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Synergism between microwave irradiation and enzyme catalysis in transesterification of ethyl-3-phenylpropanoate with n-butanol
Bioresource Technology 109 (2012) 1–6
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Synergism between microwave irradiation and enzyme catalysis in transesterification of ethyl-3-phenylpropanoate with n-butanol Ganapati D. Yadav ⇑, Sandip V. Pawar Department of Chemical Engineering, Institute of Chemical Technology, University of Mumbai,1 Matunga, Mumbai 400 019, India
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Article history: Received 14 December 2011 Received in revised form 8 January 2012 Accepted 9 January 2012 Available online 16 January 2012 Keywords: Synergism Immobilized lipase Microwave Enzyme catalysis Kinetic study
a b s t r a c t Lipase catalyzed transesterification was investigated to study the synergistic effect of microwave irradiation and enzyme catalysis. Transesterification of ethyl-3-phenylpropanoate with n-butanol was chosen as the model reaction using immobilized enzymes such as Novozyme 435, Lipozyme RMIM and Lipozyme TL IM with microwave irradiation. Novozyme 435 was the best catalyst. The effect of various parameters affecting the conversion and initial rates of transesterification were studied to establish kinetics and mechanism. There is synergism between enzyme catalysis and microwave irradiation. The analysis of initial rate data and progress curve data showed that the reaction obeys the Ping-Pong bi–bi mechanism with inhibition by n-butanol. The theoretical predictions and experimental data match very well. These studies were also extended to other alcohols such as 2-phenyl-1-propanol, n-octanol, benzyl alcohol, isoamyl alcohol, 2-hexanol and 2-pentanol under otherwise similar conditions. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Phenolic acids present in plants are hydroxylated derivatives of benzoic and cinnamic acids. Flavonoids and phenolic acids have many functions in plants. They act as cell wall support materials and as colorful attractants for birds and insects helping seed dispersal and pollination (Shahidi and Naczk, 1995). Thus, synthesis of alkyl hydroxycinnamates of natural quality is important and enzymatic routes could be used. Enzyme catalyzed reactions in non-aqueous media have gained a considerable significance (Yadav and Devi, 2001, 2002, 2004a,b; Yadav and Jadhav, 2005; Yu et al., 2007; Sathishkumar et al., 2010). Lipases are considered to be the most important group of biocatalysts for non-aqueous reactions because they possess wide substrate specificity, able to recognize the chiral centers and do not require the labile cofactors to carry out the reactions (Yadav and Sivakumar, 2004; Yadav and Borkar, 2009; Yang et al., 2009). Lipases are readily available from different sources such as bacteria and fungi and so there is a good chance of finding a suitable catalyst for biotransformation of interest in terms of catalytic activity and selectivity (Banoth et al., 2009; Acosta et al., 2011). They have been used to catalyze a variety of reactions in non-aqueous media, viz. esterification, transesterification, amidation, epoxidation, hydrolysis, thio-esterification and trans-thioesterification (Yadav ⇑ Corresponding author. Tel.: +91 22 3361 1001; fax: +91 22 3361 1020. E-mail addresses: [email protected], [email protected] Yadav). 1 Formerly, of University of Mumbai, now a separate university. 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.01.030
(G.D.
and Trivedi, 2003; Yadav and Devi, 2001, 2002, 2004a; Yadav and Lathi, 2003, 2004b; Gao et al., 2010). Hydrocinnamate esters are important intermediates in the synthesis of HIV protease inhibitor and as precursors for the synthesis of 1,3,4,9-tetrahydropyrano [3, 4-b] indole-1-acetic acid which is reported for its use as analgesic, inflammation inhibitor and antipyretic activities (Priya and Chadha, 2003; Cassani et al., 2007). Hydrocinnamates are used in the protection of chymotrypsin from denaturation by urea and ethanol (Priya and Chadha, 2003; Freidberg et al., 1969) and also find applications in flavor and fragrance industry. Esterification of hydrocinnamic acid and transesterification of hydrocinnamate esters have been studied using solvent free reactions, drying agents or in vacuo reactions (Weitkamp et al., 2006, 2008). Microwave irradiation have been accepted as efficient heating source for chemical reactions since the mid-1980s, where reactions that required several hours under conventional heating could often be completed in few minutes with high yields and reaction selectivity. Microwave assisted synthesis is an important and rapidly developing technology in green chemistry, and is environmentally benign, clean, fast and convenient which has been widely used in organic chemistry and enzyme catalyzed reactions (Kappe and Stadler, 2006; Yu et al., 2011; Zhu et al., 2006). Microwave irradiation triggers the effect which cannot be preceded by thermal heating; it leads to direct coupling of molecules by selective absorption of radiation by polar compounds (Young et al., 2008; Collins and Leadbeater, 2007). Microwave irradiation seems to improve thermal stability of the enzyme (Rejasse et al., 2006; Yadav and Borkar, 2006) as a function of enzyme hydration which can be used
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efficiently to improve the process or modify the selectivity (Sontakke and Yadav, 2011). Synergism between microwave irradiation and enzyme catalysis is known to improve reaction rate in lipase catalyzed reactions (Yadav and Lathi, 2004a; Yadav and Sajgure, 2007). The present work encompasses an environment friendly lipase catalyzed transesterification of ethyl-3-phenylpropanoate (ethylhydrocinnamte) with a variety of alcohols under microwave irradiation as a greener approach including deduction of reaction mechanism and kinetic modeling. Synergism between microwave irradiation and enzyme catalysis has been explored in order to optimize the transesterification process. The effects of different parameters including nature of biocatalyst, solvent, agitation speed, reaction temperature, molar ratio of reactants, and type of alcohols were investigated. 2. Methods 2.1. Enzymes The Novozyme 435, Lipozyme RM IM and Lipozyme TL IM were procured as gift samples from Novo Nordisk, Denmark. Novozyme 435 is Candida antarctica lipase immobilized on a macroporous polyacrylic resin beads (bead size 0.3–0.6 mm, bulk density 0.430 g cm3, water content 3%, activity of 7000 PLU g1); Lipozyme RM IM is Mucor miehei immobilized on an ionic resin with acitivity of 5–6 BAUN while Lipozyme TL IM is Thermomyces lanuginosus immobilized on silica. 2.2. Chemicals All chemicals were AR grade, procured from the firms of repute and were used without any further purification: ethyl-3-phenypropanoate (Sigma Aldrich, India), toluene, n-hexane, 1, 4 dioxane, nheptane, n-butanol, n-octanol, iso-amyl alcohol, benzyl alcohol (S. d. Fine Chemicals Pvt. Ltd., Mumbai, India). 2.3. Experimental setup 2.3.1. Conventional heating The experimental set up consisted of a 3 cm i.d. fully baffled mechanically agitated glass reactor of 50 cm3 capacity, which was equipped with four baffles and a six bladed pitched-turbine impeller. The entire reactor assembly was immersed in a thermostatic water bath maintained at the desired temperature with an accuracy of ±1 °C. In a typical experiment, the reaction mixture contained 0.56 mmol of ethyl-3-phenylpropanoate and 1.12 mmol of n-butanol, diluted to 15 ml with toluene as a solvent. It was agitated at 60 °C for 15 min at a speed of 300 rpm and a known quantity of enzyme was then added to initiate the reaction. Samples were withdrawn periodically, filtered to remove fine particles, if any, and analyzed by GC. 2.3.2. Microwave reactor The studies were carried out in a microwave reactor (Discover, CEM-SP 1245 model). The reactor was 120 ml capacity fully baffled, 4.5 cm i.d. cylindrical glass vessel with provision for mechanical stirring. A standard four bladed turbine impeller of 1.5 cm diameter was used for agitation. However, the actual reactor volume exposed to the microwave irradiation was 45 ml with 5.5 cm height. 2.4. Analysis The concentration of reactants and products were determined by GC (Chemito Gas chromatograph Model 8610) equipped with
a flame ionization detector. A 4 m 3.8 mm stainless steel column packed with 5% SE-30 on chromosorb was used for analysis. The product was confirmed by GCMS. Synthetic mixtures were prepared of pure samples and calibration was done to quantify the collected data for conversion and rate of reactions. 3. Results and discussion 3.1. Conventional heating versus microwave irradiation Transesterification of ethyl-3-phenylpropanoate with n-butanol was chosen as the model reaction. Enzymatic transesterification was performed in the presence of controlled microwave irradiation. It was found that under microwave irradiation, the reaction rate improved up to 1.4-fold and shorter period of time was needed to achieve the higher conversion as compared to that of conventional heating. This reflects that the effect may not be purely thermal and microwave absorbing character of the feed was contributing to the faster reaction rate. In the reaction mixture, n-butanol may be the good microwave absorbing material and its dipole may be reorienting quickly under microwave irradiation making the functional group more active at the interface of ethyl-3-phenylpropanoate and n-butanol. It is also be possible that enzyme may behave slightly differently and become more active, because conformational change in enzyme can facilitate the substrate to approach active site of enzyme more easily under microwave irradiation than that under conventional heating (Yu et al., 2010). The control experiments in the absence of Novozyme 435 did not show any conversion and only microwave irradiation without enzyme did not initiate the reaction. Thus there is a definite synergism between enzyme catalysis and microwave irradiation. 3.2. Effect of various biocatalysts Control experiments were performed to establish the efficacy of various enzymes in the absence of microwaves. Transesterification of ethyl-3-phenylpropanoate with n-butanol chosen as the model reaction and different supported lipase were evaluated. The conversion varied markedly with the type of lipase (Fig. 1a). The conversions with Novozyme 435 and Lipozyme RMIM were 69% and 12.5%, respectively whereas Lipozyme TL IM showed very little activity. Lipozyme TL IM is mainly intended for interesterification of bulk fats and production of frying fats (Yadav and Lathi, 2004b). Novozyme 435 is a thermostable lipase and mainly useful for synthesis of esters and amides. The purpose of using these enzymes was to find out if significant activation could be achieved through microwave irradiation, despite their known use. Since Novozyme 435 was found to be the best catalyst, it was used all in further studies. 3.3. Effect of speed of agitation In the case of immobilized catalysts, the reactant has to diffuse from the bulk liquid to the external surface of the particle and from there into the interior pores of the catalyst. External mass transfer and internal diffusion limitations can be minimized by carrying out the reaction at an optimum speed of agitation and low enzyme loading of optimum particle size. The effect of speed of agitation on conversion was studied in the range of 100–300 rpm (figure not shown). The initial rate was calculated from the knowledge of conversion and initial concentration. It was found that there was a slight increase in the rate and conversion. However, there was hardly any difference in rate and conversion at 200 and 300 rpm; above 300 rpm the catalyst particles were thrown out of the liquid phase on the reactor wall, thereby reducing the effec-
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3.5. Effect of catalyst amount
Conversion % of ethyl-3-phenylpropanoate
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The effect of catalyst loading on transesterification of ethyl 3phenylpropanoate was studied under microwave irradiation. The molar ratios of reactants were kept constant while the amount of enzyme was changed from 0.0008 to 0.0032 g cm3(Fig. 2). It was observed that the rate of reaction increased linearly with an increase in Novozyme 435 loading, after the loading of 0.0032 g cm3 of enzyme there was insignificant increase in the rate and conversion. This suggested that the amount of enzyme added was much greater than the required and the external mass transfer had limited the rate. Considering the enzyme loading of 0.0024 g cm3 to be the most efficient and optimum, it was adopted in further experiments.
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Time (min) Fig. 1a. Effect of various biocatalysts on transesterification of ethyl-3-phenylpropanoate (ethyl-3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, enzyme 0.0032 g cm3, temperature 60 °C, speed of agitation 300 rpm, toluene up to 15 ml). Novozyme 435, j Lipozyme RMIM, d Lipozyme TMIM.
3.6. Effect of temperature The effect of temperature was studied by comparing the transesterification of ethyl-3-phenylpropanoate in conventional and
Conversion % of ethyl-3-phenylpropanoate
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Conversion % of ethyl-3-phenylpropanoate
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Time (min) Fig. 1b. Effect of various solvents on transesterification of ethyl-3-phenylpropanoate (ethyl-3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, Novozyme 435 0.0032 g cm3, temperature 60 °C, speed of agitation 300 rpm, solvent up to 15 ml). Hexane, N toluene,d 1,4-dioxane, n-butanol.
Fig. 2. Effect of catalyst amount on transesterification of ethyl-3-phenylpropanoate (ethyl-3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, temperature 60 °C, speed of agitation 300 rpm, hexane up to 15 ml) 0.0008 g cm3, 0.0016 g cm3, j 0.0024 g cm3, N 0.0032 g cm3.
tive catalyst loading. Thus the optimum speed for transesterification was found to be 300 rpm. 3.4. Effect of various solvents The effect of nature of solvent was studied by choosing different solvents such as toluene, hexane, 1, 4-dioxane. It has been reported that a minimum quantity of water is essential surrounding the immobilized lipase for maintaining the enzyme activity. Therefore, hydrophobic solvents are more preferred as compared to hydrophilic solvents since the latter causes striping of the essential water layer around the enzyme, which is necessary for enzyme activity (da Silva et al., 2005; Banoth et al., 2009). It was observed that there was increase in the enzyme activity as the solvent hydrophobicity increased in the range 1.1 to 3.5. n-Hexane having hydrophobicity of 3.5 was found to be the best solvent offering maximum conversion compared to others (Fig. 1b). n-Hexane was used as a solvent for further studies at a speed of agitation of 300 rpm.
Conversion % of ethyl-3-phenylpropanoate
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Time (min) Fig. 3a. Effect of temperature (conventional heating) on transesterification of ethyl3-phenylpropanoate (ethyl 3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, Novozyme 435 0.0024 g cm3, speed of agitation 300 rpm, hexane up to 15 ml) j 40 °C, 50 °C, 60 °C, N70 °C.
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100
Conversion % of ethyl-3-phenylpropanoate
Conversion % of ethyl-3-phenylpropanoate
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Time (min) Fig. 3b. Effect of temperature (microwave irradiation) on transesterification of ethyl-3-phenylpropanoate (ethyl 3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, Novozyme 435 0.0024 g cm3, speed of agitation 300 rpm, hexane up to 15 ml) j 40 °C, 50 °C, 60 °C, N 70 °C.
microwave heating. As discussed earlier, overall conversion as well as the rate of reaction was higher under microwave irradiation (Fig. 3b) compared to than that under conventional heating (Fig. 3a). It was observed that the initial rate increased from 0.33 106 to 0.62 106 mol/l-min-g of enzyme and the conversion increased from 60% to 96% with an increase in temperature in range of 40–70 °C. An elevated temperature could help the substrate molecules to obtain adequate energy to pass over the energy barrier and enhance the reaction rate. The values of activation energy were obtained by the Arrhenius plot as 4.6 and 5.3 kcal/mol under microwave and conventional heating respectively, which are reasonable values for enzymatic reactions in the absence of diffusion limitations. 3.7. Effect of mole ratio of n-butanol The transesterification of ethyl-3-phenylpropanoate with n-butanol was studied at different mole ratios of n-butanol, keeping the moles of ethyl-3-phenylpropanoate constant with constant liquid volume using hexane as solvent. Fig. 4 shows the maximum conversion (95%) and rate of reaction were obtained at a mole ratio 1:2 of ethyl-3-phenylpropanoate: n-butanol. By increasing the mole ratio of n-butanol from 1 to 2, conversion and reaction rate increased, with further increase in n-butanol concentration there was decrease in the conversion. The decrease in the conversion and reaction rate with increase in the concentration of n-butanol could be attributed to the inhibitory effect of n-butanol on biocatalyst. The enzyme is hydrophobic in nature and n-butanol contains the hydrophobic tail and polar head. There may be a hydrophobic– hydrophobic interaction between the enzyme and n-butanol. This interaction increases the residence time of n-butanol with enzyme, which would lead to partial dehydration which may destabilize the native conformation of enzyme leading to the inhibitory effect caused by n-butanol (Yadav and Borkar, 2008). 3.8. Effect of different alcohols Transesterification of ethyl-3-phenylpropanoate was studied with various alcohols such as 2-phenyl-1-propanol, n-octanol, benzyl alcohol, iso-amyl alcohol, 2-hexanol, 2-pentanol under otherwise similar conditions with Novozyme 435. It was found that
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Time (min) Fig. 4. Effect of mole ratio of n-butanol on transesterification of ethyl-3phenylpropanoate (ethyl-3-phenylpropanoate 0.56 mmol, Novozyme 435 0.0024 g cm3, temperature 60 °C, speed of agitation 300 rpm hexane up to 15 ml) 1:01, N 1:02 1:03, j 1:04.
the conversion achieved with primary alcohol such as iso-amyl alcohol and n-octanol was 92% and 85%, respectively, whereas transestrification with secondary alcohols, 2-pentanol and 2-hexanol showed lower conversions as 59% and 51%, respectively. However, aromatic alcohols such as 2-phenyl-1-propanol and benzyl alcohol exhibited good conversions as 79% and 67% respectively, compared to than that of secondary alcohols. The variation in conversion with chain length and type of alcohol can be attributed to influence of various factors such as molecular size of alcohol, solubility in reaction media and the affinity of lipase for particular alcohol (Verma and Madras, 2010). Similar kind of effect causing variation in conversion with change of chain length, type of alcohol have been reported in the literature with respect to esterification and transesterification reactions catalyzed by lipase in the organic solvents (Yadav and Lathi, 2004a; Shintre et al., 2002). 3.9. Effect of reusability The catalyst reusability studies were carried to examine the stability of enzyme. The catalyst was filtered, washed with hexane after each use, dried at room temperature and reused. There was marginal decrease in conversion after three reuses for transesterification with n-butanol (Fig. 5) which might be due to the loss of catalyst during handling. Thus, the enzyme was quite stable for reuse. 3.10. Kinetic study The effect of concentration of both reactants on the rate of reaction was investigated systematically over a wide range. For determination of initial rates of transesterification, the concentration of n-butanol were varied from 0.56 to 2.24 mmol at different levels of ethyl-3-phenylpropanoate concentration (0.56–2.24 mmol) using 0.0024 g cm3 of novozyme 435 and the total volume was made up to 15 ml with n-hexane. The conversion was determined and quantified by using synthetic mixtures. The initial rates were determined from the quantified data. From the initial rate (r0) measurements, it was observed that the rate increased with increasing the concentration of the ethyl-3phenylpropanoate (A). When the concentration of n-butanol (B) was increased keeping the concentration of ethyl-3-phenylpropan-
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Conversion % of ethyl-3-phenylpropanoate
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Time (min) Fig. 5. Effect of reusability of biocatalyst on transesterification of ethyl-3-phenylpropanoate (ethyl 3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, Novozyme 435 0.0024 g cm3, temperature 60 °C, speed of agitation 300 rpm, hexane up to 15 ml) fresh, j 1st resuse, N 2nd reuse, 3rd reuse.
oate constant, the rate of reaction increased and reached the maximum at critical concentration. Further increase in n-butanol concentration resulted in the reaction rate to fall and thus the substrate inhibition was notable. Therefore, it may be concluded that n-butanol at higher concentrations reacts with the enzyme to form dead end inhibitory complex. There was no evidence of inhibition by ethyl-3-phenylpropanoate (A) at any concentration tested. The Lineweaver Burk plot 1/r0 versus 1/[A0] for varied initial concentrations of B gives parallel lines (Fig. 6), where r0 is the initial rate of reaction and [B0] is the initial concentration of n-butanol. The family of lines has no common intersection and therefore, a sequential mechanism can be ruled out. This is indicative of a mechanism that requires dissociation of one product before the association of second substrate to the enzyme-substrate complex. A mechanism in which the product is released between additions of two reactants is called Ping-Pong bi–bi. When one of these reactants forms a complex with the enzyme that can participate in the reaction is called Ping-Pong bi–bi with dead end inhibition. These results agree with the assumed Ping-Pong bi–bi mechanism with dead end inhibition; hence Ping-Pong bi–bi mechanism with alcohol inhibition is postulated. These assumptions are used to design a reaction mechanism that is depicted in Cleland’s notation, as shown below.
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By analogy to the classical mechanism of transesterification by lipases, it is assumed that ethyl-3-phenylpropanoate [A] first binds to the free enzyme [E] and forms a noncovalent enzyme–substrate complex [EA], which releases the first product, ethanol [P] and E0 modified enzyme. The second substrate, n-butanol [B] binds to E0 to give complex E0 B and gives the product n-butyl-3-phenylpropanoate [Q] and free enzyme [E]. Along with this, B also forms the dead-end complex [EiB] by binding to the free enzyme [E]. The rate equation is as follows:
r0 ¼
r m ½A½B K mB ½A þ K mA ½B 1 þ ½B þ ½A½B Ki
ð1Þ
where KmA is the Michaelis constant for ethyl-3-phenylpropanoate, KmB is the Michaelis constant for n-butanol, and Ki is the inhibition constant due to n-butanol. r0 and rm are the initial rate and maximum rate, respectively. To verify the application of Ping-Pong bi-bi mechanism, the same data were analyzed by non-linear regression using the software package Polymath 6.0. The kinetic parameters determined for above mechanism were obtained as: rm (mol/l-min) – 3.7 106, KmA (mol/l) – 2.7 104, KmB (mol/l) – 0.1862, Ki – 1.1 104. A parity plot of experimental rate versus simulated rate using the above parameters gave a straight line passing through the origin with excellent correlation coefficient (figure not shown). This demonstrates that the proposed model is valid. 4. Conclusion Transesterification of ethyl 3-phenylpropanoate was studied using various immobilized lipases among which Novozyme 435 was the most active. Effects of speed of agitation, nature of solvent, catalyst loading, temperature and mole ratio were studied. A kinetic model was proposed by collecting both initial rate data and concentration–time profiles for transesterification of ethyl-3-phenylpropanoate with n-butanol. The Ping-Pong bi–bi mechanism with n-butanol substrate inhibition provides support for the mechanism. The mechanism was found to fit the data well for microwave irradiated enzyme catalysis. Transesterification of ethyl-3-phenylpropanoate was also studied with 2-phenyl-1-propanol, n-octanol, benzyl alcohol, iso-amyl alcohol, 2-hexanol and 2-pentanol.
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1/Initial rate
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GDY received support from Darbari Seth Endowment, R.T. Mody Distinguished Professor Endowment and J.C. Bose National Fellowship of Department of Science and Technology, Government of India. SVP thanks the University Grant Commission for an award of SRF.
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References
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1/ [ethyl-3-phenylpropanoate] Fig. 6. Linewear–Burk plot: 1/[Initial rate] vs. 1/[A] at different concentration of [B] j 1.12 mmol, N 1.68 mmol, 2.24 mmol.
Acosta, A., Filice, M., Fernandez-Lorente, G., Palomo, J.M., Guisan, J.M., 2011. Kinetically controlled synthesis of monoglyceryl esters from chiral and prochiral acids methyl esters catalyzed by immobilized Rhizomucor miehei lipase. Bioresour. Technol. 102, 507–512. Banoth, L., Singh, M., Tekewe, A., Banerjee, U.C., 2009. Increased enantioselectivity of lipase in the transesterification of dl-(±)-3-phenyllactic acid in ionic liquids. Biocatal. Biotransform. 27, 263–270.
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Cassani, J., Luna, H., Navarro, A., Castillo, E., 2007. Comparative esterification of phenylpropanoids versus hydrophenylpropanoids acids catalyzed by lipase in organic solvent media. E. J. Biotechnol. 10, 508–513. Collins, J.M., Leadbeater, N.E., 2007. Microwave energy: a versatile tool for bioscience. Org. Biomol. Chem. 5, 1141–1150. da Silva, C.J., Queiroz, N., da Graca, N.M., Soldi, V., 2005. The use of lipases immobilized on poly (ethylene oxide) for the preparation of alkyl esters. Process Biochem. 40, 401–409. Freidberg, F., Long, J.E., Brecher, A.S., 1969. The protection of a-chymotrypsin by hydrocinnamate from denaturation by ethanol and urea. Proc. Soc. Exp. Biol. Med. 1969 (130), 1046–1047. Gao, S., Wang, W., Wang, Y., Luo, G., Dai, Y., 2010. Influence of alcohol treatments on the activity of lipases immobilized on methyl-modified silica aerogels. Bioresour. Technol. 101, 7231–7238. Kappe, C.O., Stadler, A., 2006. Microwaves in Organic and Medicinal Chemistry. Wiley-VCH, Weinheim. Priya, K., Chadha, A., 2003. Synthesis of hydrocinnamic esters by Pseudomonas cepacia lipase. Enzyme Microb. Technol. 32, 485–490. Rejasse, B., Besson, T., Legoy, M.D., Lamare, S., 2006. Influence of microwave irradiation on free Candida antartica lipase b activity and stability. Org. Biomol. Chem. 4, 3703–3707. Sathishkumar, M., Jayabalan, R., Mun, S.P., Yun, S.E., 2010. Role of bicontinuous microemulsion in the rapid enzymatic hydrolysis of (R, S)-ketoprofen ethyl ester in a micro-reactor. Bioresour. Technol. 101, 7834–7840. Shahidi, F., Naczk, M., 1995. Food Phenolics. Sources, Chemistry, Effects, Applications. Technomic Publishing Company Inc., Lancaster, USA. Shintre, M.S., Ghadge, R.S., Sawant, S.B., 2002. Kinetics of esterification of lauric acid with fatty alcohols by lipase: effect of fatty alcohol. J. Chem. Technol. Biotechnol. 77, 1114–1121. Sontakke, J.B., Yadav, G.D., 2011. Optimization and kinetic modeling of lipase catalyzed enantioselective N-acetylation of (±)-1-phenylethylamine under microwave irradiation. J. Chem. Technol. Biotechnol. 86, 739–748. Varma, M., Madras, G., 2010. Effect of chain length of alcohol on the lipase-catalyzed esterification of propionic acid in supercritical carbon dioxide. Appl. Biochem. Biotechnol. 160, 2342–2354. Weitkamp, P., Vosmann, K., Weber, N., 2006. Highly efficient preparation of lipophilic hydroxycinnamates by solvent-free lipase-catalyzed transesterification. J. Agric. Food Chem. 54, 7062–7068. Weitkamp, P., Weber, N., Vosmann, K., 2008. Lipophilic (hydroxy) phenylacetates by solvent-free lipase-catalyzed esterification and transesterification in vacuo. J. Agric. Food Chem. 56, 5083–5090. Yadav, G.D., Borkar, I.V., 2006. Kinetic modeling of microwave assisted chemoenzymatic epoxidation of styrene to styrene oxide. AICh E J. 52, 1235–1247. Yadav, G.D., Borkar, I.V., 2008. Kinetic modeling of immobilized lipase catalysis in synthesis of n-butyl levulinate. Ind. Eng. Chem. Res. 47, 3358–3363. Yadav, G.D., Borkar, I.V., 2009. Kinetic and mechanistic investigation of microwaveassisted lipase catalyzed synthesis of citronellyl acetate. Ind. Eng. Chem. Res. 48, 7915–7922.
Yadav, G.D., Devi, K.M., 2001. A kinetic model for the enzyme-catalyzed self epoxidation of oleic acid. J. Am. Oil Chem. Soc. 78, 347–351. Yadav, G.D., Devi, K.M., 2002. Enzymatic synthesis of perlauric acid using Novozyme 435. Biochem. Eng. J. 10, 93–101. Yadav, G.D., Devi, K.M., 2004a. Enzymatic synthesis of perlauric acid using Novozyme 435. Biochem. Eng. J. 10, 93–101. Yadav, G.D., Devi, K.M., 2004b. Immobilized lipase-catalysed esterification and transesterification reactions in non-aqueous media for the synthesis of tetrahydrofurfuryl butyrate: comparison and kinetic modeling. Chem. Eng. Sci. 59, 373–383. Yadav, G.D., Jadhav, S.R., 2005. Synthesis of reusable lipases by immobilization on hexagonal mesoporous silica and encapsulation in calcium alginate: Transesterification in non-aqueous medium. Micropor. Mesopor. Mater. 86, 215–222. Yadav, G.D., Lathi, P.S., 2003. Kinetics and mechanism of synthesis of butyl isobutyrate over immobilized lipases. Biochem. Eng. J. 16, 245–252. Yadav, G.D., Lathi, P.S., 2004a. Synergism between microwave and enzyme catalysis in intensification of reactions and selectivities: transesterification of methyl acetoacetate with alcohols. J. Mol. Cat. A: Chem. 223, 51–56. Yadav, G.D., Lathi, P.S., 2004b. Synthesis of citronellol laurate in organic media catalyzed by immobilized lipases: kinetic studies. J. Mol. Cat. B: Enzym. 27, 113–119. Yadav, G.D., Sajgure, A.D., 2007. Synergism of microwave irradiation and enzyme catalysis in synthesis of isoniazid. J. Chem. Technol. Biotechnol. 82, 964–970. Yadav, G.D., Sivakumar, P., 2004. Enzyme-catalysed optical resolution of mandelic acid via RS -methyl mandelate in non-aqueous media. Biochem. Eng. J. 19, 101– 107. Yadav, G.D., Trivedi, A.H., 2003. Kinetic modeling of immobilized-lipase catalyzed transesterification of n-octanol with vinyl acetate in non-aqueous media. Enzyme Microb. Technol. 32, 783–789. Yang, G., Wu, J., Xu, G., Yang, L., 2009. Improvement of catalytic properties of lipase from Arthrobacter sp. by encapsulation in hydrophobic sol–gel materials. Bioresour. Technol. 100, 4311–4316. Young, D.D., Nichols, J., Kelly, R.M., Deiters, A., 2008. Microwave activation of enzymatic catalysis. J. Am. Chem. Soc. 130, 10048–10049. Yu, D., Tian, L., Ma, D., Wu, H., Wang, Z., Wang, L., Fang, X., 2010. Microwave-assisted fatty acid methyl ester production from soybean oil by Novozym 435. Green Chem. 12, 844–850. Yu, D., Wang, C., Yin, Y., Zhang, A., Gao, G., Fang, X., 2011. A synergistic effect of microwave irradiation and ionic liquids on enzyme-catalyzed biodiesel production. Green Chem. 13, 1869–1875. Yu, D., Wang, Z., Chena, P., Jin, L., Cheng, Y., Zhou, J., Cao, S., 2007. Microwaveassisted resolution of (R, S)-2-octanol by enzymatic transesterification. J. Mol. Cat. B: Enzymatic 48, 51–57. Zhu, S., Wu, Y., Yu, Z., Zhang, X., Li, H., Gao, M., 2006. The effect of microwave irradiation on enzymatic hydrolysis of rice straw. Bioresour. Technol. 97, 1964– 1968.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Synergism between microwave irradiation and enzyme catalysis in transesterification of ethyl-3-phenylpropanoate with n-butanol Ganapati D. Yadav ⇑, Sandip V. Pawar Department of Chemical Engineering, Institute of Chemical Technology, University of Mumbai,1 Matunga, Mumbai 400 019, India
a r t i c l e
i n f o
Article history: Received 14 December 2011 Received in revised form 8 January 2012 Accepted 9 January 2012 Available online 16 January 2012 Keywords: Synergism Immobilized lipase Microwave Enzyme catalysis Kinetic study
a b s t r a c t Lipase catalyzed transesterification was investigated to study the synergistic effect of microwave irradiation and enzyme catalysis. Transesterification of ethyl-3-phenylpropanoate with n-butanol was chosen as the model reaction using immobilized enzymes such as Novozyme 435, Lipozyme RMIM and Lipozyme TL IM with microwave irradiation. Novozyme 435 was the best catalyst. The effect of various parameters affecting the conversion and initial rates of transesterification were studied to establish kinetics and mechanism. There is synergism between enzyme catalysis and microwave irradiation. The analysis of initial rate data and progress curve data showed that the reaction obeys the Ping-Pong bi–bi mechanism with inhibition by n-butanol. The theoretical predictions and experimental data match very well. These studies were also extended to other alcohols such as 2-phenyl-1-propanol, n-octanol, benzyl alcohol, isoamyl alcohol, 2-hexanol and 2-pentanol under otherwise similar conditions. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Phenolic acids present in plants are hydroxylated derivatives of benzoic and cinnamic acids. Flavonoids and phenolic acids have many functions in plants. They act as cell wall support materials and as colorful attractants for birds and insects helping seed dispersal and pollination (Shahidi and Naczk, 1995). Thus, synthesis of alkyl hydroxycinnamates of natural quality is important and enzymatic routes could be used. Enzyme catalyzed reactions in non-aqueous media have gained a considerable significance (Yadav and Devi, 2001, 2002, 2004a,b; Yadav and Jadhav, 2005; Yu et al., 2007; Sathishkumar et al., 2010). Lipases are considered to be the most important group of biocatalysts for non-aqueous reactions because they possess wide substrate specificity, able to recognize the chiral centers and do not require the labile cofactors to carry out the reactions (Yadav and Sivakumar, 2004; Yadav and Borkar, 2009; Yang et al., 2009). Lipases are readily available from different sources such as bacteria and fungi and so there is a good chance of finding a suitable catalyst for biotransformation of interest in terms of catalytic activity and selectivity (Banoth et al., 2009; Acosta et al., 2011). They have been used to catalyze a variety of reactions in non-aqueous media, viz. esterification, transesterification, amidation, epoxidation, hydrolysis, thio-esterification and trans-thioesterification (Yadav ⇑ Corresponding author. Tel.: +91 22 3361 1001; fax: +91 22 3361 1020. E-mail addresses: [email protected], [email protected] Yadav). 1 Formerly, of University of Mumbai, now a separate university. 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.01.030
(G.D.
and Trivedi, 2003; Yadav and Devi, 2001, 2002, 2004a; Yadav and Lathi, 2003, 2004b; Gao et al., 2010). Hydrocinnamate esters are important intermediates in the synthesis of HIV protease inhibitor and as precursors for the synthesis of 1,3,4,9-tetrahydropyrano [3, 4-b] indole-1-acetic acid which is reported for its use as analgesic, inflammation inhibitor and antipyretic activities (Priya and Chadha, 2003; Cassani et al., 2007). Hydrocinnamates are used in the protection of chymotrypsin from denaturation by urea and ethanol (Priya and Chadha, 2003; Freidberg et al., 1969) and also find applications in flavor and fragrance industry. Esterification of hydrocinnamic acid and transesterification of hydrocinnamate esters have been studied using solvent free reactions, drying agents or in vacuo reactions (Weitkamp et al., 2006, 2008). Microwave irradiation have been accepted as efficient heating source for chemical reactions since the mid-1980s, where reactions that required several hours under conventional heating could often be completed in few minutes with high yields and reaction selectivity. Microwave assisted synthesis is an important and rapidly developing technology in green chemistry, and is environmentally benign, clean, fast and convenient which has been widely used in organic chemistry and enzyme catalyzed reactions (Kappe and Stadler, 2006; Yu et al., 2011; Zhu et al., 2006). Microwave irradiation triggers the effect which cannot be preceded by thermal heating; it leads to direct coupling of molecules by selective absorption of radiation by polar compounds (Young et al., 2008; Collins and Leadbeater, 2007). Microwave irradiation seems to improve thermal stability of the enzyme (Rejasse et al., 2006; Yadav and Borkar, 2006) as a function of enzyme hydration which can be used
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efficiently to improve the process or modify the selectivity (Sontakke and Yadav, 2011). Synergism between microwave irradiation and enzyme catalysis is known to improve reaction rate in lipase catalyzed reactions (Yadav and Lathi, 2004a; Yadav and Sajgure, 2007). The present work encompasses an environment friendly lipase catalyzed transesterification of ethyl-3-phenylpropanoate (ethylhydrocinnamte) with a variety of alcohols under microwave irradiation as a greener approach including deduction of reaction mechanism and kinetic modeling. Synergism between microwave irradiation and enzyme catalysis has been explored in order to optimize the transesterification process. The effects of different parameters including nature of biocatalyst, solvent, agitation speed, reaction temperature, molar ratio of reactants, and type of alcohols were investigated. 2. Methods 2.1. Enzymes The Novozyme 435, Lipozyme RM IM and Lipozyme TL IM were procured as gift samples from Novo Nordisk, Denmark. Novozyme 435 is Candida antarctica lipase immobilized on a macroporous polyacrylic resin beads (bead size 0.3–0.6 mm, bulk density 0.430 g cm3, water content 3%, activity of 7000 PLU g1); Lipozyme RM IM is Mucor miehei immobilized on an ionic resin with acitivity of 5–6 BAUN while Lipozyme TL IM is Thermomyces lanuginosus immobilized on silica. 2.2. Chemicals All chemicals were AR grade, procured from the firms of repute and were used without any further purification: ethyl-3-phenypropanoate (Sigma Aldrich, India), toluene, n-hexane, 1, 4 dioxane, nheptane, n-butanol, n-octanol, iso-amyl alcohol, benzyl alcohol (S. d. Fine Chemicals Pvt. Ltd., Mumbai, India). 2.3. Experimental setup 2.3.1. Conventional heating The experimental set up consisted of a 3 cm i.d. fully baffled mechanically agitated glass reactor of 50 cm3 capacity, which was equipped with four baffles and a six bladed pitched-turbine impeller. The entire reactor assembly was immersed in a thermostatic water bath maintained at the desired temperature with an accuracy of ±1 °C. In a typical experiment, the reaction mixture contained 0.56 mmol of ethyl-3-phenylpropanoate and 1.12 mmol of n-butanol, diluted to 15 ml with toluene as a solvent. It was agitated at 60 °C for 15 min at a speed of 300 rpm and a known quantity of enzyme was then added to initiate the reaction. Samples were withdrawn periodically, filtered to remove fine particles, if any, and analyzed by GC. 2.3.2. Microwave reactor The studies were carried out in a microwave reactor (Discover, CEM-SP 1245 model). The reactor was 120 ml capacity fully baffled, 4.5 cm i.d. cylindrical glass vessel with provision for mechanical stirring. A standard four bladed turbine impeller of 1.5 cm diameter was used for agitation. However, the actual reactor volume exposed to the microwave irradiation was 45 ml with 5.5 cm height. 2.4. Analysis The concentration of reactants and products were determined by GC (Chemito Gas chromatograph Model 8610) equipped with
a flame ionization detector. A 4 m 3.8 mm stainless steel column packed with 5% SE-30 on chromosorb was used for analysis. The product was confirmed by GCMS. Synthetic mixtures were prepared of pure samples and calibration was done to quantify the collected data for conversion and rate of reactions. 3. Results and discussion 3.1. Conventional heating versus microwave irradiation Transesterification of ethyl-3-phenylpropanoate with n-butanol was chosen as the model reaction. Enzymatic transesterification was performed in the presence of controlled microwave irradiation. It was found that under microwave irradiation, the reaction rate improved up to 1.4-fold and shorter period of time was needed to achieve the higher conversion as compared to that of conventional heating. This reflects that the effect may not be purely thermal and microwave absorbing character of the feed was contributing to the faster reaction rate. In the reaction mixture, n-butanol may be the good microwave absorbing material and its dipole may be reorienting quickly under microwave irradiation making the functional group more active at the interface of ethyl-3-phenylpropanoate and n-butanol. It is also be possible that enzyme may behave slightly differently and become more active, because conformational change in enzyme can facilitate the substrate to approach active site of enzyme more easily under microwave irradiation than that under conventional heating (Yu et al., 2010). The control experiments in the absence of Novozyme 435 did not show any conversion and only microwave irradiation without enzyme did not initiate the reaction. Thus there is a definite synergism between enzyme catalysis and microwave irradiation. 3.2. Effect of various biocatalysts Control experiments were performed to establish the efficacy of various enzymes in the absence of microwaves. Transesterification of ethyl-3-phenylpropanoate with n-butanol chosen as the model reaction and different supported lipase were evaluated. The conversion varied markedly with the type of lipase (Fig. 1a). The conversions with Novozyme 435 and Lipozyme RMIM were 69% and 12.5%, respectively whereas Lipozyme TL IM showed very little activity. Lipozyme TL IM is mainly intended for interesterification of bulk fats and production of frying fats (Yadav and Lathi, 2004b). Novozyme 435 is a thermostable lipase and mainly useful for synthesis of esters and amides. The purpose of using these enzymes was to find out if significant activation could be achieved through microwave irradiation, despite their known use. Since Novozyme 435 was found to be the best catalyst, it was used all in further studies. 3.3. Effect of speed of agitation In the case of immobilized catalysts, the reactant has to diffuse from the bulk liquid to the external surface of the particle and from there into the interior pores of the catalyst. External mass transfer and internal diffusion limitations can be minimized by carrying out the reaction at an optimum speed of agitation and low enzyme loading of optimum particle size. The effect of speed of agitation on conversion was studied in the range of 100–300 rpm (figure not shown). The initial rate was calculated from the knowledge of conversion and initial concentration. It was found that there was a slight increase in the rate and conversion. However, there was hardly any difference in rate and conversion at 200 and 300 rpm; above 300 rpm the catalyst particles were thrown out of the liquid phase on the reactor wall, thereby reducing the effec-
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3.5. Effect of catalyst amount
Conversion % of ethyl-3-phenylpropanoate
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The effect of catalyst loading on transesterification of ethyl 3phenylpropanoate was studied under microwave irradiation. The molar ratios of reactants were kept constant while the amount of enzyme was changed from 0.0008 to 0.0032 g cm3(Fig. 2). It was observed that the rate of reaction increased linearly with an increase in Novozyme 435 loading, after the loading of 0.0032 g cm3 of enzyme there was insignificant increase in the rate and conversion. This suggested that the amount of enzyme added was much greater than the required and the external mass transfer had limited the rate. Considering the enzyme loading of 0.0024 g cm3 to be the most efficient and optimum, it was adopted in further experiments.
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0 0
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Time (min) Fig. 1a. Effect of various biocatalysts on transesterification of ethyl-3-phenylpropanoate (ethyl-3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, enzyme 0.0032 g cm3, temperature 60 °C, speed of agitation 300 rpm, toluene up to 15 ml). Novozyme 435, j Lipozyme RMIM, d Lipozyme TMIM.
3.6. Effect of temperature The effect of temperature was studied by comparing the transesterification of ethyl-3-phenylpropanoate in conventional and
Conversion % of ethyl-3-phenylpropanoate
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Conversion % of ethyl-3-phenylpropanoate
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Time (min)
0 0
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Time (min) Fig. 1b. Effect of various solvents on transesterification of ethyl-3-phenylpropanoate (ethyl-3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, Novozyme 435 0.0032 g cm3, temperature 60 °C, speed of agitation 300 rpm, solvent up to 15 ml). Hexane, N toluene,d 1,4-dioxane, n-butanol.
Fig. 2. Effect of catalyst amount on transesterification of ethyl-3-phenylpropanoate (ethyl-3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, temperature 60 °C, speed of agitation 300 rpm, hexane up to 15 ml) 0.0008 g cm3, 0.0016 g cm3, j 0.0024 g cm3, N 0.0032 g cm3.
tive catalyst loading. Thus the optimum speed for transesterification was found to be 300 rpm. 3.4. Effect of various solvents The effect of nature of solvent was studied by choosing different solvents such as toluene, hexane, 1, 4-dioxane. It has been reported that a minimum quantity of water is essential surrounding the immobilized lipase for maintaining the enzyme activity. Therefore, hydrophobic solvents are more preferred as compared to hydrophilic solvents since the latter causes striping of the essential water layer around the enzyme, which is necessary for enzyme activity (da Silva et al., 2005; Banoth et al., 2009). It was observed that there was increase in the enzyme activity as the solvent hydrophobicity increased in the range 1.1 to 3.5. n-Hexane having hydrophobicity of 3.5 was found to be the best solvent offering maximum conversion compared to others (Fig. 1b). n-Hexane was used as a solvent for further studies at a speed of agitation of 300 rpm.
Conversion % of ethyl-3-phenylpropanoate
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0 0
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Time (min) Fig. 3a. Effect of temperature (conventional heating) on transesterification of ethyl3-phenylpropanoate (ethyl 3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, Novozyme 435 0.0024 g cm3, speed of agitation 300 rpm, hexane up to 15 ml) j 40 °C, 50 °C, 60 °C, N70 °C.
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100
Conversion % of ethyl-3-phenylpropanoate
Conversion % of ethyl-3-phenylpropanoate
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0 0 0
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Time (min) Fig. 3b. Effect of temperature (microwave irradiation) on transesterification of ethyl-3-phenylpropanoate (ethyl 3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, Novozyme 435 0.0024 g cm3, speed of agitation 300 rpm, hexane up to 15 ml) j 40 °C, 50 °C, 60 °C, N 70 °C.
microwave heating. As discussed earlier, overall conversion as well as the rate of reaction was higher under microwave irradiation (Fig. 3b) compared to than that under conventional heating (Fig. 3a). It was observed that the initial rate increased from 0.33 106 to 0.62 106 mol/l-min-g of enzyme and the conversion increased from 60% to 96% with an increase in temperature in range of 40–70 °C. An elevated temperature could help the substrate molecules to obtain adequate energy to pass over the energy barrier and enhance the reaction rate. The values of activation energy were obtained by the Arrhenius plot as 4.6 and 5.3 kcal/mol under microwave and conventional heating respectively, which are reasonable values for enzymatic reactions in the absence of diffusion limitations. 3.7. Effect of mole ratio of n-butanol The transesterification of ethyl-3-phenylpropanoate with n-butanol was studied at different mole ratios of n-butanol, keeping the moles of ethyl-3-phenylpropanoate constant with constant liquid volume using hexane as solvent. Fig. 4 shows the maximum conversion (95%) and rate of reaction were obtained at a mole ratio 1:2 of ethyl-3-phenylpropanoate: n-butanol. By increasing the mole ratio of n-butanol from 1 to 2, conversion and reaction rate increased, with further increase in n-butanol concentration there was decrease in the conversion. The decrease in the conversion and reaction rate with increase in the concentration of n-butanol could be attributed to the inhibitory effect of n-butanol on biocatalyst. The enzyme is hydrophobic in nature and n-butanol contains the hydrophobic tail and polar head. There may be a hydrophobic– hydrophobic interaction between the enzyme and n-butanol. This interaction increases the residence time of n-butanol with enzyme, which would lead to partial dehydration which may destabilize the native conformation of enzyme leading to the inhibitory effect caused by n-butanol (Yadav and Borkar, 2008). 3.8. Effect of different alcohols Transesterification of ethyl-3-phenylpropanoate was studied with various alcohols such as 2-phenyl-1-propanol, n-octanol, benzyl alcohol, iso-amyl alcohol, 2-hexanol, 2-pentanol under otherwise similar conditions with Novozyme 435. It was found that
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Time (min) Fig. 4. Effect of mole ratio of n-butanol on transesterification of ethyl-3phenylpropanoate (ethyl-3-phenylpropanoate 0.56 mmol, Novozyme 435 0.0024 g cm3, temperature 60 °C, speed of agitation 300 rpm hexane up to 15 ml) 1:01, N 1:02 1:03, j 1:04.
the conversion achieved with primary alcohol such as iso-amyl alcohol and n-octanol was 92% and 85%, respectively, whereas transestrification with secondary alcohols, 2-pentanol and 2-hexanol showed lower conversions as 59% and 51%, respectively. However, aromatic alcohols such as 2-phenyl-1-propanol and benzyl alcohol exhibited good conversions as 79% and 67% respectively, compared to than that of secondary alcohols. The variation in conversion with chain length and type of alcohol can be attributed to influence of various factors such as molecular size of alcohol, solubility in reaction media and the affinity of lipase for particular alcohol (Verma and Madras, 2010). Similar kind of effect causing variation in conversion with change of chain length, type of alcohol have been reported in the literature with respect to esterification and transesterification reactions catalyzed by lipase in the organic solvents (Yadav and Lathi, 2004a; Shintre et al., 2002). 3.9. Effect of reusability The catalyst reusability studies were carried to examine the stability of enzyme. The catalyst was filtered, washed with hexane after each use, dried at room temperature and reused. There was marginal decrease in conversion after three reuses for transesterification with n-butanol (Fig. 5) which might be due to the loss of catalyst during handling. Thus, the enzyme was quite stable for reuse. 3.10. Kinetic study The effect of concentration of both reactants on the rate of reaction was investigated systematically over a wide range. For determination of initial rates of transesterification, the concentration of n-butanol were varied from 0.56 to 2.24 mmol at different levels of ethyl-3-phenylpropanoate concentration (0.56–2.24 mmol) using 0.0024 g cm3 of novozyme 435 and the total volume was made up to 15 ml with n-hexane. The conversion was determined and quantified by using synthetic mixtures. The initial rates were determined from the quantified data. From the initial rate (r0) measurements, it was observed that the rate increased with increasing the concentration of the ethyl-3phenylpropanoate (A). When the concentration of n-butanol (B) was increased keeping the concentration of ethyl-3-phenylpropan-
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Conversion % of ethyl-3-phenylpropanoate
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Time (min) Fig. 5. Effect of reusability of biocatalyst on transesterification of ethyl-3-phenylpropanoate (ethyl 3-phenylpropanoate 0.56 mmol, n-butanol 1.12 mmol, Novozyme 435 0.0024 g cm3, temperature 60 °C, speed of agitation 300 rpm, hexane up to 15 ml) fresh, j 1st resuse, N 2nd reuse, 3rd reuse.
oate constant, the rate of reaction increased and reached the maximum at critical concentration. Further increase in n-butanol concentration resulted in the reaction rate to fall and thus the substrate inhibition was notable. Therefore, it may be concluded that n-butanol at higher concentrations reacts with the enzyme to form dead end inhibitory complex. There was no evidence of inhibition by ethyl-3-phenylpropanoate (A) at any concentration tested. The Lineweaver Burk plot 1/r0 versus 1/[A0] for varied initial concentrations of B gives parallel lines (Fig. 6), where r0 is the initial rate of reaction and [B0] is the initial concentration of n-butanol. The family of lines has no common intersection and therefore, a sequential mechanism can be ruled out. This is indicative of a mechanism that requires dissociation of one product before the association of second substrate to the enzyme-substrate complex. A mechanism in which the product is released between additions of two reactants is called Ping-Pong bi–bi. When one of these reactants forms a complex with the enzyme that can participate in the reaction is called Ping-Pong bi–bi with dead end inhibition. These results agree with the assumed Ping-Pong bi–bi mechanism with dead end inhibition; hence Ping-Pong bi–bi mechanism with alcohol inhibition is postulated. These assumptions are used to design a reaction mechanism that is depicted in Cleland’s notation, as shown below.
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By analogy to the classical mechanism of transesterification by lipases, it is assumed that ethyl-3-phenylpropanoate [A] first binds to the free enzyme [E] and forms a noncovalent enzyme–substrate complex [EA], which releases the first product, ethanol [P] and E0 modified enzyme. The second substrate, n-butanol [B] binds to E0 to give complex E0 B and gives the product n-butyl-3-phenylpropanoate [Q] and free enzyme [E]. Along with this, B also forms the dead-end complex [EiB] by binding to the free enzyme [E]. The rate equation is as follows:
r0 ¼
r m ½A½B K mB ½A þ K mA ½B 1 þ ½B þ ½A½B Ki
ð1Þ
where KmA is the Michaelis constant for ethyl-3-phenylpropanoate, KmB is the Michaelis constant for n-butanol, and Ki is the inhibition constant due to n-butanol. r0 and rm are the initial rate and maximum rate, respectively. To verify the application of Ping-Pong bi-bi mechanism, the same data were analyzed by non-linear regression using the software package Polymath 6.0. The kinetic parameters determined for above mechanism were obtained as: rm (mol/l-min) – 3.7 106, KmA (mol/l) – 2.7 104, KmB (mol/l) – 0.1862, Ki – 1.1 104. A parity plot of experimental rate versus simulated rate using the above parameters gave a straight line passing through the origin with excellent correlation coefficient (figure not shown). This demonstrates that the proposed model is valid. 4. Conclusion Transesterification of ethyl 3-phenylpropanoate was studied using various immobilized lipases among which Novozyme 435 was the most active. Effects of speed of agitation, nature of solvent, catalyst loading, temperature and mole ratio were studied. A kinetic model was proposed by collecting both initial rate data and concentration–time profiles for transesterification of ethyl-3-phenylpropanoate with n-butanol. The Ping-Pong bi–bi mechanism with n-butanol substrate inhibition provides support for the mechanism. The mechanism was found to fit the data well for microwave irradiated enzyme catalysis. Transesterification of ethyl-3-phenylpropanoate was also studied with 2-phenyl-1-propanol, n-octanol, benzyl alcohol, iso-amyl alcohol, 2-hexanol and 2-pentanol.
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1/Initial rate
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GDY received support from Darbari Seth Endowment, R.T. Mody Distinguished Professor Endowment and J.C. Bose National Fellowship of Department of Science and Technology, Government of India. SVP thanks the University Grant Commission for an award of SRF.
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0 0.0
References
0.5
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1/ [ethyl-3-phenylpropanoate] Fig. 6. Linewear–Burk plot: 1/[Initial rate] vs. 1/[A] at different concentration of [B] j 1.12 mmol, N 1.68 mmol, 2.24 mmol.
Acosta, A., Filice, M., Fernandez-Lorente, G., Palomo, J.M., Guisan, J.M., 2011. Kinetically controlled synthesis of monoglyceryl esters from chiral and prochiral acids methyl esters catalyzed by immobilized Rhizomucor miehei lipase. Bioresour. Technol. 102, 507–512. Banoth, L., Singh, M., Tekewe, A., Banerjee, U.C., 2009. Increased enantioselectivity of lipase in the transesterification of dl-(±)-3-phenyllactic acid in ionic liquids. Biocatal. Biotransform. 27, 263–270.
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Cassani, J., Luna, H., Navarro, A., Castillo, E., 2007. Comparative esterification of phenylpropanoids versus hydrophenylpropanoids acids catalyzed by lipase in organic solvent media. E. J. Biotechnol. 10, 508–513. Collins, J.M., Leadbeater, N.E., 2007. Microwave energy: a versatile tool for bioscience. Org. Biomol. Chem. 5, 1141–1150. da Silva, C.J., Queiroz, N., da Graca, N.M., Soldi, V., 2005. The use of lipases immobilized on poly (ethylene oxide) for the preparation of alkyl esters. Process Biochem. 40, 401–409. Freidberg, F., Long, J.E., Brecher, A.S., 1969. The protection of a-chymotrypsin by hydrocinnamate from denaturation by ethanol and urea. Proc. Soc. Exp. Biol. Med. 1969 (130), 1046–1047. Gao, S., Wang, W., Wang, Y., Luo, G., Dai, Y., 2010. Influence of alcohol treatments on the activity of lipases immobilized on methyl-modified silica aerogels. Bioresour. Technol. 101, 7231–7238. Kappe, C.O., Stadler, A., 2006. Microwaves in Organic and Medicinal Chemistry. Wiley-VCH, Weinheim. Priya, K., Chadha, A., 2003. Synthesis of hydrocinnamic esters by Pseudomonas cepacia lipase. Enzyme Microb. Technol. 32, 485–490. Rejasse, B., Besson, T., Legoy, M.D., Lamare, S., 2006. Influence of microwave irradiation on free Candida antartica lipase b activity and stability. Org. Biomol. Chem. 4, 3703–3707. Sathishkumar, M., Jayabalan, R., Mun, S.P., Yun, S.E., 2010. Role of bicontinuous microemulsion in the rapid enzymatic hydrolysis of (R, S)-ketoprofen ethyl ester in a micro-reactor. Bioresour. Technol. 101, 7834–7840. Shahidi, F., Naczk, M., 1995. Food Phenolics. Sources, Chemistry, Effects, Applications. Technomic Publishing Company Inc., Lancaster, USA. Shintre, M.S., Ghadge, R.S., Sawant, S.B., 2002. Kinetics of esterification of lauric acid with fatty alcohols by lipase: effect of fatty alcohol. J. Chem. Technol. Biotechnol. 77, 1114–1121. Sontakke, J.B., Yadav, G.D., 2011. Optimization and kinetic modeling of lipase catalyzed enantioselective N-acetylation of (±)-1-phenylethylamine under microwave irradiation. J. Chem. Technol. Biotechnol. 86, 739–748. Varma, M., Madras, G., 2010. Effect of chain length of alcohol on the lipase-catalyzed esterification of propionic acid in supercritical carbon dioxide. Appl. Biochem. Biotechnol. 160, 2342–2354. Weitkamp, P., Vosmann, K., Weber, N., 2006. Highly efficient preparation of lipophilic hydroxycinnamates by solvent-free lipase-catalyzed transesterification. J. Agric. Food Chem. 54, 7062–7068. Weitkamp, P., Weber, N., Vosmann, K., 2008. Lipophilic (hydroxy) phenylacetates by solvent-free lipase-catalyzed esterification and transesterification in vacuo. J. Agric. Food Chem. 56, 5083–5090. Yadav, G.D., Borkar, I.V., 2006. Kinetic modeling of microwave assisted chemoenzymatic epoxidation of styrene to styrene oxide. AICh E J. 52, 1235–1247. Yadav, G.D., Borkar, I.V., 2008. Kinetic modeling of immobilized lipase catalysis in synthesis of n-butyl levulinate. Ind. Eng. Chem. Res. 47, 3358–3363. Yadav, G.D., Borkar, I.V., 2009. Kinetic and mechanistic investigation of microwaveassisted lipase catalyzed synthesis of citronellyl acetate. Ind. Eng. Chem. Res. 48, 7915–7922.
Yadav, G.D., Devi, K.M., 2001. A kinetic model for the enzyme-catalyzed self epoxidation of oleic acid. J. Am. Oil Chem. Soc. 78, 347–351. Yadav, G.D., Devi, K.M., 2002. Enzymatic synthesis of perlauric acid using Novozyme 435. Biochem. Eng. J. 10, 93–101. Yadav, G.D., Devi, K.M., 2004a. Enzymatic synthesis of perlauric acid using Novozyme 435. Biochem. Eng. J. 10, 93–101. Yadav, G.D., Devi, K.M., 2004b. Immobilized lipase-catalysed esterification and transesterification reactions in non-aqueous media for the synthesis of tetrahydrofurfuryl butyrate: comparison and kinetic modeling. Chem. Eng. Sci. 59, 373–383. Yadav, G.D., Jadhav, S.R., 2005. Synthesis of reusable lipases by immobilization on hexagonal mesoporous silica and encapsulation in calcium alginate: Transesterification in non-aqueous medium. Micropor. Mesopor. Mater. 86, 215–222. Yadav, G.D., Lathi, P.S., 2003. Kinetics and mechanism of synthesis of butyl isobutyrate over immobilized lipases. Biochem. Eng. J. 16, 245–252. Yadav, G.D., Lathi, P.S., 2004a. Synergism between microwave and enzyme catalysis in intensification of reactions and selectivities: transesterification of methyl acetoacetate with alcohols. J. Mol. Cat. A: Chem. 223, 51–56. Yadav, G.D., Lathi, P.S., 2004b. Synthesis of citronellol laurate in organic media catalyzed by immobilized lipases: kinetic studies. J. Mol. Cat. B: Enzym. 27, 113–119. Yadav, G.D., Sajgure, A.D., 2007. Synergism of microwave irradiation and enzyme catalysis in synthesis of isoniazid. J. Chem. Technol. Biotechnol. 82, 964–970. Yadav, G.D., Sivakumar, P., 2004. Enzyme-catalysed optical resolution of mandelic acid via RS -methyl mandelate in non-aqueous media. Biochem. Eng. J. 19, 101– 107. Yadav, G.D., Trivedi, A.H., 2003. Kinetic modeling of immobilized-lipase catalyzed transesterification of n-octanol with vinyl acetate in non-aqueous media. Enzyme Microb. Technol. 32, 783–789. Yang, G., Wu, J., Xu, G., Yang, L., 2009. Improvement of catalytic properties of lipase from Arthrobacter sp. by encapsulation in hydrophobic sol–gel materials. Bioresour. Technol. 100, 4311–4316. Young, D.D., Nichols, J., Kelly, R.M., Deiters, A., 2008. Microwave activation of enzymatic catalysis. J. Am. Chem. Soc. 130, 10048–10049. Yu, D., Tian, L., Ma, D., Wu, H., Wang, Z., Wang, L., Fang, X., 2010. Microwave-assisted fatty acid methyl ester production from soybean oil by Novozym 435. Green Chem. 12, 844–850. Yu, D., Wang, C., Yin, Y., Zhang, A., Gao, G., Fang, X., 2011. A synergistic effect of microwave irradiation and ionic liquids on enzyme-catalyzed biodiesel production. Green Chem. 13, 1869–1875. Yu, D., Wang, Z., Chena, P., Jin, L., Cheng, Y., Zhou, J., Cao, S., 2007. Microwaveassisted resolution of (R, S)-2-octanol by enzymatic transesterification. J. Mol. Cat. B: Enzymatic 48, 51–57. Zhu, S., Wu, Y., Yu, Z., Zhang, X., Li, H., Gao, M., 2006. The effect of microwave irradiation on enzymatic hydrolysis of rice straw. Bioresour. Technol. 97, 1964– 1968.