Kinetics and mechanism of esterification of isoamyl alcohol with acetic acid by immobilized lipase

Transcription

1 Indian Journal of Chemical Technology Vol. 16, May 2009, pp Kinetics and mechanism of esterification of isoamyl alcohol with acetic acid by immobilized lipase Sumbita Gogoi & N N Dutta* Chemical Engineering Division, North East Institute of Science and Technology, CSIR Jorhat , India sumbita_gogoi@yahoo.co.in Received 2 May 2008; revised 23 February 2009 In this study, the effects of the rate enhancement of isoamyl acetate formation by immobilized Mucor miehei lipase catalysed esterification of isoamyl alcohol with acetic acid were investigated. The esterification reaction was studied in ten different solvents and n-heptane was found to be most suitable. The rate of the reaction was affected by the addition of molecular sieves (3Å) and temperature. The effects of different reaction parameters such as lipase and substrate concentrations were studied. The activity of immobilized lipase was found to be maximum at lipase concentration of 12 mg/ml and at a reaction temperature of 55ºC; retained about one third of its initial activity up to seven reaction cycles after repeated use. The kinetics of the reaction can be described by Ping-Pong Bi-Bi mechanism with acid inhibition. The parameter values were estimated by non-linear regression analysis. Keywords: Immobilized, Isoamyl acetate, Lipase, Esterification Isoamyl acetate is useful in food, beverage, cosmetic and pharmaceutical industries. It has a strong banana flavour and therefore widely used in food industries and as flavouring agents. Due to several advantages of using enzyme catalysis in organic solvents, the synthesis of isoamyl acetate and other esters of isoamyl alcohol have been studied by exploiting the catalytic activities of lipases isolated from various microbial sources, such as Mucor miehei and Rhizomucor miehei lipase 1,2. The enzymatic production of flavour esters using lipases (triacylglycerol acylhydrolases, EC ) is gaining importance industrially due to the regio-, stereo- and substrate specificity s, the degree of purity of these products and they can be labeled as natural products. Lipases have been employed for direct esterification and transesterification reactions in organic solvents to produce esters of fatty acids 3,4, glycerol 5 and terpene alcohols 6. The effect of solvent is highly dependent on the nature of the enzyme and the solvent. However, it appears that there is no difference in dependence of solvent properties with enzyme activity of lipolytic and nonlipolytic enzymes 7, thus indicating the generality of enzymatic catalysis in organic solvents. The enzymes do not dissolve in most of the commonly used organic solvents and hence catalysis takes place in dispersed media, for which the efficiency depends on the amount of enzyme. Enzyme catalysis in nonaqueous media is not incompatible entities as demonstrated by catalytic activity for enzymes when dispersed in organic solvents, but substantial differences have been observed between reaction rates, maximal velocity and substrate affinity determined in water and in various organic solvents. Enzymes used in organic media require some water to achieve good catalytic activity 8. There is optimum water content which maximizes the reaction rate in organic media. Above an optimal value of water content, the enzyme activity usually decreases. The enzymatic synthesis of isoamyl acetate has been mostly carried out using acetic acid as acyl donor 9. However, high concentrations of acid lower the esterification rate due to strong acid inhibition on enzyme activity 2. Acetic acid may be an inhibitor of lipase activity causing dead-end inhibition 10. It is known that in situ water removal is a useful strategy to enhance the equilibrium of esterification reaction. The main objective of this investigation, therefore, is to examine the immobilized lipase catalyzed synthesis of isoamyl acetate carried out in an organic medium and exploiting the water removal with molecular sieve. In this paper, a comprehensive study of the kinetics of esterification of acetic acid

2 210 INDIAN J. CHEM. TECHNOL., MAY 2009 with isoamyl alcohol using immobilized M. miehei lipase has been reported. Experimental Procedure Materials Immobilized lipase from Mucor miehei (immobilized in Sol-Gel-AK) was procured from Fluka Chemicals Co, Switzerland. 1 U is the amount of immobilized enzyme which forms 1% octyl laurate from 0.05 mol lauric acid and 0.1 mol 1-octanol in 10 ml water saturated isooctane in 1 h at 20ºC. Isoamyl alcohol, acetic acid, isoamyl acetate and solvents were obtained from E-Merck (India) Limited, Mumbai. Molecular sieve 3 Å (1.5 mm diameter cylindrical pellets) was purchased from CDH, New Delhi. Methods Experiments on solvent effect were carried out under optimized reaction conditions with mol dm -3 isoamyl alcohol and mol dm -3 acetic acid dissolved in 10 ml of anhydrous solvent in presence of 10 mg ml -1 immobilized lipase from M. miehei. The reactions were carried out in a 50 ml round bottom flask by mixing the reaction substrates at a rotating speed of 150 rpm with a magnetic stirrer at 27ºC. All the kinetic experiments were carried out using n-heptane as the solvent. The effect of molecular sieve (3 Å) was studied by systematic variation of its amount in the reaction mixture. The temperature, lipase and substrate concentrations were varied systematically. The stability of the immobilized lipase was examined by reusing the recovered lipase for repeated reaction cycles. The reaction mixture consisted of mol dm -3 acetic acid and mol dm -3 isoamyl alcohol dissolved in 10 ml n-heptane in presence of 12 mg ml -1 immobilized lipase. Each reaction was performed for 8 h at 55ºC with constant stirring of the immobilized lipase. After each cycle, the enzyme was filtered and washed with solvent and allowed to dry before reuse. Aliquots of samples were withdrawn at 30 min time interval and analyzed by Gas Chromatography (GC). The concentration of isoamyl acetate was determined by GC (CHEMITO Model 8510 FID) equipped with GC data processor. A fused silica wide bore capillary column, Nukol (15 m x 0.53 mm i.d., 1.0 µm film thickness) with hydrogen flow rate of 7 ml min -1 was used for separation of the substrates and the reaction product. The column oven temperature was programmed from 50ºC (0.5 min) to 80ºC at a rate of 7ºC min -1 and then up to 135ºC at a rate of 11ºC min -1. The injector and detector port temperatures were maintained at 200ºC and 230ºC respecttively. Initial reaction rates were calculated from conversion versus time profiles corresponding to first 10% conversion, where the profiles were found to be linear. The water concentration was determined in a Karl-Fischer Titrator (Spectralab MA-101, Alfa Instruments, New Delhi). All the experiments were conducted in triplicate and reproducibility was found to be ±5% and all data points represent average values. Results and Discussion Solvent effect on initial rate The effect of solvents deals with solvent hydrophobicity (log P) as the activity of enzyme is largely affected by the polarity of organic solvents employed. The log P of the organic solvent greatly influenced the activity and substrate specificity of lipase. It is reported that solvents with higher log P produce better esterification rate 4. The solvents used for this study were selected on the basis of their log P values. They were ethyl ether (0.85), dichloromethane (1.5), benzene (2.0), chloroform (2.0), toluene (2.5), carbon tetrachloride (3.0), cyclohexane (3.2), n-hexane (3.5), n-heptane (4.0) and dioxane (-1.1). The rates of the reaction and conversions were maximum in n-heptane. Effect of stirring speed In case of immobilized catalysts, the reactants have to diffuse from the bulk liquid to the external surface of the particle and from there into the interior pores of the catalyst. So, in the micro aqueous system where the solid enzyme bound is in suspension, the quality of the suspension and the partition of water around the enzyme particles are expected to be dependent on shaking. It was therefore, necessary to study the kinetics of the reaction in the absence of both the external and internal mass transfer limitations. External and internal diffusion limitations can be minimized by carrying out the reaction at an optimum stirring speed and low enzyme loading of optimum particle size. The effect of stirring speed on the rate of the reaction was studied between rpm. The initial rate increases with the increase of the stirring speed varied up to 250 rpm reaching a plateau beyond this speed implying that the initial rates of reaction

3 GOGOI & DUTTA: KINETICS AND MECHANISM OF ESTERIFICATION OF ISOAMYL ALCOHOL WITH ACETIC ACID 211 were no longer limited by the mass-transfer limitation of immobilized enzyme above 250 rpm. This is true for reactions carried out in presence of molecular sieve 3Å probably due to enhanced rate of adsorption at higher speed through elimination of external mass transfer limitation if any. Therefore, all further reactions were carried out at 250 rpm shaking speed. Effect of molecular sieve as dehydrating medium A few esterification reactions have been reported with respect to control of water content or thermodynamic water activity during the process of the reaction by applying various methods To remove the water produced during the esterification of isoamyl alcohol, the reaction was carried out by adding molecular sieve (3 Å). The solvent and both the substrates were dried prior to use. The conversion versus time profiles of the esterification of isoamyl alcohol was obtained in the presence of molecular sieve (Fig. 1). Molecular sieve (3 Å) having water loading capacity of g/g was added to capture the water formed in situ 15. From the figure, it is seen that with the increase of the amount of molecular sieve, the rate of the reaction was increased. The increasing amount of molecular sieve apparently reduces the water content at a rate of its adsorption, which is higher than the rate of its formation and thereby increasing the rate of the reaction. After 6 h reaction with 6% (w/v) molecular sieve, 65% conversion of isoamyl alcohol to isoamyl acetate was observed whereas, only 27% conversion was obtained without any addition. The water content resulting from the reaction with addition of 6% (w/v) molecular sieve was analyzed by Karl Fischer Titrator and the same was found to be equivalent to that predicted by reaction stoichiometry. It may be noted that water formed as per reaction stoichiometry is around g, which is nearly within the total water loading capacity of the molecular sieve used in the experiments. Effect of lipase concentration The effect of lipase concentration was studied at equimolar concentration of acetic acid and isoamyl alcohol (0.025 mol dm -3 ) while varying the lipase concentration from 4 to 12 mg ml -1 (Fig. 2). It is apparent that with an increase of enzyme concentration, initial rate increased almost linearly in the low range of enzyme concentration. Then the rate increases slowly reaching a critical value at lipase concentration of 10 mg ml -1 and remained unchanged thereafter. Such an observation may be attributed to the fact that the reaction is kinetically controlled at low enzyme concentration range. It is interesting to note that the immobilized lipase gives higher reaction rate than that of free lipase, an observation identical to that reported for other lipase catalyzed reactions 16. The high activity of immobilized lipase may be attributed to hyperactivation of lipase upon contact with the adsorbed media 17. It is also due to the fact Fig. 1 Effect of different amount of molecular sieve on time versus conversion profile. (- -) : 0.6g, (- -) : 0.4g, (- -) : 0.3g, (- -) : 0.2 g. Reaction conditions: [Acetic acid] = [Isoamyl alcohol] = mol dm -3, lipase = 5 mg ml -1, temperature = 27ºC, stirring speed = 250 rpm Fig. 2 Effect of lipase concentration on time versus conversion profile. Reaction conditions: [Acetic acid] = [Isoamyl alcohol] = mol dm -3, temperature = 27ºC, molecular sieve = 0.6 g, Stirring speed = 250 rpm

4 212 INDIAN J. CHEM. TECHNOL., MAY 2009 that during immobilization from crude lipase preparation, only the purified form of the lipase is adsorbed, an observation identical to that obtained for Candida cylindracea lipase immobilized on nylon support used for the esterification and hydrolysis reactions on membrane surfaces 17,18. The present observation is also identical to that reported by Hazarika et al. 19 for transesterification reaction catalysed by Pseudomonas cepacia lipase immobilized on ceramic particles. Effect of temperature Temperature has a significant effect on the equilibrium of the reaction, and on the activity and stability of lipase. The effect of temperature was studied in the range of 25-65ºC (Fig. 3). From Fig. 3 it may be inferred that with an increase in reaction temperature from 25 to 65ºC, the initial rate increased linearly indicating a good thermostability of the immobilized lipase. However, in case of free lipase initial rate increased exponentially reaching a critical value of temperature at 55ºC and remains unchanged thereafter. Similar observation was also reported by Chowdery et al. 20 for isoamyl isovalerate synthesis catalysed by immobilized Rhizomucor miehei lipase. It is apparent that the catalytic activity of the free lipase is less than that of immobilized lipase at all the temperatures studied. The effect of temperature on the reaction rate constant (k), for this reaction was also studied. The activation energy (E a ) value was found to be 15.8 k cals mol -1. In general, the activation energy for enzyme catalyzed reactions falls within the range of 5-15 k cals mol -1, which also indicate that the reaction is kinetically controlled 21. This value of activation energy is comparable to those reported for esterification of tetrahydrofurfuryl alcohol with butyric acid and transesterification with ethyl butyrate catalysed by Candida antarctica lipase immobilized on macroporous polyacrylate resin which are reported to be 11.3 and k cals mol -1 respectively 22. The catalytic activity (%) of free and immobilized lipase with temperature was studied in this reaction system. It is evident that the free lipase was found to be inactivated after 55ºC while that of immobilized was found after 85ºC. Hence, the immobilization improved the catalytic activity as well as sensitivity to temperature. This may also be attributed to a low restriction in the diffusion of the substrate and products at higher reaction temperature. Effect of substrate concentration The effect of substrate concentration on the time course of conversion was studied by varying the substrate concentration ratios (Fig. 4). From Fig. 4, it was observed that on increasing the concentration of acetic acid at a fixed isoamyl alcohol concentration, the % conversion increased till the molar ratio of molar ratio of isoamyl alcohol to acetic acid was 1:2, thereafter an increase of isoamyl alcohol to acetic acid molar ratio caused a decrease in % conversion. The variation of the initial rate as a function of acetic acid Fig. 3 Effect of temperature on initial rate. Reaction conditions: [Acetic acid] = [Isoamyl alcohol] = mol dm -3, lipase = 12 mg ml -1, molecular sieve = 0.6 g, stirring speed = 250 rpm Fig. 4 Effect of substrate concentration ratios on time versus conversion profile. Reaction conditions: Lipase = 12 mg ml -1, [Isoamyl alcohol] = mol dm -3, [Acetic acid] : (- -) = mol dm -3, (- -) = mol dm -3, (- -) = mol dm -3 (- -) = mol dm -3

5 GOGOI & DUTTA: KINETICS AND MECHANISM OF ESTERIFICATION OF ISOAMYL ALCOHOL WITH ACETIC ACID 213 concentration is plotted in Fig. 5. The initial rate increased proportionally to a maximum, above which it decreased for all isoamyl alcohol concentrations tested. Reuse of immobilized lipase The activity of the immobilized lipase was retained up to seven reaction cycles. It was observed that up to seven repeated reaction cycles percentage conversion was found to be 85% implying higher stability of immobilized lipase 23. The higher stability and activity of the lipase may be due to the favourable interactions of the molecule of lipase with the support material. However, at higher concentration of acid, the activity of the lipase decreased rapidly after first cycle of reaction due to inactivation of the lipase by the acid forming dead-end inhibition complex. Reaction mechanism and kinetics For detailed kinetics study, n-heptane was used as the solvent. The reaction mechanism was examined using Lineweaver-Burk plot (Fig. 6) which was a reciprocal plot of initial rate and isoamyl alcohol concentration at fixed acetic acid concentrations. It is apparent that with an increase of alcohol concentration initial rate increases at a constant acid concentration. On the other hand, with an increase of acid concentration the reaction rate decreases which reflects the acid inhibition effect. Similar acid inhibition effect was observed in the isoamyl butyrate synthesis by immobilized lipase from Rhizomucor miehei 24. From the Lineweaver-Burk plot, it was observed that the lines were parallel at low concentration of acetic acid, whereas at high acetic acid concentration, the slopes of the lines increased. These results agree with the assumed Ping-Pong Bi-Bi mechanism with acid inhibition. In this mechanism, only one substrate is bound to the enzyme at any time to form the substrate-enzyme complex. As soon as one product is formed and released, the other substrate binds to the modified enzyme (substituted form of the enzyme) to form the second product 10. The reaction sequence may be given as follows k E+ A EA k2 k 1 k 1 EA E + P k3 E + B E B k4 k 4 k 3 E B E+ Q where A is the acetic acid, E the enzyme, EA the enzyme acetic acid complex, Eʹ the enzyme-acyl complex, B isoamyl alcohol, EʹB the binary complex of acyl enzyme and isoamyl alcohol, P water and Q is isoamyl acetate. According to this mechanism, the initial rate equation is as follows = V + + max v 1 Km(B) /[B](1+[A]/ Ki ) Km(A) /[A] (1) Fig. 5 Effect of initial rate on acetic acid concentration at various concentration of isoamyl alcohol Fig. 6 Reciprocal plot of isoamyl alcohol concentration and initial rate at various concentrations of acetic acid. Reaction conditions: [Acetic acid] (- -): mol dm -3, (- -): mol dm -3, (- -) : mol dm -3

6 214 INDIAN J. CHEM. TECHNOL., MAY 2009 Table 1 Estimated values of kinetic parameters Lipase V max K m(alcohol) K m(acid) K i (Acid) (mol/ min.g) (mol dm -3 ) (mol dm -3 ) (mol dm -3 ) Immobilized lipase Free lipase Table 2 Kinetic parameter for lipase catalyzed esterification of short-chain acids Reaction product Lipase Solvent V max K m(acid) K m(alcohol) K i(acid) Ref (mol/min.g) (mol dm -3 ) (mol dm -3 ) (mol dm -3 ) Isoamyl acetate Mucor miehei a n-heptane This work Butyl Isobutyrate Candida antarctica a n-heptane [22] Isoamyl butyrate Rhizomucor miehei a n-hexane [24] Tetrahydro furfuryl butyrate Candida antarctica a n-heptane [23] a Immobilized where v is the initial rate, V max is the maximum reaction rate, [A] and [B] are the initial concentrations of acetic acid and isoamyl alcohol, K m(a) and K m(b) are the respective affinity constants and K i is the inhibition constant for the acid. The kinetic parameter values were estimated by non-linear regression analysis of the model for both immobilized and free lipase (Table 1). These values of kinetic parameters estimated for immobilized lipase are comparable to those reported in literature for the synthesis of short-chain acid esters (Table 2). It is apparent from the values of the kinetic parameters, the enzyme retained its activity after immobilization than the free lipase. In general, due to mass transfer limitation and interfacial deactivation, some enzyme exhibit low activity after immobilization 25. The present system can be considered to be free from such effects. From the classical theory of internal diffusion effect in heterogeneous catalysts 26 and by considering two well-known parameters, (i) catalytic effectiveness factor (η) and (ii) Thiele modulus (φ), the internal diffusion effect has been semi-quantitatively analyzed, which could be defined by the following equation D e Effectiveness factor, η = 3 rkmρp A g and ρp AgV max Thiele modulus, φ = r D [S] e (2) (3) where D e is the effective diffusivity of the substrate (cm 2 s -1 ), A g is surface area of the enzyme particle (cm 2 ), r is radius of the particle (cm), ρ p is bulk density of the particle (g cm -3 ), [S] is concentration of acid and K m is apparent rate constant (Table 1). According to Weisz criterion 27, if (i) φ < 0.3, η 1, then the internal mass transfer limitations are insignificant and if (ii) φ > 3, η < 1, then internal mass transfer limitations are significant. In the present case, the value of effective diffusivity (D e ) was calculated from the well-known Wilke-Chang correlation 28. The values of r, A g and ρ were taken from Amano catalogue and V max was taken from Table 2. The numerical values of η and φ were found to be and 0.23 respectively, indicating that there was no significant diffusional limitation. The absence of intra-particle diffusion resistance was also confirmed by invoking the Weisz-Prater criterion, C WP 29. According to Weisz-Prater criterion C WP = -v obs ρ p r 2 / {D e [S]}, where v obs is the initial rate in mmol/min g. If (i) C WP >> 1, then the reaction is limited by severe internal diffusion resistance and if (ii) C WP << 1, then the internal diffusion effect is suggested to be absent and the reaction is intrinsically kinetically controlled. By substituting the appropriate values, C WP was found to be equal to 0.393, indicating that there was no internal diffusion resistance and the reaction was intrinsically kinetically controlled. Conclusion The esterification of acetic acid with isoamyl alcohol catalyzed by immobilized lipase from M. miehei was found to have profound effect on the reaction parameters such as lipase and substrate concentrations and temperature. In situ removal of

7 GOGOI & DUTTA: KINETICS AND MECHANISM OF ESTERIFICATION OF ISOAMYL ALCOHOL WITH ACETIC ACID 215 water from the reaction system by use of molecular sieve (3Å) adsorption exhibits conversion about 15% higher than that observed without its use under otherwise identical experimental conditions due to equilibrium shift of the reaction. The reaction was found to conform to the Ping-Pong Bi-Bi mechanism with acid inhibition. The immobilized lipase exhibits higher activity than free lipase, may also be due to adsorption of only pure part of lipase. The activity was maximum at 65ºC temperature. The present system was found to exhibit no profound effect of any pore-diffusional limitations. References 1 Harikrishna S, Manohar B, Divakar S, Prapulla S G & Karanth N G, Enzyme Microb Technol, 26 (2000) Harikrishna S, Divakar S, Prapulla S G & Karanth N G, J Biotechnol, 87 (2001) Hazarika S, Goswami P, Dutta N N & Hazarika A K, Chem Eng J, 85 (2002) Gogoi S, Hazarika S, Rao P G & Dutta N N, Biocatal Biotransform, 24 (2006) Hazarika S, Goswami P & Dutta N N, Chem Eng J, 94 (2003) 1. 6 Claon P A & Akoh C C, Enzyme Microb Technol, 16 (1994) Van Tol J B A, Stevens R M M, Veldhuizen W J, Jongejan J A & Duine J A, Biotechnol Bioeng, 47 (1995) Zaks A & Klibanov A M, J Biol Chem, 263 (1988) Welsh F W, Williams R E & Dawson K H, J Food Sci, 55 (1990) Segal I H, Enzyme Kinetics (John Wiley and Sons, New York), Wehtje E, Kaur J, Adlercreutz P, Chand S & Mattiasson B, Enzyme Microb Technol, 21 (1997) Chamouleau F, Coulon D, Girardin M & Ghoul M, J Mol Catal B: Enzym, 11 (2001) Song Q X & Wei D Z, J Mol Catal B: Enzym, 18 (2002) Ducret A, Giroux A, Trani M & Lortie R, Biotechnol Bioeng, 48 (1995) Meier W M, Molecular Sieves (American Chemical Society, Washington), Fadiloglu S & Soylemez Z, J Agric Food Chem, 46 (1998) Carta G, Gainer J L & Benton A H, Biotechnol Bioeng, 37 (1991) Malcata F X, Garcia H S, Hill C G & Amundson C H, Biotechnol Bioeng, (1992) Hazarika S & Dutta N N, Org Proc Res Dev, 8 (2004) Chowdary G V, Ramesh M N & Prapulla S G, Process Biochem, 36 (2000) Yadav G D & Devi K M, Biochem Eng J, 10 (2002) Yadav G D & Devi K M, Chem Eng Sci, 59 (2004) Yadav G D & Trivedi A H, Enzyme Microb Technol, 32 (2003) Harikrishna S & Karanth N G, Biochim Biophys Acta, 1547 (2001) Lye G J, Pavlou O P, Rosjidi M & Stucky D C, Biotechnol Bioeng, 51 (1996) Furusaki S, J Chem Eng Jpn, 18 (1988) Weisz P B, Chem Eng Prog Symp, 25 (1959) Wilke C R & Chang P, AICHE J, 24 (1955) Fogler H S, Elements of Chemical Reaction Engineering (Oxford University Press, Oxford), 1995.