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. Theesters synthesized were isoamyl acetate, isoamyl propionate, isoamyl butyrate, and isoamyloctanoate. Very low yields (3-4%) of isoamyl acetate were obtained, but high yields for the otherthree esters were obtained under both supercritical and solvent-free conditions. The yields ofesters of the even-carbon acids, isoamyl acetate, butyrate, and octanoate, increased withincreasing chain length, whereas the yield of isoamyl propionate was higher than that of isoamylbutyrate. 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 asalcohol concentration, water concentration, and enzyme loading were investigated. An increasein the water concentration decreased the conversion significantly in supercritical carbon dioxidebut not under solvent-free conditions. The optimum ratio of alcohol to acid was dependent onthe extent of inhibition by the acid. Although providing a higher apparent yield by being run ina highly concentrated medium, the overall conversion under solvent-free conditions was lowerthan that under supercritical conditions for similar enzyme concentrations, indicating that thesynthesis of esters in supercritical carbon dioxide might be a viable option.

Introduction

The modern food industry uses very large quantitiesof various flavor compounds1 as food additives toenhance the flavors of food products. Most fruit flavorsare esters of fatty acids and are synthesized chemically.Although chemical synthesis gives high yields of theesters, the flavors produced are not considered natural,and the enzymatic production of flavor compounds ispreferred. Enzymatic synthesis also offers the advan-tages of ambient reaction temperatures, increasedselectivities, and ease of downstream processing. Li-pases (triacyl glycerol hydrolases, E.C. 3.1.1.3), underconditions of reduced water, can be used for synthesizingesters. Therefore, nonpolar organic solvents such asn-hexane,2 n-heptane,3,4 and cyclohexane5 have beenused as reaction media. Solvent-free systems are sys-tems in which the reaction medium involves a reactantitself as the solvent. These systems are used com-mercially,6 and some investigations7-9 have focused onlipase-catalyzed reactions in which one of the reactants,the alcohol, acts as the solvent. Although high yieldshave been obtained with these solvents, the reactionrates are usually slow as a result of mass-transferlimitations. Lipase-catalyzed reactions are usually het-erogeneous. Whereas the substrate is soluble in thesolvent, the lipases are insoluble. Thus, the reactionoccurs at the interface between the enzyme and thesolvent. The characteristic heterogeneity of the reactionsresults in mass-transfer resistance because of the dif-fusion rates of the reactants to the active site of theenzyme.

Supercritical fluids (SCFs), defined as fluids abovetheir critical temperature and pressure, have liquidlikedensities and gaslike diffusivities. These propertiesmake them attractive as solvents for reactions with

mass-transfer limitations. Another advantage of super-critical fluids is that the solubilities of the reactants andproducts are greatly dependent on the pressure andtemperature of the system. This property of supercriticalfluids can be harnessed to integrate the reaction anddownstream processing into a single step. Further, theincreasingly stringent regulations on volatile organicshave led to the increased usage of supercritical fluids.Among SCFs, supercritical carbon dioxide (SCCO2)offers the unique advantages of being cheap, nonflam-mable, and nontoxic. It has a near-ambient criticaltemperature (31.1 °C) and moderate critical pressure(73.8 bar).

Although many lipase-catalyzed reactions, includingthat of isoamyl acetate, have been reported in SCCO2,we are unaware of any study on the synthesis of variousother esters of isoamyl alcohol in SCCO2. This study,unlike other studies, also compares the conversionsobtained from the reaction in SCCO2 to the conversionsobtained under solvent-free conditions. The objective ofthis paper is to investigate the various parameters thatinfluence the synthesis of flavor compounds of isoamylalcohol and short-chain fatty acids. Crude hog pancreaslipase (HPL) is used for synthesis because it is one ofthe least expensive lipases. Although certain lipasessuch as M. meihei demonstrate higher activities andconversions in the esterification reaction, HPL waschosen in this study because it is cheap and might beindustrially viable. The compounds synthesized wereisoamyl acetate, isoamyl propionate, isoamyl butyrate,and isoamyl octanoate. These compounds are used asbanana flavors and, therefore, are commercially impor-tant. The reactions were studied under solvent-freeconditions and in SCCO2.

Experimental Procedures

Materials. Crude hog pancreas lipase (with a statedactivity of 20 U/mg) was purchased from Fluka Chemie

* Corresponding author. Phone: 91-80-309 2321. Fax: 91-80-360 0683. E-mail: giridhar@chemeng.iisc.ernet.in.

1940 Ind. Eng. Chem. Res. 2002, 41, 1940-1945

10.1021/ie010651j CCC: $22.00 © 2002 American Chemical SocietyPublished on Web 03/19/2002

AG, Switzerland. The enzyme was stored at -15 °C anddesiccated for 12 h at room temperature before use.Isoamyl alcohol (99%, Glaxo), acetic acid (99%, RanbaxyChemicals), propionic acid (99%, E. Merck), butyric acid(97.5%, Rolex Chemicals), octanoic acid (99%, SD FineChemicals), isoamyl acetate (95%, SD Fine Chemicals),and CO2 (99%, Sicgil Gases) were used. All solventswere of HPLC grade and were distilled and filteredbefore use.

Methods. The reactions were performed in 6-cm3

stainless steel batch reactors. Each reactor, loaded withreactants and enzyme, was pressurized to an initialpressure of 68 bar at room temperature. The pressurizedreactor was then immersed in a water bath maintainedat the desired temperature (fluctuations were less than(0.5 °C). All of the reactions were conducted at aconstant density of CO2 of 0.2 g/cm3 for various tem-peratures to ensure constant solubility of the substrates.Even though the pressure was higher at every temper-ature increment, the density of the system remainedconstant. The reactors were equipped with a pressuregauge to ensure that the system operated at the samepressure throughout the reaction. After the desiredreaction time, the reactor was depressurized, and thecontents were eluted in 3 mL of methanol. The enzymewas made to settle by centrifugation, and the reactionmixture was analyzed by HPLC.

To the best of our knowledge, no literature data areavailable on the solubilities of the substrates in SCCO2in the presence of isoamyl alcohol. To ascertain whethersolubility/mixing plays a role in the reaction, a two-reactor system was designed. The acid and alcohol wereadded to one reactor, and solid enzyme powder wasadded to another reactor. This ensured that there wasno physical contact between the reactants and theenzyme. The concentrations of the reactants and theenzyme in the two-reactor setup were identical to thoseused in the single reactor. The two reactors were thenindividually pressurized. After pressurization, they wereconnected through a small steel tube, and the connect-ing valves were opened. The reactors were then incu-bated at the required temperatures for 12 h. It shouldbe pointed out that the substrates and the enzyme werenot in physical contact with each other in the two-reactor setup. Therefore, the substrate and enzymecould come into contact with each other only throughthe solvent phase, that is, the supercritical CO2. Theconversion (percent esterification) was the same as inthe single-reactor system, indicating that the system iscompletely mixed and that solubility/mixing does notplay a role in determining the conversion. We alsoperformed the reactions with agitation of the reactionsystems but found no increase in conversion.

The volumes of the substrates for the reactions werechosen so that the concentrations of the reactants werelow enough that they dissolved in SCCO2 but highenough to ensure detection without significant error.

Solvent-free reactions were performed in 5-mL cappedglass vials. The acid and alcohol were mixed in the vialsand incubated at the desired temperature for 2 min. Theenzyme was then added to the vial, and the mixture wasincubated for the desired interval of time. The reactionswere terminated by the addition of 3 mL of methanol,followed by centrifuging to remove the enzyme. Thereaction samples were analyzed by HPLC.

Analysis. The reaction samples were analyzed by aHPLC system consisting of a pump (Waters 501), a

reverse-phase column (0.39 cm × 25 cm, µBondapakC18), an injector (Rheodyne 7010, with a 250-mm3

injection loop), and a UV detector (Nulab Instruments,3010). Methanol-water solution was eluted through thesystem at a flow rate of 2 cm3/min, and the elutedcompounds were detected at a wavelength of 212 nm.A solution of 50:50 (v/v) methanol/water was used asthe eluent for isoamyl acetate, propionate, and butyrate,whereas a solution of 75:25 methanol/water was usedas the eluent for isoamyl octanoate. Isoamyl acetate(purchased) was used for calibration. Other esters weresynthesized chemically by Fischer’s method using con-centrated H2SO4 as the catalyst. Alcohol (5 cm3) and thecorresponding twice molar equivalent of acid were addedto a 50-cm3 round-bottom flask. Two drops of concen-trated H2SO4 were added to the solution, and themixture was stirred and refluxed for 24 h. The esterswere purified by first washing the reaction mixture withwater and saturated NaHCO3 solution to remove acidand then usingsilica gel chromatography. The purity ofthe synthesized esters was verified by thin-layer chro-matography and FTIR. Various amounts of estersdissolved in methanol were injected, and linear calibra-tion curves were obtained for all of the esters.

Results and Discussion

Synthesis of Isoamyl Butyrate. The temperatureprofile of the reaction was investigated by conductingreactions with 80 mm3 of butyric acid, 190 mm3 ofisoamyl alcohol, corresponding to 145 and 291 mM inSCCO2, respectively, and 20 mg of enzyme. The reactionmixtures were incubated at various temperatures for12 h. The amount of ester produced was determined byHPLC. Figure 1 depicts the effect of temperature on thesynthesis under supercritical and solvent-free condi-tions. Under solvent-free conditions, the optimum tem-perature was 35 °C, and a yield of 34% was obtained.The optimum temperature for reaction in SCCO2 was45 °C, with a 10% yield of ester.

The effect of alcohol on the yield of ester was inves-tigated by adding various amounts of alcohol to 40 mm3

of butyric acid for solvent-free reactions. The molarequivalent of alcohol is 1.185 mm3 of alcohol per mm3

of acid. After 2 min of incubation at the optimumtemperature, 20 mg of enzyme was added to the system,and the system was incubated for 12 h. For reactionsin SCCO2, various amounts of alcohol were added to 20mm3 (36 mM) of acid, with an enzyme loading of 20 mg.The reactions were performed for 12 h at the optimum

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

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temperatures, i.e., at 35 °C for solvent-free reactions and45 °C for SCCO2 reactions. The effect of alcohol on theesterification is shown in Figure 2. The conversions werelow (negligible for solvent-free reactions and 20% forSCCO2 reactions) when alcohol and acid were taken inequimolar quantities. Increasing the amount of alcoholadded increased the conversions rapidly until the con-centration of alcohol was twice the molar equivalent ofacid. A further increase in alcohol addition did not effectthe conversion. The inhibition by alcohol was almostnegligible for the range of the concentrations studied.The conversions were 68% for solvent-free reactions and37% for SCCO2 reactions. The low conversions obtainedwith equimolar alcohol quantities can possibly be at-tributed to the inhibition of the reaction by deactivation/denaturation of the enzyme by acid. The conversionsunder solvent-free conditions decreased slightly withincreasing amount of alcohol added, showing thatisoamyl alcohol is mildly inhibitory. Mensah et al.10 alsoobserved alcohol inhibition for the synthesis of isoamylpropionate in n-hexane. However, Krishna et al.11

observed a maximum conversion for alcohol/acid molarratios between 0.71 and 1.4 for the synthesis of isoamylbutyrate in n-hexane using immobilized M. meiheilipase and also observed a more severe inhibition by theacid.

The kinetics of the reaction was studied by incubating40 mm3 of acid, 95 mm3 of alcohol, and 20 mg of enzymefor various times at 35 °C under solvent-free reactions.The reactions in SCCO2 were studied with 20 mm3 (36mM) of acid, 48 mm3 (74 mM) of alcohol, and 20 mg ofenzyme at 45 °C. As shown in Figure 3, the conversionsdid not increase after 6 and 12 h under solvent-freeconditions and in SCCO2, respectively. The apparentlyfaster reaction rate observed under solvent-free condi-tions might be due to very high local enzyme concentra-tions.

The negative influence of the presence of water is two-fold. Because water is a product of the reaction, theequilibrium will shift toward the reactants, and excesswater will deactivate the enzyme. Further, water willcontribute to the mass-transfer resistance by forminga thin layer around the enzyme. Therefore, the effectof the addition of water was studied with 40 mm3 of acid,95 mm3 of alcohol, and 20 mg of enzyme for the solvent-free reactions and 20 mm3 of acid, 48 mm3 of alcohol,

corresponding to 36 mM and 74 mM, respectively, and20 mg of enzyme for the SCCO2 reactions. The reactionswere analyzed for conversions after 12 h of incubationat 35 °C for solvent-free reactions and at 45 °C forSCCO2 reactions. Figure 4 shows the effect of theaddition of water on the conversions in the two systems.Whereas the addition of water reduced the conversiononly slightly in the case of solvent-free reactions, theconversion decreased sharply in the case of reactionsin SCCO2. This might be due to different distributionsof water in the two systems. As water is not soluble inisoamyl alcohol, the water might settle as a smalldroplet and hydrate only a small part of the totalenzyme added in the solvent-free system. In the super-critical fluid system, however, the distribution of wateris uniform, and that water contributes to mass-transferresistance by forming a film around the enzyme. Oth-ers12,13 who have investigated the synthesis of esters inhexane have also observed that the water content of thesystem does not influence the conversion. Nishio et al.12

observed that the synthesis of n-butyl oleate was notaffected very much by the amount of water in themixture. Zaks and Klibanov13 showed that the watercontent does not influence the activity of M. meiheilipase.

In our previous work,14 the effect of water on theesterification of myristic acid was examined in bothsolvent-free and supercritical media. It is interesting tocompare how the addition of water affects enzymeactivity for the syntheses of isoamyl butyrate and ethylmyristate. Although the decreases in activity are verysimilar for reaction in SCCO2 (Figure 5), the trends arevery different for solvent-free conditions (Figure 6). Thisdifference arises from the different distributions of

Figure 2. Effect of addition of alcohol on synthesis of isoamylbutyrate. For solvent-free reactions: 40 mm3 of butyric acid and20 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.

Figure 3. Kinetics of the synthesis of isoamyl butyrate. Isoamylalcohol: 95 mm3 for solvent-free reactions and 48 mm3 for SCCO2reaction. Other conditions and legend are the same as in Figure2.

Figure 4. Effect of addition of water on synthesis of isoamylbutyrate. Conditions same as in Figure 3. Time ) 6 h for solvent-free reactions and 12 h for SCCO2 reactions.

1942 Ind. Eng. Chem. Res., Vol. 41, No. 8, 2002

water in the two systems. Irrespective of the alcoholinvestigated, the water-SCCO2 system is common.Therefore, it can be expected that the effects of wateron the yields in such systems will also be similar.However, the distributions of water for the solvent-freereactions are different in the two cases, depending onthe alcohol investigated. Water is soluble in ethanol andis uniformly distributed in the reaction medium for thesolvent-free synthesis of ethyl myristate. However, inthe case of isoamyl butyrate, water is insoluble inisoamyl alcohol, and therefore, it settles at the bottomof the reaction vial as a separate phase. Thus, whereasthe enzyme is uniformly affected in the case of ethylmyristate, only a small portion of the whole enzyme isaffected in the case of isoamyl butyrate. This accountsfor the different effects of water on the syntheses ofdifferent esters.

The effect of enzyme loading was studied by addingvarious amounts of enzyme to reaction mixtures withthe same reactant and enzyme concentrations as de-scribed previously. The reactions were carried out for 6and 12 h for solvent-free and SCCO2 reactions, respec-tively. Figure 7 depicts the effect of enzyme loading onthe conversions. Lower enzyme loadings led to lowconversions. Under conditions of low enzyme loading (5mg), the conversions obtained in SCCO2 (12%) werehigher than the conversions obtained under solvent-freeconditions (6%). It must be emphasized that, although

the enzyme loadings were the same for the SCCO2 andsolvent-free reactions, the enzyme concentrations wereabout 45 times lower in the case of SCCO2. Thisindicates that SCCO2 might be more commerciallyviable at low enzyme concentrations.

The maximum yields obtained under solvent-freeconditions are in agreement with the observations ofWelsh et al.,15 who obtained 68.6% yields of isoamylbutyrate with porcine pancreas lipase in hexane after48 h of incubation. The shorter time required in thepresent study might be because of the very high localenzyme concentrations in solvent-free reactions.

Isoamyl Acetate. The synthesis of isoamyl acetatewas investigated under both solvent-free and super-critical conditions for 24 h for several acid and alcoholconcentrations and temperatures. The maximum con-version was less than 4%, indicating that the acetic acidmight deactivate/denature the enzyme, thus leading tolower conversions. These results are in agreement withearlier observations by Welsh et al.,15 who obtained a3.3% conversion in 48 h with porcine pancreas lipase(PPL). This indicates that the synthesis of isoamylacetate by HPL or PPL might not be viable. However,the effects on catalysis might be enzyme-specific, be-cause other investigators2,15-17 have obtained highyields of isoamyl acetate (>80%) using M. meihei lipasewith n-hexane as the solvent.

Isoamyl Propionate. The effect of various alcoholconcentrations on the synthesis of isoamyl propionatewas studied with 20 mm3 of acid (corresponding to 45mM in SCCO2) and 15 mg of enzyme. The reactions wereperformed at 35 °C for solvent-free reactions and at 45°C for SCCO2 reactions. Figure 8 shows the effects ofthe addition of alcohol on the synthesis of isoamylpropionate. The conversions were low at equimolarconcentration (1.44 mm3 of alcohol per mm3 of acid) andincreased with increasing alcohol concentration, untilthe concentration of alcohol was 5 times the molarrequirement. A further increase in alcohol content didnot increase the conversion. This trend was observedfor both solvent-free and SCCO2 reactions, although theacid inhibition was less in the case of SCCO2, as shownby the higher yields obtained for the alcohol/acid ratiosof 1 and 2 (Figure 6). However, Mensah et al.10 obtainedhigh yields (>90%) for isoamyl propionate using im-mobilized M. meihei lipase with equimolar quantitiesof acid and alcohol (1 M each in hexane), confirming thatthe reaction is enzyme-specific.

The effect of temperature on the synthesis of isoamylpropionate was investigated with 20 mm3 of acid, 150mm3 of alcohol, and 15 mg of enzyme for 36 h. The

Figure 5. Effect of addition of water on enzyme activities inSCCO2 for the synthesis of isoamyl butyrate and ethyl myristate.Conditions similar to those in Figure 3 for isoamyl butyrate. Forethyl 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 undersolvent-free conditions for the synthesis of isoamyl butyrate andethyl myristate. For isoamyl butyrate, conditions similar to thosein Figure 3. For ethyl myristate, 15 mg of myristic acid, 100 mm3

of ethyl alcohol3, and 20 mg of enzyme. Time ) 3 h. 9, Isoamylbutyrate. O, Ethyl myristate.

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

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concentrations of the acid and alcohol in SCCO2 cor-respond to 45 and 284 mM, respectively. Because theoptimum temperatures for isoamyl butyrate were 35and 45 °C for solvent-free and SCCO2 reactions, respec-tively, the reactions in the present case were alsoinvestigated in the vicinity of these temperatures. Theconversion reached a maximum at 40 °C for solvent-free reactions (93%) and at 45 °C for SCCO2 reactions(60%), as shown in Figure 9.

Isoamyl Octanoate (Isoamyl Caprylate). Thesynthesis of isoamyl octanoate under solvent-free condi-tions and in SCCO2 was studied to investigate the effectof chain length with even-carbon acids. The effects ofthe acid/alcohol ratio and of the temperature on thesynthesis of isoamyl octanoate were investigated.

The effect of the molar alcohol/acid ratio was studiedfor 36 h with 10 mg of enzyme. To achieve an alcohol-to-acid ratio of 0.25-1, experiments were conductedwith a fixed volume of alcohol (20 mm3) and varyingamounts of added acid. To achieve alcohol-to-acid ratiosof 2-4, experiments were conducted with a fixed volumeof 20 mm3 (21 mM in SCCO2) of acid and varyingamounts of added alcohol. The reactions were performedat 40 °C for solvent-free reactions and 45 °C for SCCO2reactions. Figure 10 shows the effect of the acid/alcoholratio under the two conditions. Under solvent-freeconditions, the conversion initially increased with in-creasing alcohol/acid ratio but was constant at 96% foralcohol/acid ratios greater than 0.5. However, when thereaction was carried out in SCCO2, the conversion wasnearly independent of the alcohol/acid ratio. Thus, theeffect of the alcohol/acid ratio on the conversion toisoamyl octanoate (Figure 10) is markedly different from

the effect of this ratio on the conversiosn to isoamylbutyrate (Figure 2) and isoamyl propionate (Figure 8),where considerably higher alcohol/acid ratios are re-quired before the conversion becomes independent of theratio. This indicates that the chain length of the acidand its interactions with the substrate and enzyme playa crucial role in influencing the reaction rate. The effectof temperature was investigated with an equimolar ratioof acid/alcohol for solvent-free esterification and with a1:2 molar ratio in SCCO2 for 36 h with a 10-mg enzymeloading. The temperatures studied were 35, 40, and 45°C for solvent-free conditions and 40, 45, and 50 °C forSCCO2. The optimum temperatures were 40 and 45 °Cfor solvent-free conditions and SCCO2, respectively, asshown in Figure 11.

Summary and Conclusions

The synthesis of banana flavor esters of isoamylalcohol by crude HPL was studied under solvent-freeconditions and in SCCO2. The yields of isoamyl acetate,propionate, butyrate, and octanoate were 4, 93, 75, and97%, respectively, under solvent-free conditions and 3,60, 38, and 77%, respectively, in SCCO2. The yield ofisoamyl acetate was very low, probably because of verystrong inhibition/denaturation of enzyme by the acid.The conversions increases with increasing carbon chainlength for even-carbon acids, but the yield of isoamylpropionate (ester of C3 acid) was found to be higher thanthat of isoamyl butyrate (ester of C4 acid). The higheryield for isoamyl propionate than for isoamyl butyratemight be due to the odd number of carbon atoms in theacid employed. The optimum molar alcohol/acid ratio

Figure 8. Effect of the ratio of alcohol to acid on the synthesis ofisoamyl propionate with 20 mm3 of propionic acid and 15 mg ofenzyme at 35 °C for solvent-free synthesis and 45 °C at 90 bar forSCCO2 reactions. b, Solvent-free. 9, SCCO2.

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

Figure 10. Effect of molar alcohol/acid ratio on the synthesis ofisoamyl octanoate with 10 mg of enzyme at a temperature of 35°C for solvent-free synthesis and 45 °C for synthesis in SCCO2and a pressure of 90 bar for SCCO2 reactions. b, Solvent-free. 9,SCCO2.

Figure 11. Effect of temperature on the synthesis of isoamyloctanoate. Solvent-free reactions: 20 mm3 of isoamyl alcohol, 29mm3 of octanoic acid. SCCO2 reactions: 20 mm3 of acid, 28 mm3

of alcohol. Other conditions similar to those in Figure 8. b, Solvent-free. 9, SCCO2.

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for isoamyl propionate (5:1) was higher than that forisoamyl butyrate (2:1), indicating that this ratio mightdepend on the extent of inhibition by the acid. Higheryields for the longer-carbon-chain acids might be be-cause of the greater hydrophobicities of these acids ortheir lower inhibition of the enzyme. The optimum ratioof alcohol to acid seems to depend on the carbon chainlength. The optimum temperature under supercriticalconditions (45 °C) was higher than that under solvent-free conditions (35-40 °C), indicating greater enzymestability under supercritical conditions. Although solvent-free esterification seem to give higher reaction rates andhigh conversions, the concentrations of substrates andenzymes used are very high and unsuitable for com-mercialization. A benefit of supercritical fluids is theelimination of mass-transfer limitations, which resultsin increased conversions compared to solvent-free me-dia, especially for very low enzyme loadings. Thus, theconditions in SCCO2 appear to be more suited forcommercialization because of the high conversionsobtained with low enzyme and substrate concentrations.

Acknowledgment

The authors thank the Ministry of Human Resourcesand Development, India, and the Department of Bio-technology for financial support.

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(3) Langrand, G.; Triantaphylides, C.; Baratti, J. Lipase Cata-lyzed Formation of Flavor Esters. Biotechnol. Lett. 1988, 10, 549-554.

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(7) Goma-Doncescu, N.; Legoy, M. D. Original transesterifica-tion route for fatty acid ester production from vegetable oils in asolvent-free system. J. Am. Oil Chem. Soc. 1997, 74, 1137-1143.

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(10) Mensah, P.; Gainer, J. L.; Carta, G. Adsorptive Control ofWater in Esterification with Immobilized Enzymes: I. BatchReactor 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 byResponse Surface Methodology. J. Am. Oil Chem. Soc. 1999, 76,1483-1488.

(12) Nishio, T.; Chikano, T.; Kamimura, M. Ester Synthesis bythe Lipase from Pseudomonas fragi 22.39 B. Agric. Biol. Chem.1988, 52, 1203-1208.

(13) Zaks, A.; Klibanov, A. M. Enzyme-catalyzed processes inorganic solvents. Proc. Natl. Acad. Sci. U.S.A. 1985, 82, 3192-3196.

(14) Srivastava, S.; Madras, G.; Modak, J. M. EnzymaticEsterification of Myristic Acid in Supercritical Carbon Dioxide. J.Supercrit. Fluids, in review.

(15) Welsh, F. W.; Williams, R. E.; Dawson, K. H. LipaseMediated 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 Studyof Immobilized Lipase Catalyzing the Synthesis of Isoamyl Acetateby 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 SynthesisYields of Flavor Acetates: The Example of Isoamyl Acetate.Biotechnol. Lett. 1994, 16, 247-250.

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

Accepted January 27, 2002

IE010651J

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