Accepted Manuscript Ultrasound assisted Lipase catalysed synthesis of Cinnamyl acetate via transesterification reaction in a solvent free medium Prerana D. Tomke, Virendra K. Rathod PII: DOI: Reference:
S1350-4177(15)00115-7 http://dx.doi.org/10.1016/j.ultsonch.2015.04.022 ULTSON 2845
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
8 October 2014 14 April 2015 18 April 2015
Please cite this article as: P.D. Tomke, V.K. Rathod, Ultrasound assisted Lipase catalysed synthesis of Cinnamyl acetate via transesterification reaction in a solvent free medium, Ultrasonics Sonochemistry (2015), doi: http:// dx.doi.org/10.1016/j.ultsonch.2015.04.022
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Ultrasound assisted Lipase catalysed synthesis of Cinnamyl acetate via transesterification reaction in a solvent free medium
Prerana D. Tomke, Virendra K. Rathod*
Department of Chemical Engineering, Institute of Chemical Technology, Matunga (E), Mumbai-400019, India. *Corresponding Author: Dr. Virendra K. Rathod E-mail: [email protected] , Phone: +91-22-33612020, Fax: 91-22-33611020
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Abstract Cinnamyl acetate is known for its use as flavor and fragrance material in different industries such as food, pharmaceutical, cosmetic etc. This work focuses on ultrasound assisted lipase (Novozym 435) catalyzed synthesis of Cinnamyl acetate via transesterification of Cinnamyl alcohol and vinyl acetate in non-aqueous, solvent free system. Optimization of various parameters shows that a higher yield of 99.99 % can be obtained at Cinnamyl alcohol to vinyl acetate ratio of 1:2 with 0.2% of catalyst, at 40°C and 150 rpm, with lower ultrasound power input of 50 W (Ultrasound intensity 0.81 W/cm²), at 25 kHz frequency, 50% duty cycle. Further, the time required for the maximum conversion is reduced to 20 minutes as compared to 60 minutes of conventional process. Similarly, the enzyme can be successfully reused seven times without loss of enzyme activity. Thus, ultrasound helps to enhance the enzyme catalyzed synthesis of flavors. Keywords: Transesterification, Cinnamyl acetate, Cinnamyl alcohol, vinyl acetate, Novozym 435, Ultrasound.
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Introduction: Cinnamyl acetate is naturally found fragrance and flavoring compound from the class of phenylpropanoid. Naturally it can be found in fresh bark of cinnamon [1] and pink guava [2] as major flavoring component. It is also found in leaves of tea-tree, hyacinth and daffodil flower. It is an important ingredient used for formulation of flavors, fragrance and fine perfumes. Cinnamyl acetate is used to add flavor in beverages, condiments, toothpaste, mouthwash and also plays important role in personal care products, washing powder, perfumes. World-wide use of Cinnamyl acetate in different industries is about 100 metric ton per annum [4]. Apart from natural occurrence, Cinnamyl acetate can also be synthesized by different chemical ways like direct esterification of Cinnamyl alcohol with acetic anhydride in presence of 85-98 % p-toluene sulfonic acid which is more corrosive in nature [5]. Another process is the reaction between Cinnamyl bromide and sodium acetate in presence of tetrabutyl ammonium bromide as phase transfer catalyst at high temperature [6]. Chemical catalysts can lead to unwanted, non-specific byproducts and requires high temperature, pressure conditions to carry out the reactions. International legislation have defined that ‘‘natural’’ flavor substances can only be prepared by either physical processes (extraction from natural sources), or by enzymatic or microbial processes [7]. Thus, enzyme catalyzed esterification and transesterification are attractive alternative to chemical synthesis of fragrances and flavors. Enzyme assisted synthesis occurs at mild reaction conditions, product formed by this methods are more pure than alternative chemical methods. Since enzymes are specific in nature and no byproducts are formed, it reduces downstream processing and thus reduces overall production cost. Current researchers are targeting lipase catalyzed reaction for flavor and fragrance preparation to find out suitable biocatalyst to make this process available in large scale [8-13]. Lipases from different sources
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are currently used in different biochemical reactions including hydrolysis, esterification, transesterification, alcoholysis, acidolysis, aminolysis. Lipases can react with different substrates like lipids, glycerol, triglycerides, natural oils, esters and give various valuable products at mild conditions. Lipases possess characteristic ability of acting at the interface between aqueous and non-aqueous phases of substrates [14]. Therefore, application of these enzymes has attracted the interest of a broad range of industrial fields like Food, Pharmaceuticals and cosmetic industries [15]. Although enzymes are preferred over chemical catalyst, it is not used widely in industry due to slow reaction rate and higher cost of enzymes. Recently, various researchers have tried to improve the rate of enzymatic reaction using ultrasound [16], microwave [17], supercritical fluid [18] and ionic liquids [19]. Ultrasound is oscillating sound pressure waves with frequency higher than human hearings. Now a day, it has several applications in organic chemistry and biotechnology such as nanoparticles production, degassing, extraction, emulsification, waste water treatment etc. [20]. Enhanced physical and chemical effect of ultrasound in enzyme catalyzed reaction is due to increase in probability of collision of enzymes and substrate. Meanwhile cavitation also increases rate of mass transfer so that product diffuses faster from enzymatic sites [9-12]. It is generally called as green technology due to its high efficiency, economic performance and low instrumental requirement. Also, it significantly reduces the process time as compared to other conventional methods [13]. Hence, in the present work, the application of ultrasound for the synthesis of cinnamyl aceate has been explored. The transesterification between cinnamyl alcohol and vinyl acetate catalyzed by immobilized lipase B from Candida antarctica, commercially known as Novozym435 has been recently optimized using standard mechanical mixing [21]. Results were positive by using 4
toluene as solvent where reaction rate and reuse of biocatalyst were low to justify its use at industrial level, requires further optimization of process.
2. Materials and methods 2.1. Materials Lipase B from Candida antarctica immobilized on a macroporous resin (Novozym 435) was kindly donated as sample by Zytex, Mumbai. Vinyl acetate, Iso octane, n-butanol, oleic acid were purchased from S.D. Fine Chemicals Pvt. Ltd., Mumbai, India. Cinnamyl alcohol and ndecane were purchased from TCI Chemicals India Pvt. Ltd., Mumbai. Molecular sieves 5A0 were purchased from Racro chemicals, Mumbai. All chemicals and enzymes were used without any further modification. n-decane was used as internal standard in gas chromatography 2.1. Experimental setup The experimental set up of ultrasound assisted enzymatic transesterification reaction consisted of mechanically agitated flat bottom glass reactor of 50 cm3 capacity, equipped with four baffles and three bladed turbine impeller. The agitation is provided by means of electric motor with speed control system. The entire reactor assembly was immersed in an ultrasonic thermostatic water bath (Model No. 6-SL200H/DTC/DF – manufactured by Dakshin India Pvt. Ltd.) which was maintained at desired temperature with an accuracy of ±1°C. The reactor was kept in the ultrasonic bath in such a way that it there is a clearance of 2 cm between reactor bottom and ultrasound bath bottom [22]. A typical reaction mixture consists of 1:2 moles of cinnamyl alcohol and vinyl acetate respectively and total reaction volume is maintained at 15 cm3 without use of any solvent. The reaction mixture was agitated at 40°C for 5 min at a speed of 150 rpm then, 0.2% (w/v) enzyme
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was added with constant stirring to initiate the reaction. Very small liquid samples without any catalyst particle were periodically withdrawn from reaction mixture. After completion of the reaction remaining reaction mixture was filtered through Whatman filter paper and enzyme is washed several times with acetone. The washed enzyme was kept in a desiccator for 24 h and then further used for determination of enzyme activity. After complete transesterification, the reaction mixture consists of product cinnamyl acetate, by product acetaldehyde and any traces of reactant material. In order to check the reproducibility of experiments, all experiments were performed three times and average values with standard deviation have been reported in figures.
2.2. Analytical method 2.2.1 Gas Chromatography (GC) Liquid samples collected during reaction were analyzed by GC (Chemito 8610) equipped with flame ionization detector using 4 m × 3.8 mm stainless steel column packed with 10 % SE-30 stationary phase. Nitrogen was used as carrier gas at a flow rate of 1cm3 min-1. The temperature program used was as follows: Initial temperature 40°C for 0 min; 17°C/min up to 1500 C; 120C/min up to 1900C; then steady temperature for 1 min. The injector and detector temperatures were kept at 2900C. n-decane was used as internal standard which was added in reaction mixture. n-decane didn’t react with reaction mixture. Percentage conversion was calculated based on area under curve of limiting reactant as follows: (%) =
[(A0 /I0 ) − (A/I)] X 100 A0 /I0
whereas A0, A = area under curve of limiting reactant at time t=0 and t = t min, I0, I = area under curve of internal standard at time t=0 and t = t min.
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2.2.2 Enzyme activity To determine transesterification activity of an immobilized lipase 200 mg of vacuum dried lipase material was added to conical flask containing a mixture of 0.32 mL oleic acid, 0.27 mL dry nbutanol in 3 mL dry isooctane and 0.05 mL distilled water. The flask was kept at a temperature of 300C and shake at 250 rpm for 60 min. The reaction was stopped with addition of 10 mL methanol and immediately titrated against 0.05 M alcoholic NaOH and phenolphthalein indicator [23]. One unit of enzyme activity is defined as 1 mole of oleic acid consumed in reaction per min per mg lipase. Enzyme activity (Ea) =
" × $ × 100 E×T
Where, V= difference in volume in ml of NaOH between the blank and samples which is a measure of oleic acid consumed during reaction. M = molarity of NaOH, E = amount of enzyme employed in mg, T = time of reaction, min.
3. Results and discussion 3.1. Effect of mole ratio Various experiments were performed to obtain highest conversion of transesterification reaction with varying mole ratio of cinnamyl alcohol to vinyl acetate. Reaction was carried out at 40°C using ultrasonic water bath with 0.3% enzyme loading in a solvent free system. Throughout experiment reaction volume was kept constant at 15 cm3. Fig. 1 depicts the effect of variation in the ratio of cinnamyl alcohol to vinyl acetate which was varied from 1:1 to 1:4. It is observed 7
that conversion increases with an increase in concentration of vinyl acetate indicating no enzyme inhibition. An increase in concentration of vinyl acetate reduces the viscosity and surface tension of reaction mixture due to which molecules get scattered and reacts easily with active sites of enzyme. Thus, molecules get more exposed to ultrasonic irradiation, formation of stronger cavitation collapse occurs in less viscous solution. The circulation current and turbulence formed due to cavitation results in to proper mixing showing increase in mass transfer which reduces the reaction time [24]. This indicates that higher concentration of vinyl acetate helps to improve percent conversion. On the other hand when the reaction concentration of vinyl acetate was kept constant and cinnamyl alcohol was changed by varying the ratio of vinyl acetate to cinnamyl alcohol from 1:0.5 to 1:2, the percent conversion decreases with an increase in the concentration of cinnamyl alcohol (Fig. 2). Thus, decreases in the concentration of cinnamyl alcohol mixture increases the viscosity of reaction mixture, resulting in to covering of active sites of catalyst with increase in surface tension of reacting molecule. As cinnamyl alcohol possess higher polarity than vinyl acetate, alcohol binds with active sites of enzyme preventing the formation of ester which results in to lower present conversion [25]. 3.2 Effect of enzyme loading In the case of enzyme catalyzed synthesis, the amount of enzyme used is most important factor for industrial application as the cost of the process is depended on amount of enzyme used. Thus, effect of enzyme loading was studied for different enzyme concentration such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5% (w/v) of the reaction volume and results obtained are given in Fig. 3. As expected, the conversion increases with enzyme loading up to 0.2% and it goes on decreasing with an increase in enzyme concentration after 0.2 %. As concentration of enzyme increases it starts to form aggregates of enzyme which leads to decrease in exposed surface area to reactant
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and mass transfer. Enzyme particles present at inner side of aggregates do not react with substrate leads to mass transfer limitations [26]. Thus, 0.2 % enzyme loading was considered as a optimum. 3.3 Effect of temperature Temperature is an important parameter for enzyme catalyst synthesis either in presence or absence of ultrasound. Generally, higher temperature helps to increase the interaction between reactant molecule and catalyst by enhancing solubility of reactant and decreasing the viscosity of the reactants [27]. At lower temperature less numbers of bubbles are formed but collision intensity is relatively high, on other hand during high temperature number of bubbles formed are more but collision intensity is very low [27]. At higher temperature surface tension decreases affecting bubble formation and collision intensity which relatively decreases mass transfer [28]. Similarly, each enzyme shows highest activity at optimum temperature, above particular optimum temperature the enzyme activity has seen to be decreased due to enzyme inhibition at higher temperature [29] and hence optimum temperature for the particular reaction needs to identify. Thus, the transesterification is carried out in presence of ultrasound at different operating temperature ranging from 30°C to 60°C. Fig. 4 shows that as temperature increases from 30°C to 40°C, the percent conversion also increases from 78% to 99% respectively and further decreases with increase in temperature above 40°C. Enzyme activity determined after the reaction gives marginal decrease in its value from 8.9 U/g at 40°C to 7.3U/g at 60°C. Therefore considering all above parameters, temperature of 40°C is selected as optimum. 3.4 Effect of agitation Effect of agitation on transesterification reaction of cinnamyl alcohol and vinyl acetate was carried out in an ultrasonic bath. Agitation in heterogeneous helps to keep the catalyst in reaction
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mixture and also reduces external mass transfer resistance. Similarly, it also provides good mixing and diffusion of reagents [29-32]. For detailed agitation study numbers of experiments were performed in presence of ultrasound by changing the agitation speed in the range of 0 rpm300 rpm. It was observed from Fig. 5 that conversion gradually increases from 23% to 99.99% for 0 rpm and 150 rpm respectively while further increase in agitation speed shows no significant change in conversion. Ultrasound irradiation combined with stirring speed resulted in achieving equilibrium within 15 min with conversion of 94%. It is shown that the rate of substrate diffusion on catalyst per unit interfacial area was higher than reaction rate per unit area. External mass transfer resistance can be controlled at optimum speed and lower temperature. Therefore intra particle diffusion limitation did not show any effect on the reaction and reaction was controlled by intrinsic enzyme kinetics only [33]. However, in absence of agitation i.e.0 rpm, turbulence created by ultrasound was not sufficient to suspend the catalyst and thus lower conversion of 23% was observed. This suggests that small amount of agitation is required to suspend the catalyst particles. 3.5 Effect of ultrasonic power and frequency It is necessary to optimize energy supplied to reaction mixture for higher conversion at lowest possible energy input as well as to avoid deactivation of catalyst due to higher ultrasound power. Hence, effect of ultrasound was studied with different power inputs in synthesis of cinnamyl acetate. Mechanism of sonication reaction consists of formation of micro-bubbles contains vapors of solvent or any volatile reagent. Bubbles collapse due to shock waves leads to enormous increase in both temperature and pressure. This influences reactivity of reactive substance present in reaction leading to increasing in product formation in a short time [34]. When intensity i.e. ultrasonic power is increased amplitude of sound waves increases and more violent collapse
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of cavitation bubbles occurs. Harsher is the collapse stronger will be jet velocity resulting in to more micro-mixing between phases of Cinnamyl alcohol and vinyl acetate [35]. As power increases energy dissipation also increases which helps to increase rate of reaction. Thus, experiments were performed to determine effect of ultrasonic power ranging from 40W, 50W, 80W, 100W with duty cycle of 50% keeping other parameter constant and results are reported in Fig.6. It is observed that the conversion increases with power till 50W (Ultrasound intensity 0.81 W/cm²) while further increases in ultrasound powers i.e. 60W (Ultrasound intensity 0.97 W/cm²) to 100W (1.75W/cm²) decreases the conversion. This could be due to the fact that at higher ultrasound power, cracking and destruction of enzyme occurs while Low ultrasound power is nondestructive and non-invasive [36]. Dissipated power was also calculated by calorimetric study where it was seen that it varies from 42 to 46.5 with power inputs of 50W (Ultrasound instensity 0.81 W/cm²) to 100W (Ultrasound intensity 1.7 W/cm²) respectively. Thus, ultrasonic power of 50W was considered as optimized .This results in to finer mixing, higher mass transfer coefficient and higher cinnamyl acetate formation. Similar experiments were performed to determine the effect of ultrasound frequency. Two frequencies 25 kHz and 40 kHz were used during reaction and results are depicted in Fig.7. It was observed that at 25 kHz the percent conversion was much higher than at 40 kHz. Similarly, dissipated power at 25 kHz and 40 kHz at 50W power was 42W and 23W, respectively. Thus power dissipation was higher in case of 25 kHz which is also responsible for higher conversion. Low frequency sound waves generate large cavitation bubbles and results in high temperature and pressure in cavitation zone. Low frequency sound waves have remarkable effects on many chemical reactions [37-39]. As frequency increases cavitation zone becomes less violent. The creation of cavitation in liquids decreases with an increase in frequency. At very high frequency,
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where the rarefaction (and compression) cycles are very short, the finite time required for the rarefaction cycle is too small to permit a bubble to grow to a size sufficient to cause disruption of the liquid [39]. 4.3 Effect of duty cycle Duty cycle is percentage of time during which ultrasonic irradiation generate on reaction mixture. Since there is a possibility to degrade enzyme due to continuous exposure of ultrasonic waves, there is a need of exposure of ultrasonic waves up to particular duty time. For synthesis of Cinnamyl acetate, the range of duty cycle used was ranging from 25%, 50%, 75% and the results obtained are shown in Fig. 8. It was observed that with an increase in percent duty cycle up to 50% i.e. 2 minute on and 2 min off exposure, leads to increase in percent conversion and further increase in total duty cycle may lead to enzyme denaturation resulting in to decrease in conversion. At higher duty cycle the conversion obtained is low which may be due to denaturation of enzyme under the exposure of ultrasonic waves [40]. 4.4 Enzyme reusability In order to reduce the cost of process, the ability of catalyst to reuse it for many cycles needs to test. For reusability study after completion of each run, enzyme is filtered followed by continuous 3-4 washing with acetone. Then enzyme is kept in desiccator at room temperature (30±2°C) for whole night after complete drying enzyme was reused for next reaction. All the reusability study was performed at optimum conditions and for 20 minutes. The conversion obtained in 20 minutes reaction after 7 cycle reduces from 99.99% to 86.27% due to loss of enzyme during washing and filtration. Time required for this ultrasonic assisted reaction was much lower than conventional reaction. In this reaction exposure of enzyme to ultrasonic waves was for very short duration resulting in to very minute loss of enzyme activity. Good yield, 12
unaltered enzyme activity and higher reusability of enzyme can be obtained by using sonication [41-42]. 5. Comparative study of ultrasound exposure vs conventional: Table 1 shows the comparison between ultrasound assisted synthesis of cinnamyl acetate with conventional enzymatic synthesis. It is seen that ultrasound assisted synthesis of cinnamyl acetate can be achieved in solvent free condition in 20 min with 99.99% conversion and enzyme can be recycled for seven times. On the other hand in conventional process the total time required was more with use of solvent and only three times reusability of catalyst was observed which not at all economically feasible. It was also observed that required agitation speed during sonication is less compared to conventional process [21]. 6. Conclusion: Enzyme catalyzed transesterification of Cinnamyl acetate in a solvent free system using enzyme lipase Novozym 435 as a catalyst has been successfully performed in presence of ultrasound. All the reactant was converted into product in 20 minutes with mole ratio of 1:2 of Cinnamyl alcohol and vinyl acetate, by using 0.2% of total volume catalyst at 40°C and agitation speed of about 150 rpm. With exposure of ultrasound waves at frequency 25 kHz, power 50 W (Ultrasound intensity 0.81 W/cm²) and 50% duty cycle, enzyme can be used seven times. Time required was much lesser as compared to conventional method. Thus, it can be stated that enzyme catalyzed reaction with ultrasound technology resulted in to a green route for synthesis of cinnamyl acetate. References [1] Kamaliroosta L., Gharachorloo M., Kamaliroosta Z. and Alimohammad Zadeh K. H. Journal of Medicinal Plants Research, 2012 Vol. 6(4), 609-614.
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[2] Steinhaus, Martin, et al. Journal of agricultural and food chemistry 2009, 57(7), 2882-2888. [3] Blad, Clara Catelijne, Journal of Chemical and Pharmaceutical Research, 2011, 3(2), 403-423 [4] Bhatia S P, Wellington GA, Cocchiara J, Lalko J, Letizia C S, Api AM. Food Chem Toxicol 2007, 45, S53–57. [5] Venu Gopal Devulapelli, Hung-Shan Weng, J. Catalysis Communications 2009, 10, 1638– 1642. [6] Devulapelli V G, Weng HS. J. Catal Commun 2009, 10, 1638–42. [7] The Council of the European Communities (1988) Council Directive 88/388/of 22 June1988. [8] Suslick, Kenneth S. 1989, pp.62-68 (p.62) [9] Lee, J.; Snyder, J. K., J. Am. Chem. Soc. 1989, 111, 1522–1524. [10] Pizzuti, L.; Piovesan, L.A.; Flores, A.F.; Quina, F.H.; Pereira, J. Ultrason. Sonochem, 2009, 16, 728–731. [11] Pizzuti, L., Martins, P.L., Ribeiro, B.A., Quina, F.H.; Pinto, E. Flores, A.F. Venzke, D. Pereira, J. Ultrason. Sonochem, 2010, 17, 34–37. [12] P. R. Gogate, J. Kabadi, Biochem. Eng. 2009, 44, 60–72. [13] E.V. Rokhina, P. Lens, J. Trends Biotechnol, 2009, 27,298–306. [14] Anishetty S. Gowtham P, Lipases, AU-KBC Research Centre, (www.au-kbc.org). [15]A. Rajendran, A. Palanisamy, V. Thangavelu, J. Biol. Technol. 2009, 52, 207–219. [16] V. Kuperkar, V. G. Lade, Arushi Prakash, V. K. Rathod, J. Mol. Cat. B: Enzymatic 2014, 99,143– 149 [17] G. D. Yadav, P. A. Thorat, Journal of Molecular Catalysis B Enzymatic 83, 16–22.
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[18] Lanjekar, K., Rathod, V.K. Journal of Environmental Chemical Engineering 2013, 1,p. 1231-1236
[19] Puthli M. S, Rathod V. K, Pandit AB. Biochemical Engineering Journal. 2006; 27(3):287– 294. [20] S. Serra, C. Fuganti, E. Brenn, Trends Biotechnol. 2005, 23 193–198 [21] G. D. Yadav, S. Devendran, Process Biochemistry 47 (2012) 496–502 [22] V. M. Kulkarni, V. K. Rathod, Ultrasonics Sonochemistry 2014, 21 606–611. [23] N. Gharat, V.K. Rathod, J. Ultrason. Sonochem. 2013, 20, 900–905. [24] Farid Chemat, Zill-e-Huma, Muhammed Kamran Khan,J.Ultrasonics Sonochemistry 2011,18,813–835 [25] N.R. Sonare, V.K. Rathod, J. Mol. Catal. B: Enzym., 2010, 66 (2),142–147. [26] Puskas J E, Chiang CK, Sen MY. Weinheim: Wiley-VCH; 2011, 313–47. [27] T. W. Charpe, V. K. Rathod, Chem. Eng. Process. 2012, 54, 37-40. [28] E. Alissandrakis, D. Daferera, P. Tarantilis, M. Polissiou, P. Harizanis, J.Food Chem. 2003,8, 575–582
[29] Yang T, Rebsdorf M, Engelrud U, Xu X. J .Agric Food Chem 53,2005,1475–8.
[30] Lemoine R., Morsi, B. I., J. Chemical Engineering 2005, 114, 9–31 [31] Reichert , C., Hoell, W. H. and Franzreb, J.Powder Technology 2004, 145, 131 – 138. [32] Ruivo, R., Paiva, A. and Simões, P. C.J. Chemical Engineering and Processing 2004, 45, 224 – 231. [33] Perry RH, Green DW, McGraw-Hill; 1984, 226. [34] Lindley, J. and Mason, T.J. Sonochemistry: Part 2, 1987, 16, 275- 311. 15
[35] Singh, A.K., Fernando, S.D. and Hernandez R., J. Energy Fuels, 21, 1161-1164 (2007) [36] Price, G. Advances in Sonochemistry, 1990, 231-287. [37]Lorimer, J.P. and Mason, T. J. Sonochemistry, 1987, 16, 239-274. [38] Mason, T. J. Sonochemistry: 1990, 342. [39] N.R. Sonare, V.K. Rathod, J. Mol. Catal. B: Enzym. 2010, 66, 142–147. [40] D.N.Avhad, S.S. Niphadkar,V.K.Rathod,Ultrason.Sonochem. 2014, 21 (2), 628-633. [41] D. Yu, L. Tian, H. Wu, S. Wang, Y. Wang, M. Dongxiao, X. Fang, J. Process Biochem. 2010,45, 519–525. [42] I. Babicz, S.F. Leite, R.O.M.A. de Souza, O.A.C. Antunes,Ultrason. Sonochem. 2010, 17, 4-6.
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List of Tables Table 1: Comparison of ultrasonication with conventional stirring method
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Table 1: Comparison of ultrasonication with conventional stirring method
Time (min)
Agitation speed (rpm)
Conversion (%)
Ultrasonication with stirring
20
150
99.99
Ultrasonication without stirring
20
0
35.21
Conventional stirring method
60
200
96.00
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Figure Caption: Fig.1. Effect of concentration of vinyl acetate.(Reaction conditions: Cinnamyl alcohol to Vinyl acetate 1:1 to1:4 ;total reaction volume 15 cm3 ; speed of agitation 150 rpm; catalyst loading0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W;50% duty cycle.) Fig.2. Effect of concentration of cinnamyl alcohol.(Reaction conditions: Vinyl acetate to Cinnamyl alcohol to 0.5:1 to 2:1;total reaction volume 15 cm3; speed of agitation 150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W;50% duty cycle.) Fig.3. Effect of enzyme loading (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.1% - 0.5% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W; 50% duty cycle.) Fig.4. Effect of temperature (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 300C – 600C;Under ultrasound waves with frequency 25kHz; power 50W; 50% duty cycle.) Fig.5. Effect of speed of agitation (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation100- 300 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W; 50% duty cycle.)
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Fig.6. Effect of ultrasonic power (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 40W - 100W; 50% duty cycle. Fig.7. Effect of ultrasonic frequency(Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz – 40kHz; power 50W; 50% duty cycle. Fig.8. Effect of duty cycle (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W; 25% - 75% duty cycle
20
Conversion (%)
100 80 60 40 20 0 0
5
10 15 Time(min)
1:01
1:02
1:03
20
25
1:04
Fig. 1. Effect of concentration of vinyl acetate.(Reaction conditions: Cinnamyl alcohol to Vinyl acetate 1:1 to1:4 ;total reaction volume 15 cm3 ; speed of agitation 150 rpm; catalyst loading0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W;50% duty cycle.)
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100
Conversion (%)
80 60 40 20 0 0
5
10
15
20
25
Time (min) 0.5:1
1:01
1.5:1
2:01
Fig. 2. Effect of concentration of cinnamyl alcohol.(Reaction conditions: Cinnamyl alcohol to Vinyl acetate to 0.5:1 to 2:1;total reaction volume 15 cm3 ; speed of agitation 150 rpm; catalyst loading0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W;50% duty cycle.)
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Conversion (%)
100 80 60 40 20 0 0
5
10
15
20
25
Time (min) 0.1%EL
0.2%EL
0.3%EL
0.4%EL
0.5%EL
Fig. 3. Effect of enzyme loading (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.1% - 0.5% of total volume; temperature 400C;Under ultrasound waves with frequency 25 kHz; power 50W; 50% duty cycle.)
23
100
conversion (%)
80
60
40
20
0 0
5
10
15
20
25
Time(min) 30°C
40°C
50°C
60°C
Fig. 4. Effect of temperature (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 300C – 600C;Under ultrasound waves with frequency 25kHz; power 50W; 50% duty cycle.)
24
100
Conversion(%)
80 60 40 20 0 0
5
10
15
20
25
Time(min) 0rpm
100rpm
150rpm
300 rpm
Fig. 5. Effect of speed of agitation (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation100- 300 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W; 50% duty cycle.
25
100
Conversion(%)
80 60 40 20 0 0
5
40W
10 15 Time(min)
50W
80W
20
25
100W
Fig. 6. Effect of ultrasonic power (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 40W - 100W; 50% duty cycle.
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100
conversion(%)
80
60
40
20
0 0
5
10 Time(min) 25 kHz
15
20
25
40kHz
Fig. 7. Effect of ultrasonic frequency(Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz - 40kHz; power 50W; 50% duty cycle).
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100
conversion(%)
80 60 40 20 0 0
5
10
15
20
25
Time(min) 25% duty cycle
50% duty cycle
75% duty cycle
Fig. 8. Effect of duty cycle (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W; 25% - 75% duty cycle).
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Highlights •
Ultrasound assisted enzymatic synthesis of cinnamyl aetate is proposed
•
Process intensification in solvent free condition is carried out.
•
Various experimental parameters have been studied
•
Ultrasound helps to reduces the time of reaction without loss of activity.
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S1350-4177(15)00115-7 http://dx.doi.org/10.1016/j.ultsonch.2015.04.022 ULTSON 2845
To appear in:
Ultrasonics Sonochemistry
Received Date: Revised Date: Accepted Date:
8 October 2014 14 April 2015 18 April 2015
Please cite this article as: P.D. Tomke, V.K. Rathod, Ultrasound assisted Lipase catalysed synthesis of Cinnamyl acetate via transesterification reaction in a solvent free medium, Ultrasonics Sonochemistry (2015), doi: http:// dx.doi.org/10.1016/j.ultsonch.2015.04.022
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Ultrasound assisted Lipase catalysed synthesis of Cinnamyl acetate via transesterification reaction in a solvent free medium
Prerana D. Tomke, Virendra K. Rathod*
Department of Chemical Engineering, Institute of Chemical Technology, Matunga (E), Mumbai-400019, India. *Corresponding Author: Dr. Virendra K. Rathod E-mail: [email protected] , Phone: +91-22-33612020, Fax: 91-22-33611020
1
Abstract Cinnamyl acetate is known for its use as flavor and fragrance material in different industries such as food, pharmaceutical, cosmetic etc. This work focuses on ultrasound assisted lipase (Novozym 435) catalyzed synthesis of Cinnamyl acetate via transesterification of Cinnamyl alcohol and vinyl acetate in non-aqueous, solvent free system. Optimization of various parameters shows that a higher yield of 99.99 % can be obtained at Cinnamyl alcohol to vinyl acetate ratio of 1:2 with 0.2% of catalyst, at 40°C and 150 rpm, with lower ultrasound power input of 50 W (Ultrasound intensity 0.81 W/cm²), at 25 kHz frequency, 50% duty cycle. Further, the time required for the maximum conversion is reduced to 20 minutes as compared to 60 minutes of conventional process. Similarly, the enzyme can be successfully reused seven times without loss of enzyme activity. Thus, ultrasound helps to enhance the enzyme catalyzed synthesis of flavors. Keywords: Transesterification, Cinnamyl acetate, Cinnamyl alcohol, vinyl acetate, Novozym 435, Ultrasound.
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Introduction: Cinnamyl acetate is naturally found fragrance and flavoring compound from the class of phenylpropanoid. Naturally it can be found in fresh bark of cinnamon [1] and pink guava [2] as major flavoring component. It is also found in leaves of tea-tree, hyacinth and daffodil flower. It is an important ingredient used for formulation of flavors, fragrance and fine perfumes. Cinnamyl acetate is used to add flavor in beverages, condiments, toothpaste, mouthwash and also plays important role in personal care products, washing powder, perfumes. World-wide use of Cinnamyl acetate in different industries is about 100 metric ton per annum [4]. Apart from natural occurrence, Cinnamyl acetate can also be synthesized by different chemical ways like direct esterification of Cinnamyl alcohol with acetic anhydride in presence of 85-98 % p-toluene sulfonic acid which is more corrosive in nature [5]. Another process is the reaction between Cinnamyl bromide and sodium acetate in presence of tetrabutyl ammonium bromide as phase transfer catalyst at high temperature [6]. Chemical catalysts can lead to unwanted, non-specific byproducts and requires high temperature, pressure conditions to carry out the reactions. International legislation have defined that ‘‘natural’’ flavor substances can only be prepared by either physical processes (extraction from natural sources), or by enzymatic or microbial processes [7]. Thus, enzyme catalyzed esterification and transesterification are attractive alternative to chemical synthesis of fragrances and flavors. Enzyme assisted synthesis occurs at mild reaction conditions, product formed by this methods are more pure than alternative chemical methods. Since enzymes are specific in nature and no byproducts are formed, it reduces downstream processing and thus reduces overall production cost. Current researchers are targeting lipase catalyzed reaction for flavor and fragrance preparation to find out suitable biocatalyst to make this process available in large scale [8-13]. Lipases from different sources
3
are currently used in different biochemical reactions including hydrolysis, esterification, transesterification, alcoholysis, acidolysis, aminolysis. Lipases can react with different substrates like lipids, glycerol, triglycerides, natural oils, esters and give various valuable products at mild conditions. Lipases possess characteristic ability of acting at the interface between aqueous and non-aqueous phases of substrates [14]. Therefore, application of these enzymes has attracted the interest of a broad range of industrial fields like Food, Pharmaceuticals and cosmetic industries [15]. Although enzymes are preferred over chemical catalyst, it is not used widely in industry due to slow reaction rate and higher cost of enzymes. Recently, various researchers have tried to improve the rate of enzymatic reaction using ultrasound [16], microwave [17], supercritical fluid [18] and ionic liquids [19]. Ultrasound is oscillating sound pressure waves with frequency higher than human hearings. Now a day, it has several applications in organic chemistry and biotechnology such as nanoparticles production, degassing, extraction, emulsification, waste water treatment etc. [20]. Enhanced physical and chemical effect of ultrasound in enzyme catalyzed reaction is due to increase in probability of collision of enzymes and substrate. Meanwhile cavitation also increases rate of mass transfer so that product diffuses faster from enzymatic sites [9-12]. It is generally called as green technology due to its high efficiency, economic performance and low instrumental requirement. Also, it significantly reduces the process time as compared to other conventional methods [13]. Hence, in the present work, the application of ultrasound for the synthesis of cinnamyl aceate has been explored. The transesterification between cinnamyl alcohol and vinyl acetate catalyzed by immobilized lipase B from Candida antarctica, commercially known as Novozym435 has been recently optimized using standard mechanical mixing [21]. Results were positive by using 4
toluene as solvent where reaction rate and reuse of biocatalyst were low to justify its use at industrial level, requires further optimization of process.
2. Materials and methods 2.1. Materials Lipase B from Candida antarctica immobilized on a macroporous resin (Novozym 435) was kindly donated as sample by Zytex, Mumbai. Vinyl acetate, Iso octane, n-butanol, oleic acid were purchased from S.D. Fine Chemicals Pvt. Ltd., Mumbai, India. Cinnamyl alcohol and ndecane were purchased from TCI Chemicals India Pvt. Ltd., Mumbai. Molecular sieves 5A0 were purchased from Racro chemicals, Mumbai. All chemicals and enzymes were used without any further modification. n-decane was used as internal standard in gas chromatography 2.1. Experimental setup The experimental set up of ultrasound assisted enzymatic transesterification reaction consisted of mechanically agitated flat bottom glass reactor of 50 cm3 capacity, equipped with four baffles and three bladed turbine impeller. The agitation is provided by means of electric motor with speed control system. The entire reactor assembly was immersed in an ultrasonic thermostatic water bath (Model No. 6-SL200H/DTC/DF – manufactured by Dakshin India Pvt. Ltd.) which was maintained at desired temperature with an accuracy of ±1°C. The reactor was kept in the ultrasonic bath in such a way that it there is a clearance of 2 cm between reactor bottom and ultrasound bath bottom [22]. A typical reaction mixture consists of 1:2 moles of cinnamyl alcohol and vinyl acetate respectively and total reaction volume is maintained at 15 cm3 without use of any solvent. The reaction mixture was agitated at 40°C for 5 min at a speed of 150 rpm then, 0.2% (w/v) enzyme
5
was added with constant stirring to initiate the reaction. Very small liquid samples without any catalyst particle were periodically withdrawn from reaction mixture. After completion of the reaction remaining reaction mixture was filtered through Whatman filter paper and enzyme is washed several times with acetone. The washed enzyme was kept in a desiccator for 24 h and then further used for determination of enzyme activity. After complete transesterification, the reaction mixture consists of product cinnamyl acetate, by product acetaldehyde and any traces of reactant material. In order to check the reproducibility of experiments, all experiments were performed three times and average values with standard deviation have been reported in figures.
2.2. Analytical method 2.2.1 Gas Chromatography (GC) Liquid samples collected during reaction were analyzed by GC (Chemito 8610) equipped with flame ionization detector using 4 m × 3.8 mm stainless steel column packed with 10 % SE-30 stationary phase. Nitrogen was used as carrier gas at a flow rate of 1cm3 min-1. The temperature program used was as follows: Initial temperature 40°C for 0 min; 17°C/min up to 1500 C; 120C/min up to 1900C; then steady temperature for 1 min. The injector and detector temperatures were kept at 2900C. n-decane was used as internal standard which was added in reaction mixture. n-decane didn’t react with reaction mixture. Percentage conversion was calculated based on area under curve of limiting reactant as follows: (%) =
[(A0 /I0 ) − (A/I)] X 100 A0 /I0
whereas A0, A = area under curve of limiting reactant at time t=0 and t = t min, I0, I = area under curve of internal standard at time t=0 and t = t min.
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2.2.2 Enzyme activity To determine transesterification activity of an immobilized lipase 200 mg of vacuum dried lipase material was added to conical flask containing a mixture of 0.32 mL oleic acid, 0.27 mL dry nbutanol in 3 mL dry isooctane and 0.05 mL distilled water. The flask was kept at a temperature of 300C and shake at 250 rpm for 60 min. The reaction was stopped with addition of 10 mL methanol and immediately titrated against 0.05 M alcoholic NaOH and phenolphthalein indicator [23]. One unit of enzyme activity is defined as 1 mole of oleic acid consumed in reaction per min per mg lipase. Enzyme activity (Ea) =
" × $ × 100 E×T
Where, V= difference in volume in ml of NaOH between the blank and samples which is a measure of oleic acid consumed during reaction. M = molarity of NaOH, E = amount of enzyme employed in mg, T = time of reaction, min.
3. Results and discussion 3.1. Effect of mole ratio Various experiments were performed to obtain highest conversion of transesterification reaction with varying mole ratio of cinnamyl alcohol to vinyl acetate. Reaction was carried out at 40°C using ultrasonic water bath with 0.3% enzyme loading in a solvent free system. Throughout experiment reaction volume was kept constant at 15 cm3. Fig. 1 depicts the effect of variation in the ratio of cinnamyl alcohol to vinyl acetate which was varied from 1:1 to 1:4. It is observed 7
that conversion increases with an increase in concentration of vinyl acetate indicating no enzyme inhibition. An increase in concentration of vinyl acetate reduces the viscosity and surface tension of reaction mixture due to which molecules get scattered and reacts easily with active sites of enzyme. Thus, molecules get more exposed to ultrasonic irradiation, formation of stronger cavitation collapse occurs in less viscous solution. The circulation current and turbulence formed due to cavitation results in to proper mixing showing increase in mass transfer which reduces the reaction time [24]. This indicates that higher concentration of vinyl acetate helps to improve percent conversion. On the other hand when the reaction concentration of vinyl acetate was kept constant and cinnamyl alcohol was changed by varying the ratio of vinyl acetate to cinnamyl alcohol from 1:0.5 to 1:2, the percent conversion decreases with an increase in the concentration of cinnamyl alcohol (Fig. 2). Thus, decreases in the concentration of cinnamyl alcohol mixture increases the viscosity of reaction mixture, resulting in to covering of active sites of catalyst with increase in surface tension of reacting molecule. As cinnamyl alcohol possess higher polarity than vinyl acetate, alcohol binds with active sites of enzyme preventing the formation of ester which results in to lower present conversion [25]. 3.2 Effect of enzyme loading In the case of enzyme catalyzed synthesis, the amount of enzyme used is most important factor for industrial application as the cost of the process is depended on amount of enzyme used. Thus, effect of enzyme loading was studied for different enzyme concentration such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5% (w/v) of the reaction volume and results obtained are given in Fig. 3. As expected, the conversion increases with enzyme loading up to 0.2% and it goes on decreasing with an increase in enzyme concentration after 0.2 %. As concentration of enzyme increases it starts to form aggregates of enzyme which leads to decrease in exposed surface area to reactant
8
and mass transfer. Enzyme particles present at inner side of aggregates do not react with substrate leads to mass transfer limitations [26]. Thus, 0.2 % enzyme loading was considered as a optimum. 3.3 Effect of temperature Temperature is an important parameter for enzyme catalyst synthesis either in presence or absence of ultrasound. Generally, higher temperature helps to increase the interaction between reactant molecule and catalyst by enhancing solubility of reactant and decreasing the viscosity of the reactants [27]. At lower temperature less numbers of bubbles are formed but collision intensity is relatively high, on other hand during high temperature number of bubbles formed are more but collision intensity is very low [27]. At higher temperature surface tension decreases affecting bubble formation and collision intensity which relatively decreases mass transfer [28]. Similarly, each enzyme shows highest activity at optimum temperature, above particular optimum temperature the enzyme activity has seen to be decreased due to enzyme inhibition at higher temperature [29] and hence optimum temperature for the particular reaction needs to identify. Thus, the transesterification is carried out in presence of ultrasound at different operating temperature ranging from 30°C to 60°C. Fig. 4 shows that as temperature increases from 30°C to 40°C, the percent conversion also increases from 78% to 99% respectively and further decreases with increase in temperature above 40°C. Enzyme activity determined after the reaction gives marginal decrease in its value from 8.9 U/g at 40°C to 7.3U/g at 60°C. Therefore considering all above parameters, temperature of 40°C is selected as optimum. 3.4 Effect of agitation Effect of agitation on transesterification reaction of cinnamyl alcohol and vinyl acetate was carried out in an ultrasonic bath. Agitation in heterogeneous helps to keep the catalyst in reaction
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mixture and also reduces external mass transfer resistance. Similarly, it also provides good mixing and diffusion of reagents [29-32]. For detailed agitation study numbers of experiments were performed in presence of ultrasound by changing the agitation speed in the range of 0 rpm300 rpm. It was observed from Fig. 5 that conversion gradually increases from 23% to 99.99% for 0 rpm and 150 rpm respectively while further increase in agitation speed shows no significant change in conversion. Ultrasound irradiation combined with stirring speed resulted in achieving equilibrium within 15 min with conversion of 94%. It is shown that the rate of substrate diffusion on catalyst per unit interfacial area was higher than reaction rate per unit area. External mass transfer resistance can be controlled at optimum speed and lower temperature. Therefore intra particle diffusion limitation did not show any effect on the reaction and reaction was controlled by intrinsic enzyme kinetics only [33]. However, in absence of agitation i.e.0 rpm, turbulence created by ultrasound was not sufficient to suspend the catalyst and thus lower conversion of 23% was observed. This suggests that small amount of agitation is required to suspend the catalyst particles. 3.5 Effect of ultrasonic power and frequency It is necessary to optimize energy supplied to reaction mixture for higher conversion at lowest possible energy input as well as to avoid deactivation of catalyst due to higher ultrasound power. Hence, effect of ultrasound was studied with different power inputs in synthesis of cinnamyl acetate. Mechanism of sonication reaction consists of formation of micro-bubbles contains vapors of solvent or any volatile reagent. Bubbles collapse due to shock waves leads to enormous increase in both temperature and pressure. This influences reactivity of reactive substance present in reaction leading to increasing in product formation in a short time [34]. When intensity i.e. ultrasonic power is increased amplitude of sound waves increases and more violent collapse
10
of cavitation bubbles occurs. Harsher is the collapse stronger will be jet velocity resulting in to more micro-mixing between phases of Cinnamyl alcohol and vinyl acetate [35]. As power increases energy dissipation also increases which helps to increase rate of reaction. Thus, experiments were performed to determine effect of ultrasonic power ranging from 40W, 50W, 80W, 100W with duty cycle of 50% keeping other parameter constant and results are reported in Fig.6. It is observed that the conversion increases with power till 50W (Ultrasound intensity 0.81 W/cm²) while further increases in ultrasound powers i.e. 60W (Ultrasound intensity 0.97 W/cm²) to 100W (1.75W/cm²) decreases the conversion. This could be due to the fact that at higher ultrasound power, cracking and destruction of enzyme occurs while Low ultrasound power is nondestructive and non-invasive [36]. Dissipated power was also calculated by calorimetric study where it was seen that it varies from 42 to 46.5 with power inputs of 50W (Ultrasound instensity 0.81 W/cm²) to 100W (Ultrasound intensity 1.7 W/cm²) respectively. Thus, ultrasonic power of 50W was considered as optimized .This results in to finer mixing, higher mass transfer coefficient and higher cinnamyl acetate formation. Similar experiments were performed to determine the effect of ultrasound frequency. Two frequencies 25 kHz and 40 kHz were used during reaction and results are depicted in Fig.7. It was observed that at 25 kHz the percent conversion was much higher than at 40 kHz. Similarly, dissipated power at 25 kHz and 40 kHz at 50W power was 42W and 23W, respectively. Thus power dissipation was higher in case of 25 kHz which is also responsible for higher conversion. Low frequency sound waves generate large cavitation bubbles and results in high temperature and pressure in cavitation zone. Low frequency sound waves have remarkable effects on many chemical reactions [37-39]. As frequency increases cavitation zone becomes less violent. The creation of cavitation in liquids decreases with an increase in frequency. At very high frequency,
11
where the rarefaction (and compression) cycles are very short, the finite time required for the rarefaction cycle is too small to permit a bubble to grow to a size sufficient to cause disruption of the liquid [39]. 4.3 Effect of duty cycle Duty cycle is percentage of time during which ultrasonic irradiation generate on reaction mixture. Since there is a possibility to degrade enzyme due to continuous exposure of ultrasonic waves, there is a need of exposure of ultrasonic waves up to particular duty time. For synthesis of Cinnamyl acetate, the range of duty cycle used was ranging from 25%, 50%, 75% and the results obtained are shown in Fig. 8. It was observed that with an increase in percent duty cycle up to 50% i.e. 2 minute on and 2 min off exposure, leads to increase in percent conversion and further increase in total duty cycle may lead to enzyme denaturation resulting in to decrease in conversion. At higher duty cycle the conversion obtained is low which may be due to denaturation of enzyme under the exposure of ultrasonic waves [40]. 4.4 Enzyme reusability In order to reduce the cost of process, the ability of catalyst to reuse it for many cycles needs to test. For reusability study after completion of each run, enzyme is filtered followed by continuous 3-4 washing with acetone. Then enzyme is kept in desiccator at room temperature (30±2°C) for whole night after complete drying enzyme was reused for next reaction. All the reusability study was performed at optimum conditions and for 20 minutes. The conversion obtained in 20 minutes reaction after 7 cycle reduces from 99.99% to 86.27% due to loss of enzyme during washing and filtration. Time required for this ultrasonic assisted reaction was much lower than conventional reaction. In this reaction exposure of enzyme to ultrasonic waves was for very short duration resulting in to very minute loss of enzyme activity. Good yield, 12
unaltered enzyme activity and higher reusability of enzyme can be obtained by using sonication [41-42]. 5. Comparative study of ultrasound exposure vs conventional: Table 1 shows the comparison between ultrasound assisted synthesis of cinnamyl acetate with conventional enzymatic synthesis. It is seen that ultrasound assisted synthesis of cinnamyl acetate can be achieved in solvent free condition in 20 min with 99.99% conversion and enzyme can be recycled for seven times. On the other hand in conventional process the total time required was more with use of solvent and only three times reusability of catalyst was observed which not at all economically feasible. It was also observed that required agitation speed during sonication is less compared to conventional process [21]. 6. Conclusion: Enzyme catalyzed transesterification of Cinnamyl acetate in a solvent free system using enzyme lipase Novozym 435 as a catalyst has been successfully performed in presence of ultrasound. All the reactant was converted into product in 20 minutes with mole ratio of 1:2 of Cinnamyl alcohol and vinyl acetate, by using 0.2% of total volume catalyst at 40°C and agitation speed of about 150 rpm. With exposure of ultrasound waves at frequency 25 kHz, power 50 W (Ultrasound intensity 0.81 W/cm²) and 50% duty cycle, enzyme can be used seven times. Time required was much lesser as compared to conventional method. Thus, it can be stated that enzyme catalyzed reaction with ultrasound technology resulted in to a green route for synthesis of cinnamyl acetate. References [1] Kamaliroosta L., Gharachorloo M., Kamaliroosta Z. and Alimohammad Zadeh K. H. Journal of Medicinal Plants Research, 2012 Vol. 6(4), 609-614.
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[2] Steinhaus, Martin, et al. Journal of agricultural and food chemistry 2009, 57(7), 2882-2888. [3] Blad, Clara Catelijne, Journal of Chemical and Pharmaceutical Research, 2011, 3(2), 403-423 [4] Bhatia S P, Wellington GA, Cocchiara J, Lalko J, Letizia C S, Api AM. Food Chem Toxicol 2007, 45, S53–57. [5] Venu Gopal Devulapelli, Hung-Shan Weng, J. Catalysis Communications 2009, 10, 1638– 1642. [6] Devulapelli V G, Weng HS. J. Catal Commun 2009, 10, 1638–42. [7] The Council of the European Communities (1988) Council Directive 88/388/of 22 June1988. [8] Suslick, Kenneth S. 1989, pp.62-68 (p.62) [9] Lee, J.; Snyder, J. K., J. Am. Chem. Soc. 1989, 111, 1522–1524. [10] Pizzuti, L.; Piovesan, L.A.; Flores, A.F.; Quina, F.H.; Pereira, J. Ultrason. Sonochem, 2009, 16, 728–731. [11] Pizzuti, L., Martins, P.L., Ribeiro, B.A., Quina, F.H.; Pinto, E. Flores, A.F. Venzke, D. Pereira, J. Ultrason. Sonochem, 2010, 17, 34–37. [12] P. R. Gogate, J. Kabadi, Biochem. Eng. 2009, 44, 60–72. [13] E.V. Rokhina, P. Lens, J. Trends Biotechnol, 2009, 27,298–306. [14] Anishetty S. Gowtham P, Lipases, AU-KBC Research Centre, (www.au-kbc.org). [15]A. Rajendran, A. Palanisamy, V. Thangavelu, J. Biol. Technol. 2009, 52, 207–219. [16] V. Kuperkar, V. G. Lade, Arushi Prakash, V. K. Rathod, J. Mol. Cat. B: Enzymatic 2014, 99,143– 149 [17] G. D. Yadav, P. A. Thorat, Journal of Molecular Catalysis B Enzymatic 83, 16–22.
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[18] Lanjekar, K., Rathod, V.K. Journal of Environmental Chemical Engineering 2013, 1,p. 1231-1236
[19] Puthli M. S, Rathod V. K, Pandit AB. Biochemical Engineering Journal. 2006; 27(3):287– 294. [20] S. Serra, C. Fuganti, E. Brenn, Trends Biotechnol. 2005, 23 193–198 [21] G. D. Yadav, S. Devendran, Process Biochemistry 47 (2012) 496–502 [22] V. M. Kulkarni, V. K. Rathod, Ultrasonics Sonochemistry 2014, 21 606–611. [23] N. Gharat, V.K. Rathod, J. Ultrason. Sonochem. 2013, 20, 900–905. [24] Farid Chemat, Zill-e-Huma, Muhammed Kamran Khan,J.Ultrasonics Sonochemistry 2011,18,813–835 [25] N.R. Sonare, V.K. Rathod, J. Mol. Catal. B: Enzym., 2010, 66 (2),142–147. [26] Puskas J E, Chiang CK, Sen MY. Weinheim: Wiley-VCH; 2011, 313–47. [27] T. W. Charpe, V. K. Rathod, Chem. Eng. Process. 2012, 54, 37-40. [28] E. Alissandrakis, D. Daferera, P. Tarantilis, M. Polissiou, P. Harizanis, J.Food Chem. 2003,8, 575–582
[29] Yang T, Rebsdorf M, Engelrud U, Xu X. J .Agric Food Chem 53,2005,1475–8.
[30] Lemoine R., Morsi, B. I., J. Chemical Engineering 2005, 114, 9–31 [31] Reichert , C., Hoell, W. H. and Franzreb, J.Powder Technology 2004, 145, 131 – 138. [32] Ruivo, R., Paiva, A. and Simões, P. C.J. Chemical Engineering and Processing 2004, 45, 224 – 231. [33] Perry RH, Green DW, McGraw-Hill; 1984, 226. [34] Lindley, J. and Mason, T.J. Sonochemistry: Part 2, 1987, 16, 275- 311. 15
[35] Singh, A.K., Fernando, S.D. and Hernandez R., J. Energy Fuels, 21, 1161-1164 (2007) [36] Price, G. Advances in Sonochemistry, 1990, 231-287. [37]Lorimer, J.P. and Mason, T. J. Sonochemistry, 1987, 16, 239-274. [38] Mason, T. J. Sonochemistry: 1990, 342. [39] N.R. Sonare, V.K. Rathod, J. Mol. Catal. B: Enzym. 2010, 66, 142–147. [40] D.N.Avhad, S.S. Niphadkar,V.K.Rathod,Ultrason.Sonochem. 2014, 21 (2), 628-633. [41] D. Yu, L. Tian, H. Wu, S. Wang, Y. Wang, M. Dongxiao, X. Fang, J. Process Biochem. 2010,45, 519–525. [42] I. Babicz, S.F. Leite, R.O.M.A. de Souza, O.A.C. Antunes,Ultrason. Sonochem. 2010, 17, 4-6.
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List of Tables Table 1: Comparison of ultrasonication with conventional stirring method
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Table 1: Comparison of ultrasonication with conventional stirring method
Time (min)
Agitation speed (rpm)
Conversion (%)
Ultrasonication with stirring
20
150
99.99
Ultrasonication without stirring
20
0
35.21
Conventional stirring method
60
200
96.00
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Figure Caption: Fig.1. Effect of concentration of vinyl acetate.(Reaction conditions: Cinnamyl alcohol to Vinyl acetate 1:1 to1:4 ;total reaction volume 15 cm3 ; speed of agitation 150 rpm; catalyst loading0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W;50% duty cycle.) Fig.2. Effect of concentration of cinnamyl alcohol.(Reaction conditions: Vinyl acetate to Cinnamyl alcohol to 0.5:1 to 2:1;total reaction volume 15 cm3; speed of agitation 150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W;50% duty cycle.) Fig.3. Effect of enzyme loading (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.1% - 0.5% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W; 50% duty cycle.) Fig.4. Effect of temperature (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 300C – 600C;Under ultrasound waves with frequency 25kHz; power 50W; 50% duty cycle.) Fig.5. Effect of speed of agitation (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation100- 300 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W; 50% duty cycle.)
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Fig.6. Effect of ultrasonic power (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 40W - 100W; 50% duty cycle. Fig.7. Effect of ultrasonic frequency(Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz – 40kHz; power 50W; 50% duty cycle. Fig.8. Effect of duty cycle (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W; 25% - 75% duty cycle
20
Conversion (%)
100 80 60 40 20 0 0
5
10 15 Time(min)
1:01
1:02
1:03
20
25
1:04
Fig. 1. Effect of concentration of vinyl acetate.(Reaction conditions: Cinnamyl alcohol to Vinyl acetate 1:1 to1:4 ;total reaction volume 15 cm3 ; speed of agitation 150 rpm; catalyst loading0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W;50% duty cycle.)
21
100
Conversion (%)
80 60 40 20 0 0
5
10
15
20
25
Time (min) 0.5:1
1:01
1.5:1
2:01
Fig. 2. Effect of concentration of cinnamyl alcohol.(Reaction conditions: Cinnamyl alcohol to Vinyl acetate to 0.5:1 to 2:1;total reaction volume 15 cm3 ; speed of agitation 150 rpm; catalyst loading0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W;50% duty cycle.)
22
Conversion (%)
100 80 60 40 20 0 0
5
10
15
20
25
Time (min) 0.1%EL
0.2%EL
0.3%EL
0.4%EL
0.5%EL
Fig. 3. Effect of enzyme loading (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.1% - 0.5% of total volume; temperature 400C;Under ultrasound waves with frequency 25 kHz; power 50W; 50% duty cycle.)
23
100
conversion (%)
80
60
40
20
0 0
5
10
15
20
25
Time(min) 30°C
40°C
50°C
60°C
Fig. 4. Effect of temperature (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 300C – 600C;Under ultrasound waves with frequency 25kHz; power 50W; 50% duty cycle.)
24
100
Conversion(%)
80 60 40 20 0 0
5
10
15
20
25
Time(min) 0rpm
100rpm
150rpm
300 rpm
Fig. 5. Effect of speed of agitation (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation100- 300 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W; 50% duty cycle.
25
100
Conversion(%)
80 60 40 20 0 0
5
40W
10 15 Time(min)
50W
80W
20
25
100W
Fig. 6. Effect of ultrasonic power (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 40W - 100W; 50% duty cycle.
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100
conversion(%)
80
60
40
20
0 0
5
10 Time(min) 25 kHz
15
20
25
40kHz
Fig. 7. Effect of ultrasonic frequency(Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz - 40kHz; power 50W; 50% duty cycle).
27
100
conversion(%)
80 60 40 20 0 0
5
10
15
20
25
Time(min) 25% duty cycle
50% duty cycle
75% duty cycle
Fig. 8. Effect of duty cycle (Reaction conditions: Cinnamyl alcohol to Vinyl acetate ratio1:2; total reaction volume 15 cm3; speed of agitation150 rpm; catalyst loading 0.2% of total volume; temperature 400C;Under ultrasound waves with frequency 25kHz; power 50W; 25% - 75% duty cycle).
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Highlights •
Ultrasound assisted enzymatic synthesis of cinnamyl aetate is proposed
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Process intensification in solvent free condition is carried out.
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Various experimental parameters have been studied
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Ultrasound helps to reduces the time of reaction without loss of activity.
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