Enzymatic Synthesis of Eugenyl Acetate from Essential Oil of Clove Using Lipases in Liquid Formulation as Biocatalyst

In this research, eugenyl acetate, a compound with flavoring, antioxidant, and antimicrobial properties, was obtained from essential oil of clove (Syzygium aromaticum) via liquid lipase-mediated acetylation. Clove essential oil was extracted by drag water vapor from dry flower buds and its physic-chemical characteristics were analyzed. For the enzymatic synthesis, an extensive evaluation of reaction parameters was accomplished through employment of distinct reaction temperatures, acetic anhydride to eugenol molar ratios, enzyme loads, and three different lipases (a lyophilized enzyme produced by solid-state fermentation of sunflower seed with Penicillium sumatrense microorganism and other two commercial lipases — Lipozyme TL 100L and CALB L). The product eugenyl acetate was confirmed by 1H-NMR, 13C-NMR Distortionless Enhancement by Polarization Transfer (DEPT 135), and Heteronuclear Multiple Bond Correlation (HMBC). Through optimized conditions (55 °C, acetic anhydride to eugenol molar ratio of 1:1, 10 wt% of Lipozyme TL 100L), 91.80% of conversion after 2 h was achieved to the eugenyl acetate production. With the results obtained, it was possible to conclude that the use of lipases in liquid formulation is a promising alternative for the synthesis of essential esters largely applied on food, cosmetic, and pharmaceutical industries.


Introduction
Esters with low molecular weight represent an important chemical class of compounds derived from short-chain acids such as acetates, propionates, and butyrates [1,2]. Such importance is https://doi.org/10.1007/s12010-021-03610-z The objective of this work is to investigate a EA synthesis by liquid lipase-mediated acetylation from eugenol (extracted from essential oil of clove), using different lipases: a lyophilized enzyme produced by solid-state fermentation of sunflower seed with Penicillium sumatrense microorganism and the commercial enzymes in liquid formulation Lipozyme® TL 100L and Lipozyme® CALB L. For this proposal, statistical design was utilized to evaluate the reaction conditions that maximize the ester conversion to the process.

Materials
Dry flower buds of clove (Iremar Alimentos, Toledo, Brazil) were purchased from a local market and used as feedstock to obtain the oil employed in the assays. Regarding the biocatalysts, three enzymes were used in the acetylation reactions, a lyophilized enzyme produced by solid-state fermentation of sunflower seed with Penicillium sumatrense microorganism (enzymatic activity of 418.74 U·g −1 ). Penicillium sumatrense is an endophytic fungal strain isolated from castor bean leaves (Ricinus communis L.), identified by ribosomal DNA sequencing, according to methodology described by Stringari [25]. The optimum temperature was 30°C and pH 6.0. The protein content of lyophilized enzyme, 0.752·g mL −1 was determined by the Bradford Method.

Essential Oil of Clove (Syzygium aromaticum): Obtaining, Identification, and Quantification
Previously grounded dry flower buds were used to obtain essential oil of clove via drag water vapor distillation in a Clevenger-type distiller (Solab, model SL-76, Piracicaba, Brazil) coupled to a thermostated bath (Tecnal, model TE-2005, Piracicaba, Brazil). In all, five batches of 60 min were performed using 200 g of ground material in each operation. All the extracted oil was dried by percolating on anhydrous sodium sulfate. For this step, the mass yield of the extraction process was calculated.
For the clove oil obtained, physical-chemical characteristics were determined (color, appearance, relative density, oil refraction index, moisture by Karl Fischer, and oil solubility in ethanol 70 vol%), according to the methodology proposed by The United States Pharmacopeia -USP 41 [26].
The quantification and identification of the main constituents of the essential oil of clove were determined by gas chromatograph (7890B from Agilent® equipped with flame ionization detector (FID) and an Agilent DB-1 capillary column, 100% dimethylpolysiloxane with 30 m × 0.25 mm × 0.25 μm of coating film). Temperature program: 280°C in isotherm for 15 min, injector temperature of 290°C, and detector temperature of 290°C. Helium (99.999% purity) with a column flow rate of 1 mL·min −1 and split ratio 100:1 was used as carrier gas. Nitrogen with a flow rate of 35 mL·min −1 was used as makeup gas. Hydrogen and synthetic air with flow of 30 and 300 mL·min −1 , respectively, were used as flame gas. To identify the compounds, standard solutions of eugenol, β-caryophyllene, humulene, and eugenyl acetate (diluted in cyclohexane) were prepared. The content in % of the essential oil of clove constituents was obtained by normalization area.

Synthesis of Eugenyl Acetate
EA synthesis was performed in an orbital shaker (operating at 150 rpm) with the addition of the clove oil, lipase, and acetic anhydride until complete dissolution. Molar proportion of substrates was established considering the molar mass of acetic anhydride (102 g·mol −1 ) and the mass concentration of eugenol (molar mass of 164 g·mol −1 ) present in the clove oil previously determined (86.87 wt%). Thus, 187 g of oil contains the equivalent to 1 mol of eugenol, representing the mass of raw material applied in each assay.
After reaction finishing, the catalyst was separated from the reaction medium by filtration and the product of the enzymatic reaction was purified by washing with solution of NaOH 0.1 N and cyclohexane (25 vol%). After washing, the organic phase (rich in EA and cyclohexane) was separated from the aqueous phase (NaOH solution plus acetic acid formed in the reaction). The solvent was evaporated from the organic phase and the purified EA was stored under refrigeration for analysis.

Selection of the Ideal Biocatalyst
Preliminary tests (in triplicate) were performed with each enzyme (lipase obtained from Penicillium sumatrense microorganism, Lipozyme® TL 100L, and Lipozyme® CALB L) in order to determine which biocatalyst would return the best performance for the EA synthesis. For these tests, experimental conditions obtained by Radünz et al. [27] were used: 50°C, eugenol to acetic anhydride molar ratio of 1:3, enzyme load of 5.5 wt% (the same mass was used, but not the same enzymatic activity of each enzyme), 150 rpm, and 2 h of reaction.
After catalyst selection, a statistical design was carried out to determine the reaction conditions that optimize the EA synthesis for the enzyme chosen. The reaction conditions that were evaluated were eugenol to acetic anhydride molar ratio (mol·mol −1 ), temperature (°C), and enzyme load (wt%). A complete factorial design 2 3 with triplicate in the center point (resulting in 11 trials) was applied. Table 1 presents the real and coded variables that were tested for the enzyme selection among the three biocatalysts considered. StatSoft Statistica (version 11.0) was used to data processing using a reliability level of 95%. After determining the catalyst with better performance to the enzymatic process and the appropriate experimental conditions, a time course of the EA synthesis was accomplished.

Eugenyl Acetate Analysis and Characterization
The chromatographic methodology described in "Materials" was used to quantify the eugenol conversion in esters, following the reduction in the signal area of the limiting agent (eugenol) as well as the appearance of the peak product. The area of the reaction mixture without the catalyst (blank) and the area of the reaction were related by Eq. 1.
where QC is the amount of eugenol converted in EA, A c is the peak area of conversion, and A Blank is the area of the blank. After purification process, the EA samples were analyzed in the GC (7890B from Agilent® equipped with flame ionization detector and a Phenomenex ZB-Wax G16 column, 100% polyethylene glycol, with 30 m × 0.32 mm, 50 μm). Chromatographic system was configured using injection volume of 0.6 μL, injector temperature of 180°C , detector temperature of 250°C, and helium as carrier gas with a column flow rate of 5 mL·min −1 and split ratio 1:15. Column temperature was configured according to description: initial temperature of 40°C, 40 to 180°C in 7 min at 20°C·min −1 , and 180 to 250°C in 12 min at 50°C·min −1 with a temperature holding time of 3.5 min. To identify the compound, standard solutions of EA (diluted in cyclohexane) were prepared.
Infrared spectroscopy (FT-IR, PerkinElmer Spectrophotometer® 400 equipped with Attenuated Total Reflectance -ATR device) was used to identify the main chemical functions of the synthesized molecule. The equipment was configured for 16 scans, resolution of 2.0, and scanned in the region from 4,000 to 650 cm −1 .
Nuclear magnetic resonance (NMR, 1 H and 13 C) for 1D and 2D NMR spectra was performed on a Bruker Avance III HD 600 Spectrometer with a magnetic field of 14.1 T and 5 mm of TCI (Triple Chanel Inverse) cryoprobe. The chemical displacements of 1 H and 13 C were referenced according to the peak of the solvent dimethylsulfoxide (DMSO-d 6 ) used to solubilize the samples (δ C of 39.5).

Physical-Chemical Characteristics and Chemical Composition of the Oil
Based on the extractions process of clove oil from dry flower buds, 11.2 ± 0.9 mL of oil was obtained, representing a mass 11.7 ± 1.0 g. As the oil density was determined at 1.041 g·mL −1 , the mass yield was estimated at 5.83 ± 0.5%. In a similar study, Radünz et al. [27] reported yields ranging from 1.87 to 8.88%, using a hydrodistillation process to obtain essential oil of clove. Low yields are associated with factors such collection period of the materials, storage conditions, and climate, interfering in the oil constitution. The quality of an essential oil depends on several parameters such as solubility in different organic solvents, density, and refraction index, among others. Table 2 presents the physicalchemical parameters obtained to the essential oil of clove, according to USP 41 methodology [26]. Through verification of the parameters, it is possible to conclude that the results are satisfactory since all values of the analysis were within the specification limits provided in the regulatory standard. Table 3 presents the chemical composition of the clove oil, considering the major components that compose the samples. The mass composition relative to the eugenol, the major component of clove oil, is close to that obtained in similar researches, ranging from 83.6 to 88.4% [8].

Selection of the Lipase
Preliminary tests were carried out in order to define which lipase, among the three available enzymes (lyophilized enzyme produced Penicillium sumatrense microorganism; Lipozyme® TL 100L and Lipozyme® CALB L), would present the best response in terms of raw material conversion. At 50°C, eugenol to acetic anhydride molar ratio of 1:3, and enzyme load of 5.5 wt%, the lipase Lipozyme® TL 100L presented the higher conversion: 85.40 ± 3.2% after 2 h of reaction. In these same reaction conditions, the homemade lyophilized enzyme and Lipozyme® CALB L presented conversion of 73.4 ± 4.2% and 58.3 ± 6.0%, respectively. Based on these results of conversion, lipase Lipozyme® TL 100L was chosen as catalyst to be utilized in the optimization of the reaction process via statistical design. It is important to note the interesting conversion achieved by the lyophilized enzyme produced in laboratory, superior in comparison to the commercial lipase CALB L. This fact demonstrates that the homemade biocatalyst has potential to be applied in the process.

Influence of the Reaction Parameters in the Eugenyl Acetate Synthesis
In the evaluation of the EA production using Lipozyme® TL 100L as reaction biocatalyst, a full 2 3 factorial design with three replications of the central point was employed, investigating the effects of the eugenol to acetic anhydride molar ratio, temperature, and enzyme load in the process performance. Table 4 presents the matrix of the statistical design with coded and real values of the independent variables and the experimental reaction conversions to EA. The highest conversion into EA occurred at 55°C, eugenol to acetic anhydride molar ratio of 1:1, and 10 wt% of Lipozyme® TL 100L (assay 7), achieving 91.80% of conversion. The lowest conversion obtained occurred at 45°C, eugenol to acetic anhydride molar ratio of 1:1, and 5 wt% of Lipozyme® TL 100L (38.12% of conversion, assay 1), indicating that a catalyst load superior than 5 wt% is indicated to the reaction. Results presented in Table 4 were statistically analyzed via Pareto diagram, as shown in Figure 1. Significant variables presented p < 0.05. Coefficients with a positive sign indicate a synergic effect whereas negative coefficients indicate an antagonistic effect in the ester formation. The Pareto diagram of standardized effects confirms that the molar ratio between eugenol and acetic anhydride had no significant effect in the EA synthesis, while reaction temperature and lipase load had a significant positive effect (p < 0.05) on ester production. However, it is possible to observe that the interaction between the variables "eugenol to acetic anhydride molar ratio" and "reaction temperature" had a negative effect on ester production. Similar tendency was also observed by Silva et al. [4] that, however, used immobilized lipase to produce EA from clove oil. The statistical analysis of the results presented in Table 4 allowed the obtaining of a model for EA conversion as a function of eugenol to acetic anhydride molar ratio, temperature, and lipase load. The model was statistically validated (p < 0.05) by analysis of variance (ANOVA), as presented in Table 5, where a coefficient of determination (R 2 ) of 99.15, proving that the model obtained, with a level of reliability of 95%, is statistically valid.
Results presented in Tables 4 and 5 as well as the Pareto diagram ( Figure 1) show an interactive effect between the considered variables. Thus, an independent analysis of the parameters would not be appropriate. Therefore, contour surfaces between the parameters are necessary to find the region with the best response and thus determine the optimum   Figure 2. Figure 2a shows the conversion in EA as function of reaction temperature and lipase load, Figure 2b presents the conversion in esters as a function of eugenol to acetic anhydride molar ratio and temperature, and Figure 2c simulates the ester conversion ranging with the eugenol to acetic anhydride molar ratio and lipase load. From these results, it is possible to observe that the EA conversion is benefited with the simultaneous increase of the enzyme load and reaction temperature, especially with temperatures higher than 52°C. It was also possible to observe that at temperatures below 48°C, even an increase in the eugenol to acetic anhydride molar ratio was not enough for satisfactory EA conversions to be achieved, requiring higher temperatures to be used in the system. Similar situation was observed when the EA conversions were analyzed ranging the eugenol to acetic anhydride molar ratio and lipase load. With lipase loads inferior than 7 wt%, even using a high eugenol to acetic anhydride molar ratio, it was not possible to obtain satisfactory conversions, corroborating the hypothesis presented previously in the Pareto diagram ( Figure 1) that this variable is not significant for the EA conversion. Such finding allowed us to deduce that a minimum eugenol to acetic anhydride molar ratio of 1:1 is the ideal to be used in the process. On the other hand, as expected, an increase in the lipase load applied to the process returned satisfactory EA conversion. Therefore, based on the observed results, the reaction conditions that yielded the highest EA conversion were 55°C, eugenol to acetic anhydride molar ratio of 1:1, and 10 wt% of lipase load.

Time Course of the Eugenyl Acetate Synthesis
With the optimization of the reaction parameters considered in the EA production, the behavior of reaction temperature, Lipozyme® TL 100L load, and eugenol to acetic anhydride molar ratio were evaluated in a time course to the acetylation process. Samples at 20, 40, 60, 120, 180, 240, 300, and 360 min were collected using the optimized reaction conditions previously obtained (55°C, eugenol to acetic anhydride molar ratio of 1:1, and 10 wt% of lipase). The results obtained are presented in Figure 3.
From the results obtained, it should be noted the interesting enzymatic activity of the lipase Lipozyme® TL 100L after 1 h of process, reaching more than 60% of feedstock conversion. Between 3 and 4 h, the conversion achieved more than 80% and the maximum conversion was obtained with values higher than 90%, close to the 5 h of reaction. Data about the application of lipases in liquid formulation for EA synthesis from clove oil are rare. Few researches about this topic are published, where those that involve enzymatic process use immobilized lipases as reaction catalyst. Silva et al. [4] investigated the ability of the commercial immobilized lipase Lipozyme® TL IM to catalyze the acetylation of essential oil of clove with acetic anhydride in a solvent-free system. At 70°C, eugenol to acetic anhydride molar ratio of 1:5, and 5 wt% of lipase, a conversion of 92.86% was obtained by these authors after 3 h of reaction. In another similar research, Chiaradia et al. [28] reported data of an eugenyl acetate production by esterification of eugenol and acetic anhydride in a solvent-free system using the immobilized lipase Novozym® 435 as catalyst: at 50°C , eugenol to acetic anhydride of 1:3, and 5.5 wt% of enzyme, a conversion of 99% was achieved after 6 h of reaction.

Characterization of the Eugenyl Acetate
The chemical structure of the purified EA obtained at optimized reaction conditions was confirmed by the proton nuclear magnetic resonance ( 1 H-NMR) and infrared spectroscopy (FT-IR) analysis.  The results of the FT-IR analysis and the information related to the identified attributions and functional groups are presented in Figure 4. The evaluated spectrum shows bands at 2,939 and 2,841 cm −1 that are attributed, respectively, to the asymmetric and symmetric axial deformation of the CH 2 group, while the existing one at 3,005 cm −1 is attributed to the =C-H stretch of the aromatic ring structure. In the spectrum, it is possible to observe the characteristic presence of the carbonyl band of the ester bound with the aromatic ring at 1,761 cm −1 , suggesting that the molecule is, in fact, EA due to the addition of the acyl group in the eugenol molecule. In addition, the spectra present two axial stretch bands of the ester at 1,184 cm −1 (high intensity) and 1,267 cm −1 (medium intensity). The bond stretches of the C-O between 1,214 and 1,033 cm −1 are assigned to the methoxy group. In 1,419 and 1,368 cm −1 , there was a folding of CH 2 and CH 3 [29]. According to Engel et al. [29], the stretch of the double aliphatic/aromatic carbon bond is around 1,680-1,600 and 1,600 to 1,475 cm −1 , respectively. In Figure 4, such stretches occurred in 1,604 and 1,507 cm −1 , originating from C = C (aliphatic) and C = C (aromatic). The aromatic compound of the EA molecule presented characteristic bands around 906, 822, and 747 cm −1 , which are attributed to off-plane folding, where these occur at 900-690 cm −1 and are used to define aromatic ring replacement pattern [29]. Table 6 presents results of 1 H and 13 C NMR analysis, demonstrating the molecular structure of EA ester, obtained through acetylation reaction. The ester was submitted to detailed analysis of 1 H NMR spectra for 1 and 2 D. Through the NMR spectrum of 1 H ( Supplementary  Information 1), DEPT 135 ( Supplementary Information 2), and Table 6, a dubbing at δH 7.00 ppm ( 1 H of position 6 carbon, d, J = 8.0 Hz), a voice over at δH 6.96 ppm ( 1 H of position 3 carbon, d, J = 1.8 Hz), and a double dublet at 6.78 ( 1 H of position 5 carbon, dd, J = 8.0 and 1.8 Hz) were observed, indicating a tri-substituted aromatic ring. The position of the substituters in the aromatic ring was performed based on the values of chemical displacements, multiplicity, and long-distance correlations. The atoms of the functional group that are closer to the oxygen atom have less shielding (higher value of ppm of chemical displacement) than the atoms closest to a carbon atom (lower value of ppm chemical displacement) [29]. Thus, of the 12 carbon signals expected for EA that are observed in the DEPT 135 spectrum, the carbon signal of position 12 is highlighted, according to Table 6, which corresponds to the carbonyl group of the ester with the highest displacement of 169.0 ppm. Additionally, a dublet was observed at δH 3.38 ( 2 H of position 7 carbon, d, J = 6.8 Hz) and two multiplets in δH 6.00 ( 1 H of carbon of position 8, m) and δH 5.12 ( 2 H carbon of position 9, m) combined with the correlations of the HMBC ( Supplementary Information 3). It is possible to assign this behavior to the isoprenic unit (alilic group) as one of the substituents linked to the aromatic ring. The presence of two singlets in δH 3.76 ( 3 H of carbon from position 10, s) and 2.25 ( 3 H of carbon of position 11, s) is attributed respectively to hydrogens of the methoxy group and hydrogens of the acetoxi group, associated with data from DEPT 135. It is allowed to characterize the molecular structure of the EA obtained in the acetylation of eugenol with acetic anhydride using the enzyme Lipozyme® TL 100L as catalyst. Santos et al. [30] analyzed the molecule of EA through NMR, verifying the peaks of the additional acetyl group (C-CH 3 ) with lower chemical displacement value and -COCH 3 with higher ppm value were observed in the spectra.

Conclusions
In this work, eugenyl acetate synthesis from clove oil through liquid lipase-mediated acetylation was investigated. Among the enzymes tested, the lipase Lipozyme® TL 100 L presented better performance in terms of feedstock conversion. Via statistical design, the best condition found for the EA production was 55°C, eugenol to acetic anhydride molar ratio of 1:1, enzyme load of 10 wt%, and time of 2 h of reaction, where a conversion of 91.80% was obtained. The variables that had a significant influence on the ester synthesis were temperature and catalyst load. Infrared spectroscopy and nuclear magnetic resonance confirmed that the compound formed is eugenyl acetate. Considering that data about the enzymatic synthesis of EA using lipases in liquid formulation are scarce, the results of this work serve as a basis for future research about the topic, mainly with the enzymes used in this work. The satisfactory conversions reported support the fact that the soluble lipases-catalyzed synthesis of eugenyl acetate from clove oil is an interesting alternative to the conventional chemical route.