvalho, C. M. L.; Cabral, J. M. S. Biochimie 2000, 82, 1063.
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ronment around the enzyme can be tuned by simply changing the W0 value. It should be noted here that the microemulsions in ionic liquids were formed in the range of a W0 value less than nine. The system became two phases above this water content. The results presented in Figure 7 indicated that the catalytic activity of HRP in this new microemulsion increased with an increase in W0 values and attained an equilibrium value when W0 approached the critical value for the formation of IL microemulsions. A similar saturation trend was observed in AOT/ water/isooctane microemulsions for HRP23 and for other enzymes.11e,29 However, many research groups have reported a classical bell-shaped dependence of HRP activity with an increase in water content,11a,19a a common phenomenon obtained for most of the enzymatic reactions occurring in the w/o microemulsions.11d,28 Currently, it is not clear why the HRP activity-W0 profile shows a saturation curve. However, the most common explanation for this is that a minimum amount of water in the enzyme vicinity is necessary for maximal catalytic activity: the enzyme must be sufficiently hydrated to be fully active in microemulsions. In principal, a large fraction of water is tightly bound to the AOT head groups at the low W0 value. Consequently, less water is available to hydrate the enzyme for possible conformational changes. We believe that the above argument, which has been well established for w/o microemulsions,30 reflects the state in the new w/IL microemulsions. To obtain a better understanding of the enzyme behavior in the IL microemulsions, we investigated the kinetics parameters of the HRP-catalyzed oxidation of pyrogallol by H2O2. For this purpose, the procedure reported by Klyachko and co-workers19a was followed. The apparent Michaelis-Menten kinetic parameters (see Table 1) were obtained from Lineweaver-Burk plots using only the values of relatively small substrate (hydrogen donor) concentrations (data not shown), where inhibition is negligible. To compare the results obtained in this work, we also determined the kinetic parameters of the same reaction carried out in AOT/water/isooctane microemulsions. Although comparisons of catalytic rates in IL and isooctane microemulsions are complicated by using some different experimental conditions, it is apparent from Table 1 that HRP intrinsic activity in the new IL microemulsions was higher than those in microemulsions of AOT in isooctane under the given experimental conditions. In particular the catalysis efficiency in w/IL microemulsions is
pronounced at 0.8 mM H2O2, which is the optimal condition for the reaction rate (see Figure 4). Now the question arises why HRP showed such enhanced activity in IL microemulsions. This enhanced catalytic activity of HRP in w/IL microemulsions may be the following: first, the partition of the substrate, products, or other molecules involved in the reaction between the aqueous pseudophase and the IL continuous phase; second, a change in the enzyme microenvironment; third, the presence of 1-hexanol in the reaction medium. The solubility studies showed that both substrate (pyrogallol) and product (purpurogallin) are significantly soluble in IL [C8mim][Tf2N] whereas they are poorly soluble in isooctane. In the case of the w/IL microemulsions, it is reasonable to assume that the product concentration in the aqueous pseudophase was reduced significantly because of the favorable partitioning to the IL continuous phase. Consequently, the product inhibitory effect may be less effective in the IL systems than that in the organic solvents systems. We believe that this remarkable finding plays a significant role in accelerating the HRP oxidation of pyrogallol in w/IL microemulsions. Another reason for the high effectiveness of HRP oxidation in AOT-based w/IL microemulsions, apparently, is the change of the microenvironment (medium). In fact, the properties of new microemulsion inner cavity, such as the dynamics of the trapped water pool should differ from the nature of water pool in AOT-based w/o microemulsions. Recently, Mahiuddin and co-workers31 have reported that the superactivity of HRP encapsulated in a cationic reverse microemulsion was correlated with the slowest solvent dynamical modes of the confined water pool. Possibly, the increased activity of the enzyme in this new system is related to a change in the solvation state of its active center. A further study is required to assess the contribution of this factor to the observed improvement of the HRP oxidation of pyrogallol in this new microemulsion. The larger value of Km,app for HRP in IL microemulsions compared to that in the isooctane system is likely attributed to the increased portioning of the substrate pyrogallol to the IL continuous phase due to the high solubility of the IL which was employed. The enhanced activity of HRP in the IL microemulsions led us to examine the stability of the enzyme in this new environment. In bioprocesses, it is a very important factor to have high activities as well as to maintain these activities during long time periods. Many researchers have reported that the storage stability of enzymes is often better in ILs than in conventional organic solvents.32 However, incorporation of an enzyme into a microemulsion can change its stability because of the peculiar water structure in the core of the microemulsion as well as the interaction of the enzyme with surfactant molecules. To determine the stability of HRP in the new developed IL microemulsions, a series of samples containing a fixed amount of HRP was incubated and then the HRP stability was determined following the procedure described in the Experimental Section. As shown in Figure 8, HRP was found to retain almost 70% of its initial activity after 30 h at the given experimental conditions whereas the half-life of HRP in AOT/water/isooctane microemulsions was 33 h. This finding was somewhat puzzling, as we expected HRP to be more stable in our present study as in the case of stability of enzymes in ionic liquids.32 It is important to note that since enzymes in microemulsions are entrapped in the water pool, their stability
(29) Larsson, K. M.; Janssen, A.; Adlercreutz, P.; Mattiasson, B. Biocatalysis 1990, 4, 163. (30) (a) Avramiotis, A.; Papadimitriou, V.; Cazianis, C. T.; Xenakis, A. Colloids Surf. A 1998, 144, 295. (b) Rees, G. D.; Robinson, B. H. Biotechnol. Bioeng. 1995, 45, 344. (c) Bru, R.; Sanchez-Ferrer, A.; Garcfa-Carmona, F. Biochem. J. 1990, 268, 679.
(31) Biswas, R.; Das, A. R.; Pradhan, T.; Touraud, D.; Kunz, W.; Mahiuddin, S. J. Phys. Chem. B 2008, 112, 6620. (32) (a) Fujita, K.; Forsyth, M.; MacFarlane, D. R.; Reid, R. W.; Elliott, G. D. Biotechnol. Bioeng. 2006, 94, 1209. (b) Diego, T. De; Lozano, P.; Gmouh, S.; Vaultier, M.; Iborra, J. L. Biomacromolecules 2005, 6, 1557. (c) Lopez-Serrano, P.; Cao, L.; Van Rantwijk, F.; Sheldon, R. A. Biotechnol. Lett. 2002, 24, 1397.
Figure 7. Effect of water content (W0) on the activity of HRP encapsulated in AOT-stabilized microemulsions in IL at 35 °C. The buffer pH is 8 (50 mM Tris buffer), and the other experimental conditions are the same as described for Figure 4. The error bars are the standard deviation from three repetitions.
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Moniruzzaman et al.
Table 1. Values of Michaelis-Menten Kinetics Parameters for Pyrogallol Oxidation by Hydrogen Peroxide Catalyzed by HRP in AOT-Stabilized w/IL Microemulsion and w/o Microemulsion in Isooctanea microemulsion systems
concentration of H2O2 (mM)
kcat, app (s-1)
A B B
0.1 0.1 0.8
1.3 ( 0.25 3.2 ( 0.40 16.4 ( 2.10
Km,app (× 10-3 M) 0.60 ( 0.10 1.06 (0.11 1.50 ( 0.14
kcat,app/Km,app (× 103 M-1 s-1) 2.1 ( 0.20 3.0 ( 0.20 10.2 ( 0.45
a The overall HRP concentration is 0.1 µM. The concentration of pyrogallol (per total volume of the system) was varied from 5 × 10-5 M to 2 × 10-3 M. A: AOT (200 mM)/isooctane/water microemulsions at W0 ) 13. B: AOT (200 mM)/IL/1-hexanol/water microemulsions at W0 ) 5.
Conclusions
Figure 8. Stability of HRP in AOT/IL/1-hexanol/water (W0 ) 5) and AOT/isooctane/water (W0 ) 12) microemulsions as a function of incubation time at 28 °C. Conditions were pH 8, [HRP]T )0.1 µM, [AOT] ) 200 mM. (b) w/IL microemulsions and (2) w/o microemulsions. Errors limits are less than (10%.
is strongly dependent on the parameters of the aqueous microenvironment11e,24b (e.g., pH, W0, ionic strength) rather than the continuous phase. However, the somewhat higher stability of the HRP in IL microemulsions may arise from something unique within the new microenviroment and/or from IL continuous phase representing higher viscosity than that in nonpolar organic solvents. We believe that the stability of enzymes in the IL microemulsions might increase after optimizing the system parameters, which are now being conducted.
In summary, we explored the use of water-in-ionic liquid microemulsions as the reaction medium for the enzymatic oxidation of pyrogallol catalyzed by horseradish peroxidase. The results demonstrated that the rate of HRP-catalyzed reactions in IL microemulsions was increased significantly compared with that obtained in oil microemulsions. Therefore, a water-in-ionic liquid microemulsion may be a very promising system for performing enzymatic reactions with HRP in ILs media. We believe that these findings will be of value for the development of ionic liquids as a medium for the HRP-catalyzed oxidation of a variety of organic compounds such as phenols, biphenols, anilines, benzidines, and related heteroaromatic compounds. In parallel with this study, we are in the process of examining this new reaction medium with other enzymes in order to evaluate its application range. Acknowledgment. We gratefully acknowledge the JSPS (Japan Society for the Promotion of Science) for a JSPS Postdoctoral Fellowship (M. M. Zaman) and the necessary funding for this work. The present work is also supported by a Grantin-Aid for the Global COE Program and a Grant-in-Aid for Scientific Research (No. 20031022), “Science for Future Molecular Systems” from the Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors thank Dr. K. Nakashima for very helpful discussions. LA803118Q
