Enzyme and Microbial Technology 25 (1999) 584 590

Enzymatic synthesis of fatty esters Part I. Kinetic approach

T. Garcia, N. Sanchez, M. Martinez, J. Aracil*
Department of Chemical Engineering, Faculty of Chemistry, Complutense University, 28040 Madrid, Spain Received 26 November 1998; received in revised form 24 May 1999; accepted 1 June 1999

Abstract A kinetic study of palmitic acid esterication reaction catalyzed by immobilized lipase Novozym 435 (from Candida antarctica) has been made in a range of 6575C temperature and 37% of initial catalyst concentration and atmospheric pressure. Inuence of operating variables also was studied. Different values of temperature, initial lipase concentration, acid/alcohol molar ratio, and products concentrations were tested. Experiments were carried out in a stirred batch reactor equipped with adequate control systems. Evolution of concentration of the species involved was found by using gas-liquid chromatography. The kinetic characteristics observed in the esterication reaction with isopropanol were found to follow an ordered bi-bi mechanism with competitive inhibition by reactants and products. According to the mechanism maximum reaction rate, MichaelisMenten constants and inhibition constants were determined. A good quality of t was observed by tting experimental rate data to the kinetic model with an average error beneath the 15%. Kinetic constants, as well as enthalpy and entropy values, have been determined for the model proposed for the esterication reaction within the experimental range studied. 1999 Elsevier Science Inc. All rights reserved.
Keywords: Immobilized enzyme; Enzymatic esterication; Candida antarctica; Kinetic modeling; Fatty esters; Isopropyl palmitate

1. Introduction Interest in studies of various sources lipases-catalyzed reaction has increased dramatically during the past 10 years. Many researchers have attempted to understand the functional properties of lipase in processes that involve modication of the properties of fats and oils [1]. Particularly challenging tasks are those associated with developing rate expressions to characterize several lipase-catalyzed reactions and deciphering the mechanisms involved [2]. When dispersed in organic solvents, lipase can catalyze reactions previously unreported when the concentration of water in such systems is maintained at levels of 1% by weight [3]. Modeling of these enzyme-catalyzed organic phase reactions has received relatively little attention by the scientic community. The purpose of this study is to contribute to the clarication of the mechanisms and to obtain the corresponding rate expressions of esterication reactions catalyzed by immobilized lipase in nonpolar media.
For Part II see pp. 591597 of this issue. * Corresponding author. Tel.: 34-91-394-4167. E-mail address: jam1@eucmax.sim.ucm.es (J. Aracil)

The most extended models are based on the application of MichaelisMenten assumptions. This type of model seems to be valid for the most simple enzymatic reactions. Dehydrogenation of several alcoholic groups by using alcohol-dehydrogenase as catalyst was found to follow a kinetic model based on the hypothesis of MichaelisMenten when reaction was carried out under optimum operating conditions (pH 8.8 and 25C; [4]). Similar results were obtained for the kinetic study of hydrolysis of starch at 50C, by using Rhizopus niveus enzyme immobilized on a ceramic type support [5]. Other authors proposed a model based on a ping-pong mechanism for the kinetic study of the interesterication reaction of n-propyl acetate with geraniol at 40C, by using lipases as catalysts and different solvents in the reaction media [6]. This supposition agrees with that adopted in the study of the esterication of lauric acid with menthol at 35C by using Penicillium simplicissimum lipase [7]. Most authors agree that reaction occurs via acyl-enzyme intermediate when lipases are used as catalysts. This hypothesis was conrmed by studies that showed that the only possibility for the reaction to take place is the formation of

0141-0229/99/$ see front matter 1999 Elsevier Science Inc. All rights reserved. PII: S 0 1 4 1 - 0 2 2 9 ( 9 9 ) 0 0 0 8 2 - 4

T. Garcia et al. / Enzyme and Microbial Technology 25 (1999) 584 590

585

Nomenclature [i] concentration of component i (mol/l) initial concentration of component i (mol/l) equilibrium concentration of component i (mol/l) activation energy (cal/mol) (Kcal/Kmol) (Kcal/mol) G free Gibbs energy (cal/mol) H enthalpy (cal/mol) k rate constant (l/mol/gcat/min) K0 pre-exponential factor of Arrhenius equation (l/ mol/gcat/min) Keq equilibrium constant Kij inhibition constant for component j (mol/l) Kmi Michaelis constant for component i (mol/l) P pressure (Pa) (mmHg) R gas constant (cal/mol/K) (ri) rate of disappearance of component i (mol/l/gcat/min) (-ri)max maximum rate of disappearance of component i (mol/l/gcat/min) S entropy (cal/mol/K) t time (min) T temperature (C) (K) w amount of catalyst (g) Xi conversion of component i Xieq equilibrium conversion of component i Ci Ci0 Cieq Ea Subscript Ac fatty acid Al fatty alcohol E free enzyme E-A binary enzyme-substrate A complex E-A-B ternary substrate A-enzyme-substrate B complex Es ester f forward reaction r reverse reaction W water Greek letters , , , , : Parameters in Eq. (4).

mechanistic considerations. The validity of the model has been tested experimentally for the synthesis of isopropyl palmitate by using a commercial immobilized Candida antarctica lipase as catalyst. Isopropyl palmitate is widely used in cosmetic and topical medicinal preparations for which good absorption through the skin is required. The synthesis has been carried out in solvent-free conditions to reduce time and cost in the product purication.

2. Materials and methods 2.1. Equipment Experiments were carried out in a completely stirred tank reactor of 500 cm3 volume, 10 cm height, and 7 cm diameter. A marine-type propeller was used. The impeller speed was set at 600 rev./min to avoid mass transfer limitations. The reactor was immersed in a water bath capable of maintaining the reaction temperature 1C. Temperature, impeller speed, and pressure recorders and controllers were installed. 2.2. Materials Palmitic acid purity 98% w/w and isopropyl alcohol 98% w/w, used as reactants, were supplied by Henkel Iberica (Minneapolis, MN, USA). The catalyst used was a nonspecic triacylglycerol lipase (EC 3.1.1.3) from Candida antarctica. The commercial lipase, named Novozym 435 and supplied by Novo-Nordisk Bioindustial A/S (Copenhagen, Denmark), was immobilized on a macroporous acrylic resin with a water content of 12% w/w. The ester synthesis activity of the catalyst was 7000 Propyl Laurate Units (PLU/g). Aldrich-Sigma A/S and Fluka Chemie AG supplied other materials like standard products and solvents used in analytical procedures. 2.3. Analytical methods Reaction products were monitored by gas chromatography/mass spectrometry (GC/MS) and quantitatively determined by capillary column GC. GC/MS data were recorded on a HP 6890 Series Hewlett Packcard instrument and then treated with a Hewlett Packard Vectra series 3 computer, connected on line with the GC/MS system equipped with a HP autosampler controller. Quantitative GC analyses were performed on a Hewllet Packard HP 5890 series II instrument connected to a Hewlett Packard HP 3396 A integrator, by using a capillary column, ame ionization detector, and split-splitless injection system. The complete description of analytical method has been described in previous works [10].

an active complex between an acyl group belonging to the substrate and one of the active centers of the lipase [8,9]. However, despite the fact that several kinetic studies have been carried out, the information that allows the performance of an appropriate analysis for a later industrial scale up continues to be quite limited. On the other hand, most of the studies deal only with the synthesis of esters of low and medium molecular weight or with collecting information on enzymatic reactions with a single substrate. For these reasons, in the present study, a kinetic model for the enzymatic synthesis of esters has been deduced from

586

T. Garcia et al. / Enzyme and Microbial Technology 25 (1999) 584 590

Fig. 1. Inuence of reaction temperature on isopropyl palmitate synthesis (5% catalyst, acid/alcohol molar ratio: 1/1).

Fig. 2. Inuence of initial concentration of immobilized lipase on the esterication process (T: 70C, acid/alcohol molar ratio: 1/1).

2.4. Procedure Experiments were performed according to the procedure described by Garcia et al. in this journal [11], introducing some minor modications. Reactants were added to the reactor tted with a reux condenser. When the set pressure was reached, the reaction mixture was heated up to the desired temperature and the catalyst was introduced in the reactor. Samples were taken at regular intervals and analyzed by GC. During the experiments, the following variables remained constant: temperature, pressure, impeller speed, and acid/alcohol molar ratio. Total conversion to ester was achieved after 1 to 2 h, depending on the reaction conditions.

10% was obtained when the catalyst concentration was increased by 2%. To study the effect of the catalyst concentration and further kinetic modeling, conversion to isopropyl palmitate was plotted versus catalyst concentration for different reaction times. Fig. 3 is the plot corresponding to 70C and a Ac/Al molar ratio of 1. A linear relationship was observed for all reaction times. Similar results were obtained for all temperatures and molar ratios tested. No saturation effects were observed within the experimental range studied. This fact has been observed for analog systems of reaction [13]. 3.3. Inuence of acid/alcohol molar ratio To perform a subsequent kinetic analysis, two series of experiments were carried out at different acid/alcohol molar ratios. The rst series was carried out in excess of isopropyl alcohol to study the change in reaction rate with palmitic acid concentration. In the second group of experiments, palmitic acid was added in excess to study the dependence of the reaction rate on isopropyl alcohol concentration. In both cases, the operating temperature range used was 65C 70C75C, and the catalyst concentration was 5 wt%. Fig. 4 represents the rate of ester formation versus alcohol concentration for different acid/alcohol molar ratios, in exper-

3. Results and discussion 3.1. Inuence of temperature To study the inuence of temperature on the esterication reaction, three temperatures were studied. The lower temperature studied was 65C. Lower temperatures would result in poor conversion levels because of the relatively low enzyme activity, as indicated in the specications of the commercial lipase. Palmitic acid melting point was also a determinant value (63.1C at atmospheric pressure). The upper temperature limit was xed at 75C to avoid lipase denaturation. Both facts have been also observed in our laboratory in earlier works [12]. Fig. 1 is a plot of conversion versus time for three different temperatures. As expected, conversion was found to increase with increasing temperature. After 120 min, an 8.3% conversion increase was observed when temperature was increased from 65C to 70C. A temperature increase from 70C to 75C led to a conversion increase of 14%. 3.2. Inuence of catalyst concentration As shown in Fig. 2, conversion increased with increasing catalyst concentration. An average increase in conversion of

Fig. 3. Change in conversion with catalyst concentration at different reaction times (T: 70C, acid/alcohol molar ratio: 1/1).

T. Garcia et al. / Enzyme and Microbial Technology 25 (1999) 584 590

587

Fig. 4. Plot of reaction rate versus oleyl alcohol concentration: inuence of acid/alcohol molar ratio (5% catalyst, T: 65C).

Fig. 5. Acid conversion versus time: inuence of an excess of isopropyl palmitate (5% catalyst, T: 70C, acid/alcohol molar ratio: 1/1).

3.5. Initial rate analysis iments with an excess in acid. As observed in this plot, for alcohol concentrations larger than 0.4 mol/l, the reaction rate increases with increasing isopropyl alcohol concentration for any value of acid concentration. However, below 0.4 mol/l, when the alcohol concentration decreases, there is a strong decrease in reaction rate for large molar ratios (1/7 and 1/10). This behavior indicates the presence of inhibition effects at long reaction times. Therefore, it will be necessary to include an inhibition factor in the reaction rate expression due to the concentration of palmitic acid in the reaction mixture. A similar situation was observed when operating in an excess of isopropyl alcohol. For acid concentrations below 0.4 mol/l, the decrease in the reaction rate was larger than expected for some acid/alcohol molar ratios. This effect indicates the existence of inhibition due to the presence of isopropyl alcohol. 3.4. Study of the concentration of products in the reaction media To determine the inuence of the products on the esterication reaction, two additional groups of experiments were carried out, a series operating in excess of isopropyl palmitate and a series operating in excess of water. To compare the effect produced by the presence of each of the two products in the reaction medium, these experiments were compared with experiments in the same operating conditions but without initial addition of products. As shown in Fig. 5, a strong negative effect was observed by the presence of an excess of ester because conversion decreased drastically when the product was added. A similar effect was observed for experiments in which water was added at the beginning of the reaction. Although this behavior would be expected for the chemical equilibrium of the species, the drastic decrease observed indicates the possibility of inhibition effects due to the concentration of both species. The maximum initial rate of substrates consumption (rAc)max and MichaelisMenten constants (Km) were determined by linear regression using the LineweaverBurk approach. Fig. 6a and b shows these plots at different Ac/Al molar ratios. Both slopes and intercepts then were plotted versus the inverse of the specie in excess concentration. This way, we can obtain the above constants. These values were given as initial values in the subsequent kinetic modeling.

Fig. 6. LineweaverBurk plots. (A) Excess of isopropyl alcohol. (B) Excess of palmitic acid (5% catalyst, T: 70C).

588

T. Garcia et al. / Enzyme and Microbial Technology 25 (1999) 584 590

ena. Being the most frequent inhibition type to be considered, the so-called competitive inhibition, where competition for the formation of a free lipase complex, or the formation of ternary complexes with the most active complex, enzyme-acid appear. Thus, the general mechanism for this kind of reactions would consist of two groups of steps. Main steps E Ac 7 E Ac E Ac Al 7 E AcAl E AcAl 7 E EsW E EsW 7 E W E s
Fig. 7. General mechanisms for the enzymatic esterication.

K mAc K iAc K mAl K iAl

f r K eqr Ac max r Ac max

K mEs K iEs K mW K iW K iAl

EW 7 E W Inhibition steps: E Al 7 E Al

3.6. Kinetic modeling The esterication of palmitic acid with isopropyl alcohol can be considered in this research as an enzymatic reaction system, which involves two reactants and two products. The reaction mechanism was studied on this basis. Three possibilities were taken into account: (1) a random-order mechanism, (2) a ping-pong mechanism, and (3) an ordered bi-bi mechanism. Fig. 7 shows the general reaction schemes for the three possibilities. According to the studies of some researchers [14], the active conformation of this type of lipase comes via an acyl-enzyme intermediate. That is the reason because the reaction sequence must be started with the bond formation between the carboxyl group of fatty acid. Then, random mechanism can be considered as neglected. On the other hand, several authors [15,16] considered the ping-pong mechanism as the most appropriate to explain lipase catalysis becoming very common to describe by this mean the interesterication and transesterication reactions. However, in the case of a simple esterication reaction, it seems much more adequate to consider an ordered bi-bi mechanism, in which two reactants and two products are involved. The fatty acid would be the rst reactant involved in the reaction mechanism producing the active enzymeacid complex; in a next step, the alcohol would be incorporated to form the ternary complex alcohol-enzyme-acid, followed by the chemical reaction step, i.e. the transformation of the ternary complex into an ester-enzyme-water complex. In the next step, an ester molecule would be released, and nally water would be released. The above constitute the simple reaction mechanism. However, it is necessary to add inhibition steps corresponding to formation of binary and ternary complexes. The most frequent inhibition phenomena in this type of reactions involve the formation of either (1) binary complexes between the free enzyme and the alcohol or the ester or (2) inactive ternary complexes between the fatty acid or the ester and the active enzyme-acid complex. Therefore, both reactants and products are involved in inhibition phenom-

E Ac Ac 7 E AcAcT K iAc By taking into account the steps involved in the above reaction scheme, a general kinetic equation for the esterication reaction can be proposed: r Ac

f r rAc max rAc max CAc CAl r rAc max Ki AcKm Al 1

CAl CAc r rAc max Km AlCAc 1 KiAl KiAc C Al K iAl

r r Ac max K mAcC Al 1 f r Ac max K mWC Es

CEs CW Keq

K eq

f r Ac max K mEsC W

K eq
f r Ac max K mWC AcC Es

f r Ac max C AcC Al

K eqK iAc

r r r Ac max C EsC W r Ac max)K mAcC AlC W K eq K iW r f (r Ac) max C AcC AlC Es r Ac) max C AlC EsC W K iEs K iAlK eq

(1)

For a 1/1 molar ratio acid/alcohol, C Ac0 C Al0 X Ac0 X Al0 C Ac0 X Ac0 C Ac0 X Al0 C Ac C Al C Ac0 1 X Ac C Es C w C Ac0 X Ac And boundary conditions, (2)

T. Garcia et al. / Enzyme and Microbial Technology 25 (1999) 584 590

589

t 0 C Ac C Ac0 X Ac 0 t t C Ac C Ac X Ac C Ac0 C Ac / C Ac0

(3)

Table 1 Kinetic parameters obtained for the enzymatic esterication of palmitic acid and isopropyl alcohol Parameter (rAc)max (l/mol min) K mAc (mol/l) K mAl (mol/l) K iAc (mol/l) K iAl (mol/l) K iAc (mol/l) K iAl (mol/l) K eq K mEs (mol/l) K mW (mol/l) K iEs (mol/l) K iW (mol/l) 65C 0.1251 0.3411 0.8282 0.0653 0.0022 0.0053 0.6479 2.1584 3.744 104 0.0288 2.524 104 0.1268 70C 0.1750 0.2048 0.6481 0.0526 3.96 104 0.0041 0.5242 3.3011 2.436 104 0.0168 1.388 104 0.0768 75C 0.2042 0.1344 0.5244 0.0432 2.65 104 0.0028 0.3708 6.3561 3.644 105 0.0047 7.682 105 0.0404

By integrating the general equation, the following expression is obtained: t . Ln X Ac K eq 1 K eq K eq 1 K eq 1 K eq 1 X Ac K eq 1 K eq

. Ln X 2 Ac K eq 1 2 K eq X Ac K eq . Ln K eq 1 . LnK eq . X 2 Ac . X Ac (4)

where greek symbols are the coefcients dened by mathematical relations which involve the kinetic constants. The entire development of kinetic model can be seen in a recent work [17]. The general owchart used to obtain the kinetic

parameters and to optimize the model is shown in Fig. 8. Multiple linear regression and nonlinear regression methods based on Marquardts algorithm were applied for the estimation of parameters and are given in Table 1 for the three temperatures tested. These parameters dene the complete kinetic model for the esterication reaction. As observed in Table 1, the values of all the parameters decrease with increasing temperature, except for the equilibrium constant. This fact indicates that high temperatures lead to a decrease in the formation of inhibition complexes and to larger ester conversions. To know the inuence of temperature on the parameters, the standard free energy of reaction as a function of the equilibrium constant and the enthalpy and entropy change were considered. This makes possible the estimatation of thermodynamic parameters from experimental data of temperature and equilibrium constant. The effect produced by the variation of temperature on the equilibrium constant is given by Vant Hoffs equation, which allows the estimation of enthalpy changes. The activation energy can be estimated from Arrhenius equation. In Table 2, the values obtained for activation energy, pre-exponential factor, and enthalpy and entropy changes are given. The thermodynamic parameters obtained from Michaelis constants and inhibition constants are summarized in Table 3. To obtain the preexponential factor and activation energy, the Arrhenius equation has been employed from the kinetic constant values for different temperatures. Enthalpy variations have been obtained from the MichaelisMenten and inhibition constants applying the Vant Hoff equation and the entropy variations from the second law of the thermodinamics.
Table 2 Thermodynamic parameters, pre-exponential factor and activation energy H 0 (Kcal/mol) S 0 (cal/mol K) k 0 (l/mol/gcat/min) E a (Kcal/mol) 25.22 0.51 76.06 1.5 1.469 105 2.9 103 11.66 0.23

Fig. 8. Flowchart of kinetic study.

590

T. Garcia et al. / Enzyme and Microbial Technology 25 (1999) 584 590

Table 3 Thermodynamic parameters for MichaelisMenten and inhibition constants Parameter K mAc K mAl K mEs K mW K iAc K iAl K iEs K iW K iAc K iAl -H 0 (Kcal/mol) 21.66 9.89 54.62 52.28 10.80 42.89 24.69 26.75 14.35 13.03 S 0 (cal/mol K) 57.30 25.21 138.97 111.92 22.83 107.42 82.88 28.04 28.04 35.23

describe the kinetics of the esterication of palmitic acid and isopropyl alcohol over an immobilized lipase. References
[1] Gandhi NN. Applications of lipase. J Am Oil Chem Soc 1997;74: 62134. [2] Malcata FX, Reyes HR, Garcia HS, Hill CG, Amundson CH. Kinetics and mechanisms of reaction catalysed by immobilised lipases. Enzyme Microb Technol 1992;14:426 46. [3] Halling PJ. Thermodynamic predictions for biocatalysis in nonconventional media: theory, test and recommendations for experimental design and analysis. Enzyme Microb Technol 1994;16:178 206. [4] Khmelnitsky YL, Neverova IN, Polyakov VI, Grinberg VY, Levashov AV, Martinek K. Kinetic theory of enzymatic reactions in reversed micellar systems. Application of the pseudophase approach for partitioning substrates. Eur J Biochem 1990;190:15559. [5] Shiraishi F. Experimental evaluation of the usefulness of equations describing the apparent maximum reaction rate and apparent Michaelis constant of an immobilized enzyme reaction. Enzyme Microb Technol 1993;15:150 4. [6] Chulalaksananukul W, Condoret JS, Combes D. Geranyl acetate synthesis by lipase-catalyzed transesterication in supercritical carbon dioxide. Enzyme Microb Technol 1993;15:691 8. [7] Stamatis H, Xenakis A, Menge U, Kolisis FN. Estericacion reactions catalyzed by lipases in microemulsions: The role of enzyme localization in relation to its selectivity. Biotechnol Bioeng 1993;42: 10310. [8] Guit RPM, Kloosterman M, Meindersma GW, Mayer M, Meijer EM. Lipase kinetics: hydrolysis of triacetin by lipase from Candida cylindracea in a hollow-ber membrane reactor. Biotechnol Bioeng 1991; 38:72732. [9] Lortie R, Trani M, Ergan F. Kinetic study of the lipase-catalyzed synthesis of triolein. Biotechnol Bioeng 1993;41:1021 6. [10] Garcia T, Coteron A, Martinez M, Aracil J. Optimization of the enzymatic synthesis of isobutyl palmitate using a central composite design. Trans Chem 1995;73:140 4. [11] Garc a T, Mart nez M, Aracil J. Enzymatic synthesis of an analogue of jojoba oil: optimisation by statistical analysis. Enzyme Microb Technol 1993;15:60710. [12] Garcia D, Garcia T, Martinez M, Aracil J. Enzymatic synthesis of esters of high molecular weigh: optimisation of the synthesis of a sperm whale analogue. Ind Crops Prod 1995;4:10511. [13] Garc a T, Mart nez M, Aracil J. Kinetics of the enzymatic synthesis of an analogue of jojoba oil. Trans Chem 1993;71:4751. [14] Brzozowski AM, Derewenda U, Derewenda ZS, Godson GG, Lawson DM, Turkenburg JP, Bjorkling F, HugeJensen B, Patkar SA, Thim L. A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex. Nature (London) 1991;351: 491 4. [15] Tsai SW, Chiang CL. Kinetics, mechanism and time course analysis of lipase-catalysed hydrolysis of high concentration olive oil in aotisooctane reversed micelles. Biotech Bioeng 1991;38:206 11. [16] Chulalaksananukul W, Condoret JS, Combes D. Kinetics of geranyl acetate synthesis by lipase-catalyzed transesterication in n-hexane. Enzyme Microb Technol 1992;14:293 8. [17] Garcia T, Coteron A, Martinez M, Aracil J. Kinetic of esterication reactions catalysed by immobilized lipases. Chem Eng Sci 1996;51: 2841 6.

4. Conclusions The enzymatic synthesis of isopropyl palmitate can be well described by a bi-bi ordered kinetic model. The estimated rate constants show an Arrhenius dependence on temperature. The activation energy calculated for the overall reaction is 11.66 Kcal/mol. The model developed in the present work was used to simulate the esterication process and to evaluate the temperature, enzyme concentration, and molar ratio effect on the reaction. Fig. 9 shows one of the simulation curves from the kinetic model and the corresponding experimental results. The quality of t was estimated by residual analysis. In most cases, the residuals were lower than 15%, and a random error distribution was obtained, which indicated a good t between the models and the experimental results. The average error for all the experiments was 8.55%. These results indicate that the models developed are adequate to

Fig. 9. Simulation of the esterication reaction for isopropyl palmitate (P: 101325 Pa, 5% catalyst, T: 70C, acid/alcohol molar ratio: 1/1).