Ind. Eng. Chem. Res. 2005, 44, 9631-9635
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Comparison of the Esterification of Fructose and Palmitic Acid in Organic Solvent and in Supercritical Carbon Dioxide Sasˇ a S ˇ abeder, Maja Habulin, and Z ˇ eljko Knez* University of Maribor, Faculty of Chemistry and Chemical Engineering, Laboratory for Separation Processes, Smetanova 17, SI-2000 Maribor, Slovenia
Sugar fatty acid esters are nonionic surfactants, which are used for personal care products, cosmetic applications, and as emulsifiers for food. In recent years, enzymatic synthesis of sugar fatty acid esters is attracting keen attention as a new manufacturing method for future application. Reaction parameters of lipase-catalyzed synthesis of fructose fatty acid esters in organic solvent were optimized in a batch reactor at atmospheric pressure. Optimum conditions for reaction performed in organic solvent at atmospheric pressure were 10% (w/w of substrates) of lipase from Candida antarctica B, 12.14% (w/w of reaction mixture) of molecular sieves at 60 °C, and stirring rate of 600 rpm. Esterification of fructose and palmitic acid was performed in supercritical carbon dioxide with and without addition of cosolvent (organic solvent) at 60 °C. Effect of pressure on enzyme activity was studied. 1. Introduction Fatty acid sugar esters are widely used in the food, cosmetic, detergent, and pharmaceutical industry, especially as W/O emulsifiers in food products.1 Sugar fatty acid esters used in food products, such as ice cream, soup, and mayonnaise, are marked as E 473. They are also widely used in the cosmetic industry, in toothpaste, lotions, shampoos, and lipsticks. Fructose monoesters synthesized by lipases are used as antibacterial agents that suppress the cell growth of Streptococcus mutans, which has been implicated as a causative organism of dental caries.2 Therefore, enzymatic synthesis of fructose esters has a potential for developing antibacterial agents applicable to food additives. Fatty acid sugar esters are nontoxic, nonskin irritant, odorless, and tasteless compounds, which are produced from renewable and inexpensive substances and completely biodegradable under aerobic and anaerobic conditions.3-7 Enzymatic synthesis of fatty acid sugar esters in organic media enables one to obtain pure products due to enzyme specificity. The enzymatic catalysis is conducted under mild reaction conditions, which minimize side reactions compared to the chemical process. However, water generated during the esterification must be removed by addition of molecular sieves, which is not practical on a larger scale. The use of molecular sieves increases the reactor volume, and mass transfer limitations can occur due to difficult stirring.1 Furthermore, the use of toxic organic solvents in food and pharmaceutical industry is being progressively restricted; even traces of an organic solvent may be unacceptable.8,9 The supercritical state of some fluids, such as carbon dioxide, may provide an interesting alternative. Carbon dioxide has several advantages over organic solvents as reaction media because it is nontoxic, nonflammable, has near-ambient critical temperature (31 °C) and moderate critical pressure (7.3 MPa), and is available * To whom correspondence should be addressed. Tel.: +386 2 22 94 461. Fax: +386 2 25 27 774. E-mail: zeljko.knez@ uni-mb.si.
Figure 1. Fructose palmitate.
at low cost. Because supercritical carbon dioxide (SC CO2) is gaseous at room temperature, the solvent can be easily removed without leaving any residues in the product. Easy separation of the unreacted fatty acid from the sugar ester product seems to be feasible in this system. The most important characteristic of SC CO2 is the great variability of its density and therefore of its solvent power as a function of temperature and pressure. After the reaction is performed in SC CO2, it is possible to recover products easily, with great selectivity by operating a series of depressurizations. SC CO2 has also a high diffusivity and low viscosity and surface tension, which allow easy penetration into macro- and microporous materials. This makes it attractive when mass transfer limitations are high.10-14 The stability of immobilized enzymes in SC CO2 has proven to be good and similar to that obtained in liquid organic solvents.11 A limitation of SC CO2 systems is that only nonpolar compounds are soluble at an acceptable level. Because of that, various methods have been suggested, such as addition of cosolvents or preadsorption of the polar compound onto an inert material with a high internal surface, like silica gel.9 In present research, reaction parameters of lipasecatalyzed synthesis of fructose palmitate in organic solvent were optimized in a batch reactor at atmospheric pressure. Fructose palmitate is shown in Figure 1. At optimized reaction conditions, esterification of fructose and palmitic acid was performed in supercritical carbon dioxide with and without the addition of an organic solvent, serving as cosolvent. 2. Materials and Methods 2.1. Enzymes and Chemicals. Immobilized lipase Novozym 435 from Candida antarctica B was kindly
10.1021/ie050266k CCC: $30.25 © 2005 American Chemical Society Published on Web 09/17/2005
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Figure 2. Schematic diagram of the SC CO2 apparatus: (1) magnetic stirrer and heater; (2) reactor; (P) high-pressure pump; (PI) pressure indicator.
donated from Novo Nordisk AS (Copenhagen, Denmark). D-(-) fructose (g98%; Cat. No. 47745) and molecular sieves (3 Å; Cat. No. 69832) were purchased from Fluka (Buchs, Switzerland), palmitic acid (min 98%; Cat. No. 27734) was obtained from Riedel de Hae¨n (Seelze, Germany), and 2-methyl-2-butanol (99%) was supplied from Aldrich (Deisenhofen, Germany). Sodium hydroxide solution (0.1 N) was from Merck (Darmstadt, Germany), and phenolphthalein was from Kemika (Zagreb, Croatia). Carbon dioxide 4.5 (purity 99.995 vol. %) was supplied by Messer MG Ruse, Solvenia. 2.2. Reactions in Organic Solvent at Atmospheric Pressure. The reaction mixture consisted of an equimolar (20 mmol) mixture of fructose and fatty acid and 24 mL of organic solvent as adjuvant. 5.735.6% (w/w of reaction mixture) of molecular sieve (3 Å) was added for the absorption of water generated during esterification. Synthesis of fructose palmitate was performed in a 100 mL round-bottom flask, placed in a water or oil bath, heated to the desired operating temperature, and stirred by a magnetic stirrer. The esterification was started by addition of the lipase. Samples were taken from the reaction mixture at defined intervals and analyzed by thin-layer chromatography (TLC) and a volumetric titration. A control without the lipase addition was also considered for reaction mixtures, and it was treated the same way as the reaction mixture. 2.3. Reactions in SC CO2. The reaction mixture consisted of an equimolar (10 mmol) mixture of fructose and palmitic acid and 10% (w/w of substrates) lipase. Esterification was performed in a 78 mL high-pressure batch stirred-tank reactor at a defined temperature with a stirring rate 600 rpm. Cooled liquid carbon dioxide was pumped into the reactor up to the desired pressure. When the reaction was performed in the presence of the cosolvent, samples were taken from the reaction mixture at defined time intervals using a sampling valve. A schematic diagram of the high-pressure device is shown in Figure 2. Reaction was also performed without organic solvent; at various time intervals, the reaction was terminated by depressurization of SC CO2 and the reaction mixture was analyzed by volumetric titration. 2.4. Analyses. Samples were analyzed qualitatively by thin-layer chromatography (TLC). TLC analysis was performed on silica gel plates 60F254 (Merck, Germany) using chloroform:methanol:acetic acid:water (70:20:8:
Figure 3. Conversion of palmitic acid at different temperatures. Reaction conditions: 20 mmol of fructose, 20 mmol of palmitic acid, 24 mL of 2-methyl-2-butanol, 10% lipase, 12.1% molecular sieve, 600 rpm, 72 h. Table 1. Initial Rates for Synthesis of Fructose Palmitatea temperature [°C]
initial rate [mmol/g/min/g enzyme]
30 40 50 60 70 80 90 100 110
0.7 × 10-3 1.7 × 10-3 2.6 × 10-3 4.9 × 10-3 9.8 × 10-3 40.6 × 10-3 46.8 × 10-3 34.7 × 10-3 23.3 × 10-3
a Reaction conditions: 20 mmol of fructose, 20 mmol of palmitic acid, 24 mL of 2-methyl-2-butanol, 10% lipase, 12.1% molecular sieve, 600 rpm.
2) as the mobile phase. The plates were sprayed with 50% sulfuric acid and heated at 110 °C for 5 min to develop sugar and sugar ester spots.3 The ester content was quantified by calculating the residual fatty acid amount in the reaction mixture, which was determined by volumetric method. 0.1 g of sample of the reaction mixture was diluted in 20 mL of 0.1 wt % phenolphthalein solution in absolute ethanol and then titrated with standardized sodium hydroxide solution of 0.1 mol/L in water.15 3. Results and Discussion 3.1. Fructose Palmitate Production at Atmospheric Pressure. For the synthesis of fructose palmitate in organic solvent, experimental conditions, such as temperature, biocatalyst concentration, stirring rate, and molecular sieve concentration, were optimized at atmospheric pressure. First, the effect of temperature on immobilized lipase activity was studied. Esterification of fructose with palmitic acid was performed in 2-methyl-2-butanol at temperatures from 30 to 110 °C. The highest conversion of 78% was obtained at 60 °C after 72 h of reaction performance (Figure 3). At lower temperatures, poor conversions were obtained because of the low enzyme activity. At temperatures higher than 60 °C, a decrease in conversion was observed after 72 h of reaction performance. The initial rates of fructose palmitate formation are presented in Table 1. The initial rates of fructose palmitate formation increased from 0.7 × 10-3 to 46.8 × 10-3 mmol/(g‚min) per g enzyme with increasing temperature from 30 to
Ind. Eng. Chem. Res., Vol. 44, No. 25, 2005 9633 Table 2. Thermodynamic Properties for Synthesis of Fructose Palmitate magnitude
value
Ea ∆Hd Kd ∆Gd ∆Sd
67.5 × 103 kJ/mol 107.8 × 103 kJ/mol 1.89 -1.9 × 103 kJ/mol 302 kJ/(mol K)
90 °C. At temperatures higher than 90 °C, thermal deactivation occurred. In the studies of fructose oleate synthesis, catalyzed by Candida antarctica B lipase, it was observed that the initial rate and the concentration of fructose oleate after 10 h of reaction performance increased with increasing the temperature up to 90 °C.16 The effect of temperature was also studied for the esterification of fructose with lauric acid (in ratio of 1:3) in acetone catalyzed by the same lipase. The results showed that temperature affects both reaction rate and selectivity.17 Activation energy and deactivation enthalpy were calculated from the Arrhenius plot, in which ln vi is graphed against 1/T giving a straight line with a slope of -Ea/R for temperatures up to 90 °C and another straight line with a slope approximately equal to ∆H (Ea/R) for temperatures above 90 °C. Using a simple model of reversible thermal deactivation, assuming that the enzyme exists in its active and inactive forms in equilibrium, deactivation entropy ∆Sd and deactivation Gibb’s free energy ∆Gd were estimated.18 The values for activation energy Ea, deactivation enthalpy ∆Hd, equilibrium constant Kd, Gibb’s free energy of deactivation ∆Gd, and entropy of deactivation ∆Sd are presented in Table 2. The deactivation enthalpy is higher than the activation energy at temperatures above 90 °C. At this temperature, enzyme deactivation predominates in the system, causing a great decrease of activity with temperature rise from 90 to 110 °C. The free energy during deactivation is negative, indicating that thermal deactivation occurred at temperatures above 90 °C. The use of higher temperatures causes an increase in enzyme activity and a decrease in stability, taking into account the period of time the enzyme is used. To study the effect of biocatalyst concentration on the reaction rate, experiments were performed varying enzyme loading from 8% to 20% (w/w of substrates). Synthesis of fructose palmitate was conducted in 2-methyl-2-butanol at 60 °C and at atmospheric pressure. Experiments were performed to determine the minimum lipase concentration, which maximizes the concentration of fructose palmitate synthesized during the reaction performance. The effect of lipase concentration on ester concentration during 72 h of reaction performance is shown in Figure 4. Fructose palmitate concentration increased with increasing the lipase concentration until 10% (w/w of substrates). At higher added biocatalyst concentration, a decrease in ester concentration occurred, which may be due to an increase in the produced water content. Excessive production of water, which is the byproduct of the reversible esterification reaction, can cause the reversible reaction, which results in decreasing of the ester concentration. Moreover, at loadings higher than 10% (w/w of substrates), visual observations indicated that it was impossible to maintain a uniform reaction suspension, which resulted in limited mass transfer.
Figure 4. Fructose palmitate concentration at different lipase concentrations. Reaction conditions: 20 mmol of fructose, 20 mmol of palmitic acid, 24 mL of 2-methyl-2-butanol, 12.1% molecular sieve, 60 °C, 600 rpm.
Figure 5. Conversion of palmitic acid at different concentrations of molecular sieves. Reaction conditions: 20 mmol of fructose, 20 mmol of palmitic acid, 24 mL of 2-methyl-2-butanol, 10% lipase, 60 °C, 600 rpm.
The highest ester concentration (1.082 mmol/g) was obtained at an enzyme concentration of 10% after 72 h of reaction performance, which resulted in 75% conversion. Similar results were published for the synthesis of glucose palmitate in hexane using Palatase M,8 for the synthesis of glucose palmitate in acetone, catalyzed by Novozym 435,19 and for myristyl glucose synthesis, catalyzed by Candida antarctica lipase.20 The effect of molecular sieve concentration on the reaction rate was studied, as well. Water, generated during the esterification, was removed by adding different concentrations of molecular sieves, varying from 5.7% to 35.6% (w/w of reaction mixture). The direct esterification of fructose palmitate in 2-methyl-2-butanol was performed at optimized reaction conditions: 60 °C, 600 rpm, and 10% (w/w of substrates) of lipase addition. The effect of the concentration of molecular sieves on the conversion is shown in Figure 5. After 72 h of reaction performance at 60 °C, the highest conversion of 78% was obtained when the concentration of molecular sieves was 12.1%. The conversion of 65% was obtained after 24 h of reaction performance, which means that the conversion increase was less than 13% within the next 48 h of reaction performance. With further increase in molecular sieve concentration, a decrease in conversion was observed, which probably happened due to excessive removing of essential water for enzyme activity from its vicinity. The final fructose palmitate concentration after 3 days of reaction performance at 12.1% of molecular sieve loading was 1.202 mmol/g per g enzyme.
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Figure 6. Conversions for esterification reactions of palmitic acid and fructose in organic solvent, catalyzed by untreated lipase and lipase that was previously exposed to SC CO2 at 60 °C and 10 MPa.
Similar results about the optimal molecular sieve concentration were reported for the synthesis of glucose palmitate in hexane using Palatase M as biocatalyst.8 The addition of molecular sieves is not practical on a larger scale. Therefore, the synthesis of fructose palmitate was performed in SC CO2, where their addition is not required. 3.2. Activity and Stability of the Lipase in SC CO2. The activity and stability of Candida antarctica B lipase in SC CO2 was studied with intention to perform lipase-catalyzed reaction in this medium. Several studies on the activity and stability of different lipases have been carried out in supercritical fluids. A decreased relative lipase activity in SC CO2 has been reported for esterification reactions of n-butyric acid and ethanol catalyzed by Rhizopus niveus, Rhizopus javanicus, Pseudomonas fluorescens, and porcine pancreas lipase.21 A decreased lipase activity in SC CO2 is attributed to interactions between SC CO2 and the biocatalyst. Another reason could be that SC CO2 removed the essential water for enzyme activity from its vicinity, causing enzyme deactivation. For enzyme activation, at least a monolayer of water per enzyme molecule is necessary. Lipase from Candida antarctica B was incubated in SC CO2 at 10 MPa and 60 °C. After 24 h, lipase was recovered from the reactor after slow depressurization and used as a biocatalyst for esterification of fructose with palmitic acid in organic solvent (2-methyl-2-butanol) at 60 °C and atmospheric pressure. For the comparison, the same reaction, catalyzed by nonincubated lipase, was performed in 2-methyl-2-butanol at the same temperature. From the difference between conversion of preincubated-lipase catalyzed synthesis and conversion of fresh-lipase catalyzed synthesis, lipase stability in CO2 was determined. Stability data for lipase from Candida antarctica are shown in Figure 6. The results showed that the lipase remained stable in the first hours of reaction performance after being exposed to SC CO2 for 24 h. Conversion of palmitic acid slightly increased after 2 h when the reaction was catalyzed by lipase, which was previously treated in SC CO2 compared to the reaction catalyzed by nontreated lipase. Under supercritical conditions, changes in protein structure may occur. The tendency of carbon dioxide to
Figure 7. Pressure effect on the palmitic acid conversion in SC CO2. Reaction conditions: 20 mmol of fructose, 20 mmol of palmitic acid, 10% lipase, 60 °C, 600 rpm, 24 h.
influence the enzyme activity depends on the solubility of water in carbon dioxide. For catalytic activity of the enzyme, a certain amount of water is essential. Dry carbon dioxide may alter the concentration of the aqueous layer around the lipase.19 3.3. Fructose Palmitate Production in SC CO2. Lipase from Candida antarctica B (Novozym 435), which was previously treated in SC CO2, showed increased activity and stability compared to the reaction catalyzed by nontreated (fresh) lipase. Therefore, it was used as biocatalyst in reactions, performed in SC CO2. 3.3.1. Effect of Pressure on Ester Production. To study the pressure effect on the activity of Candida antarctica lipase in SC CO2, synthesis of fructose palmitate was performed in a high-pressure batch stirred-tank reactor at pressure ranging from 8 to 20 MPa. The temperature was 60 °C, and the rotational speed was 600 rpm. The effect of pressure on esterification rate after 24 h of reaction performance is shown in Figure 7. The conversion of palmitic acid slightly increased with increasing pressure to 10 MPa. Final conversion at 60 °C after 24 h of reaction performance was 61%. Published results regarding Candida antarctica lipase catalyzing reaction of methyl-β-D-fructofuranoside with caprylic acid in SC CO2 showed that the equilibrium conversion rate was similar (about 52%) at pressure ranging from 14 to 26 MPa, which is in agreement with presented results.22 3.3.2. Addition of Organic Cosolvent. A limitation of SC CO2 systems is that only nonpolar compounds, such as fatty acids, are soluble at the acceptable levels. Therefore, the addition of cosolvents could be used to overcome the solubility of polar substances, such as sugars. The effect of organic cosolvent was studied for the reaction performed in SC CO2. Esterification of fructose palmitate was performed at 60 °C and 600 rpm. 2-Methyl-2-butanol as cosolvent (20 mol %) was added to the reaction mixture, and the reaction rate was compared with the reaction performed without any addition of cosolvent. The effect of 2-methyl-2-butanol as organic cosolvent on palmitic acid conversion is shown in Figure 8. Reactions performed in SC CO2 with and without addition of cosolvent (organic solvent) were compared. Esterification of fructose with palmitic acid in SC CO2 resulted in conversion yield of 54% after 48 h of reaction
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Figure 8. Effect of 2-methyl-2-butanol as cosolvent on the conversion of palmitic acid in SC CO2. Reaction conditions: 10 mmol of fructose, 10 mmol of palmitic acid, 10% lipase, 60 °C, 600 rpm.
performance. Esterification was also performed at 16 MPa with addition of cosolvent (2-methy-2-butanol). The effect of the reaction mixture with added cosolvent concentration for the synthesis of fructose palmitate performed in SC CO2 was investigated. When 20 mol % of 2-methyl-2-butanol was added to the reaction mixture in SC CO2, conversion increased for about 5% after 48 h of reaction performance compared to the reaction performed in pure SC CO2. 4. Conclusion Lipase-catalyzed esterification of fructose with palmitic acid was performed in 2-methyl-2-butanol at atmospheric pressure and in SC CO2 with and without addition of cosolvent. The highest conversion of 78% was obtained at 60 °C after 72 h of reaction performance at atmospheric pressure. High conversions after 24 h of reaction performance were also achieved in SC CO2 at 60 °C and 10 MPa without addition of organic solvent (60%). SC CO2 is a promising alternative to conventional organic media for the lipase-catalyzed synthesis of fatty acid sugar esters. Reactions performed in SC CO2 do not require any addition of molecular sieves. Because only palmitic acid is soluble in the supercritical phase, the separation of the fructose palmitate from remaining substrates and enzyme could easily be achieved when reaction is performed in pure SC CO2. When 2-methyl2-butanol is used as a cosolvent in SC CO2, the separation becomes more difficult. Therefore, the optimization of reaction parameters for the synthesis of fructose palmitate in SC CO2 without addition of organic solvent will be further studied. Acknowledgment We would like to thank the Slovenian Ministry of Higher Education, Science, and Technology (contract no. P2 0046-07946, Program group “Separation processes”) and Centre of Excellence (contract no. 3311-04-855013, “Supercritical fluids”). Literature Cited (1) Yan, Y.; Bornscheuer, U. T.; Cao, L.; Schmid, R. D. Lipasecatalyzed solid-phase synthesis of sugar fatty acid esters. Enzyme Microb. Technol. 1999, 25, 725. (2) Watanabe, T.; Katayama, S.; Matsubara, M.; Honda, Y.; Kuwahara, M. Antibacterial carbohydrate monoesters suppressing cell growth of Streptococcus mutans in the presence of sucrose. Curr. Microbiol. 2000, 41, 210.
(3) Cao, L.; Fischer, A.; Bornscheuer, U. T.; Schmid, R. D. Lipase-catalyzed solid-phase synthesis of sugar fatty acid ester. Biocatal. Biotransform. 1996, 14, 269. (4) Coulon, D.; Ismail, A.; Girardin, M.; Rovel, B.; Ghoul, M. Effect of different biochemical parameters on the enzymatic synthesis of fructose oleate. J. Biotechnol. 1996, 51, 115. (5) Coulon, D.; Girardin, M.; Ghoul, M. Enzymatic synthesis of fructose monooleate in a reduced pressure pilot scale reactor using various acyl donors. Process Biochem. 1999, 34, 913. (6) Degn, P.; Pedersen, L. H.; Duus, J.; Zimmermann, W. Lipase-catalysed synthesis of glucose fatty acid esters in tertbutanol. Biotechnol. Lett. 1999, 21, 275. (7) Soultani, S.; Engasser, J.-M.; Ghoul, M. Effect of acyl donor chain length and sugar/acyl donor molar ratio on enzymatic synthesis of fatty fructose esters. J. Mol. Catal. B 2001, 11, 725. (8) Tarahomjoo, S.; Alemzadeh, I. Surfactant production by an enzymatic method. Enzyme Microb. Technol. 2003, 33, 33. (9) Tsitsimpikou, C.; Stamatis, H.; Sereti, V.; Daflos, H.; Kolisis, F. N. Acylation of glucose catalyzed by lipases in supercritical carbon dioxide. J. Chem. Technol. Biotechnol. 1998, 71, 309. (10) Marty, A.; Chulalaksananukul, W.; Willemot, R. M.; Condoret, J.-S. Kinetics of lipase-catalyzed esterification in supercritical CO2. Biotechnol. Bioeng. 1992, 39, 273. (11) Marty, A.; Combes, D.; Condoret, J.-S. Continuous reaction-separation process for enzymatic esterification in supercritical carbon dioxide. Biotechnol. Bioeng. 1994, 43, 497. (12) Romero, M. D.; Calvo, L.; Alba, C.; Habulin, M.; Primoz _ic _, M.; Knez, Zˇ . Enzymatic synthesis of isoamyl acetate with immobilized Candida antarctica lipase in supercritical carbon dioxide. J. Supercrit. Fluids 2005, 33, 77. (13) Srivastava, S.; Madras, G.; Modak, J. Esterification of myristic acid in supercritical carbon dioxide. J. Supercrit. Fluids 2003, 27, 55. (14) Vija, H.; Telling, A.; Tougu, V. Lipase-catalyzed esterification in supercritical carbon dioxide and in hexane. Bioorg. Med. Chem. Lett. 1997, 7, 259. (15) Habulin, M.; Knez, Zˇ . The influence of water on the synthesis of n-butyl oleate by immobilized Mucor miehei lipase. J. Am. Oil Chem. Soc. 1990, 67, 775. (16) Coulon, D.; Girardin, M.; Engasser, J.-M.; Ghoul, M. Investigation of keys parameters of fructose oleate enzymatic synthesis catalyzed by an immobilized lipase. Ind. Crop. Prod. 1997, 6, 375. (17) Arcos, J. A.; Bernabe, M.; Otero, C. Quantitative enzymatic production of 1,6-diacyl fructofuranoses. Enzyme Microb. Technol. 1998, 22, 27. (18) Bailey, J. E.; Ollis, D. F. Biochemical engineering fundamentals; McGraw-Hill: New York, 1986. (19) Arcos, J. A.; Hill, C. G.; Otero, C. Kinetics of the lipasecatalyzed synthesis of glucose esters in acetone. Biotechnol. Bioeng. 2001, 73, 104. (20) Degn, P.; Zimmermann, W. Optimization of carbohydrate fatty acid ester synthesis in organic media by a lipase from Candida antarctica. Biotechnol. Bioeng. 2001, 74, 483. (21) Habulin, M.; Knez, Zˇ . Activity and stability of lipases from different sources in supercritical carbon dioxide and near-critical propane. J. Chem. Technol. 2001, 76, 1260. (22) Heo, J.-H.; Kim, S. Y.; Kim, H.-S.; Yoo, K.-P. Enzymatic preparation of a carbohydrate ester of medium-chain fatty acid in supercritical carbon dioxide. Biotechnol. Lett. 2000, 22, 995. (23) Dumont, T.; Barth, D.; Corbier, C.; Branlant, G.; Perrut, M. Enzymatic reaction kinetic: Comparison in an organic solvent and in supercritical carbon dioxide. Biotechnol. Bioeng. 1992, 39, 329. (24) Lozano, P.; Villora, G.; Gomez, D.; Gayo, A. B.; SanchezConesa, J. A.; Rubio, M.; Iborra, J. L. Membrane reactor with immobilized Candida antarctica lipase B for ester synthesis in supercritical carbon dioxide. J. Supercrit. Fluids 2004, 29, 121. (25) Matsuda, T.; Watanabe, K.; Harada, T.; Nakamura, K. Enzymatic reactions in supercritical CO2: carboxylation, asymmetric reduction and esterification. Catal. Today 2004, 96, 103.
Received for review February 28, 2005 Revised manuscript received June 15, 2005 Accepted June 20, 2005 IE050266K
