Enzymatic synthesis of benzyl benzoate using different acyl donors: Comparison of solvent-free reaction techniques

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Enzymatic synthesis of benzyl benzoate using different acyl donors: Comparison of solvent-free reaction techniques Alessandra Cristina de Meneses, Manuela Balen, Elaine de Andrade Jasper, Ilka Korte, Pedro Henrique Hermes de Araújo, Claudia Sayer, Débora de Oliveira* Department of Chemical and Food Engineering, Federal University of Santa Catarina (UFSC). 88040-900, Florianópolis, SC, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Benzyl benzoate Enzymatic acylation Ultrasound Batch Fed-batch

In this study, benzyl benzoate was successfully synthesized via enzymatic acylation using three immobilized enzymes as biocatalysts. Different acyl donors (benzoic acid and benzoic anhydride), operation regimes (batch, fed-batch), mixing modes (conventional mechanical stirring and ultrasound), process parameters (temperature, substrate molar ratio of acyl donor to acyl acceptor), presence or absence of solvents, enzyme amount and type were evaluated. Benzoic acid is a solid that is difficult to solubilize and, thus, was not efficient as acyl donor for the synthesis of benzyl benzoate. On the other hand, benzoic anhydride was very effective for the acylation of benzyl benzoate, and the presence of an excess of benzyl alcohol was essential to ensure the solute-solvent intermolecular attractions and good substrate solubilization, allowing the ester synthesis to be performed in the absence of organic solvents. The ultrasound was effective in increasing increase the initial reaction rate and the final conversion (88 %). However, the Lipozyme TL-IM and RM-IM supports were damaged, and the reuse was unfeasible. The batch and fed-batch approaches in conventional stirring ensured high conversions of 92 and 90 %, respectively, for batch (anhydride: alcohol 1:6) and fed-batch (1:3) using the Lipozyme TL-IM as biocatalyst. The controlled addition of the anhydride (fed-batch) allowed the reduction of alcohol molar ratio but decreased the reaction rates, and the maximum conversions were reached only after 24 h, while the batch approach had 92 % of conversion after 6 h. The yield of benzyl benzoate was high at 6 wt.% of enzyme, low temperature (50 °C), and simple reactor operation (batch). Results show the feasibility of the synthesis of benzyl benzoate via acylation using a green process that may be an alternative route to the chemical synthesis.

1. Introduction

The availability of benzyl benzoate in nature is low [1], and its synthesis is strict to the chemical route [6]. The industrial demand for more sustainable and environmentally-friendly routes encourages the researchers to develop sustainable pathways for the production of important organic compounds such as benzyl benzoate [7]. Thus, the enzymatic route using lipases seems to be a competitive alternative to traditional chemical synthesis and has been successfully applied in the catalysis of several aromatic esters such as eugenyl acetate [8–10], cinnamyl acetate [11–13], cinnamyl propionate [14], geranyl cinnamate [15,16], isoamyl acetate [17–19], among several others. Immobilized lipases are non-toxic, biodegradable, accelerate the rate of reactions under mild conditions of temperature and pressure, ease recyclability and processing, and ability to succeed many organic transformations in various reaction media with reduced residues generation [2,20–24]. These characteristics integrate several principles of the green chemistry [25], and the aroma esters produced by microbial

Benzyl benzoate (C14H12O2) is an aromatic ester that is found in traces in some plants like babaco fruit (Carica pentagona Heilborn), caper (Capparis spinosa), and celery (Apium graveolens), and possesses a balsamic, herb, oily smell [1,2]. Benzyl benzoate is one of the oldest medicines used for the treatment of scabies, a highly contagious skin infection. The mite Sarcoptes scabiei hominis burrows into the skin and consumes the epidermis, resulting in inflammation, allergy reactions, and pruritic lesions [3]. In the 1930s the topical application of this compound treated successfully thousands of scabies cases in Denmark, since then, the benzyl benzoate body lotion (25 %) has been used in emerging countries for scabies treatment due to the lower cost as compared to other topical treatments like monosulfiram, malathion, lindane, crotamiton, and sulfur in petrolatum, and drugs like permethrin and ivermectin [4,5].

⁎ Corresponding author at: Universidade Federal de Santa Catarina (UFSC), Departamento Engenharia Química e Engenharia de Alimentos (EQA), Laboratório de Controle de Processos – Campus Trindade, Cx. Postal 476, Brazil. E-mail address: [email protected] (D. de Oliveira).

https://doi.org/10.1016/j.procbio.2020.01.018 Received 16 September 2019; Received in revised form 16 January 2020; Accepted 18 January 2020 1359-5113/ © 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: Alessandra Cristina de Meneses, et al., Process Biochemistry, https://doi.org/10.1016/j.procbio.2020.01.018

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or enzymatic methods can be labeled as “natural” following the United States and European Legislations, thereby satisfying the consumer trend towards natural products in various industries [2,10,11,26]. As mentioned, these catalysts perform well the synthesis of aroma esters, but there is a lack of information in the literature about the enzymatically catalyzed synthesis of benzyl benzoate. The only study reporting its synthesis did not show a viable reaction condition using benzyl alcohol and methyl benzoate as substrates for the transesterification and Novozym 435 as biocatalyst (80 % of yield in 100 h) [21]. In this way, more studies are needed to investigate the viability of the enzymatically catalyzed synthesis for benzyl benzoate using different acyl donors and well-established immobilized enzymes. The choice of the right substrates interferes directly with the global enzyme performance, and studies have shown that different acyl donors for esterification, transesterification, and acylation may affect the global conversion and reaction rates [17,18,27,28]. In the enzymatically catalyzed synthesis of aroma esters, the anhydrous acyl donors may possess several benefic effects in comparison to the acidic acyl donors. The main positive effect include the fact the anhydride does not acidify the microaqueous layer present in the lipase active site, reducing the problems of enzyme deactivation [17], the undissociated anhydride is a source of acyl groups, leading to high ester yields [17], and the acylation has moderate to fast reaction rates [18]. Moreover, the enzymecatalyzed systems can be carried out as an equilibrium-controlled (thermodynamically controlled) or as a kinetically controlled process [29,30]. The use of acid as the acyl donor implies in an equilibriumcontrolled process, and the hydrolysis of the ester may occur due to the water generated as a side-product, stimulating the enzymatic hydrolysis. On the other hand, the use of activated acyl donors as anhydrides in kinetically controlled reactions may circumvent this event since the leaving group is, in this case, a weak nucleophile that cannot attack the ester formed [31]. There are several reports using acylation as a route for aroma esters synthesis [17,18,32,33]. However, at present, no information is available in the literature depicting benzyl benzoate acylation or esterification using benzoic anhydride and benzoic acid as an acyl donor. The use of anhydride for ester synthesis is associated with a complex reaction [18], as shown in Fig. 1. The anhydride possesses two acyl groups and, consequently, one acyl group is employed to form the ester, and the other one leads to the formation of one acid molecule. The acid generated as a byproduct may react with the remaining alcohol, and both acylation and esterification can co-occur. The present study deals with the benzyl benzoate synthesis using

two different acyl donors (benzoic anhydride and benzoic acid) mediated by different immobilized enzymes (Novozym 435, Lipozyme RMIM and Lipozyme TL-IM) in the presence or absence of solvents. In order to find the best reaction configuration, tests were conducted using ultrasound, conventional stirring, and batch and fed-batch operation regimes. Finally, process variables like substrate molar ratio (acyl donor to acyl acceptor), amount of catalyst, and reaction temperature were investigated to ensure high global productivity. 2. Material and methods 2.1. Enzymes and chemicals Benzyl alcohol (Neon), benzoic acid (Lafan) and benzoic anhydride (Acros) were used as substrates. Enzymatic catalysts were the commercial immobilized lipases Novozym 435 (Candida antarctica fraction B lipase), Lipozyme TL-IM (Thermomyces lanuginosus lipase), Lipozyme RM-IM (Rhizomucor miehei lipase), kindly donated by Novozymes®. Tert-butyl alcohol (Sigma-Aldrich) and 2-propanol (Neon) were used as solvents of the reaction system. Dichloromethane (Quimis) was used in gas chromatography injections, and acetone (Dinâmica) was utilized to wash the enzyme for reuse tests. 2.2. Enzymatic synthesis of benzyl benzoate in the presence and absence of organic solvents Initially, the solubility of the substrates (benzoic acid and benzoic anhydride, as acyl donors, and benzyl alcohol, as acyl receptor) was tested using tert-butyl alcohol, cyclohexane, n-heptane, n-octane, npentane, 2-propanol, and isooctane as solvents. In addition to these solubility tests, excess of benzyl alcohol was used to enable the reaction in a solvent-free system (SFS), which would represent a possible economic advantage for the industrial applications. The substrates benzoic acid: benzyl alcohol (esterification) or benzoic anhydride: benzyl alcohol (acylation) in a molar ratio of 1:9 were added in 2 mL reactors. In sequence, when used, 1 mL of solvent was added in each reactor (1:9:30 M ratio of anhydride: alcohol: solvent). The enzyme Novozym 435 was used as biocatalyst (10 % w/w), and at the end of 24 h of reaction at 50 °C, the reaction media were centrifuged (10,000 rpm for 5 min), aliquots of the supernatant were withdrawn and suitably diluted in dichloromethane for quantification analysis. All experiments were carried out in duplicate.

Fig. 1. Benzyl benzoate enzymatic synthesis using benzoic anhydride as acyl donor and benzyl alcohol as acyl acceptor. Main reaction: acylation; Secondary reaction: esterification. 2

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2.3. Variation of the molar ratio of the acylation substrates

2.7. Effect of enzyme amount

The synthesis of benzyl benzoate by enzymatic acylation was performed under different molar ratios of benzoic anhydride: benzyl alcohol from 3:1 to 1:9 using 2-propanol as solvent for solubilization of the substrates. The solvent-free system (SFS) was also evaluated using a high excess of benzyl alcohol (1:9) that plays the role of solvent for the solubilization of benzoic anhydride. The biocatalysts tested in this assay were Lipozyme RM-IM, Lipozyme TL-IM, and Novozym 435. The reaction parameters temperature, amount of catalyst, and reaction time were fixed at 50 °C, 10 w/w% catalyst, and 24 h, respectively. The reactions were conducted by adding the substrates in the adequate proportion of 3:1, 1:1, 1:3, and 1:9 when used, 1 mL of 2-propanol was added to the reactions with solvent. Reactions were performed under mild mechanical stirring, and with controlled temperature and at the end of the reaction time, an aliquot of the product was centrifuged (10,000 rpm for 5 min) and adequately diluted for quantification. All experiments were carried out in duplicate.

The effect of enzyme amount in benzyl benzoate synthesis was evaluated at different Lipozyme TL-IM amounts of 6, 8, and 10 w/w % with the temperature fixed at 50 °C. The reactions involved the addition of an adequate amount of biocatalyst, benzoic anhydride, and benzyl alcohol (molar ratio of 1:6) in glass reactors and placed in a mechanical shaker under mild agitation. The sample collection and preparation were the same as those described in the previous paragraph. 2.8. Benzyl benzoate quantification The ester quantification was carried out as described previously elsewhere with some modifications [34]. Gas chromatography (GC, Shimadzu 2010), with the automatic coupled injector (Shimadzu AOC 5000), was used. A DB-5 column (27 m length ×0.25 mm internal diameter ×0.25 μm thickness) was employed for the separation of the analytes, and the detection was performed by flame ionization detector (FID). The column temperature ramp was programmed to 100 °C (2 min), 100−135 °C (3 °C/min), 135−250 °C (10 °C/min) and 250 °C for 15 min to ensure proper separation of all components. The temperatures of the injector and detector were kept at 250 °C, and a split ratio of 1:100 was maintained to sample injection, the injection volume was 10 μL, and the carrier gas was nitrogen (N2, 56 K Pa). The conversion was calculated based on the benzyl benzoate corresponding peak of each chromatogram and a previously prepared benzyl benzoate (99 % purity) calibration curve (R² = 0.99).

2.4. Effect of ultrasound on the enzymatically catalyzed synthesis of benzyl benzoate In order to investigate a non-conventional method of homogenization, reactions performed in an ultrasonic bath were compared with those performed with conventional mechanical stirring. The SFS containing benzoic anhydride: benzyl alcohol (1:9) and 10 w/w% of catalyst (Lipozyme RM-IM, Lipozyme TL-IM or Novozym 435) were added into glass reactors of 50 mL and conducted either in an ultrasonic bath (Unique, Model USC-1800A, 40 kHz, 132 W) or in a conventional heating bath with mechanical agitation (Dist), both previously heated at 50 °C. Aliquots of the reaction were collected at times 0, 1, 2, 4, 6, and 8 h. Subsequently, the samples were centrifuged (10,000 rpm for 5 min), aliquots of the supernatant were diluted correctly in dichloromethane and stored under refrigeration for further analysis.

2.9. Enzyme reuse The reusability study was conducted to investigate the enzyme performance on the benzyl benzoate synthesis following the washing procedure described elsewhere with minor modifications [11]. After each reaction cycle, the enzyme was recovered by vacuum filtration using glass funnel and filter paper. The enzyme was washed with acetone for three times to remove any product or subtract. Then, the enzyme was dried during 24 h at 50 °C and reused in a new reaction cycle. This procedure was repeated for each reuse cycle.

2.5. Benzyl benzoate synthesis in batch and fed-batch mode The batch and fed-batch syntheses of benzyl benzoate were performed with conventional stirring. Different substrate molar ratios (1:3, 1:6 and 1:18, anhydride: acid) and 10 w/w% of enzyme (Lipozyme TLIM) were added to glass batch reactors of 100 mL. The reactions were carried out at 150 rpm and 50 °C, and at reaction times of 0, 1, 2, 3, 4, 5, 6, 8, 10, and 24 h samples were taken and diluted correctly for further quantification. For the fed-batch reactions, only benzyl alcohol and 10 w/w% of enzyme (Lipozyme TL-IM) were added as initial charge in a jacketed glass reactor of 100 mL previously heated at 50 °C. Benzoic anhydride was fed into the reaction media every 1 h during the first 6 h, totaling six additions. The final benzoic anhydride: benzyl alcohol molar ratios were 1:3, 1:6, and 1:18. The remaining conditions were the same as those described in the previous paragraph for the respective batch reactions.

2.10. Initial reaction rate The initial reaction rate (r) was calculated as follows:

dC r = ⎛ ester ⎞ ⎝ dt ⎠ t = 0

(1)

Where r is the initial rate of reaction (h−1); Cester conversion of benzoic anhydride into the ester (wt.%) at time t; and t is the reaction time (h) [35]. 3. Results and discussion 3.1. Effect of solvents Benzoic acid is the simplest aromatic carboxylic acid that can be produced in large quantities, with high purity and low-cost. It is a colorless crystalline solid of difficult solubilization, and research has been conducted to understand its solubility in different organic solvents [36–40]. The use of this compound as a substrate for enzymaticallycatalyzed reactions may present some drawbacks related to the solubility and diffusion problems. Lipases are stable in many solvents. However, some organic solvents may reduce the lipase catalytic activity [12,17,41,42], and the choice of a suitable solvent for solid substrates like benzoic acid is fundamental in biocatalysis to ensure proper performance of the catalyst. Initially, a solubilization test was conducted with organic solvents like cyclohexane, heptane, octane, pentane, isooctane, tert-butyl

2.6. Effect of temperature The effect of reaction temperature on the enzymatically-catalyzed synthesis of benzyl benzoate was studied at 40, 50, and 60 °C. The reactions involved the addition of 10 w/w% of Lipozyme TL-IM as biocatalyst, benzoic anhydride, and benzyl alcohol (molar ratio of 1:6) in glass reactors and placed in a mechanical shaker under mild agitation. Aliquots were taken at times 0, 1, 2, 4, 6, 8, 10, and 24 h. Then, the samples were centrifuged (10,000 rpm for 5 min), and the supernatant was collected, adequately diluted, and stored correctly under refrigeration for further analysis. All experiments were carried out in duplicate. 3

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Table 1 Effect of organic solvents on the synthesis of benzyl benzoate using different acyl donors and benzyl alcohol as acyl acceptor. Reaction conditions fixed at molar ratio acyl donor: acceptor 1:9, 50 °C, 10 w/w% of Novozym 435 and 24 h of reaction. Acyl donor

Solvent

Log P

Conversion (%)

Benzoic acid

Tert- butyl alcohol 2-propanol Tert- butyl alcohol 2-propanol Benzyl alcohol

0.35 0.05 0.35 0.05 1.10

1.2 0.4 30.6 71.3 60.3

Benzoic anhydride

alcohol, 2-propanol, and benzyl alcohol and the benzoic acid was visually soluble in tert-butyl alcohol and 2-propanol, solvents usually employed for enzyme-catalyzed reactions [17,41,43–50]. Table 1 shows the conversion into ester using Novozym 435 as catalyst and benzoic acid as acyl donor, and both solvents tested were inefficient in the intermolecular attractions between solute-solvent, reaching the very low conversion. On the other hand, benzoic anhydride showed positive results as the acyl donor for benzyl benzoate synthesis in the presence of the solvents tert-butyl alcohol and 2-propanol. The results also indicate that an excess of benzyl alcohol in the reaction media was efficient in the anhydride solubilization and presented good conversion as a solvent-free system (SFS). The synthesis of benzyl benzoate using benzoic anhydride is a complex reaction since it possesses two acyl groups. The first acyl donor is employed to form the benzyl benzoate and the second one leads to the formation of benzoic acid. The acylation is the primary reaction and the acid formed as a byproduct may react with the excess of alcohol and, consequently, a secondary reaction of esterification may occur (Fig. 1). However, the benzoic acid was not soluble in the benzyl alcohol, and the 2-propanol and tert-butyl alcohol were not suitable solvents for the esterification reaction between both substrates. The results strongly indicate that the acylation is the leading and only reaction occurring in the benzyl benzoate synthesis mediated by lipases. The benzyl benzoate acylation using solvents with log P > 4, like isooctane and heptane, 4.5 and 4, respectively, reached no conversion (data not shown) and the solvents tert-butanol, 2-propanol and benzyl alcohol that possess very low log P (Table 1) showed the best conversion results using anhydride as substrate. The log P represents the partitioning of a given solvent between water and octanol in a twophase system, and values above 4 are associated with higher enzyme activity and better ester synthesis [2,17,44,51]. The benzoic anhydride is a white solid, soluble in more hydrophilic solvents (low Log P) [52] and the conversion results strongly indicate a correlation between solute solubilization and diffusion problems, i.e., if an inadequate solvent is used the poor solubilization of the anhydride prevents its diffusion through the Novozym 435 pores, and the substrates never reach the enzyme active site. Based on these results, the commercial immobilized enzymes Novozym 435, Lipozyme TL-IM, and Lipozyme RM-IM were tested under a fixed condition using 2-propanol, tert-butyl alcohol and SFS (alcohol in excess) as organic media for the benzyl alcohol acylation. The solvent tert-butyl alcohol showed the lowest ester conversions, probably due to the weak solute-solvent intermolecular attractions associated with the reduced benzoic anhydride solubility. Both SFS and 2propanol showed to be efficient in the substrates and products solubilization, reaching conversions above 60, 47, and 66 % respectively for Novozym 435, Lipozyme RM-IM and Lipozyme TL-IM. These enzymes are broadly used in the biocatalytic synthesis of aroma ester [11,13,34,53–60] and showed an excellent performance (conversions close to 100 %) in other acylation reactions mediated by these lipases for eugenyl acetate [9,33], isoamyl acetate [17,18] synthesis (Fig. 2).

Fig. 2. Benzyl benzoate acylation using different biocatalysts (Novozym 435, Lipozyme RM IM and TL IM) in solvent-free system (SFS) or tert-butyl alcohol and 2-propanol as solvents, using fixed reaction conditions of substrate MR 1:9 (anhydride :alcohol), 50 °C, 10 w/w% of enzyme and 24 h of reaction.

3.2. Effect of substrate molar ratio The equilibrium of esterification reactions can be pushed forward by either using an excess of the nucleophile (alcohol) or by removing products from the reaction mixture [17,44], and reports showed that the excess of alcohol reduces the adverse effects associated the acid like lipase inhibition and inactivation [34,61]. The acylation generally occurs successfully in equimolar concentrations of substrates since the anhydride substrates are not associated with hydrolysis or lipase inhibition reaching conversion around 100 % [17,18] and an excess of alcohol stimulates the secondary reaction of esterification between the acid generated as byproduct of the primary reaction of acylation with the alcohol resulting in ester and water [18]. In order to investigate the effect of substrate molar ratio on benzyl benzoate synthesis using benzoic anhydride as acyl donor, tests were carried out varying the substrate molar ratio from 3:1 to 1:9 (anhydride: alcohol) using 2-propanol as the solvent and the lipases Lipozyme TL-IM, Lipozyme RM-IM and Novozym 435 as biocatalysts. A higher concentration of alcohol (acyl acceptor/nucleophile) usually leads to higher levels of conversion due to the availability of nucleophiles in excess for acyl transfer, and commonly, both reactions of acylation and esterification may occur due to the use of anhydride substrate [17]. As previously reported by Romero et al. [18], an equimolar concentration of acetic anhydride to isoamyl alcohol reached the maximum acylation percentage of 100 % in 3 h of reaction. However, when a molar ratio of 1:3 (anhydride: alcohol) was used, the anhydride promptly reacted with isoamyl alcohol, producing isoamyl acetate and acetic acid. Then, a new reaction started, much slower than the first one, in which the produced acetic acid reacted with the alcohol surplus, forming again isoamyl acetate and water. A maximum esterification extent of 192 %, referred to initial acetic anhydride content, was obtained at that time of 3 h. However, the previous results showed that the benzoic acid did not react with benzyl alcohol in esterification using 2-propanol as solvent (Table 1). As acylation was the only reaction in progress, the excess of benzyl alcohol could be beneficial for the benzoic anhydride solubilization, and Fig. 3 shows the dependency of great amounts of alcohol to ensure high conversions. Moreover, the SFS with benzyl alcohol in excess (same molar ratio conditions 1:9) also ensures high conversions for all enzymes tested, which indicates that strong solute-solvent intermolecular attractions lead to improved benzoic anhydride solubility and diffusion through the pores of the Lipozyme TL-IM and RM-IM and, consequently, to the highest results of conversion. Another work showed a different perspective in the synthesis of menthyl propionate and menthyl butyrate using acid anhydride on a lipase-catalyzed enantioselective system with hexane as solvent [62]. The reactions yields 4

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Fig. 3. Impact of the substrate molar ratio on the benzyl benzoate acylation using Novozym 435, Lipozyme RM IM and TL IM as biocatalysts in a system containing 2-propanol as solvent (anhydride: alcohol: solvent 3:1:30, 1:1:30, 1:3:30, 1:9:30) and in a solvent-free system (SFS) for the condition 1:9 (excess of alcohol). The experiments were conducted at a fixed reaction condition of 50 °C, 10 w/w% of enzyme, and 24 h of reaction.

containing menthol to acyl donor from 1: 0.5 to 1:2 had increased with the increase of the acyl donor. Thus, results showed in this work indicate the excess of benzyl alcohol is indispensable to ensure suitable solubilization of the benzoic anhydride, assisting the diffusion of the substrates and product molecules through the enzyme pores. Based on these results, only SFS was employed in the next experiments.

Fig. 4. Benzyl benzoate acylation (a) and initial reaction rate (b) under conventional (mechanical agitation) and non-conventional (ultrasound) agitation using three different biocatalysts, considering fixed reaction condition in solvent-free system with molar ratio anhydride to alcohol of 1:9, 10 w/w% of enzyme and 50 °C.

3.3. Effect of conventional and non-conventional methods

RM-IM and TL-IM. Novozym 435 presented the lowest conversion (above 60 %) for both homogenization methods showing not to be the best choice for the enzymatic synthesis of benzyl benzoate. Novozym 435 is one of the most used biocatalysts for aroma ester synthesis, and other works reported that the enzyme had enhanced activity reaching 100 % of conversion in ester and operational stability in ultrasound over several cycles [11,69]. The influence of the ultrasound on the performance of the enzymes can also be seen in Fig. 4b that shows an increase in the initial reaction rate of the acylation when the non-conventional method was employed, as already reported by other authors [41,68,69,73]. Despite the positive effects of conversion and initial reaction rates, and reports indicating that Lipozyme TL-IM is resistant to the ultrasound cavitation [41,68], the enzyme support was destroyed under the presented conditions of reaction with no possibility of recuperation and reuse. The same behavior was found for Lipozyme RM-IM. These results hamper the use of ultrasound in the benzyl benzoate synthesis, and the conventional agitation was employed in the subsequent reactions using Lipozyme TLIM as biocatalyst. The enzyme showed to be efficient in the benzyl alcohol acylation in all the systems studied and is the cheapest enzyme as compared to the other two immobilized lipases, justifying its use.

The immobilization of enzymes to an inert and resistant polymeric support offers many process advantages, like lower production cost, increased activity, specificity and selectivity, improved structural stability, reduction of inhibition, and the heterogeneous characteristic of the biocatalyst facilitates the separation, recovery and further reuse [63–66]. On the other hand, the immobilization of an enzyme means a deliberate restriction of its mobility, which can also affect the solutes mobility [22] and various phenomena associated with mass transfer effects can lead to a reduced reaction rate, and an efficient stirring is crucial to avoid diffusion limitations and improve catalyst performance [67]. Generally, conventional stirring (mechanical or orbital) is efficient in avoiding internal or external diffusion drawbacks associated with the immobilized enzymes. However, some emergent techniques like ultrasound have been successfully used for flavor and aromatic ester synthesis [11,63,68–71]. Ultrasound is a high-energy technique considered as a green technology with high efficiency, economic performance, and low instrumental requirement [2,69]. The cavitation energy accelerates the reaction rate due to the vibratory gas bubble formed by the acoustic field and generates around it a circulatory liquid motion referred to as a microstreaming. This stream favors the flux of reagents to the active site of the enzyme and the products to the medium and, consequently, increasing the turnover number and the rate of the process [72], reducing the reaction time as compared with other conventional mixing techniques [2]. Tests were conducted using conventional (mechanical agitation) and non-conventional (ultrasound) agitation methods using Novozym 435, Lipozyme TL-IM, and Lipozyme RM-IM as biocatalysts of the benzyl alcohol acylation under SFS (molar ratio anhydride: alcohol 1:9). Fig. 4a shows the anhydride conversion over time, and the enzyme Lipozyme RM-IM showed the highest conversions (88 %) after 8 h of reaction in ultrasound-assisted acylation, followed by Lipozyme TL-IM (76 %). Both enzymes allowed reaching high conversions after 24 h under conventional agitation 73 and 75 %, respectively, for Lipozyme

3.4. Effect of batch and fed-batch operation regimes The heterogeneous enzymatic processes can be conducted in batch, semi-batch, or continuous mode and fully satisfy the requirements of the green chemistry and the interests of the organic synthesis industries. The batch reactors are the most simple configuration to conduct organic reactions, and combined with the favorable global productivities, make this configuration the most used over the industry. Although the fedbatch reactors need more considerable engineering to allow the controlled addition of the substrates along with the reaction, this configuration presents many positive effects related to the enzyme performance and the maintenance of enzyme activity over several recycles. 5

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reuse. The enzyme showed suitable performance over three reaction cycles with a loss of 20 w/w% of the enzyme activity after the third cycle (data not shown). The high conversions reached and the operational stability of the enzyme showed that both batch and fed-batch configurations could be used for benzyl alcohol acylation despite the lower initial reaction rates found for the fed-batch reactors (Fig. 5b) that can be associated with the controlled addition of the anhydride during the first six hours of reaction, limiting the reaction rate. The batch reactions had higher reaction rates and conversions with maximum values reached in only six hours (molar ratio 1:6), which improved the global productivity and simplified the process conditions. Based on the results, the batch configuration with a molar ratio 1:6 was employed for the next experiments. 3.5. Effect of temperature and enzyme amount The impact of the reaction temperature is crucial for lipase-mediated reactions since each enzyme has an optimal range of temperature, and an adequate increase in the temperature may lead to an increase in the kinetic constants, consequently, higher conversions. On the other hand, the treatment with high temperatures may lead to a negative effect as the indiscriminate increase can lead to the modification in the tertiary structure resulting in enzyme inactivation [78]. Furthermore, the temperature is a crucial parameter as it assists the solubilization of reactants and boosts the molecular-collision interface [2,28]. Experiments were performed to find out the most suitable temperature for benzyl alcohol acylation, considering similar reaction conditions. Fig. 6 shows that the increase in the temperature from 40 to 50 °C led to an increase in the initial reaction rates, but the final conversions (after 24 h) remained similar. A further increase in the temperature from 50 to 60 °C also led to an increase in the initial reaction rate while global conversions (91 % after 6 h of reaction) were similar for both temperatures (Fig. 6), and all temperatures showed maximum conversions between 90 and 100 % after 24 h of reaction. Thus, to keep the minimum possible temperature and reaction time, the temperature of 50 °C was used for the next experiments. The amount of biocatalyst is another crucial parameter from the industrial and economic point of view and is essential to achieve high conversion at low enzyme concentration. The effect of different Lipozyme TL-IM concentrations on the benzyl anhydride conversions with fixed substrate molar ratio and temperature was investigated. Fig. 7 showed the effect of Lipozyme TL IM concentrations of 6, 8, and 10 w/w% in the production of benzyl benzoate, considering conversion (primary graph) and initial rate (secondary graph). The higher amount of biocatalyst increased the lipase active sites available to the substrates and accelerated the reaction rate, reaching a value of 92 % of conversion in just 6 h (10 w/w% of biocatalyst and 50 °C). However, the

Fig. 5. Benzyl benzoate acylation (a) and initial reaction rate (b) of a solventfree system under batch and fed-batch configuration at substrates molar ratio of 1:3, 1:6 and 1:18 using 10 w/w% of Lipozyme TL IM as biocatalyst and 50 °C.

Some studies have been showing positive results for esterification [34,61], which had some acids or alcohols as substrates that are responsible for inhibition, deactivation, or adsorption events. Generally, aromatic esters are successfully synthesized in batch reactions [16,54–56,74–77], but a fed-batch configuration (controlled addition of the acyl donor) may directly influence the enzyme performance and assist the solubilization of solid or acidic substrates. In this way, both configurations were tested with different anhydride: alcohol molar ratios using Lipozyme TL-IM as biocatalyst for the benzyl benzoate acylation. Fig. 5a shows good conversion for both reactor configurations. The effect of the molar ratio of the substrates on the final conversions (24 h) did not follow a clear tendency as the concentration of the alcohol was increased in the reaction media. The molar ratio of 1:3 in fed-batch mode reached a similar conversion to 1:6 in batch mode. The results showed that the slow addition of the anhydride during the initial hours improved its solubilization in the reaction media, improving the final conversion at lower molar ratios (anhydride: alcohol). Some works used the fed-batch configuration to overcome acid inhibition effects caused by short-chain acids in esterification reactions. The controlled feed of the acyl donor was essential to ensure high aroma ester conversion and improve enzyme performance in the synthesis of benzyl butyrate [34] and benzyl propionate [61]. Several reports in the literature showed that the lipase-catalyzed acylation in batch reactors occurs at moderate reaction rates and reach maximum conversion of 100 % within 6 h [9,18,33,78]. The drawbacks related to the benzoic anhydride solubilization may be responsible for the incomplete or low acyl donor consumption and, consequently, conversions around 90 %. The operational stability of the Lipozyme TL-IM was tested under the benzyl alcohol acylation for both batch and fed-batch modes. The enzyme reusability was carried out using the recovered enzyme over three subsequent cycles. After each cycle, the enzyme was filtered out, washed with fresh solvent, and allowed to drain out the solvent before

Fig. 6. Effect of temperature in the benzyl benzoate acylation using the Lipozyme TL IM as biocatalyst in a solvent-free system with substrate molar ratio of 1:6 (anhydride to alcohol) and 10 w/w% of enzyme. 6

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[2]

[3]

[4] [5]

[6]

Fig. 7. Effect of Lipozyme TL IM concentration (6, 8 and 10 w/w%) in the production of benzyl benzoate, considering substrates molar ratio of 1:6 (anhydride to alcohol) and 50 °C.

[7]

[8]

results of conversion and reaction rate were quite similar to the concentration of 6 and 8 % with maximum conversion in 10 h of reaction, showing the lower amount of enzyme is very efficient to catalyze the benzyl benzoate acylation.

[9]

[10]

4. Conclusions [11]

In the present work, benzyl benzoate was successfully synthesized via enzymatic catalysis in a solvent-free system. The benzoic acid is a solid of difficult solubilization, and the benzyl benzoate esterification did not occur under the studied conditions. Controversially, the acylation was very effective in the benzyl benzoate synthesis using benzoic anhydride as acyl donor. The results showed that acylation was the main and only reaction occurring at the present conditions, reaching high conversions. The solvent 2-propanol was efficient in the substrates solubilization at increased benzyl alcohol molar ratios; however, the free solvent system with benzyl alcohol in excess was very efficient in the benzoic anhydride solubilization, and it was the adequate system to the benzyl benzoate acylation using the Lipozyme TL-IM as biocatalyst. A considerable amount of acyl acceptor was essential to ensure good solubilization of the benzoic anhydride. The use of ultrasound damaged the Lipozyme TL-IM and RM-IM supports, avoiding the enzyme reuse under the tested conditions. The controlled addition of benzoic anhydride to the reaction medium under conventional homogenization was efficient at lower benzyl alcohol molar ratios, but the reaction rate was decreased, and the maximum conversions were reached at the end of the reaction period. On the other hand, the batch reactions showed an increased initial reaction rate reaching maximum conversion at the initial hours. The benzyl alcohol acylation mediated by lipases in batch reactors had high reaction rates and conversions using an excess of benzyl alcohol, allowing the ester synthesis in the absence of organic solvents with good global productivity and a rather simple process conditions.

[12]

[13]

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[16]

[17]

[18]

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Declaration of Competing Interest

[24]

None.

[25]

Acknowledgements

[26]

The authors thank the financial support from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico).

[27]

[28]

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