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Ultrasound assisted lipase catalysed synthesis of isoamyl butyrate
Accepted Manuscript Title: Ultrasound assisted lipase catalysed synthesis of Isoamyl butyrate Author: Sneha R. Bansode Virendra K. Rathod PII: DOI: Reference:
S1359-5113(14)00266-9 http://dx.doi.org/doi:10.1016/j.procbio.2014.04.018 PRBI 10129
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
14-1-2014 15-4-2014 22-4-2014
Please cite this article as: Bansode SR, Rathod VK, Ultrasound assisted lipase catalysed synthesis of Isoamyl butyrate, Process Biochemistry (2014), http://dx.doi.org/10.1016/j.procbio.2014.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ultrasound assisted lipase catalysed synthesis of Isoamyl butyrate
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Sneha R. Bansode, Virendra K. Rathod
*Corresponding author Dr. V. K. Rathod
Department of Chemical Engineering Institute of Chemical Technology, Matunga (E), Mumbai-400019, India. E-mail: [email protected] ,
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Phone: +91-22-33612020, Fax: 91-22-33611020.
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Abstract: The present work illustrates the incorporation of ultrasound and its improved impact in the lipase catalysed esterification. Synthesis of isoamyl butyrate from isoamyl alcohol and
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butyric acid using immobilized Novozym 435, has been carried out in the presence of ultrasound. The optimisation of various parameters affecting the synthesis of ester in
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presence of ultrasound was done. The systematic experimentation involves change of one
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working parameter at one time while keeping the others constant. For the maximum conversion, optimum parameters such as the ultrasound of 25 kHz frequency with power of
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70 W, at the temperature of 60˚C with stirring speed of 80 rpm, mole ratio of alcohol: acid followed as 2:1, use of molecular sieves weighing 2g, with immobilized enzyme loading of
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2% (m/v) and duty cycle of 83%, were obtained. The optimum parameters collectively, gave 96% conversion of the product in 3 h as compared with 10 h in absence of ultrasound. The
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immobilized biocatalyst, Novozym 435 has an added benefit of reusability till 7 repetitive
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cycles. Besides, the synthesis is executed in the solvent free system that contributes the
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production of flavour in greener way.
Keywords: Isoamyl butyrate, Ultrasound, immobilised lipase, Novozym 435, Fruity Banana flavour, optimisation
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1. Introduction: Short chain esters derived from the alcohol and an acid form flavour and perfume that have their essence in food industry, pharmaceutical industry, cosmetics etc. [1]. Subsequently, the
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natural flavours are costly due to limited availability of raw material, preservation and processing cost and the traditionally extracted flavours cannot meet the increasing demands
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due to their low yield [2]. Thus, the artificial flavours are in demand as they are synthesised
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easily and cheaply available. Isoamyl butyrate, a fruity banana flavour is well known in food industry especially in fruit jams, fruit punch and analogous preparations [3]. The annual
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demand for isoamyl butyrate reported in 1989 was 13,792 kg/annum from around the globe by welsh et al, the figure now reached 2,00,000 kg/year in Columbia alone. The ester,
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isoamyl butyrate can be obtained from chemical interaction of the two substrate, isoamyl alcohol and butyric acid in presence of catalyst. If the ester happens to be a synthetic
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flavouring agent in food components, it must be free of any harmful acidulous catalyst and
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accede with the food grade implementations [4]. Esterification from catalysis using P-toulene
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sulphonic acid [5], Amberlyst [6] and P tolune sulphonic acid calix [n] arenes [7] has been tried out. Triacyl acyl hydrolases, (EC 3.1.1.3) or precisely known as lipases have surfaced its applications due to targeted cut down of side product and production of desired product in plenty. Consumption of higher substrate concentration can replace solvent to be added in turn reducing the cost of recovery and recycling, thus improving the productivity [8]. Lipases are familiar to promote the esterification trans-esterification [9] and hydrolysis chemical reactions [2] [10]. Thus, the step for isolation or fractionation within the products is abolished. Thus, enzymatic synthesis can solve the problem of using other lethal chemicals and also reduces the separation cost.
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The added advantage of immobilised lipase over the chemical synthesis is requirement of mild conditions to carry out reaction, ease of recovery, reusability for next batch and fetching stereoselective product with better purity [11]. The characteristics such as activity, selectivity,
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specificity and stability of lipase as a catalyst are extensively improved with immobilising it on different supports having different hydrophobicities and internal morphologies [11]. The
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lipase catalysed synthesis of isoamyl acetate is executed by M. D. Romero et al 2002 to give 96% conversion in hexane as a medium in 48 h by conventional methodology [8]. Likewise
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the synthesis of isoamyl butyrate had been carried out in hexane as a solvent using lipozyme
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IM as a biocatalyst by S. Hari Krishna et al at 1999 [12] and 2001 [13] and also using Rhizomucor miehei lipase[14]. The further studies was carried out by Rui Zhao et al, 2003
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[15] synthesizing isoamyl butyrate from isoamyl alcohol and butyraldehyde using zeolite TS1 in air as a catalyst. Similar efforts were done by Andreia et al 2010 using lipozyme TL IM
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yielded 92% conversion in the period of 48 hours [5] and also reported by Rodrigues et al
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2004 using Rhizopus Sp. lipase as biocatalyst [16]. Thus, although the enzyme catalysed esterification has various advantages over chemical catalyst, it suffers from major drawback
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of exorbitant enzyme cost and slow rate of reaction which further limits its efficacy in Industry. Recent development in the fermentation technology with enzyme modification techniques has curtailed the overall cost of enzymatic process. Apparently, if the free enzyme is entrapped or encapsulated on an intact support then the immobilised form can be used in succession for numerous processes to yield the product of interest which helps to solve the problem of cost [17]. However, the slower rate of reaction still remains an unresolved concern. Thus, there is entail to improvise novel technology for accretion of the rate of the enzyme catalysed reaction. Ultrasound synthesis or Sonochemistry is currently known for its novel advent as new process technology and process intensification genre [18]. Ultrasound when passed generates waves in fluid creating successive compressions and rarefactions. The
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frequency of ultrasound (20,000 cycles/second) travels through a medium to advent bubble formation. These bubbles attain growth and gradually disrupts. There is formation of void as the negative pressure of the rarefaction cycle exceeds the attractive forces between the
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molecules of the liquid (C. Leonelli et al, 2010). The bubble takes up ample vapour from the vicinity and grows to form tiny cavitational chamber before it implodes. Considering the
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violent collapse of enormous bubbles simultaneously, tremendous generation of heat and pressure occur. The micro jet are also formed which help to create turbulence in the reaction
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mixture[18]. Thus, these mechanical effects can be best exploited for chemical synthesis[19].
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There are very few reports available with respect to the production of esters, emphasising the work on ultrasound assisted[20] enzyme catalysed reaction[21][22]. To the best of our
catalysed synthesis of isoamyl butyrate.
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knowledge, there is no research paper available reporting the ultrasound assisted and lipase
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The objective of the work is to explore the application of ultrasound for synthesis isoamyl
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butyrate using enzyme lipase from Candida antarctica to catalyse isoamyl alcohol and butyric acid in a solvent free system and the optimization of various parameters affecting this
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enzyme catalysed reaction in presence of ultrasound to obtain maximum conversion.
2. Experimental 2.1 Materials
Isoamyl alcohol and butyric acid (98%) were purchased from Loba Chemie pvt. Ltd, Mumbai, India. Potassium hydroxide was procured from sigma Aldrich. Ethanol and methanol were obtained from S.D Fine chemicals pvt. Ltd, Mumbai, India. Novozym 435 source Candida antarctica; lipase B immobilised on the macro porous poly acrylic resin
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support having activity of 9.32 U was procured as gift samples from Zytex India. All other
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reagents used were of analytical grade and used up as procured.
2.2 Determination of Enzyme activity
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The methodology for evaluating the activity of enzyme was followed from S. Harikrishna et
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al 2002 [23]. The mixture of 85.95 mL n-heptane containing (0.16 M) 1.35 mL and (0.33 M) 2.7 mL butyric acid and butanol respectively, constituted stock solution. The stock solution
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3ml with weighed amount of lipase was put together in an Erlenmeyer flask mounted on the incubated shaker at 40˚C, 50˚C and 60˚C for an hour (60 min) at stirring speed of 150
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rotations per minute (rpm). The flask without the enzyme certainly a blank was to investigate the relative activity of enzyme. After an hour of incubation, the mixture was quenched with
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solution.
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1mL of ethanol and titrated with 0.02 M NaOH solution, indication with phenolphthalein
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The esterification activity of lipase thus given by equation (1),
……………………………… (Eq.1)
V0- volume of blank titrated against 0.02 N NaOH V- Volume of the samples titrated against the alkali M- Molarity of the NaOH from standardisation A- Weight of the enzyme in the analyte T- Time (minutes) required for the reaction or incubation time.
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The butyric acid of 1µmol utilised by the reaction per min per mg enzyme is stated as the esterification activity of the lipase, (denoted as U for convenience). The determined activity of the fresh immobilised lipase was 9.32U.
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2.3 Experimental setup and procedure
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The experimental set up consists of an ultrasonic bath equipped with a transducers purchased from Dakshin pvt.ltd. Mumbai, India. The ultrasonic unit can be operated at two different
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frequencies of 25 kHz and 40 kHz with maximum input power of 200 W. The temperature of the ultrasonic bath can be controlled using heater provided and by circulation of cooling
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water. Similarly the reactor assembly consisted of a four baffled flat bottom reactor vessel of 50 mL capacity having the internal diameter of 4 cm employed with blade four agitating
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impeller, three necked lid and a condenser was mounted to it to provide the reflux of the evaporating mixture. The experimental optimisation was performed by varying one parameter
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and keeping all the others constant. The required amount of reactant and enzyme (alcohol
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(24.6 mL), acid (10.4 mL) and immobilised enzyme) were added in the reactor assembly.
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Reaction set up as depicted in figure 1. The total reacting volume was 35 mL. The entire assembly is then put in the ultrasonic bath containing water. The bottom of the reactor was kept 2.5 cm above the bottom of the bath. The position of reactor was selected based on earlier work [24]. Ultrasound was passed for stipulated time and small amount of samples from the reaction mixture were taken after certain fixed time intervals. The samples thus, withdrawn were analysed with standard acid value method and GC analysis. 2.4 Analysis of Sample Titration of the aliquots was done using alcoholic KOH solution to determine the amount of unconverted butyric acid in terms of its acid value. While in the instrumental analysis by gas chromatography, Gas Chemito model no 8610 equipped with flame ionised detector along 8
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with steel packed column (3m × 0.32 mm and 10% stationary phase) OV 17 column was used. The injected volume of 2 µL was observed throughout. Nitrogen was used as carrier gas at pressure 0.8 bar. The temperature program was customized in the following steps: 70°C for
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1 min; 5 °C/min up to 120°C; then steady temperature for 1 min. Calibration of the standard
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product to compare with its synthetic desired product, was done to quantify the conversion.
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3. Result and discussion
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3.1. Effect of time
In order to study the progress of ultrasound assisted enzyme catalysed esterification reaction,
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experiment was performed using isoamyl alcohol to butyric acid ratio of 2:1, at the temperature of 60˚C and agitation speed of 100 rpm. While the ultrasound parameters used
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were ultrasound power 100 W, frequency 24 kHz and duty cycle of 83%. The enzyme
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loading of 1% (m/v) was used to initiate the reaction and results are reported in Figure 2. Low
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water content is necessity for esterification reaction. Water being the by-product in the reaction, it becomes essential to control its formation or removal to avoid the reaction to drive in backward direction to hydrolysis and yielding back the substrates. It is also reported that presence of water and acid can decay support matrix by affecting its pH and deteriorate the activity and enzyme [25]. The solubility of water in isoamyl alcohol is poor, it can settle or form thin film encircling the enzyme contributing to mass transfer resistances [26]. Molecular sieves in this case can prove a promising solution in the process [20]. In the absence of molecular sieves the conversion do not exceed 52% (result not presented) which proves the above discussion and in the literature. It is observed that during the study of time optimisation, the conversion obtained at the early stage increases linearly with time till 50 % conversion and further conversion increases slowly and almost 130 minutes are required for 9
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next 43% conversion. The maximum conversion of 96% was observed in the time span of 180 min and thus all further experiments were performed till 180 minutes (3 h). 3.2. Effect of reaction temperature
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Since enzyme shows higher activity at particular temperature range, the reaction was carried at different temperatures ranging as 30˚C, 40˚C, 50˚C 60̊ C and 70˚C. The molar ratio of
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alcohol to acid of 2:1, enzyme loading 1% (m/v), molecular sieves 2 g, the frequency of 25
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kHz, power as 100 W and duty cycle 83%, was kept constant for all temperatures. The effect of temperature can be seen in figure 3 which shows that the temperature of 60˚C was
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observed as the optimal temperature in terms of increase in conversion. The conversion of desired ester attained 88% in an hour at 60˚C compared to the room temperature (30±2ºC)
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that showed 65% conversion and the conversion is reduced at 70°C. The result proves that the activity of immobilized Novozym 435 is stable and its efficiency is higher 60˚C. The
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reduction in conversion above 60°C is possibly due to denaturation of enzyme at higher
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temperature [27]. Also, ultrasound effect reduces with an increase in temperature. At lower
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temperature, few bubbles are formed but they collapse with relatively high intensity which enhances the mass transfer while at higher temperature although more bubbles are created, they collapse with less intensity. Surface tension also decreases with the increase of temperature which affects the bubble formation and collapse. Thus, the mass transfer enhancement due to cavitation reduces at higher temperature since bubbles collapse with less intensity [28]. Ultrasound irradiation gives remarkable increase in product formation in little time owing to its enhanced mass transfer effect due to cavitation. V.G. Deshmane et al. 2009 [29], reported similar effect of rise in reaction conversion with rise in temperature from 30˚C to 60˚C. The reaction was carried in ultrasound for the synthesis of isopropyl esters to attain 80% product conversion at optimised temperature of 60˚C.
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Thus 60˚C is determined as the optimum temperature for the synthesis of Isoamyl butyrate
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flavour and it is followed for further experiments.
3.3 Effect of molar ratio
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The mole ratio of the reacting species is an important parameter as the rate of forward
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reaction can be increased by manipulating the concentration of one of the reactants. The investigation of this parameter was done by increasing the alcohol ratio from 1:1, 2:1and 3:1.
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The effect of increased acid ratio was also observed by altering the concentration of acid: alcohol by 2: 1 and the results are depicted in figure 4. All other working parameters were
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kept constant such as temperature of reacting mixture 60˚C, molecular sieves 2 g, duty cycle 83%, enzyme loading 1% (m/v), power input 100 W, ultrasound frequency 25 kHz and
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agitation 80 rpm. From the data, it was cleared that the alcohol in excess drives forward the
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reaction as the higher concentration of acid gave lower conversion of product. In most of the esterification instances the reaction proceeds well with the excess of one component. Thus,
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excess of alcohol or the nucleophile shifts the equilibrium of the esterification in forward pathway to obtain ester [30]. It is elucidated from improved conversion of ester as 50% and 77% using ratio as 1:1 and 1:2 of acid: alcohol respectively in an hour. Surplus concentration of the acid at the vicinity of enzyme leads to acidification of the lipase and its inactivation [31]. It is observed that excessive acidification causes ill-effects to alter enzyme stability or denaturation at higher concentration of acid [22]. At higher molar concentration of acid, the conventional method could reach only 9% conversion in 3 h, whereas under sonication at similar conditions 42 % conversion was gained. Cavitational effect towards the reaction mixture is helpful in uniform mixing of acid without accumulating at the micro environment of the enzyme. Thus it could be inferred that ultrasonic irradiation towards acidic substrates is
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effective in solvent free reactions without destabilising the biocatalyst assisting in efficient mass transfer. Inhibitory action of the acid could be explained as formation of dead end complex when butyric acid binds with enzyme to form acyl enzyme complex. This acyl
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enzyme complex owing to dead end complex finally cannot yield ester. Additionally, polar nature of butyric acid lowers the pH at the micro-environment of lipase and eventually
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deactivates it. Also as alcohol ratio is taken three times the conversion is lowered, due to the fact that even alcohol inhibition may have occur though not prominent and effective as acid
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[13]. The excess of alcohol more than critical concentration results in the binding action
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towards the enzyme [31]. Thus, the molar ratio of the substrates isoamyl alcohol to butyric acid as 2:1 is observed as optimum and kept consistent throughout. The optimum mole ratio
for production in supercritical conditions.
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for isoamyl alcohol and butyric acid mole ratio 1:2 are also quoted by S. Srivastava et al [26]
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3.4. Effect of enzyme loading
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To study the effect of enzyme concentration, two batches were put up simultaneously, one
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with the enzyme and the other without the enzyme called as blank. Practically, no reaction took place in the blank batch as no conversion was observed. Thus, it can be stated that for an esterification reaction to take place cavitation alone is not sufficient. The ultrasonic waves assist to accelerate the rate of reaction. The pressure released from many continuous bubble formation and collapse result in emergence of cavitation. This eliminates the mass transfer resistances thus increasing the events of collision and contact of surfaces of substrates. Relevantly, it is physical parameters which accelerates the mass transfer phenomena and suffice mixing throughout the different phases.[32]. However, the concentration of enzyme plays an important role to obtain higher concentration of the product. Thus, experiments were performed for different concentrations ranging from 0.5 % to 3% at temperature of 60˚C, molecular sieves (size 4A˚) weighing 2 g, the ratio of acid to alcohol as 1:2, ultrasound 12
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frequency 25 kHz and the power of 100 W. Figure 5 shows the effect of enzyme concentration on ester conversion. The loading of enzyme with 2% (m/v) fetch optimum results as the conversion reached 90% in 120 min. The further loading proved abortive in
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terms of accelerating the rate or converting the substrate in shorter stipulated time [30]. The internal diffusion and relevant acquaintance of the substrate with the active sites could
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hamper definitely when the enzyme concentration exceeds. The effect is also profound due to saturation of the enzyme concentration with respect to the substrate concentration beyond and
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it indicates no relative change.
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3.5 Effect of agitation speed
The speed of agitation has profound effect on the enzyme as well as conversion. In
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conventional enzyme catalysed reaction, very high speed is required for higher mass transfer.
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But higher agitation speed causes physical damage to immobilized enzyme affecting the rate of the reaction. Here, one experiment was performed without agitation and other experiments
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were performed for different agitation at 80 rpm, 120 rpm and 200 rpm. The temperature of 60˚C, alcohol to acid mole ratio of 2:1, enzyme loading of 2% (m/v), molecular sieves 2g, power of 70 W, frequency of 25 kHz were kept as constant. The result is illustrated in figure 6 which indicates that the optimum speed of agitation was found to be 80 rpm, from the wide range that was chosen to find out the effect of stirring. There is a remarkable reduction in the conversion when the speed exceeds 120 rpm. At 0 rpm, 80 rpm, 120 rpm and 200 rpm the fall activity was experimentally determined as 9.1U, 8.8U, 7.8U and 6.2U respectively. The measured activity of the enzyme subsides from 9.3U to 6.27U as the speed of agitation increase from 80 rpm to 200 rpm. Illustrated in the figure 6, in absence of mechanical stirring, only 82.6 % conversion was obtained compared to 97.6 % employed with stirring of
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80 rpm. Thus, this shows that small mechanical agitation helps to improve the conversion in presence of ultrasound. Similar results are quoted by Nikhil et al (2012) [33]. The reaction tends to form a turbid mixture indicating the abrasive enzyme denaturation. The higher
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mechanical shear force (more than 100 rpm) employed with overhead stirring cause denaturation of the enzyme affecting its catalytic property. Besides the reusability is
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relevantly affected if the immobilised lipase does not remain intact on its support [34]. The range from 0 rpm – 200 rpm for the study was included with the other working parameters.
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Nevertheless, the agitation also needs more energy consumption when increased further.
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3.6 Effect of frequency
To evaluate the impact of frequency on the synthesis of ester, two frequencies lower being 25
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kHz and higher at 40 kHz were included. It is observed that, at lower frequency 25 kHz the
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yield of the flavour was found to be maximum than at the higher frequency 40 kHz. The calorimetric study was performed to analyse the power dissipation taking place at two
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different frequencies (figure 7). From the experimentation it was calculated that at 40 kHz frequency the power dissipation (30.8 W) is less than that of 25 kHz (46.2 W) at input power of 100 W. Theoretically, the cavitation and the bubbles size is deliberately reduced with increased frequency, which result in subsequent decrease of the intensity and the energy liberated though the implosion of the smaller bubbles [35]. As compared to lower frequency the higher frequency have profound effect but higher frequency has proved to hamper the catalytic activity [36]. In this way higher frequency could not fetch higher product conversion due to less power dissipation, insufficient cavitation phenomenon and inactivation of biocatalyst [37]. Thus, frequency of 25 kHz was chosen for further experimentation and optimisation study.
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3.7. Effect of Input power The power has an added impact on the reaction and higher ultrasound power gives higher
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conversion. In the case of enzyme catalysed reaction, the conversion increased with the
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ultrasound power due to improvement in enzyme activity. On the other the hand, higher ultrasonic power may deactivate the enzyme [38]. Thus, experiments were performed by
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varying ultrasonic power from 20-140 W to identify the optimum ultrasound power. From the experiment, it is inferred that higher power of more than 100 W when subjected to the
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mixture, perturb the process enormously. This contrasting result is produced due the fact that the biocatalyst is not able to survive at higher input power leading to deactivation or
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reduction in catalytic activity [22]. The lower conversion or slow reaction rate conclude that conformational changes or desorption and disruption n of the immobilised lipase take place
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when exposed to more harsh sonication [39]. An optimal power of 70 W is found to carry the
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reaction in the forward trend. Specified in the figure 8 is the effect of power for ester
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formation. Additionally the calorimetric analysis for power dissipation was carried out. The power dissipation values obtained for 25 kHz at power 40, 70 100, 140 and 200 W were 36, 43.4, 46.2, 57.2, 72.7 W respectively. There was no remarkable increase in the conversion even when the input power of more than 100 W was employed. Although power dissipation was higher for 140 W it results in deactivation of the lipase. Thus the power input of 70 W was found to the normalised input power in terms of conversion, activity of the catalysed used, power dissipated and energy economical terms.
3.8. Effect of Duty cycle
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The duty cycle that was engaged all throughout the experimentation was 83%. It indicates On time of 10 min and Off time of 2 min. For the study of the effect of duty cycle the two turns including 83% and 50% (On and off timing of 6 min each) were taken into consideration. The
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other conditions were kept similar, the temperature 60˚C, alcohol to acid mole ratio of 2:1, molecular sieves 2 g, enzyme loading of 2% m/v, power of 70 W and frequency of 25 kHz. It
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was justified, that the duty cycle of 83% provided more efficiency than 50%. Naturally, the exposed time of ultrasound irradiation to the reacting mixture when increased, the product
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formation subsequently increased. The figure 9 depicts the explanation. Although
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ultrasonication employed for more period of time improve the production of flavour, the continuous operation without pulse can damage the transducers responsible to produce
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ultrasonic waves [38]. Moreover the synthesis is not expected to carry for longer time in presence of ultrasound as prolonged exposure will deactivate the enzyme catalyst and the
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3.9. Effect of reusability
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temperature of the system may uncontrollably shoot up resulting in charring of the substrates.
The immobilised enzyme, Novozym 435 used, in the reaction was further separated by means of filtration, washed with hexane and reused. Ablution with hexane is crucial step to remove accumulation of water and acid content from the environment of the immobilised biocatalyst [40]. The residue of acid and water may block active sites of lipase and hamper the next successive reaction cycles yielding lower conversions [2]. Several repetitions for the reaction with the treated immobilised enzyme were performed. It was seen that there was a steady decrease in the fall of the activity of the enzyme from initial cycle till the sixth and seventh recurring cycle. There was reduction of activity till eighth cycle up to 38%. The reason could be attributed to losses during recurring cycles and inactivation cause due to substrate
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inhibition [27]. It can be concluded that the enzyme can be reused for 6 to 7 cycles the reduction from the initial activity being 35% and further till 10 cycles wherein the activity fall observed to 48%. As mentioned earlier consistent exposure of ultrasound can bring about
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conformational changes in lipase and lead to gradual decline in its activity [36]. The Figure
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10 depicts the fall of activity of the biocatalyst with repetitive use in consecutive cycles.
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3.10 Comparison between conventional and ultrasound assisted reaction
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In order to compare the effect ultrasound on esterification, reaction was carried out in a batch reactor with enzyme in absence of ultrasound. It is found that the time required for the
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esterification was 10 h to attain conversion of 90 % at the temperature of 60˚C and the amount of isoamyl alcohol double to that of butyric acid (2:1) with addition of molecular
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sieves 2g. The speed employed for agitation was 300 rpm to enhance the mass transfer. The
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results obtained using conventional stirred reactor, the ultrasonic assisted reaction with and without agitation are depicted in the figure 11. It has been observed that the incorporation of
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ultrasound irradiation enhances the conversion from about 85% from 13% without the ultrasound in 1 h. Thus, due to the nature of cavitation generated in ultrasound system, the mass transfer resistance is eliminated providing essentially desired product. It also helps in the uniform mixing assisted by the cavitation and turbulence created by overhead stirrer. Further, the micro jets formed when bubble implodes helps for interface renewal. All these effects are responsible for the enhancement in the conversion. However, when reaction was performed in absence of agitation, lower conversion of desired ester was achieved with only 80% in 3 h. This is due to fact that small agitation keeps the particles suspended in reaction mixture and prohibits the coagulation of enzyme particles and also prevents them from settling down.
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Conclusion The present work was emphasised on intensifying the reaction process of the enzyme
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catalysed synthesis of 3-methyl butyl butanoate, an ester possessing commercial value as
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fruity flavour which is widely used in several industries. Incorporating ultrasonication was an added novelty to the conventional method of synthesis of isoamyl butyrate from isoamyl
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alcohol and butyric acid. Under optimised conditions, maximum conversion of 96.7 % is obtained in 3 hour as compared to 10 h of conventional enzyme catalysed reaction. The
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immobilised lipase Novozym 435 also proved useful up to 7 cycles with only 30% reduction in the fall of its initial activity. It is clear from the experiments that the immobilised enzyme
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could sustain ultrasonic environment serving improved yield in less time. This data shows
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potential application of ultrasound for enzymatic synthesis of flavour compounds.
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Rodrigues RC. Effect of immobilization protocol on optimal conditions of ethyl
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Krishna SH, Sattur AP, Karanth NG. Lipase-catalyzed synthesis of isoamyl isobutyrate
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butyrate synthesis catalyzed by lipase B from Candida antarctica. J Chem Technol
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— optimization using a central composite rotatable design. Process Biochem
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Chapuis C, Jacoby D. Catalysis in the preparation of fragrances and flavours. Appl
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Fernandes SA, Natalino R, Gazolla PAR, da Silva MJ, Jham GN. p-Sulfonic acid calix[n]arenes as homogeneous and recyclable organocatalysts for esterification reactions. Tetrahedron Lett 2012;53:1630–3. Romero MD, Calvo L, Alba C, Daneshfar a., Ghaziaskar HS. Enzymatic synthesis of
ip t
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isoamyl acetate with immobilized Candida antarctica lipase in n-hexane. Enzyme
Sonare NR, Rathod VK. Transesterification of used sunflower oil using immobilized
Rathod VK, Pandit AB. Effect of various additives on enzymatic hydrolysis of castor oil. Biochem Eng J 2009;47:93–9.
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Microb Technol 2005;37:42–8.
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Fernandez-Lafuente R,Rodrigues RC. Improved production of butyl butyrate with lipase from Thermomyces lanuginosus immobilized on styrene-divinylbenzene beads.
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Bioresour Technol 2013;134:417–22.
[12] Krishna SH, Manohar B, Divakar S, Karanth NG. Lipase-catalyzed synthesis of isoamyl butyrate: Optimization by response surface methodology. J Am Oil Chem Soc 1999;76:1483–8.
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Krishna SH, Karanth NG. Lipase-catalyzed synthesis of isoamyl butyrate A kinetic
study. Biochimica et biophysia Acta 2001;1547:262–7. [14]
Hari Krishna S, Prapulla SG, Karanth NG. Enzymatic synthesis of isoamyl butyrate using immobilized Rhizomucor miehei lipase in non-aqueous media. J Ind Microbiol Biotechnol 2000;25:147–54. 20
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Zhao R, Ding Y, Peng Z, Wang X, Suo J. One-step synthesis of isoamyl butyrate from isoamyl alcohol and n -butyraldehyde over TS-1 in air. Catalysis Letters 2003;87:81– 83. Macedo GA, Pastore GM, Rodrigues MI. Optimising the synthesis of isoamyl butyrate
ip t
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using Rhizopus sp . lipase with a central composite rotatable design. Process Biochem
Ben Salah R, Ghamghui H, Miled N, Mejdoub H, Gargouri Y. Production of butyl
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acetate ester by lipase from novel strain of Rhizopus oryzae. J Biosci Bioeng 2007;103:368–72.
Leonelli C, Mason TJ. Microwave and ultrasonic processing: Now a realistic option
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Chemat F, Zill-e-Huma, Khan MK. Applications of ultrasound in food technology:
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for industry. Chem Eng Process Process Intensif 2010;49:885–900.
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Processing, preservation and extraction. Ultrason Sonochem 2011;18:813–35. [20]
Fallavena LP, Antunes FHF, Alves JS, Paludo N, Ayub MAZ, Fernandez-Lafuente R,
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Ultrasound technology
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molecular sieves improve the
thermodynamically controlled esterification of butyric acid mediated by immobilized lipase from Rhizomucor miehei. RSC Adv 2014;4:8675.
[21]
Hobuss CB, Venzke D, Pacheco BS, Souza AO, Santos MZ, Moura S, Quina FH,
Fiametti KG, Oliveira JV, Pereira CMP Ultrasound-assisted synthesis of aliphatic acid esters at room temperature. Ultrason Sonochem 2012;19:387–9.
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Martins AB, Schein MF, Friedrich JLR, Fernandez-Lafuente R, Ayub MAZ, Rodrigues RC. Ultrasound-assisted butyl acetate synthesis catalyzed by Novozym 435: enhanced activity and operational stability. Ultrason Sonochem 2013;20:1155–60. Kiran KR, Krishna SH, Babu CVS, Karanth NG, Divakar S. An esterification method
ip t
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[24]
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for determination of lipase activity Biotechnology letters 2000;20:1511–4.
Kulkarni VM, Rathod VK. Mapping of an ultrasonic bath for ultrasound assisted
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extraction of mangiferin from Mangifera indica leaves. Ultrason Sonochem
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2013;21:606–11.
Martins B, Friedrich JLR, Rodrigues RC, Garcia-galan C, Fernandez-lafuente R, Ayub
M
MAZ. Optimized Butyl Butyrate Synthesis Catalyzed by Thermomyces lanuginosus
Srivastava S, Modak J, Madras G. Enzymatic Synthesis of Flavors in Supercritical
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Lipase. Biotechnol Prog, 2013;29:6 2013.
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Carbon Dioxide. Ind Eng Chem Res 2002;41:1940–5. [27]
Xun E, Lv X, Kang W, Wang J, Zhang H, Wang L, Zhang H, Wang L& Wang Z.
Immobilization of Pseudomonas fluorescens lipase onto magnetic nanoparticles for resolution of 2-octanol. Appl Biochem Biotechnol 2012;168:697–707.
[28]
Alissandrakis E. Ultrasound-assisted extraction of volatile compounds from citrus
flowers and citrus honey. Food Chem 2003;82:575–82.
[29]
Deshmane VG, Gogate PR, Pandit AB. Ultrasound assisted synthesis of isopropyl esters from palm fatty acid distillate. Ultrason Sonochem 2009;16:345–50.
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[30]
Lorenzoni ASG, Graebin NG, Martins AB, Fernandez-Lafuente R, Ayub MAZ, Rodrigues RC. Optimization of pineapple flavour synthesis by esterification catalysed by immobilized lipase from Rhizomucor miehei. Flavour Fragr J 2012;27:196–200. Guvenc A, Kapucu N, Mehmetoglu U. The production of isoamyl acetate using
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immobilized lipases in a solvent-free system. Process Biochemistry 2002;379-386.
Jadhav D, Rekha BN, Gogate PR, Rathod VK. Extraction of vanillin from vanilla
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Food Eng 2009;93:421–6.
Gharat N, Rathod VK. Ultrasound assisted enzyme catalyzed transesterification of
Kuperkar V V, Lade VG, Prakash A, Rathod VK. Synthesis of isobutyl propionate
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waste cooking oil with dimethyl carbonate. Ultrason Sonochem 2013;20:900–5.
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Mol Catal B Enzym 2014;99:143–9. [35]
Gogate PR. Cavitational reactors for process intensification of chemical processing
applications : A critical review .Chemical Engineering and Processing 2008;47:515– 27.
[36]
Zheng M-M, Wang L, Huang F-H, Dong L, Guo P-M, Deng Q-C, Zheng C, Wan C-H.
Ultrasound irradiation promoted lipase-catalyzed synthesis of flavonoid esters with unsaturated fatty acids. J Mol Catal B Enzym 2013;95:82–8.
[37]
Zheng M-M, Wang L, Huang F-H, Guo P-M, Wei F, Deng Q-C,Li W-L Zheng C, Ultrasonic pretreatment for lipase-catalyed synthesis of phytosterol esters with different acyl donors. Ultrason Sonochem 2012;19:1015–20. 23
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[38]
Avhad DN, Rathod VK. Ultrasound stimulated production of a fibrinolytic enzyme. Ultrason Sonochem 2014;21:182–8.
[39]
Fiametti KG, Sychoski MM, De Cesaro A, Furigo A, Bretanha LC, Pereira CMP,
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Treichel H, de Oliveira D, Oliveira VJ. Ultrasound irradiation promoted efficient solvent-free lipase-catalyzed production of mono- and diacylglycerols from olive oil.
cr
Ultrason Sonochem 2011;18:981–7.
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[40] Martins AB, Graebin NG, Lorenzoni ASG, Fernandez-Lafuente R, Ayub MAZ,
an
Rodrigues RC. Rapid and high yields of synthesis of butyl acetate catalyzed by Novozym 435: Reaction optimization by response surface methodology. Process
Ac ce p
te
d
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Biochem 2011;46:2311–6.
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List of Figures: Fig 1 Schematic of experimental set up of esterification of Isoamyl alcohol and butyric acid catalysed with enzyme (Novozym 435) assisted with ultrasonic system.
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Fig2. Effect of time on the esterification with the impact of ultrasound irradiation Reaction conditions: Novozym 435 1% (m/v), Temperature 60˚C, stirring speed 100
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rpm, Frequency 25 kHz, duty cycle 83%, Power 100 W Fig 3 Effect of temperature on the esterification of Isoamyl butyrate
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Reaction conditions: Novozym 435 (lipase)1% (m/v), Acid: Alcohol 1:2, stirring speed 80 rpm, frequency 25 kHz, duty cycle 83%, power 100 W
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Fig 4 Effect of mole ratio of Butyric acid and Isoamyl alcohol on conversion Reaction conditions: Novozym 435 1% (m/v), Temperature 60˚C, stirring speed
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80rpm, Frequency 25 kHz, duty cycle 83%, Power 100 W
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Fig 5 Effect of enzyme loading on conversion
Reaction conditions: Temperature 60˚C, stirring speed 80 rpm, acid alcohol ratio 1:2,
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Frequency 25 kHz, Duty cycle 83% and Power 100 W
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Fig 6 Effect of stirring speed on conversion Reaction conditions: Temperature 60˚C, acid alcohol ratio 1:2, enzyme loading 2% (m/v) Frequency 25 kHz, Duty cycle 83% and power 100 W
Fig 7 Effect of frequency of the ultrasound irradiation on conversion Reaction conditions: temperature 60˚C, acid: alcohol1:2, stirring speed 80 rpm, enzyme concentration 2% (m/v) duty cycle 83% and power 100 W
Fig 8 Effect of power of the ultrasound irradiation on conversion Reaction conditions: temperature 60˚C, acid: alcohol 1:2, stirring speed 80 rpm, enzyme loading 2% (m/v), frequency 25 kHz, and duty cycle 83% Fig 9 Effect of duty cycle input during the reaction in progress
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Reaction condition: temperature 60˚C acid: alcohol1:2, stirring speed 80 rpm, enzyme loading 2% (m/v), frequency 25 kHz, Power 70 W Fig 10 Reusability of immobilised Novozym 435 for repeated cycles Fig 11 The Comparison for synthesis of Isoamyl butyrate through conventional synthesis
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(CS) and ultrasound irradiation (UI) The reaction conditions CS: temperature 60˚C, molecular sieves 2g, acid: alcohol 1:2, enzyme loading 2% (m/v), stirring 300 rpm.
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UI: temperature 60˚C, molecular sieves 2g, acid; alcohol 1:2, stirring speed 80 rpm,
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enzyme loading 2% (m/v), frequency 25 kHz, duty cycle 83%, Power 70 W with ultrasonic irradiation
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.
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1. Overhead Stirrer 1
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2. Condenser
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3. Reaction mixture
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4. Ultrasonic Bath
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5. Transducers
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Ultrasound Generator
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Fig 1 Schematic of experimental set up of esterification of Isoamyl butyrate from Isoamyl alcohol and butyric acid catalysed with enzyme (Novozym 435) assisted with ultrasonic system.
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Fig 2 Effect of time on the esterification with the impact of ultrasound irradiation Reaction conditions: Novozym 435 1% (m/v), Temperature 60˚C, stirring speed 100 rpm, molecular sieves 2g, Frequency 25 kHz, duty cycle 83% and power 100 W
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Fig 3 Effect of temperature on the esterification of Isoamyl butyrate Reaction conditions: Novozym 435 (lipase)1% (m/v), Acid: Alcohol 1:2, stirring speed 80 rpm, molecular sieves 2g, frequency 25 kHz, duty cycle 83%, power 100 W
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Fig 4 Effect of mole ratio of Butyric acid and Isoamyl alcohol on conversion Reaction conditions: Novozym 435 1% (m/v), Temperature 60˚C, stirring speed 80 rpm, molecular sieves 2g, Frequency 25 kHz, duty cycle 83%, Power 100 W
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Fig 5 Effect of enzyme loading on conversion Reaction conditions: Temperature 60˚C, stirring speed 80 Rpm, molecular sieves 2g,
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acid alcohol ratio 1:2, Frequency 25 kHz, duty cycle 83% and Power 100 W
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Fig 6 Effect of stirring speed on conversion
Reaction conditions: Temperature 60˚C, acid alcohol ratio 1:2, enzyme loading 2%
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(m/v), molecular sieves 2g, Frequency 25 kHz, duty cycle 83% and power 100 W
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Fig 7 Effect of frequency of the ultrasound irradiation on the conversion
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Reaction conditions: temperature 60˚C, acid: alcohol 1:2, stirring speed 80 rpm, enzyme concentration 2% (m/v), molecular sieves 2g, duty cycle 83% and power
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100W
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Fig 8 Effect of power input of the ultrasound irradiation on conversion Reaction conditions: temperature 60˚C, acid: alcohol 1:2, stirring speed 80 Rpm,
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molecular sieves 2g, enzyme loading 2% (m/v), frequency 25 kHz, and duty cycle 83%
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Fig 9 The Effect of duty cycle employed during the reaction in progress
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Reaction condition: temperature 60˚C acid: alcohol1:2, stirring speed 80 rpm, molecular sieves 2g, enzyme loading 2% (m/v), frequency 25 kHz, Power 70 W
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Fig 10 The reusability of immobilised Novozym 435 for repeated cycles
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Fig 11 The Comparison for synthesis of Isoamyl butyrate through conventional synthesis (CS) and ultrasound irradiation (UI) The reaction conditions CS: temperature 60˚C,
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molecular sieves 2g, acid: alcohol 1:2, enzyme loading 2% (m/v), stirring 300 Rpm. UI: temperature 60˚C, molecular sieves 2g, acid; alcohol 1:2, stirring speed 80 rpm, enzyme loading 2% (m/v), duty cycle 83 %, frequency 25 kHz, Power 70 W with ultrasonic irradiation.
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Highlights Ultrasound assisted lipase catalyzed synthesis of Isoamyl butyrate was performed. The reaction parameters needed to drag the reaction to forward pathway were optimized.
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Ultrasonication reduced reaction time than conventional enzymatic synthesis.
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an
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The immobilized lipase could be use repetitively for 7 cycles.
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S1359-5113(14)00266-9 http://dx.doi.org/doi:10.1016/j.procbio.2014.04.018 PRBI 10129
To appear in:
Process Biochemistry
Received date: Revised date: Accepted date:
14-1-2014 15-4-2014 22-4-2014
Please cite this article as: Bansode SR, Rathod VK, Ultrasound assisted lipase catalysed synthesis of Isoamyl butyrate, Process Biochemistry (2014), http://dx.doi.org/10.1016/j.procbio.2014.04.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Ultrasound assisted lipase catalysed synthesis of Isoamyl butyrate
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Sneha R. Bansode, Virendra K. Rathod
*Corresponding author Dr. V. K. Rathod
Department of Chemical Engineering Institute of Chemical Technology, Matunga (E), Mumbai-400019, India. E-mail: [email protected] ,
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Phone: +91-22-33612020, Fax: 91-22-33611020.
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Abstract: The present work illustrates the incorporation of ultrasound and its improved impact in the lipase catalysed esterification. Synthesis of isoamyl butyrate from isoamyl alcohol and
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butyric acid using immobilized Novozym 435, has been carried out in the presence of ultrasound. The optimisation of various parameters affecting the synthesis of ester in
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presence of ultrasound was done. The systematic experimentation involves change of one
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working parameter at one time while keeping the others constant. For the maximum conversion, optimum parameters such as the ultrasound of 25 kHz frequency with power of
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70 W, at the temperature of 60˚C with stirring speed of 80 rpm, mole ratio of alcohol: acid followed as 2:1, use of molecular sieves weighing 2g, with immobilized enzyme loading of
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2% (m/v) and duty cycle of 83%, were obtained. The optimum parameters collectively, gave 96% conversion of the product in 3 h as compared with 10 h in absence of ultrasound. The
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immobilized biocatalyst, Novozym 435 has an added benefit of reusability till 7 repetitive
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cycles. Besides, the synthesis is executed in the solvent free system that contributes the
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production of flavour in greener way.
Keywords: Isoamyl butyrate, Ultrasound, immobilised lipase, Novozym 435, Fruity Banana flavour, optimisation
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1. Introduction: Short chain esters derived from the alcohol and an acid form flavour and perfume that have their essence in food industry, pharmaceutical industry, cosmetics etc. [1]. Subsequently, the
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natural flavours are costly due to limited availability of raw material, preservation and processing cost and the traditionally extracted flavours cannot meet the increasing demands
cr
due to their low yield [2]. Thus, the artificial flavours are in demand as they are synthesised
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easily and cheaply available. Isoamyl butyrate, a fruity banana flavour is well known in food industry especially in fruit jams, fruit punch and analogous preparations [3]. The annual
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demand for isoamyl butyrate reported in 1989 was 13,792 kg/annum from around the globe by welsh et al, the figure now reached 2,00,000 kg/year in Columbia alone. The ester,
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isoamyl butyrate can be obtained from chemical interaction of the two substrate, isoamyl alcohol and butyric acid in presence of catalyst. If the ester happens to be a synthetic
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flavouring agent in food components, it must be free of any harmful acidulous catalyst and
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accede with the food grade implementations [4]. Esterification from catalysis using P-toulene
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sulphonic acid [5], Amberlyst [6] and P tolune sulphonic acid calix [n] arenes [7] has been tried out. Triacyl acyl hydrolases, (EC 3.1.1.3) or precisely known as lipases have surfaced its applications due to targeted cut down of side product and production of desired product in plenty. Consumption of higher substrate concentration can replace solvent to be added in turn reducing the cost of recovery and recycling, thus improving the productivity [8]. Lipases are familiar to promote the esterification trans-esterification [9] and hydrolysis chemical reactions [2] [10]. Thus, the step for isolation or fractionation within the products is abolished. Thus, enzymatic synthesis can solve the problem of using other lethal chemicals and also reduces the separation cost.
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The added advantage of immobilised lipase over the chemical synthesis is requirement of mild conditions to carry out reaction, ease of recovery, reusability for next batch and fetching stereoselective product with better purity [11]. The characteristics such as activity, selectivity,
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specificity and stability of lipase as a catalyst are extensively improved with immobilising it on different supports having different hydrophobicities and internal morphologies [11]. The
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lipase catalysed synthesis of isoamyl acetate is executed by M. D. Romero et al 2002 to give 96% conversion in hexane as a medium in 48 h by conventional methodology [8]. Likewise
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the synthesis of isoamyl butyrate had been carried out in hexane as a solvent using lipozyme
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IM as a biocatalyst by S. Hari Krishna et al at 1999 [12] and 2001 [13] and also using Rhizomucor miehei lipase[14]. The further studies was carried out by Rui Zhao et al, 2003
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[15] synthesizing isoamyl butyrate from isoamyl alcohol and butyraldehyde using zeolite TS1 in air as a catalyst. Similar efforts were done by Andreia et al 2010 using lipozyme TL IM
d
yielded 92% conversion in the period of 48 hours [5] and also reported by Rodrigues et al
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2004 using Rhizopus Sp. lipase as biocatalyst [16]. Thus, although the enzyme catalysed esterification has various advantages over chemical catalyst, it suffers from major drawback
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of exorbitant enzyme cost and slow rate of reaction which further limits its efficacy in Industry. Recent development in the fermentation technology with enzyme modification techniques has curtailed the overall cost of enzymatic process. Apparently, if the free enzyme is entrapped or encapsulated on an intact support then the immobilised form can be used in succession for numerous processes to yield the product of interest which helps to solve the problem of cost [17]. However, the slower rate of reaction still remains an unresolved concern. Thus, there is entail to improvise novel technology for accretion of the rate of the enzyme catalysed reaction. Ultrasound synthesis or Sonochemistry is currently known for its novel advent as new process technology and process intensification genre [18]. Ultrasound when passed generates waves in fluid creating successive compressions and rarefactions. The
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frequency of ultrasound (20,000 cycles/second) travels through a medium to advent bubble formation. These bubbles attain growth and gradually disrupts. There is formation of void as the negative pressure of the rarefaction cycle exceeds the attractive forces between the
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molecules of the liquid (C. Leonelli et al, 2010). The bubble takes up ample vapour from the vicinity and grows to form tiny cavitational chamber before it implodes. Considering the
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violent collapse of enormous bubbles simultaneously, tremendous generation of heat and pressure occur. The micro jet are also formed which help to create turbulence in the reaction
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mixture[18]. Thus, these mechanical effects can be best exploited for chemical synthesis[19].
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There are very few reports available with respect to the production of esters, emphasising the work on ultrasound assisted[20] enzyme catalysed reaction[21][22]. To the best of our
catalysed synthesis of isoamyl butyrate.
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knowledge, there is no research paper available reporting the ultrasound assisted and lipase
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The objective of the work is to explore the application of ultrasound for synthesis isoamyl
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butyrate using enzyme lipase from Candida antarctica to catalyse isoamyl alcohol and butyric acid in a solvent free system and the optimization of various parameters affecting this
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enzyme catalysed reaction in presence of ultrasound to obtain maximum conversion.
2. Experimental 2.1 Materials
Isoamyl alcohol and butyric acid (98%) were purchased from Loba Chemie pvt. Ltd, Mumbai, India. Potassium hydroxide was procured from sigma Aldrich. Ethanol and methanol were obtained from S.D Fine chemicals pvt. Ltd, Mumbai, India. Novozym 435 source Candida antarctica; lipase B immobilised on the macro porous poly acrylic resin
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support having activity of 9.32 U was procured as gift samples from Zytex India. All other
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reagents used were of analytical grade and used up as procured.
2.2 Determination of Enzyme activity
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The methodology for evaluating the activity of enzyme was followed from S. Harikrishna et
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al 2002 [23]. The mixture of 85.95 mL n-heptane containing (0.16 M) 1.35 mL and (0.33 M) 2.7 mL butyric acid and butanol respectively, constituted stock solution. The stock solution
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3ml with weighed amount of lipase was put together in an Erlenmeyer flask mounted on the incubated shaker at 40˚C, 50˚C and 60˚C for an hour (60 min) at stirring speed of 150
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rotations per minute (rpm). The flask without the enzyme certainly a blank was to investigate the relative activity of enzyme. After an hour of incubation, the mixture was quenched with
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solution.
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1mL of ethanol and titrated with 0.02 M NaOH solution, indication with phenolphthalein
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The esterification activity of lipase thus given by equation (1),
……………………………… (Eq.1)
V0- volume of blank titrated against 0.02 N NaOH V- Volume of the samples titrated against the alkali M- Molarity of the NaOH from standardisation A- Weight of the enzyme in the analyte T- Time (minutes) required for the reaction or incubation time.
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The butyric acid of 1µmol utilised by the reaction per min per mg enzyme is stated as the esterification activity of the lipase, (denoted as U for convenience). The determined activity of the fresh immobilised lipase was 9.32U.
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2.3 Experimental setup and procedure
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The experimental set up consists of an ultrasonic bath equipped with a transducers purchased from Dakshin pvt.ltd. Mumbai, India. The ultrasonic unit can be operated at two different
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frequencies of 25 kHz and 40 kHz with maximum input power of 200 W. The temperature of the ultrasonic bath can be controlled using heater provided and by circulation of cooling
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water. Similarly the reactor assembly consisted of a four baffled flat bottom reactor vessel of 50 mL capacity having the internal diameter of 4 cm employed with blade four agitating
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impeller, three necked lid and a condenser was mounted to it to provide the reflux of the evaporating mixture. The experimental optimisation was performed by varying one parameter
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and keeping all the others constant. The required amount of reactant and enzyme (alcohol
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(24.6 mL), acid (10.4 mL) and immobilised enzyme) were added in the reactor assembly.
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Reaction set up as depicted in figure 1. The total reacting volume was 35 mL. The entire assembly is then put in the ultrasonic bath containing water. The bottom of the reactor was kept 2.5 cm above the bottom of the bath. The position of reactor was selected based on earlier work [24]. Ultrasound was passed for stipulated time and small amount of samples from the reaction mixture were taken after certain fixed time intervals. The samples thus, withdrawn were analysed with standard acid value method and GC analysis. 2.4 Analysis of Sample Titration of the aliquots was done using alcoholic KOH solution to determine the amount of unconverted butyric acid in terms of its acid value. While in the instrumental analysis by gas chromatography, Gas Chemito model no 8610 equipped with flame ionised detector along 8
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with steel packed column (3m × 0.32 mm and 10% stationary phase) OV 17 column was used. The injected volume of 2 µL was observed throughout. Nitrogen was used as carrier gas at pressure 0.8 bar. The temperature program was customized in the following steps: 70°C for
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1 min; 5 °C/min up to 120°C; then steady temperature for 1 min. Calibration of the standard
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product to compare with its synthetic desired product, was done to quantify the conversion.
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3. Result and discussion
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3.1. Effect of time
In order to study the progress of ultrasound assisted enzyme catalysed esterification reaction,
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experiment was performed using isoamyl alcohol to butyric acid ratio of 2:1, at the temperature of 60˚C and agitation speed of 100 rpm. While the ultrasound parameters used
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were ultrasound power 100 W, frequency 24 kHz and duty cycle of 83%. The enzyme
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loading of 1% (m/v) was used to initiate the reaction and results are reported in Figure 2. Low
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water content is necessity for esterification reaction. Water being the by-product in the reaction, it becomes essential to control its formation or removal to avoid the reaction to drive in backward direction to hydrolysis and yielding back the substrates. It is also reported that presence of water and acid can decay support matrix by affecting its pH and deteriorate the activity and enzyme [25]. The solubility of water in isoamyl alcohol is poor, it can settle or form thin film encircling the enzyme contributing to mass transfer resistances [26]. Molecular sieves in this case can prove a promising solution in the process [20]. In the absence of molecular sieves the conversion do not exceed 52% (result not presented) which proves the above discussion and in the literature. It is observed that during the study of time optimisation, the conversion obtained at the early stage increases linearly with time till 50 % conversion and further conversion increases slowly and almost 130 minutes are required for 9
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next 43% conversion. The maximum conversion of 96% was observed in the time span of 180 min and thus all further experiments were performed till 180 minutes (3 h). 3.2. Effect of reaction temperature
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Since enzyme shows higher activity at particular temperature range, the reaction was carried at different temperatures ranging as 30˚C, 40˚C, 50˚C 60̊ C and 70˚C. The molar ratio of
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alcohol to acid of 2:1, enzyme loading 1% (m/v), molecular sieves 2 g, the frequency of 25
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kHz, power as 100 W and duty cycle 83%, was kept constant for all temperatures. The effect of temperature can be seen in figure 3 which shows that the temperature of 60˚C was
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observed as the optimal temperature in terms of increase in conversion. The conversion of desired ester attained 88% in an hour at 60˚C compared to the room temperature (30±2ºC)
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that showed 65% conversion and the conversion is reduced at 70°C. The result proves that the activity of immobilized Novozym 435 is stable and its efficiency is higher 60˚C. The
d
reduction in conversion above 60°C is possibly due to denaturation of enzyme at higher
te
temperature [27]. Also, ultrasound effect reduces with an increase in temperature. At lower
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temperature, few bubbles are formed but they collapse with relatively high intensity which enhances the mass transfer while at higher temperature although more bubbles are created, they collapse with less intensity. Surface tension also decreases with the increase of temperature which affects the bubble formation and collapse. Thus, the mass transfer enhancement due to cavitation reduces at higher temperature since bubbles collapse with less intensity [28]. Ultrasound irradiation gives remarkable increase in product formation in little time owing to its enhanced mass transfer effect due to cavitation. V.G. Deshmane et al. 2009 [29], reported similar effect of rise in reaction conversion with rise in temperature from 30˚C to 60˚C. The reaction was carried in ultrasound for the synthesis of isopropyl esters to attain 80% product conversion at optimised temperature of 60˚C.
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Thus 60˚C is determined as the optimum temperature for the synthesis of Isoamyl butyrate
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flavour and it is followed for further experiments.
3.3 Effect of molar ratio
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The mole ratio of the reacting species is an important parameter as the rate of forward
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reaction can be increased by manipulating the concentration of one of the reactants. The investigation of this parameter was done by increasing the alcohol ratio from 1:1, 2:1and 3:1.
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The effect of increased acid ratio was also observed by altering the concentration of acid: alcohol by 2: 1 and the results are depicted in figure 4. All other working parameters were
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kept constant such as temperature of reacting mixture 60˚C, molecular sieves 2 g, duty cycle 83%, enzyme loading 1% (m/v), power input 100 W, ultrasound frequency 25 kHz and
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agitation 80 rpm. From the data, it was cleared that the alcohol in excess drives forward the
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reaction as the higher concentration of acid gave lower conversion of product. In most of the esterification instances the reaction proceeds well with the excess of one component. Thus,
Ac ce p
excess of alcohol or the nucleophile shifts the equilibrium of the esterification in forward pathway to obtain ester [30]. It is elucidated from improved conversion of ester as 50% and 77% using ratio as 1:1 and 1:2 of acid: alcohol respectively in an hour. Surplus concentration of the acid at the vicinity of enzyme leads to acidification of the lipase and its inactivation [31]. It is observed that excessive acidification causes ill-effects to alter enzyme stability or denaturation at higher concentration of acid [22]. At higher molar concentration of acid, the conventional method could reach only 9% conversion in 3 h, whereas under sonication at similar conditions 42 % conversion was gained. Cavitational effect towards the reaction mixture is helpful in uniform mixing of acid without accumulating at the micro environment of the enzyme. Thus it could be inferred that ultrasonic irradiation towards acidic substrates is
11
Page 11 of 38
effective in solvent free reactions without destabilising the biocatalyst assisting in efficient mass transfer. Inhibitory action of the acid could be explained as formation of dead end complex when butyric acid binds with enzyme to form acyl enzyme complex. This acyl
ip t
enzyme complex owing to dead end complex finally cannot yield ester. Additionally, polar nature of butyric acid lowers the pH at the micro-environment of lipase and eventually
cr
deactivates it. Also as alcohol ratio is taken three times the conversion is lowered, due to the fact that even alcohol inhibition may have occur though not prominent and effective as acid
us
[13]. The excess of alcohol more than critical concentration results in the binding action
an
towards the enzyme [31]. Thus, the molar ratio of the substrates isoamyl alcohol to butyric acid as 2:1 is observed as optimum and kept consistent throughout. The optimum mole ratio
for production in supercritical conditions.
M
for isoamyl alcohol and butyric acid mole ratio 1:2 are also quoted by S. Srivastava et al [26]
d
3.4. Effect of enzyme loading
te
To study the effect of enzyme concentration, two batches were put up simultaneously, one
Ac ce p
with the enzyme and the other without the enzyme called as blank. Practically, no reaction took place in the blank batch as no conversion was observed. Thus, it can be stated that for an esterification reaction to take place cavitation alone is not sufficient. The ultrasonic waves assist to accelerate the rate of reaction. The pressure released from many continuous bubble formation and collapse result in emergence of cavitation. This eliminates the mass transfer resistances thus increasing the events of collision and contact of surfaces of substrates. Relevantly, it is physical parameters which accelerates the mass transfer phenomena and suffice mixing throughout the different phases.[32]. However, the concentration of enzyme plays an important role to obtain higher concentration of the product. Thus, experiments were performed for different concentrations ranging from 0.5 % to 3% at temperature of 60˚C, molecular sieves (size 4A˚) weighing 2 g, the ratio of acid to alcohol as 1:2, ultrasound 12
Page 12 of 38
frequency 25 kHz and the power of 100 W. Figure 5 shows the effect of enzyme concentration on ester conversion. The loading of enzyme with 2% (m/v) fetch optimum results as the conversion reached 90% in 120 min. The further loading proved abortive in
ip t
terms of accelerating the rate or converting the substrate in shorter stipulated time [30]. The internal diffusion and relevant acquaintance of the substrate with the active sites could
cr
hamper definitely when the enzyme concentration exceeds. The effect is also profound due to saturation of the enzyme concentration with respect to the substrate concentration beyond and
an
us
it indicates no relative change.
M
3.5 Effect of agitation speed
The speed of agitation has profound effect on the enzyme as well as conversion. In
d
conventional enzyme catalysed reaction, very high speed is required for higher mass transfer.
te
But higher agitation speed causes physical damage to immobilized enzyme affecting the rate of the reaction. Here, one experiment was performed without agitation and other experiments
Ac ce p
were performed for different agitation at 80 rpm, 120 rpm and 200 rpm. The temperature of 60˚C, alcohol to acid mole ratio of 2:1, enzyme loading of 2% (m/v), molecular sieves 2g, power of 70 W, frequency of 25 kHz were kept as constant. The result is illustrated in figure 6 which indicates that the optimum speed of agitation was found to be 80 rpm, from the wide range that was chosen to find out the effect of stirring. There is a remarkable reduction in the conversion when the speed exceeds 120 rpm. At 0 rpm, 80 rpm, 120 rpm and 200 rpm the fall activity was experimentally determined as 9.1U, 8.8U, 7.8U and 6.2U respectively. The measured activity of the enzyme subsides from 9.3U to 6.27U as the speed of agitation increase from 80 rpm to 200 rpm. Illustrated in the figure 6, in absence of mechanical stirring, only 82.6 % conversion was obtained compared to 97.6 % employed with stirring of
13
Page 13 of 38
80 rpm. Thus, this shows that small mechanical agitation helps to improve the conversion in presence of ultrasound. Similar results are quoted by Nikhil et al (2012) [33]. The reaction tends to form a turbid mixture indicating the abrasive enzyme denaturation. The higher
ip t
mechanical shear force (more than 100 rpm) employed with overhead stirring cause denaturation of the enzyme affecting its catalytic property. Besides the reusability is
cr
relevantly affected if the immobilised lipase does not remain intact on its support [34]. The range from 0 rpm – 200 rpm for the study was included with the other working parameters.
an
us
Nevertheless, the agitation also needs more energy consumption when increased further.
M
3.6 Effect of frequency
To evaluate the impact of frequency on the synthesis of ester, two frequencies lower being 25
d
kHz and higher at 40 kHz were included. It is observed that, at lower frequency 25 kHz the
te
yield of the flavour was found to be maximum than at the higher frequency 40 kHz. The calorimetric study was performed to analyse the power dissipation taking place at two
Ac ce p
different frequencies (figure 7). From the experimentation it was calculated that at 40 kHz frequency the power dissipation (30.8 W) is less than that of 25 kHz (46.2 W) at input power of 100 W. Theoretically, the cavitation and the bubbles size is deliberately reduced with increased frequency, which result in subsequent decrease of the intensity and the energy liberated though the implosion of the smaller bubbles [35]. As compared to lower frequency the higher frequency have profound effect but higher frequency has proved to hamper the catalytic activity [36]. In this way higher frequency could not fetch higher product conversion due to less power dissipation, insufficient cavitation phenomenon and inactivation of biocatalyst [37]. Thus, frequency of 25 kHz was chosen for further experimentation and optimisation study.
14
Page 14 of 38
3.7. Effect of Input power The power has an added impact on the reaction and higher ultrasound power gives higher
ip t
conversion. In the case of enzyme catalysed reaction, the conversion increased with the
cr
ultrasound power due to improvement in enzyme activity. On the other the hand, higher ultrasonic power may deactivate the enzyme [38]. Thus, experiments were performed by
us
varying ultrasonic power from 20-140 W to identify the optimum ultrasound power. From the experiment, it is inferred that higher power of more than 100 W when subjected to the
an
mixture, perturb the process enormously. This contrasting result is produced due the fact that the biocatalyst is not able to survive at higher input power leading to deactivation or
M
reduction in catalytic activity [22]. The lower conversion or slow reaction rate conclude that conformational changes or desorption and disruption n of the immobilised lipase take place
d
when exposed to more harsh sonication [39]. An optimal power of 70 W is found to carry the
te
reaction in the forward trend. Specified in the figure 8 is the effect of power for ester
Ac ce p
formation. Additionally the calorimetric analysis for power dissipation was carried out. The power dissipation values obtained for 25 kHz at power 40, 70 100, 140 and 200 W were 36, 43.4, 46.2, 57.2, 72.7 W respectively. There was no remarkable increase in the conversion even when the input power of more than 100 W was employed. Although power dissipation was higher for 140 W it results in deactivation of the lipase. Thus the power input of 70 W was found to the normalised input power in terms of conversion, activity of the catalysed used, power dissipated and energy economical terms.
3.8. Effect of Duty cycle
15
Page 15 of 38
The duty cycle that was engaged all throughout the experimentation was 83%. It indicates On time of 10 min and Off time of 2 min. For the study of the effect of duty cycle the two turns including 83% and 50% (On and off timing of 6 min each) were taken into consideration. The
ip t
other conditions were kept similar, the temperature 60˚C, alcohol to acid mole ratio of 2:1, molecular sieves 2 g, enzyme loading of 2% m/v, power of 70 W and frequency of 25 kHz. It
cr
was justified, that the duty cycle of 83% provided more efficiency than 50%. Naturally, the exposed time of ultrasound irradiation to the reacting mixture when increased, the product
us
formation subsequently increased. The figure 9 depicts the explanation. Although
an
ultrasonication employed for more period of time improve the production of flavour, the continuous operation without pulse can damage the transducers responsible to produce
M
ultrasonic waves [38]. Moreover the synthesis is not expected to carry for longer time in presence of ultrasound as prolonged exposure will deactivate the enzyme catalyst and the
Ac ce p
3.9. Effect of reusability
te
d
temperature of the system may uncontrollably shoot up resulting in charring of the substrates.
The immobilised enzyme, Novozym 435 used, in the reaction was further separated by means of filtration, washed with hexane and reused. Ablution with hexane is crucial step to remove accumulation of water and acid content from the environment of the immobilised biocatalyst [40]. The residue of acid and water may block active sites of lipase and hamper the next successive reaction cycles yielding lower conversions [2]. Several repetitions for the reaction with the treated immobilised enzyme were performed. It was seen that there was a steady decrease in the fall of the activity of the enzyme from initial cycle till the sixth and seventh recurring cycle. There was reduction of activity till eighth cycle up to 38%. The reason could be attributed to losses during recurring cycles and inactivation cause due to substrate
16
Page 16 of 38
inhibition [27]. It can be concluded that the enzyme can be reused for 6 to 7 cycles the reduction from the initial activity being 35% and further till 10 cycles wherein the activity fall observed to 48%. As mentioned earlier consistent exposure of ultrasound can bring about
ip t
conformational changes in lipase and lead to gradual decline in its activity [36]. The Figure
cr
10 depicts the fall of activity of the biocatalyst with repetitive use in consecutive cycles.
us
3.10 Comparison between conventional and ultrasound assisted reaction
an
In order to compare the effect ultrasound on esterification, reaction was carried out in a batch reactor with enzyme in absence of ultrasound. It is found that the time required for the
M
esterification was 10 h to attain conversion of 90 % at the temperature of 60˚C and the amount of isoamyl alcohol double to that of butyric acid (2:1) with addition of molecular
d
sieves 2g. The speed employed for agitation was 300 rpm to enhance the mass transfer. The
te
results obtained using conventional stirred reactor, the ultrasonic assisted reaction with and without agitation are depicted in the figure 11. It has been observed that the incorporation of
Ac ce p
ultrasound irradiation enhances the conversion from about 85% from 13% without the ultrasound in 1 h. Thus, due to the nature of cavitation generated in ultrasound system, the mass transfer resistance is eliminated providing essentially desired product. It also helps in the uniform mixing assisted by the cavitation and turbulence created by overhead stirrer. Further, the micro jets formed when bubble implodes helps for interface renewal. All these effects are responsible for the enhancement in the conversion. However, when reaction was performed in absence of agitation, lower conversion of desired ester was achieved with only 80% in 3 h. This is due to fact that small agitation keeps the particles suspended in reaction mixture and prohibits the coagulation of enzyme particles and also prevents them from settling down.
17
Page 17 of 38
Conclusion The present work was emphasised on intensifying the reaction process of the enzyme
ip t
catalysed synthesis of 3-methyl butyl butanoate, an ester possessing commercial value as
cr
fruity flavour which is widely used in several industries. Incorporating ultrasonication was an added novelty to the conventional method of synthesis of isoamyl butyrate from isoamyl
us
alcohol and butyric acid. Under optimised conditions, maximum conversion of 96.7 % is obtained in 3 hour as compared to 10 h of conventional enzyme catalysed reaction. The
an
immobilised lipase Novozym 435 also proved useful up to 7 cycles with only 30% reduction in the fall of its initial activity. It is clear from the experiments that the immobilised enzyme
M
could sustain ultrasonic environment serving improved yield in less time. This data shows
Ac ce p
te
d
potential application of ultrasound for enzymatic synthesis of flavour compounds.
18
Page 18 of 38
[1]
ip t
References: Gubicza L, Kabiri-Badr A, Keoves E, Belafi-Bako K. Large-scale enzymatic
cr
production of natural flavour esters in organic solvent with continuous water removal.
[2]
us
J Biotechnol 2001;84:193–6.
Friedrich JLR, Pena FP, Garcia-Galan C, Fernandez-Lafuente R, Ayub MAZ,
an
Rodrigues RC. Effect of immobilization protocol on optimal conditions of ethyl
Biotechnol 2013;88:1089–95.
Krishna SH, Sattur AP, Karanth NG. Lipase-catalyzed synthesis of isoamyl isobutyrate
d
[3]
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butyrate synthesis catalyzed by lipase B from Candida antarctica. J Chem Technol
[4]
Ac ce p
2001;37:9–16.
te
— optimization using a central composite rotatable design. Process Biochem
Anschau A, Aragão VC, Porciuncula BDA, Kalil SJ, Burkert CA V, Burkert JFM.
Enzymatic Synthesis Optimization of Isoamyl Butyrate. J Braz Chem Soc 2011;22:2148–56.
[5]
Chapuis C, Jacoby D. Catalysis in the preparation of fragrances and flavours. Appl
Catal A Gen 2001;221:93–117. [6]
Santhanakrishnan A, Shannon A, Peereboom L, Lira CT, Miller DJ. Kinetics of Mixed Ethanol / n - Butanol Esterification of Butyric Acid with Amberlyst 70 and p - Toluene Sulfonic Acid. Ind. Eng. Chem. Res. 2013, 52, 1845−1853
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[7]
Fernandes SA, Natalino R, Gazolla PAR, da Silva MJ, Jham GN. p-Sulfonic acid calix[n]arenes as homogeneous and recyclable organocatalysts for esterification reactions. Tetrahedron Lett 2012;53:1630–3. Romero MD, Calvo L, Alba C, Daneshfar a., Ghaziaskar HS. Enzymatic synthesis of
ip t
[8]
isoamyl acetate with immobilized Candida antarctica lipase in n-hexane. Enzyme
Sonare NR, Rathod VK. Transesterification of used sunflower oil using immobilized
Rathod VK, Pandit AB. Effect of various additives on enzymatic hydrolysis of castor oil. Biochem Eng J 2009;47:93–9.
Martins AB, Friedrich JLR, Cavalheiro JC, Garcia-Galan C, Barbosa O, Ayub MAZ,
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an
enzyme. J Mol Catal B Enzym 2010;66:142–7.
us
[9]
cr
Microb Technol 2005;37:42–8.
te
Fernandez-Lafuente R,Rodrigues RC. Improved production of butyl butyrate with lipase from Thermomyces lanuginosus immobilized on styrene-divinylbenzene beads.
Ac ce p
Bioresour Technol 2013;134:417–22.
[12] Krishna SH, Manohar B, Divakar S, Karanth NG. Lipase-catalyzed synthesis of isoamyl butyrate: Optimization by response surface methodology. J Am Oil Chem Soc 1999;76:1483–8.
[13]
Krishna SH, Karanth NG. Lipase-catalyzed synthesis of isoamyl butyrate A kinetic
study. Biochimica et biophysia Acta 2001;1547:262–7. [14]
Hari Krishna S, Prapulla SG, Karanth NG. Enzymatic synthesis of isoamyl butyrate using immobilized Rhizomucor miehei lipase in non-aqueous media. J Ind Microbiol Biotechnol 2000;25:147–54. 20
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[15]
Zhao R, Ding Y, Peng Z, Wang X, Suo J. One-step synthesis of isoamyl butyrate from isoamyl alcohol and n -butyraldehyde over TS-1 in air. Catalysis Letters 2003;87:81– 83. Macedo GA, Pastore GM, Rodrigues MI. Optimising the synthesis of isoamyl butyrate
ip t
[16]
using Rhizopus sp . lipase with a central composite rotatable design. Process Biochem
Ben Salah R, Ghamghui H, Miled N, Mejdoub H, Gargouri Y. Production of butyl
us
[17]
cr
2004;39:687–92.
an
acetate ester by lipase from novel strain of Rhizopus oryzae. J Biosci Bioeng 2007;103:368–72.
Leonelli C, Mason TJ. Microwave and ultrasonic processing: Now a realistic option
M
[18]
Chemat F, Zill-e-Huma, Khan MK. Applications of ultrasound in food technology:
te
[19]
d
for industry. Chem Eng Process Process Intensif 2010;49:885–900.
Ac ce p
Processing, preservation and extraction. Ultrason Sonochem 2011;18:813–35. [20]
Fallavena LP, Antunes FHF, Alves JS, Paludo N, Ayub MAZ, Fernandez-Lafuente R,
Rodrigues
RC.
Ultrasound technology
and
molecular sieves improve the
thermodynamically controlled esterification of butyric acid mediated by immobilized lipase from Rhizomucor miehei. RSC Adv 2014;4:8675.
[21]
Hobuss CB, Venzke D, Pacheco BS, Souza AO, Santos MZ, Moura S, Quina FH,
Fiametti KG, Oliveira JV, Pereira CMP Ultrasound-assisted synthesis of aliphatic acid esters at room temperature. Ultrason Sonochem 2012;19:387–9.
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[22]
Martins AB, Schein MF, Friedrich JLR, Fernandez-Lafuente R, Ayub MAZ, Rodrigues RC. Ultrasound-assisted butyl acetate synthesis catalyzed by Novozym 435: enhanced activity and operational stability. Ultrason Sonochem 2013;20:1155–60. Kiran KR, Krishna SH, Babu CVS, Karanth NG, Divakar S. An esterification method
ip t
[23]
[24]
cr
for determination of lipase activity Biotechnology letters 2000;20:1511–4.
Kulkarni VM, Rathod VK. Mapping of an ultrasonic bath for ultrasound assisted
us
extraction of mangiferin from Mangifera indica leaves. Ultrason Sonochem
[25]
an
2013;21:606–11.
Martins B, Friedrich JLR, Rodrigues RC, Garcia-galan C, Fernandez-lafuente R, Ayub
M
MAZ. Optimized Butyl Butyrate Synthesis Catalyzed by Thermomyces lanuginosus
Srivastava S, Modak J, Madras G. Enzymatic Synthesis of Flavors in Supercritical
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[26]
d
Lipase. Biotechnol Prog, 2013;29:6 2013.
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Carbon Dioxide. Ind Eng Chem Res 2002;41:1940–5. [27]
Xun E, Lv X, Kang W, Wang J, Zhang H, Wang L, Zhang H, Wang L& Wang Z.
Immobilization of Pseudomonas fluorescens lipase onto magnetic nanoparticles for resolution of 2-octanol. Appl Biochem Biotechnol 2012;168:697–707.
[28]
Alissandrakis E. Ultrasound-assisted extraction of volatile compounds from citrus
flowers and citrus honey. Food Chem 2003;82:575–82.
[29]
Deshmane VG, Gogate PR, Pandit AB. Ultrasound assisted synthesis of isopropyl esters from palm fatty acid distillate. Ultrason Sonochem 2009;16:345–50.
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[30]
Lorenzoni ASG, Graebin NG, Martins AB, Fernandez-Lafuente R, Ayub MAZ, Rodrigues RC. Optimization of pineapple flavour synthesis by esterification catalysed by immobilized lipase from Rhizomucor miehei. Flavour Fragr J 2012;27:196–200. Guvenc A, Kapucu N, Mehmetoglu U. The production of isoamyl acetate using
ip t
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[32]
cr
immobilized lipases in a solvent-free system. Process Biochemistry 2002;379-386.
Jadhav D, Rekha BN, Gogate PR, Rathod VK. Extraction of vanillin from vanilla
us
pods: A comparison study of conventional soxhlet and ultrasound assisted extraction. J
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an
Food Eng 2009;93:421–6.
Gharat N, Rathod VK. Ultrasound assisted enzyme catalyzed transesterification of
Kuperkar V V, Lade VG, Prakash A, Rathod VK. Synthesis of isobutyl propionate
d
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waste cooking oil with dimethyl carbonate. Ultrason Sonochem 2013;20:900–5.
te
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Ac ce p
Mol Catal B Enzym 2014;99:143–9. [35]
Gogate PR. Cavitational reactors for process intensification of chemical processing
applications : A critical review .Chemical Engineering and Processing 2008;47:515– 27.
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Zheng M-M, Wang L, Huang F-H, Dong L, Guo P-M, Deng Q-C, Zheng C, Wan C-H.
Ultrasound irradiation promoted lipase-catalyzed synthesis of flavonoid esters with unsaturated fatty acids. J Mol Catal B Enzym 2013;95:82–8.
[37]
Zheng M-M, Wang L, Huang F-H, Guo P-M, Wei F, Deng Q-C,Li W-L Zheng C, Ultrasonic pretreatment for lipase-catalyed synthesis of phytosterol esters with different acyl donors. Ultrason Sonochem 2012;19:1015–20. 23
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[38]
Avhad DN, Rathod VK. Ultrasound stimulated production of a fibrinolytic enzyme. Ultrason Sonochem 2014;21:182–8.
[39]
Fiametti KG, Sychoski MM, De Cesaro A, Furigo A, Bretanha LC, Pereira CMP,
ip t
Treichel H, de Oliveira D, Oliveira VJ. Ultrasound irradiation promoted efficient solvent-free lipase-catalyzed production of mono- and diacylglycerols from olive oil.
cr
Ultrason Sonochem 2011;18:981–7.
us
[40] Martins AB, Graebin NG, Lorenzoni ASG, Fernandez-Lafuente R, Ayub MAZ,
an
Rodrigues RC. Rapid and high yields of synthesis of butyl acetate catalyzed by Novozym 435: Reaction optimization by response surface methodology. Process
Ac ce p
te
d
M
Biochem 2011;46:2311–6.
24
Page 24 of 38
List of Figures: Fig 1 Schematic of experimental set up of esterification of Isoamyl alcohol and butyric acid catalysed with enzyme (Novozym 435) assisted with ultrasonic system.
ip t
Fig2. Effect of time on the esterification with the impact of ultrasound irradiation Reaction conditions: Novozym 435 1% (m/v), Temperature 60˚C, stirring speed 100
cr
rpm, Frequency 25 kHz, duty cycle 83%, Power 100 W Fig 3 Effect of temperature on the esterification of Isoamyl butyrate
us
Reaction conditions: Novozym 435 (lipase)1% (m/v), Acid: Alcohol 1:2, stirring speed 80 rpm, frequency 25 kHz, duty cycle 83%, power 100 W
an
Fig 4 Effect of mole ratio of Butyric acid and Isoamyl alcohol on conversion Reaction conditions: Novozym 435 1% (m/v), Temperature 60˚C, stirring speed
M
80rpm, Frequency 25 kHz, duty cycle 83%, Power 100 W
d
Fig 5 Effect of enzyme loading on conversion
Reaction conditions: Temperature 60˚C, stirring speed 80 rpm, acid alcohol ratio 1:2,
te
Frequency 25 kHz, Duty cycle 83% and Power 100 W
Ac ce p
Fig 6 Effect of stirring speed on conversion Reaction conditions: Temperature 60˚C, acid alcohol ratio 1:2, enzyme loading 2% (m/v) Frequency 25 kHz, Duty cycle 83% and power 100 W
Fig 7 Effect of frequency of the ultrasound irradiation on conversion Reaction conditions: temperature 60˚C, acid: alcohol1:2, stirring speed 80 rpm, enzyme concentration 2% (m/v) duty cycle 83% and power 100 W
Fig 8 Effect of power of the ultrasound irradiation on conversion Reaction conditions: temperature 60˚C, acid: alcohol 1:2, stirring speed 80 rpm, enzyme loading 2% (m/v), frequency 25 kHz, and duty cycle 83% Fig 9 Effect of duty cycle input during the reaction in progress
25
Page 25 of 38
Reaction condition: temperature 60˚C acid: alcohol1:2, stirring speed 80 rpm, enzyme loading 2% (m/v), frequency 25 kHz, Power 70 W Fig 10 Reusability of immobilised Novozym 435 for repeated cycles Fig 11 The Comparison for synthesis of Isoamyl butyrate through conventional synthesis
ip t
(CS) and ultrasound irradiation (UI) The reaction conditions CS: temperature 60˚C, molecular sieves 2g, acid: alcohol 1:2, enzyme loading 2% (m/v), stirring 300 rpm.
cr
UI: temperature 60˚C, molecular sieves 2g, acid; alcohol 1:2, stirring speed 80 rpm,
us
enzyme loading 2% (m/v), frequency 25 kHz, duty cycle 83%, Power 70 W with ultrasonic irradiation
Ac ce p
te
d
M
an
.
26
Page 26 of 38
ip t cr
1. Overhead Stirrer 1
us
2. Condenser
2
3. Reaction mixture
an
4. Ultrasonic Bath
M
5. Transducers
4
Ultrasound Generator
5
Ac ce p
te
d
3
Fig 1 Schematic of experimental set up of esterification of Isoamyl butyrate from Isoamyl alcohol and butyric acid catalysed with enzyme (Novozym 435) assisted with ultrasonic system.
27
Page 27 of 38
ip t cr us an M d te
Ac ce p
Fig 2 Effect of time on the esterification with the impact of ultrasound irradiation Reaction conditions: Novozym 435 1% (m/v), Temperature 60˚C, stirring speed 100 rpm, molecular sieves 2g, Frequency 25 kHz, duty cycle 83% and power 100 W
28
Page 28 of 38
ip t cr us an M d te
Ac ce p
Fig 3 Effect of temperature on the esterification of Isoamyl butyrate Reaction conditions: Novozym 435 (lipase)1% (m/v), Acid: Alcohol 1:2, stirring speed 80 rpm, molecular sieves 2g, frequency 25 kHz, duty cycle 83%, power 100 W
29
Page 29 of 38
ip t cr us an M d te
Ac ce p
Fig 4 Effect of mole ratio of Butyric acid and Isoamyl alcohol on conversion Reaction conditions: Novozym 435 1% (m/v), Temperature 60˚C, stirring speed 80 rpm, molecular sieves 2g, Frequency 25 kHz, duty cycle 83%, Power 100 W
30
Page 30 of 38
ip t cr us an M d
te
Fig 5 Effect of enzyme loading on conversion Reaction conditions: Temperature 60˚C, stirring speed 80 Rpm, molecular sieves 2g,
Ac ce p
acid alcohol ratio 1:2, Frequency 25 kHz, duty cycle 83% and Power 100 W
31
Page 31 of 38
ip t cr us an M d
te
Fig 6 Effect of stirring speed on conversion
Reaction conditions: Temperature 60˚C, acid alcohol ratio 1:2, enzyme loading 2%
Ac ce p
(m/v), molecular sieves 2g, Frequency 25 kHz, duty cycle 83% and power 100 W
32
Page 32 of 38
ip t cr us an M d
Fig 7 Effect of frequency of the ultrasound irradiation on the conversion
te
Reaction conditions: temperature 60˚C, acid: alcohol 1:2, stirring speed 80 rpm, enzyme concentration 2% (m/v), molecular sieves 2g, duty cycle 83% and power
Ac ce p
100W
33
Page 33 of 38
ip t cr us an M d
te
Fig 8 Effect of power input of the ultrasound irradiation on conversion Reaction conditions: temperature 60˚C, acid: alcohol 1:2, stirring speed 80 Rpm,
Ac ce p
molecular sieves 2g, enzyme loading 2% (m/v), frequency 25 kHz, and duty cycle 83%
34
Page 34 of 38
ip t cr us an M d te
Fig 9 The Effect of duty cycle employed during the reaction in progress
Ac ce p
Reaction condition: temperature 60˚C acid: alcohol1:2, stirring speed 80 rpm, molecular sieves 2g, enzyme loading 2% (m/v), frequency 25 kHz, Power 70 W
35
Page 35 of 38
ip t cr us an M d te
Ac ce p
Fig 10 The reusability of immobilised Novozym 435 for repeated cycles
36
Page 36 of 38
ip t cr us an M d
te
Fig 11 The Comparison for synthesis of Isoamyl butyrate through conventional synthesis (CS) and ultrasound irradiation (UI) The reaction conditions CS: temperature 60˚C,
Ac ce p
molecular sieves 2g, acid: alcohol 1:2, enzyme loading 2% (m/v), stirring 300 Rpm. UI: temperature 60˚C, molecular sieves 2g, acid; alcohol 1:2, stirring speed 80 rpm, enzyme loading 2% (m/v), duty cycle 83 %, frequency 25 kHz, Power 70 W with ultrasonic irradiation.
37
Page 37 of 38
Highlights Ultrasound assisted lipase catalyzed synthesis of Isoamyl butyrate was performed. The reaction parameters needed to drag the reaction to forward pathway were optimized.
ip t
Ultrasonication reduced reaction time than conventional enzymatic synthesis.
Ac ce p
te
d
M
an
us
cr
The immobilized lipase could be use repetitively for 7 cycles.
38
Page 38 of 38