Enzymatic Synthesis of Flavors in Supercritical Carbon Dioxide

Shireesh Srivastava, Jayant Modak, and Giridhar Madras*. Department of Chemical Engineering, Indian Institute of Science, Bangalore 560 012, India...
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Enzymatic Synthesis of Flavors in Supercritical Carbon Dioxide Shireesh Srivastava, Jayant Modak, and Giridhar Madras* Department of Chemical Engineering, Indian Institute of Science, Bangalore 560 012, India

Commercially important flavor esters of isoamyl alcohol, catalyzed by crude hog pancreas lipase (HPL), were synthesized under solvent-free conditions and in supercritical carbon dioxide. The esters synthesized were isoamyl acetate, isoamyl propionate, isoamyl butyrate, and isoamyl octanoate. Very low yields (3-4%) of isoamyl acetate were obtained, but high yields for the other three esters were obtained under both supercritical and solvent-free conditions. The yields of esters of the even-carbon acids, isoamyl acetate, butyrate, and octanoate, increased with increasing chain length, whereas the yield of isoamyl propionate was higher than that of isoamyl butyrate. The optimum temperature of the reaction was higher under supercritical conditions (45 °C) than under solvent-free conditions (35-40 °C). The effects of other parameters such as alcohol concentration, water concentration, and enzyme loading were investigated. An increase in the water concentration decreased the conversion significantly in supercritical carbon dioxide but not under solvent-free conditions. The optimum ratio of alcohol to acid was dependent on the extent of inhibition by the acid. Although providing a higher apparent yield by being run in a highly concentrated medium, the overall conversion under solvent-free conditions was lower than that under supercritical conditions for similar enzyme concentrations, indicating that the synthesis of esters in supercritical carbon dioxide might be a viable option. Introduction The modern food industry uses very large quantities of various flavor compounds1 as food additives to enhance the flavors of food products. Most fruit flavors are esters of fatty acids and are synthesized chemically. Although chemical synthesis gives high yields of the esters, the flavors produced are not considered natural, and the enzymatic production of flavor compounds is preferred. Enzymatic synthesis also offers the advantages of ambient reaction temperatures, increased selectivities, and ease of downstream processing. Lipases (triacyl glycerol hydrolases, E.C. 3.1.1.3), under conditions of reduced water, can be used for synthesizing esters. Therefore, nonpolar organic solvents such as n-hexane,2 n-heptane,3,4 and cyclohexane5 have been used as reaction media. Solvent-free systems are systems in which the reaction medium involves a reactant itself as the solvent. These systems are used commercially,6 and some investigations7-9 have focused on lipase-catalyzed reactions in which one of the reactants, the alcohol, acts as the solvent. Although high yields have been obtained with these solvents, the reaction rates are usually slow as a result of mass-transfer limitations. Lipase-catalyzed reactions are usually heterogeneous. Whereas the substrate is soluble in the solvent, the lipases are insoluble. Thus, the reaction occurs at the interface between the enzyme and the solvent. The characteristic heterogeneity of the reactions results in mass-transfer resistance because of the diffusion rates of the reactants to the active site of the enzyme. Supercritical fluids (SCFs), defined as fluids above their critical temperature and pressure, have liquidlike densities and gaslike diffusivities. These properties make them attractive as solvents for reactions with * Corresponding author. Phone: 91-80-309 2321. Fax: 9180-360 0683. E-mail: [email protected].

mass-transfer limitations. Another advantage of supercritical fluids is that the solubilities of the reactants and products are greatly dependent on the pressure and temperature of the system. This property of supercritical fluids can be harnessed to integrate the reaction and downstream processing into a single step. Further, the increasingly stringent regulations on volatile organics have led to the increased usage of supercritical fluids. Among SCFs, supercritical carbon dioxide (SCCO2) offers the unique advantages of being cheap, nonflammable, and nontoxic. It has a near-ambient critical temperature (31.1 °C) and moderate critical pressure (73.8 bar). Although many lipase-catalyzed reactions, including that of isoamyl acetate, have been reported in SCCO2, we are unaware of any study on the synthesis of various other esters of isoamyl alcohol in SCCO2. This study, unlike other studies, also compares the conversions obtained from the reaction in SCCO2 to the conversions obtained under solvent-free conditions. The objective of this paper is to investigate the various parameters that influence the synthesis of flavor compounds of isoamyl alcohol and short-chain fatty acids. Crude hog pancreas lipase (HPL) is used for synthesis because it is one of the least expensive lipases. Although certain lipases such as M. meihei demonstrate higher activities and conversions in the esterification reaction, HPL was chosen in this study because it is cheap and might be industrially viable. The compounds synthesized were isoamyl acetate, isoamyl propionate, isoamyl butyrate, and isoamyl octanoate. These compounds are used as banana flavors and, therefore, are commercially important. The reactions were studied under solvent-free conditions and in SCCO2. Experimental Procedures Materials. Crude hog pancreas lipase (with a stated activity of 20 U/mg) was purchased from Fluka Chemie

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AG, Switzerland. The enzyme was stored at -15 °C and desiccated for 12 h at room temperature before use. Isoamyl alcohol (99%, Glaxo), acetic acid (99%, Ranbaxy Chemicals), propionic acid (99%, E. Merck), butyric acid (97.5%, Rolex Chemicals), octanoic acid (99%, SD Fine Chemicals), isoamyl acetate (95%, SD Fine Chemicals), and CO2 (99%, Sicgil Gases) were used. All solvents were of HPLC grade and were distilled and filtered before use. Methods. The reactions were performed in 6-cm3 stainless steel batch reactors. Each reactor, loaded with reactants and enzyme, was pressurized to an initial pressure of 68 bar at room temperature. The pressurized reactor was then immersed in a water bath maintained at the desired temperature (fluctuations were less than (0.5 °C). All of the reactions were conducted at a constant density of CO2 of 0.2 g/cm3 for various temperatures to ensure constant solubility of the substrates. Even though the pressure was higher at every temperature increment, the density of the system remained constant. The reactors were equipped with a pressure gauge to ensure that the system operated at the same pressure throughout the reaction. After the desired reaction time, the reactor was depressurized, and the contents were eluted in 3 mL of methanol. The enzyme was made to settle by centrifugation, and the reaction mixture was analyzed by HPLC. To the best of our knowledge, no literature data are available on the solubilities of the substrates in SCCO2 in the presence of isoamyl alcohol. To ascertain whether solubility/mixing plays a role in the reaction, a tworeactor system was designed. The acid and alcohol were added to one reactor, and solid enzyme powder was added to another reactor. This ensured that there was no physical contact between the reactants and the enzyme. The concentrations of the reactants and the enzyme in the two-reactor setup were identical to those used in the single reactor. The two reactors were then individually pressurized. After pressurization, they were connected through a small steel tube, and the connecting valves were opened. The reactors were then incubated at the required temperatures for 12 h. It should be pointed out that the substrates and the enzyme were not in physical contact with each other in the tworeactor setup. Therefore, the substrate and enzyme could come into contact with each other only through the solvent phase, that is, the supercritical CO2. The conversion (percent esterification) was the same as in the single-reactor system, indicating that the system is completely mixed and that solubility/mixing does not play a role in determining the conversion. We also performed the reactions with agitation of the reaction systems but found no increase in conversion. The volumes of the substrates for the reactions were chosen so that the concentrations of the reactants were low enough that they dissolved in SCCO2 but high enough to ensure detection without significant error. Solvent-free reactions were performed in 5-mL capped glass vials. The acid and alcohol were mixed in the vials and incubated at the desired temperature for 2 min. The enzyme was then added to the vial, and the mixture was incubated for the desired interval of time. The reactions were terminated by the addition of 3 mL of methanol, followed by centrifuging to remove the enzyme. The reaction samples were analyzed by HPLC. Analysis. The reaction samples were analyzed by a HPLC system consisting of a pump (Waters 501), a

Figure 1. Effect of temperature on synthesis of isoamyl butyrate with 80 mm3 of butyric acid, 190 mm3 of isoamyl alcohol, and 20 mg of enzyme at a constant density (0.2 g/cm3) for 12 h. b, Solventfree. 9, SCCO2.

reverse-phase column (0.39 cm × 25 cm, µBondapak C18), an injector (Rheodyne 7010, with a 250-mm3 injection loop), and a UV detector (Nulab Instruments, 3010). Methanol-water solution was eluted through the system at a flow rate of 2 cm3/min, and the eluted compounds were detected at a wavelength of 212 nm. A solution of 50:50 (v/v) methanol/water was used as the eluent for isoamyl acetate, propionate, and butyrate, whereas a solution of 75:25 methanol/water was used as the eluent for isoamyl octanoate. Isoamyl acetate (purchased) was used for calibration. Other esters were synthesized chemically by Fischer’s method using concentrated H2SO4 as the catalyst. Alcohol (5 cm3) and the corresponding twice molar equivalent of acid were added to a 50-cm3 round-bottom flask. Two drops of concentrated H2SO4 were added to the solution, and the mixture was stirred and refluxed for 24 h. The esters were purified by first washing the reaction mixture with water and saturated NaHCO3 solution to remove acid and then usingsilica gel chromatography. The purity of the synthesized esters was verified by thin-layer chromatography and FTIR. Various amounts of esters dissolved in methanol were injected, and linear calibration curves were obtained for all of the esters. Results and Discussion Synthesis of Isoamyl Butyrate. The temperature profile of the reaction was investigated by conducting reactions with 80 mm3 of butyric acid, 190 mm3 of isoamyl alcohol, corresponding to 145 and 291 mM in SCCO2, respectively, and 20 mg of enzyme. The reaction mixtures were incubated at various temperatures for 12 h. The amount of ester produced was determined by HPLC. Figure 1 depicts the effect of temperature on the synthesis under supercritical and solvent-free conditions. Under solvent-free conditions, the optimum temperature was 35 °C, and a yield of 34% was obtained. The optimum temperature for reaction in SCCO2 was 45 °C, with a 10% yield of ester. The effect of alcohol on the yield of ester was investigated by adding various amounts of alcohol to 40 mm3 of butyric acid for solvent-free reactions. The molar equivalent of alcohol is 1.185 mm3 of alcohol per mm3 of acid. After 2 min of incubation at the optimum temperature, 20 mg of enzyme was added to the system, and the system was incubated for 12 h. For reactions in SCCO2, various amounts of alcohol were added to 20 mm3 (36 mM) of acid, with an enzyme loading of 20 mg. The reactions were performed for 12 h at the optimum

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Figure 2. Effect of addition of alcohol on synthesis of isoamyl butyrate. For solvent-free reactions: 40 mm3 of butyric acid and 20 mg of enzyme at 35 °C for 12 h. For SCCO2 reactions: 20 mm3 of butyric acid and 20 mg of enzyme for 12 h at 90 bar and 45 °C. b, Solvent-free. 9, SCCO2.

temperatures, i.e., at 35 °C for solvent-free reactions and 45 °C for SCCO2 reactions. The effect of alcohol on the esterification is shown in Figure 2. The conversions were low (negligible for solvent-free reactions and 20% for SCCO2 reactions) when alcohol and acid were taken in equimolar quantities. Increasing the amount of alcohol added increased the conversions rapidly until the concentration of alcohol was twice the molar equivalent of acid. A further increase in alcohol addition did not effect the conversion. The inhibition by alcohol was almost negligible for the range of the concentrations studied. The conversions were 68% for solvent-free reactions and 37% for SCCO2 reactions. The low conversions obtained with equimolar alcohol quantities can possibly be attributed to the inhibition of the reaction by deactivation/ denaturation of the enzyme by acid. The conversions under solvent-free conditions decreased slightly with increasing amount of alcohol added, showing that isoamyl alcohol is mildly inhibitory. Mensah et al.10 also observed alcohol inhibition for the synthesis of isoamyl propionate in n-hexane. However, Krishna et al.11 observed a maximum conversion for alcohol/acid molar ratios between 0.71 and 1.4 for the synthesis of isoamyl butyrate in n-hexane using immobilized M. meihei lipase and also observed a more severe inhibition by the acid. The kinetics of the reaction was studied by incubating 40 mm3 of acid, 95 mm3 of alcohol, and 20 mg of enzyme for various times at 35 °C under solvent-free reactions. The reactions in SCCO2 were studied with 20 mm3 (36 mM) of acid, 48 mm3 (74 mM) of alcohol, and 20 mg of enzyme at 45 °C. As shown in Figure 3, the conversions did not increase after 6 and 12 h under solvent-free conditions and in SCCO2, respectively. The apparently faster reaction rate observed under solvent-free conditions might be due to very high local enzyme concentrations. The negative influence of the presence of water is twofold. Because water is a product of the reaction, the equilibrium will shift toward the reactants, and excess water will deactivate the enzyme. Further, water will contribute to the mass-transfer resistance by forming a thin layer around the enzyme. Therefore, the effect of the addition of water was studied with 40 mm3 of acid, 95 mm3 of alcohol, and 20 mg of enzyme for the solventfree reactions and 20 mm3 of acid, 48 mm3 of alcohol,

Figure 3. Kinetics of the synthesis of isoamyl butyrate. Isoamyl alcohol: 95 mm3 for solvent-free reactions and 48 mm3 for SCCO2 reaction. Other conditions and legend are the same as in Figure 2.

Figure 4. Effect of addition of water on synthesis of isoamyl butyrate. Conditions same as in Figure 3. Time ) 6 h for solventfree reactions and 12 h for SCCO2 reactions.

corresponding to 36 mM and 74 mM, respectively, and 20 mg of enzyme for the SCCO2 reactions. The reactions were analyzed for conversions after 12 h of incubation at 35 °C for solvent-free reactions and at 45 °C for SCCO2 reactions. Figure 4 shows the effect of the addition of water on the conversions in the two systems. Whereas the addition of water reduced the conversion only slightly in the case of solvent-free reactions, the conversion decreased sharply in the case of reactions in SCCO2. This might be due to different distributions of water in the two systems. As water is not soluble in isoamyl alcohol, the water might settle as a small droplet and hydrate only a small part of the total enzyme added in the solvent-free system. In the supercritical fluid system, however, the distribution of water is uniform, and that water contributes to mass-transfer resistance by forming a film around the enzyme. Others12,13 who have investigated the synthesis of esters in hexane have also observed that the water content of the system does not influence the conversion. Nishio et al.12 observed that the synthesis of n-butyl oleate was not affected very much by the amount of water in the mixture. Zaks and Klibanov13 showed that the water content does not influence the activity of M. meihei lipase. In our previous work,14 the effect of water on the esterification of myristic acid was examined in both solvent-free and supercritical media. It is interesting to compare how the addition of water affects enzyme activity for the syntheses of isoamyl butyrate and ethyl myristate. Although the decreases in activity are very similar for reaction in SCCO2 (Figure 5), the trends are very different for solvent-free conditions (Figure 6). This difference arises from the different distributions of

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Figure 5. Effect of addition of water on enzyme activities in SCCO2 for the synthesis of isoamyl butyrate and ethyl myristate. Conditions similar to those in Figure 3 for isoamyl butyrate. For ethyl myristate, 15 mg of myristic acid, 100 mm3 of ethyl alcohol3, and 20 mg of enzyme. Time ) 3 h, CO2 density ) 0.2 g/cm3. 9, Isoamyl butyrate. O, Ethyl myristate.

Figure 6. Effect of addition of water on enzyme activities under solvent-free conditions for the synthesis of isoamyl butyrate and ethyl myristate. For isoamyl butyrate, conditions similar to those in Figure 3. For ethyl myristate, 15 mg of myristic acid, 100 mm3 of ethyl alcohol3, and 20 mg of enzyme. Time ) 3 h. 9, Isoamyl butyrate. O, Ethyl myristate.

water in the two systems. Irrespective of the alcohol investigated, the water-SCCO2 system is common. Therefore, it can be expected that the effects of water on the yields in such systems will also be similar. However, the distributions of water for the solvent-free reactions are different in the two cases, depending on the alcohol investigated. Water is soluble in ethanol and is uniformly distributed in the reaction medium for the solvent-free synthesis of ethyl myristate. However, in the case of isoamyl butyrate, water is insoluble in isoamyl alcohol, and therefore, it settles at the bottom of the reaction vial as a separate phase. Thus, whereas the enzyme is uniformly affected in the case of ethyl myristate, only a small portion of the whole enzyme is affected in the case of isoamyl butyrate. This accounts for the different effects of water on the syntheses of different esters. The effect of enzyme loading was studied by adding various amounts of enzyme to reaction mixtures with the same reactant and enzyme concentrations as described previously. The reactions were carried out for 6 and 12 h for solvent-free and SCCO2 reactions, respectively. Figure 7 depicts the effect of enzyme loading on the conversions. Lower enzyme loadings led to low conversions. Under conditions of low enzyme loading (5 mg), the conversions obtained in SCCO2 (12%) were higher than the conversions obtained under solvent-free conditions (6%). It must be emphasized that, although

Figure 7. Effect of enzyme loading on the synthesis of isoamyl butyrate. Conditions similar to those in Figure 3. b, Solvent-free. 9, SCCO2.

the enzyme loadings were the same for the SCCO2 and solvent-free reactions, the enzyme concentrations were about 45 times lower in the case of SCCO2. This indicates that SCCO2 might be more commercially viable at low enzyme concentrations. The maximum yields obtained under solvent-free conditions are in agreement with the observations of Welsh et al.,15 who obtained 68.6% yields of isoamyl butyrate with porcine pancreas lipase in hexane after 48 h of incubation. The shorter time required in the present study might be because of the very high local enzyme concentrations in solvent-free reactions. Isoamyl Acetate. The synthesis of isoamyl acetate was investigated under both solvent-free and supercritical conditions for 24 h for several acid and alcohol concentrations and temperatures. The maximum conversion was less than 4%, indicating that the acetic acid might deactivate/denature the enzyme, thus leading to lower conversions. These results are in agreement with earlier observations by Welsh et al.,15 who obtained a 3.3% conversion in 48 h with porcine pancreas lipase (PPL). This indicates that the synthesis of isoamyl acetate by HPL or PPL might not be viable. However, the effects on catalysis might be enzyme-specific, because other investigators2,15-17 have obtained high yields of isoamyl acetate (>80%) using M. meihei lipase with n-hexane as the solvent. Isoamyl Propionate. The effect of various alcohol concentrations on the synthesis of isoamyl propionate was studied with 20 mm3 of acid (corresponding to 45 mM in SCCO2) and 15 mg of enzyme. The reactions were performed at 35 °C for solvent-free reactions and at 45 °C for SCCO2 reactions. Figure 8 shows the effects of the addition of alcohol on the synthesis of isoamyl propionate. The conversions were low at equimolar concentration (1.44 mm3 of alcohol per mm3 of acid) and increased with increasing alcohol concentration, until the concentration of alcohol was 5 times the molar requirement. A further increase in alcohol content did not increase the conversion. This trend was observed for both solvent-free and SCCO2 reactions, although the acid inhibition was less in the case of SCCO2, as shown by the higher yields obtained for the alcohol/acid ratios of 1 and 2 (Figure 6). However, Mensah et al.10 obtained high yields (>90%) for isoamyl propionate using immobilized M. meihei lipase with equimolar quantities of acid and alcohol (1 M each in hexane), confirming that the reaction is enzyme-specific. The effect of temperature on the synthesis of isoamyl propionate was investigated with 20 mm3 of acid, 150 mm3 of alcohol, and 15 mg of enzyme for 36 h. The

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Figure 8. Effect of the ratio of alcohol to acid on the synthesis of isoamyl propionate with 20 mm3 of propionic acid and 15 mg of enzyme at 35 °C for solvent-free synthesis and 45 °C at 90 bar for SCCO2 reactions. b, Solvent-free. 9, SCCO2.

Figure 9. Effect of temperature on the synthesis of isoamyl propionate with 150 mm3 of isoamyl alcohol. Other conditions similar to those in Figure 6. b, Solvent-free. 9, SCCO2.

concentrations of the acid and alcohol in SCCO2 correspond to 45 and 284 mM, respectively. Because the optimum temperatures for isoamyl butyrate were 35 and 45 °C for solvent-free and SCCO2 reactions, respectively, the reactions in the present case were also investigated in the vicinity of these temperatures. The conversion reached a maximum at 40 °C for solventfree reactions (93%) and at 45 °C for SCCO2 reactions (60%), as shown in Figure 9. Isoamyl Octanoate (Isoamyl Caprylate). The synthesis of isoamyl octanoate under solvent-free conditions and in SCCO2 was studied to investigate the effect of chain length with even-carbon acids. The effects of the acid/alcohol ratio and of the temperature on the synthesis of isoamyl octanoate were investigated. The effect of the molar alcohol/acid ratio was studied for 36 h with 10 mg of enzyme. To achieve an alcoholto-acid ratio of 0.25-1, experiments were conducted with a fixed volume of alcohol (20 mm3) and varying amounts of added acid. To achieve alcohol-to-acid ratios of 2-4, experiments were conducted with a fixed volume of 20 mm3 (21 mM in SCCO2) of acid and varying amounts of added alcohol. The reactions were performed at 40 °C for solvent-free reactions and 45 °C for SCCO2 reactions. Figure 10 shows the effect of the acid/alcohol ratio under the two conditions. Under solvent-free conditions, the conversion initially increased with increasing alcohol/acid ratio but was constant at 96% for alcohol/acid ratios greater than 0.5. However, when the reaction was carried out in SCCO2, the conversion was nearly independent of the alcohol/acid ratio. Thus, the effect of the alcohol/acid ratio on the conversion to isoamyl octanoate (Figure 10) is markedly different from

Figure 10. Effect of molar alcohol/acid ratio on the synthesis of isoamyl octanoate with 10 mg of enzyme at a temperature of 35 °C for solvent-free synthesis and 45 °C for synthesis in SCCO2 and a pressure of 90 bar for SCCO2 reactions. b, Solvent-free. 9, SCCO2.

Figure 11. Effect of temperature on the synthesis of isoamyl octanoate. Solvent-free reactions: 20 mm3 of isoamyl alcohol, 29 mm3 of octanoic acid. SCCO2 reactions: 20 mm3 of acid, 28 mm3 of alcohol. Other conditions similar to those in Figure 8. b, Solventfree. 9, SCCO2.

the effect of this ratio on the conversiosn to isoamyl butyrate (Figure 2) and isoamyl propionate (Figure 8), where considerably higher alcohol/acid ratios are required before the conversion becomes independent of the ratio. This indicates that the chain length of the acid and its interactions with the substrate and enzyme play a crucial role in influencing the reaction rate. The effect of temperature was investigated with an equimolar ratio of acid/alcohol for solvent-free esterification and with a 1:2 molar ratio in SCCO2 for 36 h with a 10-mg enzyme loading. The temperatures studied were 35, 40, and 45 °C for solvent-free conditions and 40, 45, and 50 °C for SCCO2. The optimum temperatures were 40 and 45 °C for solvent-free conditions and SCCO2, respectively, as shown in Figure 11. Summary and Conclusions The synthesis of banana flavor esters of isoamyl alcohol by crude HPL was studied under solvent-free conditions and in SCCO2. The yields of isoamyl acetate, propionate, butyrate, and octanoate were 4, 93, 75, and 97%, respectively, under solvent-free conditions and 3, 60, 38, and 77%, respectively, in SCCO2. The yield of isoamyl acetate was very low, probably because of very strong inhibition/denaturation of enzyme by the acid. The conversions increases with increasing carbon chain length for even-carbon acids, but the yield of isoamyl propionate (ester of C3 acid) was found to be higher than that of isoamyl butyrate (ester of C4 acid). The higher yield for isoamyl propionate than for isoamyl butyrate might be due to the odd number of carbon atoms in the acid employed. The optimum molar alcohol/acid ratio

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for isoamyl propionate (5:1) was higher than that for isoamyl butyrate (2:1), indicating that this ratio might depend on the extent of inhibition by the acid. Higher yields for the longer-carbon-chain acids might be because of the greater hydrophobicities of these acids or their lower inhibition of the enzyme. The optimum ratio of alcohol to acid seems to depend on the carbon chain length. The optimum temperature under supercritical conditions (45 °C) was higher than that under solventfree conditions (35-40 °C), indicating greater enzyme stability under supercritical conditions. Although solventfree esterification seem to give higher reaction rates and high conversions, the concentrations of substrates and enzymes used are very high and unsuitable for commercialization. A benefit of supercritical fluids is the elimination of mass-transfer limitations, which results in increased conversions compared to solvent-free media, especially for very low enzyme loadings. Thus, the conditions in SCCO2 appear to be more suited for commercialization because of the high conversions obtained with low enzyme and substrate concentrations. Acknowledgment The authors thank the Ministry of Human Resources and Development, India, and the Department of Biotechnology for financial support. Literature Cited (1) Welsh, F. W.; Murray, W. D.; Williams, R. E. Microbiological and Enzymatic Production of Flavor and Fragrance Chemicals. CRC Crit. Rev. Biotechnol. 1989, 9, 105-169. (2) Krishna, S. H.; Manohar, B.; Divakar, S.; Prapulla, S. G.; Karanth, N. G. Optimization of Isoamyl Acetate Production by Using Immobilized Lipase from Mucor meihei by Response Surface Methodology. Enzyme Microb. Technol. 2000, 26, 131-136. (3) Langrand, G.; Triantaphylides, C.; Baratti, J. Lipase Catalyzed Formation of Flavor Esters. Biotechnol. Lett. 1988, 10, 549554. (4) Langrand, G.; Rondot, N.; Triantaphylides, C.; Baratti, J. Short Chain Flavor Esters Synthesis by Microbial Lipases. Biotechnol. Lett. 1990, 12, 581-586. (5) Norin, M.; Boutelje, J.; Holmberg, E.; Hult, K. Lipase Immobilized by Adsorption: Effect of Support Hydrophobicity on

the Reaction Rate of Ester Synthesis in Cyclohexane. Appl. Microbiol. Biotechnol. 1988, 28, 527-530. (6) Ison, A. P.; Macrae, A. R.; Smith, C. G.; Bosley, J. Mass transfer effects in solvent-free fat interesterification reactions: Influences on catalyst design. Biotechnol. Bioeng. 1994, 43, 122130. (7) Goma-Doncescu, N.; Legoy, M. D. Original transesterification route for fatty acid ester production from vegetable oils in a solvent-free system. J. Am. Oil Chem. Soc. 1997, 74, 1137-1143. (8) Trani, M.; Ergan, F.; Andre, G. Lipase-catalyzed production of wax esters. J. Am. Oil Chem. Soc. 1991, 68, 20-22. (9) De, B. K.; Bhattacharyya, D. K.; Bandhu, C. Enzymatic synthesis of fatty alcohol esters by alcoholysis. J. Am. Oil Chem. Soc. 1999, 76, 451-453. (10) Mensah, P.; Gainer, J. L.; Carta, G. Adsorptive Control of Water in Esterification with Immobilized Enzymes: I. Batch Reactor Behavior. Biotechnol. Bioeng. 1998, 60, 434-444. (11) Krishna, S. H.; Manohar, B.; Divakar, S.; Karanth, N. G. Lipase-Catalyzed Synthesis of Isoamyl Butyrate: Optimization by Response Surface Methodology. J. Am. Oil Chem. Soc. 1999, 76, 1483-1488. (12) Nishio, T.; Chikano, T.; Kamimura, M. Ester Synthesis by the Lipase from Pseudomonas fragi 22.39 B. Agric. Biol. Chem. 1988, 52, 1203-1208. (13) Zaks, A.; Klibanov, A. M. Enzyme-catalyzed processes in organic solvents. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 31923196. (14) Srivastava, S.; Madras, G.; Modak, J. M. Enzymatic Esterification of Myristic Acid in Supercritical Carbon Dioxide. J. Supercrit. Fluids, in review. (15) Welsh, F. W.; Williams, R. E.; Dawson, K. H. Lipase Mediated Synthesis of Low Molecular Weight Flavor Esters. J. Food Sci. 1990, 55, 1679-1682. (16) Rizzi, M.; Stylos, P.; Reik, A.; Reuss, M. A Kinetic Study of Immobilized Lipase Catalyzing the Synthesis of Isoamyl Acetate by Transesterification in n-Hexane. Enzyme Microb. Technol. 1992, 14, 709-714. (17) Razafindralambo, H.; Blecker, C.; Lognay, G.; Marlier, M.; Wathelet, J. P.; Severin, M. Improvement of Enzymatic Synthesis Yields of Flavor Acetates: The Example of Isoamyl Acetate. Biotechnol. Lett. 1994, 16, 247-250.

Received for review July 30, 2001 Revised manuscript received January 25, 2002 Accepted January 27, 2002 IE010651J