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Ind. Eng. Chem. Res. 2004, 43, 1568-1573
Enzymatic Synthesis of Ethyl Palmitate in Supercritical Carbon Dioxide Rajnish Kumar, Giridhar Madras,* and Jayant Modak Department of Chemical Engineering, Indian Institute of Science, Bangalore 560012, India
The esterification of palmitic acid with ethanol was investigated at various temperatures (3570 °C) in the presence of three lipases (Novozym 435, Lipolase 100T, and hog pancreas lipase) in supercritical carbon dioxide (SCCO2) and under solvent-free conditions. All enzymes showed an optimum temperature of 55 °C under both conditions. The effect of water and ethanol addition on the esterification of acid was also investigated. The conversion obtained in SCCO2 and under solvent-free conditions with Novozym at optimal conditions was 74 and 97%, respectively. Although a higher apparent yield was obtained under the solvent-free conditions due to higher substrate and enzyme concentrations, the reaction in supercritical carbon dioxide is better because of lower enzyme loading, higher reaction rates and easier downstream processing. 1. Introduction Many pharmaceutical and biotech companies have concentrated their research on phytochemistry. Phytochemicals, termed as “better than vitamins”,1 are blazing a new frontier in the arena of cancer-prevention research.1 Ethyl palmitate is a phytochemical found in some herbs, such as licorice root2 and chamomile,2 and is used for treatment of various diseases, including cancer.2 Apart from its applications in pharmaceuticals, ethyl palmitate is used in the wine industry for enhancing flavors and as an additive in tobacco for cigarette making and in the food industry for processing nut products.2 Ethyl palmitate is a GRAS (generally recognized as safe for food use) grade product. The synthesis of ethyl palmitate promoted by enzymes is considered “natural”. Enzymatic synthesis also offers the advantage of low reaction temperature, increased selectivity and ease of downstream processing. Lipases (triacyl glycerol hydrolases, E.C. 3.1.1.3) can be used for synthesizing esters under conditions of low water availability. Therefore, nonpolar organic solvents, such as hexane,3 heptane4,5 and cyclohexane,6 have been used as the reaction medium for the synthesis of esters of different fatty acids. Synthesis under solvent-free conditions, defined as the situation in which a reactant itself acts as the solvent, has been used commercially,7 and some investigations have focused on the study of solvent-free reactions.8-10 Although high yields have been obtained with solvent-free reactions, the reaction rates are masstransfer-limited.8-10 Lipase-catalyzed reactions are usually heterogeneous because the substrate is soluble in the solvent but the lipases are insoluble in the solvent. Thus, the reaction occurs at the interface between the enzyme and the solvent. The heterogeneity of the reactions results in mass transfer resistance because of the low diffusion rates of the reactant to the active sites of the enzymes. Supercritical fluids (SCFs), defined as fluids above their critical temperature and pressure, have liquid-like densities and gaslike diffusivities. These properties * To whom correspondence should be addressed. Tel: 9180-394-2321.Fax: 91-80-360-0683.E-mail:
[email protected]. ernet.in.
make them attractive as solvents for reactions with mass transfer limitations. The solubilities of the reactants and products are greatly dependent on the pressure and temperature of the system, and this property can be harnessed to integrate the reaction and downstream processing into a single step.10 The increasingly stringent regulations on volatile organics have led to the increased usage of SCFs. Among SCFs, supercritical carbon dioxide (SCCO2) offers unique advantages of being cheap, nonflammable, and nontoxic. It has a nearambient critical temperature (31.1 °C) and moderate critical pressure (73.8 bar). Although many lipase-catalyzed reactions in SCCO2 have been reported,4,11-15 there are no studies on the synthesis of ethyl palmitate under solvent-free and SCCO2 conditions. The objective of the work reported here was to investigate the various parameters that influence the synthesis of ethyl palmitate. The reactions were catalyzed by three different lipases, Novozym (immobilized), Lipolase (immobilized) and hog pancreas lipase (free enzyme). This study also compared the conversions obtained in SCCO2 with the conversions obtained under solvent-free conditions. 2. Experimental Methods 2.1. Materials. Absolute ethanol (99.9%) (Les Alcohols De Commerce Inc. Ontario, Canada). palmitic acid LR (S.D.Fine-chem Ltd, Mumbai, India) and the lipases were stored at -15 °C and desiccated for 12 h at room temperature before use. Carbon dioxide (98%, Vinayaka Gases, Bangalore) was dehydrated by passing it through silica gel before use. Novozym 435 and Lipolase are of commercial grades and were received as gifts from Novo Nordisk, Denmark. The hog pancreas lipase (specific activity, 147 PLU/g) was procured from Fluka Chemie AG, Switzerland. Among the enzymes employed, Lipolase and Novozym 435 are immobilized, and hog pancreas is a free enzyme. It is important to note that all of the enzymes are of low cost. All solvents were of HPLC grade and were distilled and filtered before use. 2.2. Methods. Reactions were performed in a 6-cm3 stainless steel batch reactor containing the requisite amount of reactants and enzyme. The reactor was pressurized with CO2 and then immersed in a water
10.1021/ie034032h CCC: $27.50 © 2004 American Chemical Society Published on Web 02/27/2004
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bath maintained at the desired temperature within (0.5 °C. The time required for the addition of reactants and for the reactor to attain thermal equilibrium was ∼10 min. All of the reactions were conducted at a constant density of CO2 of 200 kg/m3 for various temperatures. Increase of the initial pressure so that the density of carbon dioxide was 700 kg/m3 showed only a marginal increase in conversions. Upon completion of the reaction period, the reactions were terminated by quickly depressurizing the reactor. The influence of water on the reaction kinetics was investigated by adding water with the reactants and letting the system equilibriate before pressurizing the system. Solvent-free reactions were performed in 4-cm3 covered glass vials with lateral stirring at 100 oscillations/min (OPM). The vials were tightly sealed to prevent evaporation. The reactants were incubated at the desired temperature for 2 min to ensure proper dissolution of the acid in alcohol and that the mixture had attained the incubation temperature. The enzyme was then added. In both the systems, 2 cm3 of acetonitrile was added to the reactor at the end of the reaction time, and the enzyme was removed by centrifugation at 4000 rpm for 4 min. The supernatant liquid was used for the analysis. 2.3. Analysis. The reaction samples were analyzed using an HPLC system consisting of a pump (Waters 501), reversed-phase column (0.39 × 25 cm, µBondapak C18), an injector (Rheodyne 7010, with an injection loop of 250 mm3) and a UV detector (Nulab Instruments, 3010) at 212 nm. The eluent solvent was acetonitrile/ water (80:20 v/v) at a flow rate of 1 cm3/min. The ester was chemically synthesized by the Fischer method. It was washed with water and saturated aqueous sodium bicarbonate solution to remove acid and further purified by silica gel chromatography. The final purified product was injected into the HPLC at various concentrations to construct a calibration curve. Multiple experiments indicated that the error in the determination of the percent esterification was ∼2%. Although the solubility and phase equilibria data of palmitic acid in supercritical carbon dioxide is available, to the best of our knowledge, no literature is available on the solubilities of the palmitic acid in the presence of ethanol. Therefore, a two-reactor system was designed to investigate the effect of solubility/mixing in the reaction. The enzyme was added to one reactor, and acid and alcohol were added to the other reactor. Thus, there was no physical contact between the reactants and the enzyme. The concentration of the reactants and the enzyme in the two-reactor setup was taken to be identical to the concentrations in the single reactor. The reactors were then individually pressurized. After pressurization, they were connected through a small steel tube, and the connecting valves were opened and incubated at the required temperatures for 12 h. It should be pointed out that the substrates and the enzyme are not in physical contact with each other in the two-reactor setup. Therefore, the substrate and enzyme can come in contact with each other only through the solvent phase, namely, the supercritical CO2. The conversion was identical to the conversion obtained in the single reactor system, indicating the system is completely mixed and solubility does not play a role in determining the conversion of the substrates. 3. Results and Discussion 3.1. Synthesis of Ethyl Palmitate. The temperature profiles of the reaction were investigated by
Figure 1. Effect of temperature on the esterification of palmitic acid (a) in SCCO2 for 6 h, (b) under solvent-free conditions for 12 h. 15 mg palmitic acid (8.3 mM), 40 mm3 ethanol (97.3 mM) with 2 mg enzyme. Legends: 9 Novozym, b, Lipolase, 2 HPL.
conducting reactions with 15 mg of palmitic acid; 40 mm3 of ethanol (alcohol to acid molar ratio of 12:1), corresponding to 8.3 and 97.3 mM in SCCO2, respectively; and 2 mg of enzyme. Solvent-free reactions were conducted in 4-cm3 glass vials. Substrate concentrations under solvent-free conditions were 1.1 and 13.0 M, respectively. A 2-mg portion of enzyme was used in every case. This indicates that the substrates and enzyme concentrations are substantially higher in solvent-free conditions than in SCCO2. The reactions in SCCO2 were conducted at a constant density of 200 kg/ m3. Even though the pressure was higher at every temperature increment, the density of the system remained constant. The reaction mixture was incubated in a water bath at various temperatures for 6 h in SCCO2 condition and for 12h under solvent-free conditions. The reactor was equipped with a pressure gauge to ensure that the system operated at the same pressure throughout the reaction. Figure 1a,b shows the effect of temperature on the synthesis under supercritical and solvent-free conditions for three different enzymes. The optimum temperature for reactions in SCCO2 was 5560 °C, with a yield of 74, 44, and 40% in the presence of Novozym, Lipolase, and HPL, respectively. The optimum temperature under solvent-free conditions was 50-55 °C for all of the enzymes, and the maximum percentage conversion was 97, 66, and 40%, respectively, in the presence of Novozym, Lipolase, and HPL. This
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Figure 2. Effect of alcohol concentration on the esterification reaction (a) in SCCO2 at 80 bar for 6 h and (b) under solvent-free conditions for 12 h. 15 mg of palmitic acid (8.3 mM), 2 mg of enzyme at 55 °C. Legends are the same as in Figure 1.
suggests that for this particular reaction, Novozym is a better catalyst than the other two enzymes, both in the absence of solvent and in SCCO2. Lipolase and HPL showed similar activity in SCCO2, whereas in the solvent-free case, Lipolase was better than HPL for promoting the esterification of palmitic acid. The effect of alcohol concentration on the conversion of acid was investigated by adding various amounts of alcohol to 15 mg (8.3 mM) of palmitic acid. The reactions were done at optimum temperature of 55 °C both for SCCO2 and solvent-free conditions. The effect of alcohol concentration on the reactions is shown in Figure 2a and b, respectively. The conversion increases with increasing alcohol concentration until 40 mm3 (97.3 mM) alcohol has been added, and then the conversion decreases with further addition of alcohol. This indicates that deactivation of the enzyme occurs when the quantity of ethanol present in the reaction system exceeds 40 mm3. This is similar to the previous observation16 for the synthesis of geranyl acetate promoted by Mucor miehei. In that case, excess geraniol was found to decrease the rate of reaction after a critical concentration level. Excess ethanol has been shown to deactivate Mucor miehei (Lypozyme)17 for the esterification of oleic acid by ethanol. Deactivation of Novozym 435 with excess isopropyl alcohol in the synthesis of fatty esters has also been reported;18 however, earlier work has shown excess alcohol (isoamyl alcohol) does not have any effect on conversion obtained for the synthesis of isoamyl
Figure 3. Kinetics of the esterification reaction in (a) SCCO2 at 80 bar (b) under solvent-free conditions. 15 mg of palmitic acid (8.3 mM), 40 mm3 of ethanol (97.3 mM) with 2 mg of enzyme at 55 °C. Legends are the same as in Figure 1.
esters.11 In this case, deactivation of the enzymes by alcohol is similar in solvent-free reactions as well as in reactions conducted in SCCO2. The maximum conversion obtained in SCCO2 was 74, 44, and 40%, for Novozym-, Lipolase-, and HPL promoted esterification, respectively. Progress of reaction as a function of time was investigated by treating 15 mg (8.3 mM) of palmitic acid with 40 mm3 (97.3 mM) ofethanol in the presence of 2 mg of enzyme at 55°C under solvent-free conditions and in SCCO2. As shown in Figure 3a,b, conversion did not increase appreciably after 6 h for the reactions in SCCO2 for all the enzymes. However, Lipolase- and HPLcatalyzed reactions under solvent-free conditions show an increase in conversion for 12 h; therefore, all the reactions in SCCO2 and under solvent-free conditions were carried out for 6 and 12 h, respectively. Results show that the rate of the reaction was higher under solvent-free conditions. The result obtained is in accordance with previous results,14,19,20 for which the esterification rates were significantly lower in SCCO2 than that in n-hexane as solvent. Water plays a vital role in the noncovalent interactions that allow the enzyme to retain its native conformation. In the complete absence of water, enzymes cannot maintain an active conformation, thus hindering their ability to function as catalysts.11,21,22 The amount of water needed is specific to each solvent-substrate-
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Figure 4. Effect of added water on the enzyme-catalyzed esterification (a) in SCCO2 at 80 bar for 6 h. (b) under solvent-free conditions for 12 h. Other parameters and the legends are the same as in Figure 3.
Figure 5. Effect of enzyme loading on the enzyme-catalyzed esterification (a) in SCCO2 at 80 bar for 6 h and (b) under solventfree conditions for 12 h. Other parameters and the legends are the same as in Figure 3.
enzyme system. In genera,l it has been shown that enzymes exhibit increased specific activity when water is added to the system;10 however, an excess of water can hinder the synthesis of esters by promoting hydrolysis.11 Therefore, it is vital to determine the optimum water content for the reaction system. Reactions were carried out with 40 mm3 of ethanol and 15 mg of palmitic acid in the presence of 2 mg of enzymes in SCCO2 and in the absence of solvent. Water was added to the system at the start of the reaction. Because the reactants and carbon dioxide contain no water, the x axis of Figure 4 represents the water present in the system (without accounting for the water formed in the reaction). Figure 4a,b shows the optimum water concentration needed for the esterification of ethyl palmitate promoted by all three enzymes, in SCCO2 and in the absence of solvent. As shown in Figure 4a, the optimum water requirement for the reaction promoted by Novozym in SCCO2 is 3 mm3 (23.6 mM) and 1 mm3 (7.9 mM) for those promoted by Lipolase and HPL. In the solvent-free system, the optimum water requirement was found to be around 5 mm3 (5.3 M) for Novozym- and Lipolase-catalyzed reactions and 4 mm3 (4.2 M) for HPL catalysis. The optimum water content of the reaction is very low, and conversion decreases as the amount of water beyond the optimum amount is added to the system. This is in accordance with earlier
results suggesting that excess water leads to lower conversion to esters.11,23,24 The effect of enzyme loading was studied by adding various amounts of enzyme to reaction mixtures with 40 mm3 (97.3 mM) of ethanol, 15 mg (8.3 mM) of palmitic acid, and an optimum amount of water. Figure 5a,b shows the effect of enzyme loading on the conversions. As may be seen, 2 mg of enzyme is the minimum amount of enzyme to effect maximum conversion in solvent-free conditions and in SCCO2. A further increase in the amount of enzyme did not increase the conversion appreciably. The effect of stirring on the conversion to ethyl palmitate was studied under solvent-free conditions and in SCCO2 in the presence of Novozym 435. Stirring was achieved by lateral movement of the reactors at different oscillations. Figure 6a,b indicates that the effect of stirring has no effect on conversion for reaction in SCCO2. However, the conversion increased with an increase in stirring rate for solvent-free reactions. Stirring decreases the mass transfer resistance by ensuring better contact of reactants and enzyme. However, stirring has no effect on the conversion in SCCO2 because the diffusion coefficient of the reactants (∼10-3 cm3/s) is almost 100 times greater than the diffusion rate of the reactant in a typical organic solvent (∼10-5 cm3/s). It should be emphasized that the molar concentration (mM) of the substrates is extremely high in the solvent-
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Acknowledgment The authors thank the Ministry of Human Resources and Development, Department of Bio-technology, India, for financial support and Novo Nordisk, Denmark for donating the enzymes. Literature Cited
Figure 6. Effect of stirring on the esterification reaction in (a) SCCO2 at 80 bar for 6 h and (b) under solvent-free conditions. Other parameters are the same as in Figure 3. Legends: 9 Novozym.
free reactions, because the total volume of the reactants is extremely small (e110 mm3), unlike reactions inSCCO2, in which the reaction volume is 6 cm3. Though the molar concentration of substrates and the enzymes were much higher in the solvent-free system, conversions were only marginally higher. 4. Conclusion The esterification of palmitic acid in SCCO2 and under solvent-free conditions were conducted in the presence of two mg of three different enzymes Novozym 435, Lipolase 100T, and HPL. The effects of temperature, water content in the reaction medium, alcohol concentration, and enzyme loading on conversion were determined. The optimum reaction temperature was determined to be 55 °C. The maximum conversion obtained in SCCO2 and under solvent-free conditions was 74 and 97%, respectively. Small amounts of water were found to increase the conversion of acid to ester. Higher amounts of water (>6 mL) decreased the conversion to ester. A large amount of ethanol was found to decrease the conversion of the acid. Although higher conversions were obtained under solvent-free conditions, this was because of the very high local substrate and enzyme concentration with respect to reactions carried out in SCCO2. Among the enzymes studied, Novozym 435 was found to be the best catalyst for these reactions.
(1) Begley, S.; Springen, K.; Hager, M. Beyond Vitamins. Newsweek 1994, 123 (17), 45. (2) Mann, C.; Staba, J. The Chemistry, Pharmacology, and Commercial Formulations of Chamomile. In Herbs, Spices, and Medicinal Plants. Recent Advances in Botany, Horticulture, and Pharmacology; Craker, L. E., and Simon, J. E., Eds,; Oryx Press: Phoenix, AZ, 1986, p 233. (3) Krishna, S. H.; Manohar, B.; Divakar, S.; Prapulla, S. G.; Karnath, N. G. Optimization of Isoamyl Acetate Production by Using Immobilized Lipase from Mucor mehei by Response Surface Methodology. Enzyme Microb. Technol. 2000, 26, 131. (4) Langrand, G.; Triantaphylides, C.; Barrati, J. Lipase Catalyzed Formation of Flavor Esters. Biotechnol. Lett. 1988, 10, 549. (5) Langrand, G.; Rondot, N.; Triantaphylides, C.; Barrati, J. Short Chain Flavor Ester Synthesis by Microbial Lipases. Biotechnol. Lett. 1990, 12, 581. (6) Norin, M.; Boutelje, J.; Holmberg, E.; Hult, K. Lipase Immobilized by Adsorption: Effect of Support Hydrophobicity on the Reaction Rate of Esters Synthesis in Cyclohexane. Appl. Microbiol. Biotechnol. 1998, 28, 527. (7) Ison, A. P.; Macrae, A. R.; Smith, C. G.; Bosley, J. Mass transfer effect in solvent free fat interesterification reactions: Influences on catalyst design. Biotechnol. Bioeng. 1994, 43, 122. (8) Goma-Doncescu, N.; Legoy, M. D. An original transesterification route for fatty acid ester production from vegetable oils in a solvent-free system. J. Am. Oil Chem. Soc. 1997, 74, 11371143. (9) Trani, M.; Ergan, F.; Andre, G. Lipase-catalyzed production of wax esters. J. Am. Oil Chem. Soc. 1991, 68, 20. (10) De, B. K.; Bhattacharyya, D. K.; Bandhu, C. Enzymatic synthesis of fatty alcohol esters by alcoholysis. J. Am. Oil Chem. Soc. 1999, 76, 451. (11) Srivastava, S.; Modak, J. M.; Madras, G. Enzymatic synthesis of flavors in supercritical carbon dioxide. Ind. Eng. Chem. Res. 2002, 41, 1940. (12) Capewell, A.; Wendel, V.; Bornscheuer, U.; Meyer, H. H.; Scheper, T. Lipase-catalyzed kinetic resolution of 3-hydroxy esters in organic solvents and supercritical carbon dioxide. Enzyme Microb. Technol. 1996, 19, 181. (13) Erickson, J. C.; Schyns, P.; Cooney, C. L. Effect of pressure on an enzymatic-reaction in a supercritical fluid. AIChE. J. 1990, 36, 299. (14) Chulalaksananukul, W.; Jean-Stephane, C.; Combes, D. Grenyl acetate synthesis by lipase-catalysed transesterification in supercritical carbon dioxide. Enzyme Microb. Technol. 1993, 15, 691. (15) Chaudhary, A. K.; Beckman, E. J.; Russell, A. J. Rational control of polymer molecular weight and dispersity during enzymecatalyzed polyester synthesis in supercritical fluids. J. Am. Chem. Soc. 1995, 117, 3728. (16) Chulalaksananukul, W.; Jean-Stephane, C.; Combes, D. Kinetics of grenyl acetate synthesis by lipase-catalysed transesterification in n-hexane. Enzyme Microb. Technol. 1992, 14, 293. (17) Marty, A.; Chulalaksananukul, W.; Willemot, R. M.; Condoret, J. M. Kinetics of Lipase catalyzed Esterification in supercritical CO2. Biotechno. Bioeng. 1992, 39, 273. (18) Gracia, T.; Sanchen, N.; Martinez, M.; Aracil, J. Enzymatic synthesis of fatty esters. Part I. Kinetic approach. Enzyme Microb. Technol. 1999, 25, 584. (19) Kamat, S.; Barrera, J.; Beckman, E. J.; Russell, A. J. Biocatalytic synthesis of acrylates in organic solvents and supercritical fluids. I. Optimization of enzyme environment. Biotechnol. Bioeng. 1992, 40, 158.
Ind. Eng. Chem. Res., Vol. 43, No. 7, 2004 1573 (20) Dumont, T.; Barth, D.; Corbier, C.; Branlant, G.; Perrut, M. Enzymatic reaction kinetics: Comparison in an organic solvent and supercritical carbon dioxide. Biotechnol. Bioeng. 1992, 39, 329. (21) Mesiano, A. J.; Beckman, E. J.; Russell, A. J. Supercritical biocatalysis. Chem. Rev. 1999, 99, 623. (22) Zaks, A.; Klibanov, A. M. Enzymatic catalysis in nonaqueous solvents J. Biol. Chem. 1988, 263, 3194. (23) Miller, D. A.; Blanch, H. W.; Prausnitz, J. M. Enzymecatalyzed interesterification of triglycerides in supercritical carbon dioxide. Ind. Eng. Chem. Res. 1991, 30, 939.
(24) Dumont, T.; Barth, D.; Perrut, M. Proceedings of the 2nd International Symposium on Supercritical Fluids; Johns Hopkins University, Baltimore, MD, 1991; McHugh, M. A., Ed.; p 150.
Received for review July 30, 2003 Revised manuscript received November 5, 2003 Accepted December 17, 2003 IE034032H
