An intensified technique for lipase catalysed amide synthesis

Chemical Engineering & Processing: Process Intensification 143 (2019) 107605

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An intensified technique for lipase catalysed amide synthesis Sneha R. Bansode, Virendra K. Rathod

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Chemical Engineering Department, Institute of Chemical Technology, Matunga, Mumbai, 400 019, India

A R T I C LE I N FO

A B S T R A C T

Keywords: Propyl acetamide Amidation Lipase Ultrasonication Substrate inhibition

The enzymatic synthesis of propyl acetamide under the influence of sonication has been explored. The effect of various reaction parameters that drive the reaction was studied and optimised under solvent free condition to achieve maximum yield. At temperature 60 °C, mole ratio of propyl amine to butyl acetate 1:1.5, 3% (w/v) of lipase loading with ultrasound (US) frequency as 22 kHz, 100 W power of ultrasonic bath and with agitation speed of 100 rpm, 98% conversion was obtained in 2 h. Kinetic modeling of lipase catalysed amidation illustrated the substrate inhibition by propylamine and the values determined are Vmax = 9.9 M/min/g of catalyst and Kia = 1.45 M. The thermodynamic study revealed that US lowered the energy of activation thus making it more feasible than the conventional technique. The reusability studies were also performed and immobilised lipase was reusable for 5 successive cycles with 50% loss from its initial activity. Thus, ultrasound successfully intensified the amidation reaction as compared to conventional mechanical stirring by two folds.

1. Introduction Sonochemical synthesis is gaining attention in recent times due to its intensification phenomenon and several other advantages. The prime phenomenon induced from ultrasound irradiation is cavitation [1]. The cavitation phenomenon generates numerous bubbles in a medium which undergo consecutive formation, growth, and finally implodes. These bubbles on collapse can generate magnificent localised hot spots (temperature) and pressure more than 1000 atm [2]. The implosion of bubble not only creates immense temperature and pressure but also produces shockwaves, microstreaming, and jet formation that provide efficient mixing and improved contact of the species undergoing irradiation. Owing to these advantages, the sonication has been deliberately applied during reactions in various industries for the synthesis of chemicals [3]. With the advancement of other chemical synthesis, enzymatic synthesis also attracted the interest of the researchers. The physical changes occur due to ultrasound irradiation in enzymatic reactions offer improved results and intensifications with respect to conventional chemical catalysed reaction. It is identified that the ultrasonic waves can perturb the enzymatic structure, which is responsible for enhanced exposure of active sites and their effectual contact with reactive species [4]. Most of the times, enzyme assisted reactions are preferred owing to the greener route, few side products, and maintenance of mild reaction conditions. Besides, it suffers a major drawback of slow reaction rate. Hence, there was a need to accelerate this rate of reaction by

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incorporating newer techniques, and ultrasound irradiation is one of the alternatives [5]. There are numerous compounds such as drugs, food additives, or other consumables that are not only synthesised enzymatically under mild reaction conditions but also directly consumed by individuals which fall under food safety regulations. Thus, enzyme catalysed routes can be suitable in industrial processes or products that should devoid of injurious chemicals or catalysts [6]. Reactions viz, hydrolysis, esterification, transesterification, and aminolysis, etc. are well known and reported reactions which are carried out under mild reaction conditions using lipases as biocatalysts [7]. Lipases [EC 3.1.1.3] belong to hydrolyses group of enzymes, and their structure constitutes of surface loop and lid like structure. The three major residues viz; serine, histidine, and aspartate or glutamate comprise as the active catalytic site of lipase as biocatalyst [8]. Ultrasound irradiation is identified to activate lipase by flipping the lid- surface thus exposing the active binding site [9]. Overall, ultrasound assisted lipase catalysed synthesis has paved a broader aspect from conventional systems by intensifying reaction processes. Amide synthesis is gaining importance due to its soaring demands from various industries and particularly pharmaceutical industry where amides are precursors in many compounds of medicinal significance. The amidation is carried out using amines and carboxylic acids or carboxylic esters as starting materials to obtain respective amides [10]. The work carried out on amides so far includes enzymatic synthesis at 30 °C using di-iso-propyl ether as solvent from phenolic esters and phenolic amines. The formation of amides such as benzyl lauryl amide

Corresponding author. E-mail address: [email protected] (V.K. Rathod).

https://doi.org/10.1016/j.cep.2019.107605 Received 18 March 2019; Received in revised form 15 July 2019; Accepted 17 July 2019 Available online 18 July 2019 0255-2701/ © 2019 Elsevier B.V. All rights reserved.

Chemical Engineering & Processing: Process Intensification 143 (2019) 107605

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Notation US E DES ES P v Vmax Ka Kb

Kia Kib T A Ea R Kb h ΔH ΔS ΔG

Ultrasound Free enzyme Dead end complex Enzyme substrate complex Product Rate of reaction Maximum rate of reaction Michaelis Menten constant for propyl amine Michaelis Menten constant for butyl acetate

Inhibitory constant of amine Inhibitory constant of butyl acetate Temperature (Kelvin K). Pre-exponential factor Energy of activation (Jmol−1) universal gas constant (8.314 Jmol−1 K) Boltzmann constant Planck’s constants change in enthalpy change in entropy change in Gibbs free energy

bath having internal dimensions 300 cm x 150 cm x 150 cm and provided with five transducers at the bottom along the length of the bath. The reaction position was fixed in the ultrasound bath at the place of maximum cavitation intensity, which was investigated from previous work [15]. The reagents viz; propyl amine 3 mL (0.1 mol), butyl acetate 10 mL (0.2 mol), dodecane as internal standard (2 mL) and weighed quantity of biocatalyst were charged in a stirred batch reactor without addition of solvent. The ultrasound frequency of 22 kHz was applied in a pulsating mode at a stirring speed of 100 rpm and aliquots of samples were drawn to determine the progress of reaction through gas chromatography (GC) analysis.

and benzyl caprylate amide in conventional technique required more than 5 h for 50% conversion [11]. Enzymatic amidation of carboxylic acid through liquid ammonia [12] and ammonium salts (ammonium carbamate) in different solvents [13] [14] are also reported in presence of solvents such as ter amyl alcohol and methyl isobutyl ketone. Toluene as solvent using Novozym 435 as a catalyst to produce amide from benzyl acetate and butylamine was carried out at 500 rpm and 55 °C using conventional stirring method to yield 46% in 8 h [10]. Amidation catalyzed by Novozym 435 was carried out through continuous column (plug flow reactor) in a various solvent using oleic acid with the addition of ammonium carbamate and liquid ammonia. Terbutanol at 60 °C was found to yield 75% amidation product through the study. Though vast literature is available on amidation reactions, detailed study related to effects of reaction parameters underlying the synthesis is hardly explored for ultrasound assisted solvent free amidation. Thus, this present investigation explains the synthesis of propyl amide catalysed using immobilised lipase as biocatalyst with intensification under ultrasound irradiation. To the best of authors knowledge, the synthesis of propyl acetamide is not carried out in ultrasonic system using lipase as biocatalyst or reported. Hence, to achieve maximum conversion and make the synthesis route of propyl amide greener immobilised lipase Candida antarctica was used as biocatalyst, and use of solvent was avoided.

2.3. Analysis of sample GC analysis was carried out to determine the concentration of product in the reaction mixture. Dodecane was added as an internal standard in the reaction and conversion of reactants was calculated with reference to the peak area of internal standard. Sample volume of 2 μL was injected in Gas Chemito model no 8610 equipped with a flame ionised detector (FID) along with steel packed column OV 17 column (3 m ×0.32 mm and 10% stationary phase). Nitrogen was used as carrier gas at pressure 1.2 bar. The temperature program was customised in the following steps: 70 °C for 1 min; 10 °C/min up to 120 °C; then steady temperature for 1 °C; further 280 °C was programmed at a rate of 20 °C/min. The injector and detector temperature were maintained at 150 °C and 250 °C, respectively.

2. Materials and methods 2.1. Materials

2.4. Spectrophotometric assay of lipase 2.1.1. Biocatalyst Lipase as biocatalyst Fermase CALB 10,000, a commercial Candida antarctica lipase B (CALB) immobilized on support comprised of glycidyl methacrylate terdivinylbenzene-ter-ethylene glycol dimethacrylate (particle size of 150–300 μm, pore volume of 1.32 cm3/g, bulk density of 0.54 g/cm3) was generously gifted by Fermenta Biotech Ltd., Mumbai, India.

The assay of Lipase, Fermase CALB was done with p-Nitrophenyl acetate (p-NPA) as substrate and determined spectrophotometrically. The reaction mixture is prepared with 2.0 mL of buffer with the composition of 0.15 M NaCl, 1 g L−1 gum arabic, and 0.1 M sodium phosphate (pH 7.0) and 0.1 mL of 0.1 M p-NPA dissolved in 2-propanol as substrate. In the next step, weighed amount of catalyst (0.05 mg) was added in the above reaction mixture kept at 40 °C. The mixture is allowed to incubate for 5 min, and after the incubation period, the solution was decanted carefully to record its absorbance at 405 nm under UV/vis spectrophotometer [16]. The activity of enzyme is calculated from its absorbance and one enzyme unit is defined as amount of the enzyme that releases 1 μmol of p-nitrophenol per minute at pH 7.0 and 40 °C and is expressed as 9.78 U (U for convenience).

2.1.2. Reagents Chemicals including Propyl Amine (purity 98%), butyl acetate (purity 98%), and Dodecane (purity 99%) as internal standard, (hexane 98%) and isopropyl alcohol (98%) were bought from Thomas Baker Pvt. Ltd., Mumbai. Other chemicals for lipase assay including NaCl, gum arabic, sodium phosphate and p- nitro phenyl acetate were purchased from S.D. Fine Chemicals Pvt. Ltd., Mumbai. All the reagents mentioned above were used as procured without further treatment.

2.5. Determination of initial rates The initial rates for propyl amide were determined mainly for temperature and mole ratio. Mole ratio of propyl amine: butyl acetate was varied from 1:1, 1:1.5, 1:2, 1:2.5, and 2:1 at constant temperature of 60 °C, frequency 22 kHz, 100 W power, 75% duty cycle, stirring speed 100 rpm and enzyme loading of 3% (w/v). The amidation reaction between butyl acetate (ester) and propyl amine is catalysed by lipase to form propyl acetamide along with butanol (schematic

2.2. Synthesis of lipase assisted propyl acetamide The reaction was carried out in a flat bottom stirred batch reactor of 50 mL capacity having 4.5 cm internal diameter. It was covered with three necked lid to which various parts, i.e., four-blade turbine impeller, condenser for reflux and sample port, were fixed. The assembly of this batch reactor was immersed in a thermostatic ultrasonic water 2

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representation in Fig. 1). To study the progress of rate of reaction, the samples were withdrawn and analysed through GC analysis. 3. Result and discussion The ultrasonic technique has both positive and negative impact on enzymatic reactions. The physical outcomes give rise to tremendous changes in the reaction environment with respect to temperature, pressure and shear forces that can generate a deleterious effect on biocatalyst as well as reaction rates. Accordingly, it is necessary to optimise the reaction parameters so that the adverse conditions are avoided to yield better product and process is intensified and benefitted in positive mode. 3.1. Influence of temperature Fig. 2. Influence of temperature on amidation; reaction conditions: lipase 3% (w/v), molar ratio propyl amine: butyl acetate 1:2, ultrasound frequency 22 kHz, input power 100 W, duty cycle 75% and stirring speed 100 rpm.

Lipase catalysed synthesis is temperature dependent since the activity of enzyme can be affected with a slight change in the temperature of the system. The requirement of mild temperature condition during the process is the additional advantage of enzyme catalyst [17]. Thus, it is crucial to study the optimum temperature in order to obtain improved conversion and maintain active form of the biocatalyst. It is well known that at elevated temperatures, the kinetic energy of the molecules increases that lead to faster and efficient collisions [18]. Here, the temperature range of 40 °C to 70 °C was studied with a mole ratio of propyl amine and butyl acetate 1:2, lipase loading 3%, frequency of 22 kHz, input power of 100 W and stirring speed of 100 rpm. Fig. 2 shows that as the temperature is elevated from 40 to 70 °C, the conversion and the rate of reaction increases. With the rise in temperature, there is an increased rate of diffusion since the viscosity of system is lowered, especially in the heterogeneous reaction mixtures [18]. However, temperature of 60 °C is preferred as optimal for amidation reaction since it is observed and reported that lipase form candida antarctica is mostly active and exhibit its higher catalytic activity at 60 °C. For the synthesis of D-isoascorbyl palmitate, maximum 94% conversion was reported at 61 °C when 30 to 70 °C temperature range was studied in the presence of Novozym 435 as a catalyst and 137 W ultrasonic power [19]. It is best to avoid temperatures above 60 °C since the prolonged exposure of lipase at higher temperatures may hamper the durability of the immobilised enzyme [20]. The final conversion obtained at 60 °C and 70 °C at the end of 2 h was 94% and 88% respectively. This can be attributed that at 70 °C there is a reduction of lipase activity with prolonged time. On the other hand, at higher temperatures, there is trapping of vapours in the growing cavitational bubble that results in cushioning effect during the implosion of these bubbles and exhorts diminished sonochemical effect on the system [21]. Another thermal impact that influences the catalytic activity of lipase emerging from ultrasonic heat and moderate pressure is called as mano-thermosonication (MTS). MTS can alter the activity of lipase moiety and also a profound effect of MTS interferes with chemical or physical interaction among the substrate and enzyme [22]. The experiments performed by Zheng et al. for the synthesis of phytosterol esters using Canadia sp. 99–125 lipase (LS-20) as biocatalyst also reported maximum conversion at 60 °C [23].

Fig. 3. Influence of molar ratio (propyl amine: butyl acetate) on amidation; reaction conditions: lipase 3% (w/v), temperature 60 °C, ultrasound frequency 22 kHz, input power 100 W, duty cycle 75% and stirring speed 100 rpm.

to 1:2 at 60 °C, 3% (w/v) of lipase loading with overhead stirring of 100 rpm, engaging frequency and power of ultrasonic bath 22 kHz and 100 W respectively. From Fig. 3 it can be perceived that the conversion improved with a slight excess of butyl acetate than propyl amine. The conversion obtained with a mole ratio of 1:1.5 (propyl amine to butyl acetate) was 98% as compared to 73% for 1:1 in 3 h. On the contrary, there was a slight decrease in the conversion (90%) when the mole ratio of 1:2 was used. Further, an increase in a mole ratio to 1:2.5 the conversion decreased to 85% due to the inhibitory action of butyl acetate ester. It is mostly preferred that a slight excess of one of the substrates can assist to increase the rate of forward reaction [24]. Nevertheless, with an excess of butyl acetate there was no significant change in reaction rate which can be attributed to two reasons a) higher concentration of butyl ester could be responsible for inhibitory action on lipase and b) excess butyl ester can form dead end complex with the lipase that further does not yield any product. With the increased concentration of propyl amine, the reaction could not proceed for more than 30% due to the decay of immobilised lipase owing to the denaturation in a basic environment (data not shown). The exact cause of lower conversion at high concentration of

3.2. Influence of ratio of substrates The mole ratio of propyl amine and butyl acetate was varied as 1:1

Fig. 1. Schematic representation of chemical reaction for amidation of Propyl acetamide catalyzed from lipase.

3

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creates less and ineffective cavitational impact leading to a lower rate of conversion. Similarly, with an increase in power intensity from 110 W and 120 W, the conversion also declined to 72% and 61% respectively from 95% (at 100 W) in 2 h. At high power, there is a very high implosion of larger bubbles that can cause excessive heating that denatures the enzyme and transduces are prone to damage. Also, it has been found that severe conditions emerging due to high intensities can disrupt the lipase by breaking down the intra-molecular hydrogen bonding thus rendering the biocatalyst useless [9]. Hydrolysis of waste cooking oil catalysed by immobilised Candida antarctica was also performed under 100 W power intensity at ultrasound frequency of 22 kHz to obtain 75% conversion in 2 h [33]. The enzyme activity test was also carried out at 120 W and 22 kHz to confirm the decline from 9.7 to 8U. Further, in order to investigate actual ultrasound power dissipation, the calorimetric study was performed at 22 kHz and 40 kHz. The values of power dissipated at an input power of 50, 100 and 150 W were 42 W, 46.5 W and 61.8 W while the intensity was found to be 6687.89 W/m2, 7356.68 W/m2, and 9808.91 W/m2 respectively. Similarly at 40 kHz frequency power dissipation found was 18.2 W, 30.8 W and 43.4 W respectively. The power dissipation study indicated that higher ultrasound frequency lowered the dissipation of power. At higher frequency, the time cycle of compression and rarefaction is reduced; thus, cavitational bubble size is reduced, and it implodes with less intensity and energy liberated through these bubbles is comparatively less than at lower frequency. Consequently, the overall effect of cavitation is reduced at high frequency and power [21]. Hence, 22 kHz ultrasound frequency with 100 W power input was found to be optimum with consideration of the rate of reaction and damage to enzyme catalytic sites and the impact of cavitational intensity.

amine is not identified, but it can be related to the denaturation of lipase at basic pH (enzymes are pH sensitive) [9]. The optimum pH for candida species is 6.5–7.5 where it exhibits its maximum activity, with an increase in the concentration of propyl amine the pH shifts from 7.5 to 9.5 thus the activity is hampered and conversion is lowered drastically [25]. Propyl amine is a basic primary amine and may bring about adverse structural change that curtails the catalytic function of lipase [26]. 3.3. Influence of lipase loading The effect of enzyme loading was studied by varying concentration of immobilised lipase from 1% to 4% (w/v) of the total reaction mixture. It was depicted in Fig. 4 that with an increase in the concentration of lipase, there was an increase in the conversion and rate of reaction. As the lipase loading was increased from 2% to 3%, the conversion also improved significantly from 87% to 98% in 2 h. In US environment, micro-stirring and turbulence created from cavitation lead to efficient mass transport, thus improving the overall rate of reaction as compared to the mechanical stirring method [21]. It is suggested that mild frequency of ultrasound causes the flipping of the active catalytic conformation of lipase that improves exposure and interaction of substrates and hence enhances the rate of reaction [27]. Moreover, cavitation causes unfolding of the enzyme structure, thus accessing the active sites to the substrate molecules easily [28]. With further increase in loading of catalyst from 3% to 4% (w/v), there was a marginal improvement in conversion or rate of reaction. Thus 3% (w/v) was selected as optimum enzyme loading. One of the possible reasons for decrement of the conversion is the aggregation of immobilized catalyst at higher concentration and limiting the exposure of active sites [29]. With high enzyme loading crowding of enzyme particles can limit the internal diffusion and mass transfer in the reaction, which results in lower conversion. Similarly, more quantity of solid particles in US is reported to affect the dissipation energy and may inhibit the scattering of sound waves in the medium [30]. Considering the cost and recycle of immobilised lipase 3% (w/v) was used in further experiments.

3.5. Influence of duty cycle The prolonged continuous irradiation of ultrasound may lead to excessive heating of the reaction system, which poses many harmful effects in the case of enzymatic reactions. The pulse mode of ultrasonic irradiation is preferred over continuous mode in ultrasound irradiation system to avoid denaturation of lipase that occurs from the generation of excessive heat and also to increase the life of the transducers [16]. The duty cycle of 50% (6 min ON 6 min OFF) 75% (9 min ON 3 min OFF) and 91% (11 min ON 1 min OFF) were studied to understand its effect and obtain maximum conversion depicted in Fig. 7. The conversion attained at 75% duty cycle was 98% as compared to 75% and 85% conversion obtained at 50% and 91% duty cycle respectively. The results are in good agreement with work published by Adulkar et al., for hydrolysis of dairy waste catalysed by lipase under ultrasound

3.4. Influence of ultrasound frequency and power It is very crucial to determine the optimal frequency, and power of ultrasonic device for enzyme assisted synthesis as the reaction rate, conversion and activity of lipase depend upon the overall cavitational impact and intensity of implosion of cavitating bubbles. At lower (20–35 kHz) frequency and power, large cavitational bubbles are formed that implode with high intensity leading to generation of high temperature and pressure areas that induce catalytic activity and interaction of substrate molecules enhancing the reaction rates [31]. The ultrasonic bath under study can be set up at two frequencies (22 kHz and 40 kHz) within the power range of 40–200 W. Fig. 5 depicts that the final conversion and rate is higher at 22 kHz (98%) than 40 kHz (83%), which indicates that at high frequency, the cavitational effect was reduced leading to decreased reaction rates. Though the generation of bubbles at a higher frequency is large, their growth and implosion intensity is comparatively less than lower frequency, and this may be a possible reason for the decline in conversion. The synthesis of cinnamyl propionate reported by Badgujar et al. with immobilised lipase Pseudomonas cepacia at 40 kHz and intensity of 100 W also showed 24% reduction in conversion as compared at 33 kHz. [32]. Similarly, Fig. 6 indicated that maximum conversion was achieved at 100 W(98%) at the end of 3 h, from a wide range of power input (60–120 W). At the lower power intensity than 100 W, there was a reduction in rate and conversion. At an input power of 60 W and 80 W, the conversion obtained at the end of 2 h was 70% and 81% respectively compared to 95% at 100 W. At lower power intensity, the formation of cavitational bubbles is few and small, which on implosion

Fig. 4. Influence of biocatalyst loading for amidation; reaction conditions, molar ratio propyl amine: butyl acetate 1:1.5, temperature 60 °C, ultrasound frequency 22 kHz, input power 100 W, duty cycle 75% and stirring speed 100 rpm. 4

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Fig. 5. Influence of ultrasound frequency for amidation; reaction conditions, lipase 3% (w/v), molar ratio propyl amine: butyl acetate 1:1.5, temperature 60 °C, input power 100 W, duty cycle 75% and stirring speed 100 rpm.

Fig. 8. Influence of speed of agitation for amidation; reaction conditions, molar ratio propyl amine: butyl acetate 1:1.5, temperature 60 °C, lipase loading 3% (w/v) ultrasound frequency 22 kHz, input power 100 W and duty cycle 75%.

destroying its catalytic activity [35]. With continuous US irradiation, there is a possibility of generation of excessive bubble formation and cavitational effects associated with it. The resulting cloud formation from excessive bubbles can cause lower cavitational effects and barrier to dissipative energy transfer [31]. The lipase activity analysed at 91% duty cycle also show a decrease in activity from 9.7 U to 8.2 U. It is also described that along with disruption of the biocatalyst in ultrasonic systems, continual operation without pulse mode damages the transducers [36]. 3.6. Influence of stirring speed The reaction mixture was agitated with overhead stirrer and effect of stirring speed was investigated at 0 rpm, 100 rpm 150 rpm and 200 rpm. The conversion increased considerably from 0 to 150 rpm, but as the mixture was agitated further at 200 rpm, a significant reduction in conversion was observed (Fig. 8). Agitation at 100 rpm and 150 rpm showed no significant difference in the conversion (95%) and rate of reaction. Hence 100 rpm was carefully chosen as the optimised speed of agitation owing to energy consumption and decay of catalyst due to shear force within the batch reactor. Generally, the idea behind agitation is to provide better mixing of the reaction mixture and reduce mass transfer resistances effective contact of substrates with a catalyst [37]. One of the reasons for lower conversion at 200 rpm and higher is that there is an emergence of shear forces that damages the immobilised lipase particles which causes loss of activity.

Fig. 6. Influence of ultrasound power for amidation; reaction conditions, lipase 3% (w/v), molar ratio propyl amine: butyl acetate 1:1.5, temperature 60 °C, ultrasound frequency 22 kHz, duty cycle 75% and stirring speed 100 rpm.

3.7. Reusability of immobilised lipase As a biocatalyst, it is necessary that it should easily regenerate at the end of the process, but in the case of enzymes, the separation and regeneration was a difficult task. Hence, taking into account the concerns of the production cost of enzymes, it's regeneration and regaining of its activity, immobilisation of enzymes on solid and rigid support solved related troubles tremendously. Immobilisation technique not only offered easy regeneration but also enhanced the activity of biocatalyst and its reusability [38]. Reusability is the recycling step where the immobilised enzyme can be separated from the first process and reused again for a reaction. The biocatalyst thus separated from the reaction mixture through filtration, was washed with hexane and then reused for the next successive cycle. The main objective to wash the immobilised enzyme is to remove any residual substrates, or unwanted molecules adhering to it [39]. It was detected that immobilised lipase exhibited fair activity for every consecutive cycle and could be reused for more than 6 cycles. The activity retained at 6th cycle was nearly 50% of the fresh lipase. The retention of lipase activity also depends on numerous

Fig. 7. Influence of duty cycle for amidation; reaction conditions, lipase 3% (w/ v), molar ratio propyl amine: butyl acetate 1:1.5, temperature 60 °C, ultrasound frequency 22 kHz, input power 100 W, and stirring speed 100 rpm.

frequency 25 kHz and 66% duty cycle [34]. This suggests that the time of exposure is also an essential factor like frequency and intensity in driving sono-enzymatic process. The plausible cause for the decline in conversion at more than 91% duty cycle is the disruption of lipase under prolonged exposure. With 91% duty cycle, the effect of sonication is increased to an extent where the continuous shock of ultrasound waves causes conformational changes in the lipase structure, thus 5

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are mentioned in Table. 1 As enzyme (E) interacts with the substrate [S], enzyme substrate [ES] complex is generated which further dissociates to give corresponding product [P] and [E]. The inhibition action takes place when there is blocking of active sites of catalyst by the dead end complex [DES]. The DES complex is the ES complex, which could not further detach as product (P) and Enzyme (E). This renders the biocatalyst inactive and lowers the conversion owing to dead end complex inhibition [47]. Subsequently, inhibition occurs either reversibly or irreversibly by altering the amino acid sequence responsible for active catalytic activity. US assisted synthesis has a major role in lowering the phenomenon of inhibition. With the incorporation of US, there is enhanced micro-stirring due to shockwaves in reacting mixture, and hence, consequences of inhibition are fairly minimized than in conventional system [28]. In US assisted enzymatic reaction the cavitation energy is thought to accelerate the reaction rate, but the mechanism by which this occurs is unclear. With the incorporation of US there is formation of large cavities and their implosion create microjets, microstreaming and micro-stirring in the reaction medium [21]. This can lead to enhanced molecular motion and the rate of substrates binding, formation of ES complex and also the release of products formed. Thus, with cavitation phenomenon the rate of reaction is enhanced by increasing the movement of liquid molecules, the substrate’s access to the active sites of lipase is increased. The micro turbulence and mass transfer produced from cavitation could allow the release of product from ES and obstruct complete DES inhibition, thus minimising its effect [27]. Thus, mole ratio of 1:1.5 for propyl amine to butyl acetate successfully yield 98% conversion without the use of any solvent other than substrates and a further increase in the concentration of propyl amine leads to inhibitory effects. Moreover, the overall enzyme kinetics under ultrasonic irradiation is enhanced since it aids in lowering the energy barrier to overcome the formation of ES complex. The formation of ES complex is fast and reversible, but under sonication, this ES is stabilised owing to less energy requirement for the formation of this complex and the backward reaction rate is also minimized [48]. Thus, the kinetic model for propyl amide synthesis proved useful in interpreting that high concentration of propyl amine leads to substrate inhibition and the model proposed is expressed as [49];

aspects that include establishing adverse conditions taking part to drive the reaction such as temperature, frequency, intensity, and extent of US exposure time to the catalyst. Most of the time, the biocatalyst loses their catalytic activity by deformation of the structure under high intensity ultrasound, which is primarily responsible for thermal denaturation [40]. The other reason for decline in the activity of lipase is the leaching of enzyme from the immobilised support due to above enumerated aspects. The reusability studied were conducted under optimised parameters (temperature 60 °C, mole ratio of propyl amine and butyl acetate 1:1.5, lipase loading 3% (w/v), frequency of 22 kHz and input power of 100 W and stirring speed of 100 rpm). Fig. 9, clearly shows a marginal reduction in the activity of lipase after every reuse. Yu et al. also reported reusability of lipase from Candida antarctica in the ultrasonic system for more than 4 successive cycles with a decline of only 4% loss from the initial activity at 4th reuse [41]. Similar results were also obtained by Michelin et al., for production of FAME from Macauba oil in ultrasound technique using Novozym 435 in solvent free condition and enzyme was reusable for 5 cycles [42]. 4. Kinetic study 4.1. Kinetic modeling Kinetic investigation of the reaction along with experimental parameter optimisation is also necessary as it is one of the requirements for the design of the reactor. The analysis of reaction rate parameters and velocity of reaction can comprehend the enzyme- substrate interaction, and also the factors that govern them [43]. The simulation or kinetics of reaction gives an account of mechanism of any reaction, effect of sonication on catalyst and several other characteristics of reactor position and designing, which can further contribute to the improvement of yield. Michaelis Menten assumptions are devised and widely accepted assumptions for the enzymatic reactions [44]. According to MichaliesMenten, initial rates are derived with respect to time, an increase in the concentration of product or decrease in the concentration of a substrate. The model also proposes that product (P) generated from enzymesubstrate [ES] complex is formed when substrate (S) binds to the active site of lipase (E). The formation of ES is a reversible process while the liberation of product is slow or also called as the rate determining step [45]. There are several models proposed for enzymatic reactions out of which Michalies-Menten forms the basis of most models. Likewise, Ping Pong model for lipase catalysed reactions is extensively used since it not only takes in account the concentration of two or more substrates or products but also their respective inhibition actions and formation of dead end complexes. Ping Pong Bi Bi model illustrates the participation of two or more substrates and release of first product before the second substrate attaches to the enzyme active site to release the subsequent product [46]. The expression for basic Ping Pong model is as follows:

v=

Vmax [A][B] Kb [A] + K a [B] + [A][B]

Vmax [A][B]

v= Kb [A]{1 +

[A] } Kia

+ K a [B] + [A][B]

(2)

4.2. Thermodynamic evaluation For determining thermodynamic constants, the activation energies for amidation reaction catalysed by CALB, in both ultrasound (US) and conventional techniques were calculated by applying the Arrhenius equation at different temperatures ranging from 323, 333 and 343 K. To

(1)

The substrate concentration of propyl amine and butyl acetate was varied as 1:1, 1:1.5, 1:2, 1:2.5, and 2:1. The initial rates were determined by analysing reaction mixture in every 15 min through GC. The model parameters were evaluated from the equations devised by ping pong model by means of non-linear regression with the least sum of square of errors (SSE) method (Microsoft Excel). In the present study (i) Ping Pong Bi Bi; (ii) Ping Pong Bi Bi with butyl acetate inhibition; (iii) Ping Pong Bi Bi with propyl amine inhibition; and (iv) Ping Pong Bi Bi with both butyl acetate and propyl amine inhibition were explored, and Ping Pong Bi Bi with propyl amine inhibition was found to fit well. From Fig. 10 it is clear that propyl amine inhibition model could be the best fit among all and the values for Vmax = 9.9 M/min/g of catalyst, Ka = 160 M Kb = 33 M and Kia = 1.45 M and other constants evaluated

Fig. 9. Reusability of immobilized Novozym 435 for successive cycles. 6

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Fig. 10. Comparison of experimental initial rates with predicted values for different Ping Pong models. Reaction conditions: temperature 60 °C, enzyme loading 3% (w/v), molar ratio propyl amine: butyl acetate 1:1.5, ultrasound frequency 22 kHz, input power 100 W, duty cycle 75% and stirring speed 100 rpm. Table 1 Comparison of experimental values for Ping Pong models obtained for ultrasound assisted synthesis of propyl acetamide in solvent free system. Kinetic constants

Vmax Ka Kb Kia Kib

Units

M/min/g catalyst M M M M

Kinetic models Basic Ping Pong

Ping Pong with propyl Amine inhibition

Ping Pong with butyl acetate inhibition

Ping Pong with both propyl amine and butyl acetate inhibition

1.12 15 13 – –

9.9 160 33 1.45 –

8.23 0.395 0.69 – 0.021

0.138 0.12 0 3 5.6

Fig. 11. Arrhenius plots for amidation in US and conventional systems catalysed by CALB at enzyme loading 3%(w/v), Reaction conditions: temperature 60 °C, molar ratio propyl amine: butyl acetate 1:1.5, ultrasound frequency 22 kHz, input power 100 W, duty cycle 75%, stirring speed 100 rpm (US) and stirring at 300 rpm (conventional system).

equations. Further values of change in enthalpy (ΔH), change in entropy (ΔS) and change in Gibbs free energy (ΔG) of the reactions were evaluated by substituting values of kinetic constant (k) and energy of activation (Ea) in thermodynamic relations.

determine the activation energy (Ea), a graph between ln k on y-axis and 1/T on x-axis was plotted to find the slope (−Ea/R) and ln A as yintercept. The thermodynamic study was conducted using the Eyring equations combined with basic relations between thermodynamic 7

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translational and rotational energies with loss of entropy [52,53]. The Gibbs free energy change (ΔG) indicated no significant difference in values at different temperatures or even at US and conventional reaction system.

Table 2 Determination of thermodynamic constants for ultrasound assisted synthesis of Propyl acetamide at different temperatures. US

Units

T ΔH ΔS ΔG

K kJ/mol kj/mol K kJ/mol

323 15.196 −0.278 105.019

333 15.113 −0.277 107.211

343 15.030 −0.275 109.479

Conventional T ΔH S ΔG

K kJ/mol kj/mol K kJ/mol

323 28.746 −0.282 119.752

333 28.663 −0.280 121.807

343 28.579 −0.277 123.441

5. Comparison of amidation in US and conventional system Sonication produces cavitational effects, which has a profound impact on reaction rates. The impact is due to improved activation of biocatalyst, faster and successful collisions and interaction between substrates and enzyme, effectual mixing and micro stirring action, enhanced diffusion and mass transfer rates, and increment in reaction kinetics by lowering the energy of activation. Thus ultrasonic irradiation intensifies the same conventional reaction system by several folds. It is necessary to optimise the reaction parameters of US so that the adverse effects on lipase as biocatalyst are prevented from intense irradiation and in case of immobilised lipase also preserves its reusability. The synthesis of propyl acetamide was also performed in a mechanical stirring system without sonication (conventional technique) in the presence of 3% (w/v) of lipase as a catalyst, 60 °C temperature, mole ratio of 1:1.5 for propylamine and butyl acetate agitated at 300 rpm. The conversion obtained in conventional treatment at 2 h was 53%, and maximum conversion reached only 81% at the end of 8 h whereas with a combination of US power 100 W at 22 kHz frequency, a maximum conversion of 98% was achieved in 2 h (depicted in Fig. 12). Moreover, the reaction in the ultrasonic system holds an advantage over the conventional technique that the quantity of biocatalyst consumed in ultrasound is less than the conventional system. The lipase loading requirement in conventional technique was 4% (w/v) to achieve 80% in 8 h. Hence, it can be inferred that through sonication, the reaction was successfully intensified by 2-folds than the mechanical stirring method. Ciu et al. also acquired interesting results for synthesis D- isoascorbyl palmitate where US intensified the process with 4-folds than in conventional system and lipase loading, mole ratio as well as the time required were also very low in US system [19]. Another notable result published by Martins et al. suggested reusability of Novozym 435 in both conventional and US treatments. The productivity of Novozym 453 in US was 2.5 times higher than in mechanical stirring method [54].

All reactions need the energy to establish equilibrium, this amount of energy can be expressed for both reactants and products through Gibbs free energy (G), enthalpy (H) and entropy (S) involved in a reaction. For the formation of transition state, reactants need to overcome the energy barrier, which is called activation energy denoted as Ea. This Ea is positive since the molecules for enzyme-substrate transition state require energy to overcome this energy barrier. The cavitation occurs with US irradiation where bubble implosion and rise in the local temperature attribute to increase in kinetic energy of molecules that also lead to more collisions of the reacting species. Hence, the rate of reaction increases effectively as the energy required by the molecules is lowered, the activation energy also decreases. The thermodynamics evaluation was performed and compared for both conventional and ultrasonic systems. The activation energy for esterification of propyl acetamide was calculated from Arrhenius plot and depicted in Fig. 11. The values for amidation reaction determined in US and conventional treatment are 17.8 and 31.4 kJ mol−1, respectively. Thus it can be inferred that sonication accelerates the rate of reaction by stabilizing the transition state, as the energy barrier is reduced than in conventional reaction [50]. The values for kinetic constants are summarised in Table 2. The change in enthalpy (ΔH) signifies randomness of reaction. The ΔH values calculated for both US and conventional system were positive, which suggest that enzymatic amidation is of endothermic nature [51]. The ΔH value for US technique at temperature 323 K, 333 K, and 343 K is 15 kJ mol-1, is nearly doubled than the conventional technique. Correspondingly the change in entropy ΔS shows spontaneity of reacting molecules and expressed in negative values because of formation of ES there is a liberation of both

Fig. 12. Comparison of synthesis of propyl acetamide with reaction conducted in US with no agitation (0 rpm), US with 100 rpm agitation, conventional mechanical (300 rpm) stirring. Reaction conditions: temperature 60 °C, enzyme loading 3% (w/v), molar ratio propyl amine: butyl acetate 1:1.5, ultrasound frequency 22 kHz, input power 100 W, duty cycle 75% and stirring speed 100 rpm.

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6. Conclusion

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