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Green Enzymatic Synthesis of Geranyl Butyrate: Process

Optimization and Mechanistic Insights
Francisco Simão Neto, Paulo Gonçalves de Sousa Junior, Carlos José Alves da Silva Filho,
Lucas Pinheiro Coutinho, Rafael Leandro Fernandes Melo, Javier Rocha-Martin,
Maria Alexsandra de Sousa Rios, Ada Amélia Sanders Lopes, Norberto de K. V. Monteiro,
Marcos Carlos de Mattos, Leonardo Farias Serafim, and José Cleiton Sousa dos Santos*
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sı Supporting Information

ABSTRACT: Flavor esters are organic compounds widely used in the food
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industry to enhance the aroma and taste of products. However, most chemical
processes for the production of these flavoring compounds use toxic organic
solvents. Some organic solvents derived from petroleum can leave behind
residual traces in food products, which may raise concerns about potential
health risks and contamination. In this study, we employ Eversa Transform 2.0,
a commercial lipase derived from the lipase from Thermomyces lanuginosus, to
produce geranyl butyrate in aqueous media. The chemical process was
optimized using the Taguchi method, and a conversion of 93% was obtained at
the optimal reaction conditions of: 1:5 molar ratio (v/v), 15% biocatalyst load
(w/w), at 50 °C, in 6 h. Classic (molecular dynamics) and quantum (density
functional theory) simulations unveiled amino acid residues involved in the
stabilization of the enzyme−substrate complex. Detailed QM/MM mechanistic
studies identified the nucleophilic attack of the deacylation reaction as the rate-limiting step of the entire mechanism, which has a
free energy barrier of 14.0 kcal/mol.

1. INTRODUCTION floral scent reminiscent of fresh and tropical fruits such as

Flavor esters are organic compounds widely used in the pineapple, peach, and strawberry.6 It is formed through the
industry to enhance the aroma of products such as soaps, esterification of geraniol, a naturally occurring compound
deodorants, and perfumes.1 These compounds play an found in various fruits and plants, with butyric acid, as shown
in eq 1.
essential role in the food industry, where they are used as
flavoring agents due to their ability to replicate the natural
flavors found in fruits, flowers, and other natural sources.2 In
addition to their versatility, flavor esters contribute to the The rate of esterification reactions can vary depending on
overall sensory appeal of food and beverages.3 By using these several factors including: the chemical nature of the reactants,
compounds, manufacturers can tune their product’s taste and their respective concentrations, the temperature and pressure
aroma profiles, making them more appealing to consumers of the reaction medium, and other factors. Generally,
since the unique scent of ester compounds can trigger esterification reactions are relatively slow compared with
pleasurable associations and evoke memories, creating a other chemical reactions. To overcome this challenge, using
sense of familiarity with the product. However, it is worth enzymatic catalysis is essential for the feasibility of some
noticing that the excessive usage of flavor esters can result in an industrial processes.7 These biomolecules work as natural
artificial or overpowering final taste of the product. Regulatory catalysts, increasing the speed of chemical reactions while
bodies governing food safety and quality control have reducing the formation of unwanted byproducts. Additionally,
established guidelines to ensure the responsible and safe use
of flavor esters in food production.4
Flavor esters can be synthesized through the esterification Received: October 26, 2023
reaction between an alcohol (acyl acceptor) and an organic Revised: January 24, 2024
acid (acyl donor). The characteristic flavor and scent of the Accepted: January 31, 2024
esters produced by this chemical route are directly associated Published: April 1, 2024
with the chemical nature of the reactants used in the
synthesis.5 For instance, geranyl butyrate exhibits a fruity and
© 2024 The Authors. Published by
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these catalysts are considered environmentally friendly, and Eversa Transform 2.0 (ET2) is the commercial name of the
their biochemical reactions deliver natural products in terms of lipase from Thermomyces lanuginosus (TlL) (previously
their origin, which is very attractive to the food industry.8 Humicola lanuginosa), produced by Novozymes through the
Moreover, when enzymes are disposed of in the environment, submerged fermentation of a genetically modified strain of
they are readily broken down into amino acids by natural Aspergillus oryzae, with high specificity for raw materials and
processes such as microbial activity, oxidation, and hydrolysis.9 high activity at mild process conditions.15 The ET2 enzyme is
Among all types of enzymes, those belonging to the lipase an extremophilic lipase capable of functioning under extreme
family, also classified as hydrolases, are very commonly applied temperature and pressure conditions. It has a low production
to biotechnological processes.10 Lipases are enzymes that cost (15 US$/kg) and contains 269 amino acid residues, a
catalyze the hydrolysis, esterification, and transesterification of molecular mass of ∼30 kDa, and an activity of 9100 IU/mL.16
lipids and are commonly found in many living organisms. The This enzyme has been used as a catalyst in many chemical
remarkable affinity of lipases for different types of substrates reactions, such as the enzymatic esterification of glycerol,17 the
and their ability to function in acidic or alkaline conditions synthesis of biolubricants,18 the production of biodiesel from
make these biocatalysts ideal for synthesizing biofuels, the esterification of babassu oil (Orbignya sp.),19 and the
detergents, food, and other products.11 Regarding their production of esters using tucuman oil (Astrocaryum
structure, most lipases have an additional hydrophobic lid vulgaris).20
domain, an α/β hydrolase fold, and a Ser-His-Asp/Glu Along with the utilization of biocatalysts, experimental
catalytic triad, as shown in Scheme 1.12 planning is an essential aspect of optimizing chemical
processes, as it allows the determination of the most efficient
Scheme 1. (a) Opening of the Lid Domain of the Lipase experimental conditions. The Taguchi methodology is an
(TlL) Exposing the Active Site to Substrates. (b) General experimental planning and process organization technique that
Acylation and Deacylation Mechanisms of Lipases aims to improve product quality and production efficiency.21 It
was developed by the Japanese engineer Taguchi in the 1950s
and has gained widespread popularity in various industries due
to its effectiveness in achieving robustness and cost reduction
while minimizing the number of experiments. The first step of
this methodology involves the identification of key perform-
ance metrics or response variables that must be optimized.
Next, the factors, which are the variables or parameters that
can potentially influence the response variables, are selected.
Each factor is then assigned a set of levels or values, chosen
based on the range of practical values and the desired
sensitivity used during the experimentation. Depending on the
number of factors and levels considered, an orthogonal array is
chosen to ensure an efficient and balanced allocation of factor
combinations. Using the chosen orthogonal array, experiments
are conducted to collect data on the response variables. Each
experiment consists of a specific combination of factor levels,
and multiple replications are typically performed to account for
variability. Finally, signal-to-noise ratio (S/N) and variance
(ANOVA) analyses are performed to interpret the exper-
imental data and identify the significant factors affecting the
response variables. Once the optimal factor level is identified, a
confirmation experiment is conducted to validate the results
and ensure the robustness of the obtained solution.
Since some molecular factors controlling chemical reactions
cannot be determined experimentally, computational chemistry
provides useful tools to study chemical reactions and predict
important thermodynamic and kinetic parameters.22 One
Under minimal substrate concentration, the lid domain, powerful approach in computational chemistry for studying
which envelops the active site in the resting state, moves apart enzymatic reactions is the quantum mechanics/molecular
via conformational changes at the oil/water interface, making mechanics (QM/MM) method.23 This technique combines
the active site accessible to substrates.13 Once at the active site, the accuracy of quantum mechanical calculations with the
the substrate can undergo catalysis through a mechanism efficiency of molecular mechanics force fields to capture the
comprised of two consecutive processes: acylation and electronic and structural features of the active site of the
deacylation.14 In the acylation process, the serine amino acid enzyme and its surrounding environment. In QM/MM
residue from the active site promotes a nucleophilic attack on calculations, the active site where the reaction occurs is treated
the electrophilic carbon atom of the substrate. This process quantum mechanically to accurately describe the electronic
fixes the substrate through a covalent bond, generating an behavior of the reacting species. In contrast, the rest of the
acyl−enzyme complex intermediate. In the second step enzyme and solvent molecules are treated using molecular
(deacylation), a free nucleophile (HO-R) promotes a mechanics to account for their bulk effects. This hybrid
nucleophilic attack onto the acyl−enzyme complex intermedi- approach accurately represents the enzyme’s active site and the
ate, releasing the products and regenerating the enzyme. surrounding environment, providing insights into the reaction
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mechanism, transition states, reaction energetics, and various point was analyzed by high-performance liquid chromatog-
aspects of enzyme catalysis.24 raphy using a Shimadzu LC-18 column at a temperature of 25
This work presents an optimized synthetic route to produce °C equipped with a UV detector. A retention time of 5.5 min
the flavor ester geranyl butyrate from the enzymatic was observed for the product in the injected sample (1 mg/
esterification of geraniol and butyric acid catalyzed by the mL), as shown in Figure S1. The eluent was a mixture of
Eversa Transform 2.0 (ET2). In contrast to previous reports, acetonitrile and water in a 4:1 (v/v) ratio, with a flow rate of
we performed an esterification reaction rather than an acyl− 3.0 mL/min for 5 min. The wavelength of 220 nm was chosen
transfer reaction.5,7 By applying the Taguchi method, the effect for the analysis, which showed the highest absorption. The
of the molar ratio (MR) of the reactants, the temperature, the one-dimensional hydrogen (1H NMR) and carbon (13C NMR)
reaction time, and the biocatalyst load on the conversion was NMR spectra were recorded in a Bruker spectrometer, model
evaluated. The reaction product was characterized by multiple Advance DRX-300, belonging to the northeast center for
chromatographic techniques and nuclear magnetic resonance application and use of NMR, located at the Federal University
(NMR) spectroscopy. Finally, using a combination of classic of Ceara (CENAUREMN-UFC). The experiment was carried
[molecular docking and molecular dynamics (MD)] and out at a hydrogen atom frequency of 300 MHz and a carbon
quantum [density functional theory (DFT)] computational atom frequency of 75 MHz. All samples were dissolved in
methods, structural, kinetic, and thermodynamic data of the deuterated chloroform (CDCl3) and analyzed in 5 mm NMR
esterification reaction were obtained and are in excellent tubes.
accordance with the experimental observations. 2.4. Experimental Design and Statistical Analysis. An
experimental design based on the Taguchi method was used
2. MATERIALS AND METHODS with a standard L9 orthogonal matrix (the “L” and “9”
2.1. Biocatalyst and Chemical Reagents. The commer- represent the Latin square and the number of experiments,
cial lipase Eversa transform 2.0 (ET2) from T. lanuginosus respectively) to distribute four factors in three levels to
(TlL), produced by Novozymes through the submerged maximize the conversion. The four independent factors: the
fermentation of a genetically modified strain of A. oryzae, molar ratio between organic acid and alcohol (MR), the
was purchased from Sigma-Aldrich Brazil Ltd. (Cotia, São biocatalyst load (Cat), the temperature (T), and the reaction
Paulo, Brazil). The chemical reagents used were analytical time (t), as well as their corresponding levels, are shown in
grade from Synth (São Paulo, Brazil) and Vetec (São Paulo, Table 1. The biocatalyst load was calculated from the reaction
Brazil). The Statistica 10 software (Statsoft, USA) was used for
the experimental design based on the Taguchi method. Table 1. Experimental Procedure Levels and the Range of
2.2. Enzymatic Activity Assay. Following the exper- Independent Factorsa
imental design assessments, the activity assay was carried out levels MR Cat (% w/w) T (°C) t (h)
with reaction mixtures containing different ratios of reagents, at
level 1 (L1) 1:1 5 30 2
different temperatures, and for different time intervals. Vials of
level 2 (L2) 1:5 10 40 4
2 mL were used to perform biochemical reactions under a
level 3 (L3) 1:9 15 50 6
controlled incubator temperature, in addition to maintaining a
an orbital agitation of 200 rpm. After completion of the The chosen parameters were the molar ratio of acid: alcohol (MR),
the biocatalyst load (Cat), the temperature (T), and the reaction time
esterification reaction, the samples were analyzed in duplicate
(t).
in two Erlenmeyer flasks with 0.2 g of the sample, 5 mL of
standard ethyl alcohol, and 3 drops of phenolphthalein as the
indicator. Each sample was titrated with a 0.1 M NaOH volume after the calculation of the MR. The values of S/N
solution until the color changed to a subtle pink. After ratios (signal-to-noise) corresponding to the conversions were
titration, the total consumed volume was used in eq 2 to obtain calculated using the characteristics of the “bigger is better”
the acidity index (AI). function since the objective of this study is to maximize the
response (flavor ester production). In the Taguchi method, the
MWNaOH·MNaOH ·f ·VNaOH S/N ratio measures quality characteristics and the deviation
AI =
m (2) from the desired value. Thus, using the S/N ratio to analyze
the results reduces the system’s sensitivity to sources of
In eq 1, MWNaOH is the molecular mass of NaOH, MNaOH is variation, resulting in good performance. The value of the S/N
the molarity of the NaOH solution used in the titration, f is the ratio for each experiment was calculated according to eq 4.
correction factor determined by NaOH standardization, VNaOH
is the total volume of NaOH consumed during the titration, ij 1 n 1 yz
and m is the mass of the analyzed sample. The conversion (X) S/N = 10 logjjjj zz
2zzz
jn y
of butyric acid into the flavor ester geranyl butyrate is then k i=1 i { (4)
given by eq 3, where AI0 represents the initial acidity value due In eq 4, y is the fatty acid conversion for the corresponding
to the starting concentration of acid in the sample, and AI sample, i is the number of replicates, and n is the number of
represents the final acidity value, equivalent to the remaining responses for the combination of factor levels in any given
acid in the solution, not consumed during the enzymatic
reaction. parametric combination. The predicted signal-to-noise (S/N)
ratio under optimal conditions for obtaining the maximum
AI0 AI conversion was estimated by eq 5.
X (%) = × 100
AI 0 (3) n ij S S yzzz
S jj
S/N = + jj z
2.3. Quantification and Characterization of the N j Nj N zz
Products. The sample obtained from the optimal conversion i=1 k { (5)

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In eq 5, S̅ /N is the arithmetic mean of all S/N ratios, S/Nj is correlation DFT functional. The 6-31G(d,p) basis set was used
the S/N ratio at the sweet spot for each factor, and n is the to implement the computations, and Grimme’s function with
number of factors that significantly affect the process. the Becke−Johnsons damping effect (GD3BJ) was used to take
2.5. Computational Modeling and MD Simulations. into account the dispersive effects. Hessians were calculated at
The computational model of ET2 was based on the original the same level of theory as those of the optimizations to
structure of the lipase from T. lanuginosus (TlL, PDB confirm the nature of the stationary points along the reaction
identification code: 1EIN).25 The geometry of the substrates coordinate. The transition states were confirmed to have only
butyric acid and geraniol was optimized at the B3LYP/6-311G one imaginary frequency corresponding to the reaction
+(d,p)/D3 level of the DFT without any geometric constraints coordinates. Single-point calculations were performed using
using the Gaussian software package.26 Their charges and the larger 6-311+G(d,p) basis set with the B3LYP and M06-2X
electrostatic potentials (ESP) were computed at the same level functionals (Figure S4). In these calculations, the electrostatic
of theory, and their force field parameters were developed interactions between the QM and the MM parts were treated
using the antechamber software from the AmberTools by electronic embedding. Zero-point vibrational, thermal, and
package.27 The enzyme−substrate complexes were built entropy corrections were added to the final energies (at 298.15
through molecular docking using AutoDock Vina 1.5.6 K and 1 atm). The MM region was modeled using the AMBER
software.28 In the rigid docking protocol utilized, the structure force field as implemented in Gaussian software.
of the enzyme was kept fixed, but the substrates were allowed
to adopt any conformations. This procedure provided 20 3. RESULTS AND DISCUSSION
poses, which were ranked based on their energies. The three 3.1. Process Optimization by the Taguchi Method.
poses with the lowest energies for each substrate were selected The application of the Taguchi method for the optimization of
for all-atom MD simulations. the biochemical process allowed for the determination of all
The MD simulations were performed using the GRO- parameters affecting the conversion of the chemical reaction by
MACS29,30 program utilizing the AMBER ff19SB force field.27 performing a minimum number of experiments. In addition, a
The enzyme−substrate complex was placed in a cubic box with variation in the interaction between each parameter was also
dimensions of 10 × 10 × 10 nm. The shortest distance from evaluated to determine the best level of each parameter that
the edge of the box to the surface of the complex was less than ensures the optimal conversion point within the chosen
1 nm. The particle mesh Ewald method was used to compute variable range. Table 2 presents, in an expanded form, the
the electrostatic interactions, and for both Coulombic and van
der Waals interactions, a 1.2 nm cutoff distance was Table 2. Experimental Design of the Taguchi L9 Plana
established. The TIP3P31 water model was used as the solvent,
and Na+ and Cl− ions were added to neutralize the total charge experiment MR Cat (% w/w) T (°C) t (h) X (%) S/R
of the system. The system was then energy-minimized for 3000 1 1:1 5 30 2 15.2 ± 0.5 23.61
steps using the steepest descent algorithm before each MD 2 1:1 10 40 4 56.5 ± 0.7 35.04
simulation. The MD simulations were performed using a 3 1:1 15 50 6 90.1 ± 0.2 39.09
constant number of particles (N), pressure (P), and temper- 4 1:5 5 40 6 75.8 ± 0.6 37.59
ature (T) as an NPT ensemble for a total of 100 ns. The 5 1:5 10 50 2 50.9 ± 0.3 33.95
trajectories were computed for each model with a time step of 6 1:5 15 30 4 53.8 ± 0.3 34.61
1 fs. Finally, cluster analysis was performed to obtain the most 7 1:9 5 50 4 66.1 ± 0.8 36.40
representative structure. 8 1:9 10 30 6 64.6 ± 1.5 36.20
2.6. Hybrid Quantum Mechanics and Molecular 9 1:9 15 40 2 33.8 ± 0.2 30.59
Mechanics Calculations. The hybrid two-layer QM/MM a
All chemical reactions were performed in triplicate to ensure that the
(ONIOM) calculations were performed by using the Gaussian error was within the standard limits. A constant orbital agitation of
software package. This method utilizes a subtractive method in 190 rpm was maintained during the reaction process.
which the MM energy [EMM(model)] of the QM(model) part
is subtracted from the sum of the QM energy of the model
[EQM(model)] and MM energy [EMM(total)] of the whole results of the experimental planning, including the conversion
(total) system, as shown in eq 6. values for each point analyzed. By inspecting Table 2, it is easy
to notice that the reaction time was the parameter that most
EQM/MM(total) = E MM(total) EMM(model)
influenced the process, positively affecting the formation of the
+ EQM(model) (6)
flavor ester. As can be inferred from experiments 3 and 4, a
long reaction time is associated with a high conversion rate
This subtraction method corrects the artifacts introduced by since both experiments performed for 6 h displayed the highest
using link atoms. The ONIOM optimization procedure uses conversions among all 9 experiments. On the contrary,
macro/microinteractions, and the electrostatic interactions experiments 5 and 9, with a total reaction time of 2 h,
between the QM and the MM part were treated by mechanical displayed a very low reaction yield despite the use of a high
embedding. The QM region (model) was defined as the catalyst load (10 and 15% w/w). A similar trend can be
catalytic triad (Ser146, His258, and Asp201), the second observed for the temperature. For instance, experiments 3 and
coordination shell residue Tyr21, and the substrates. It contains 7, which exhibited the highest and the third highest conversion
a total of 51 atoms in the acylation mechanism and 77 atoms in (90.1 and 66.1%, respectively), were performed at the highest
the deacylation mechanism, with a −1 charge on each system. temperature level of 50 °C. These results emphasize the
The remaining atoms of the system were treated in the classic significance of the temperature in increasing the system’s
MM region. The QM region was optimized without any kinetic energy, enabling a more significant fraction of the
geometric constraint by using the hybrid B3LYP exchange− molecules to surpass the activation energy barrier. It also
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indicates that the catalyst employed can withstand high

temperatures without losing catalytic activity.
On the other hand, the MR and the catalyst load were the
factors that least influenced the conversion. In most enzymatic
processes, the employed concentration of acyl donors is higher
than the concentration of acyl acceptors. However, a high
concentration of acid (acyl donor) in esterification reactions
may lead to reduced activity or even inactivation of the enzyme
due to the changes in pH, which is a known disadvantage of
this chemical route. For this reason, the concentration of the
acid was kept constant while the concentration of the alcohol
was increased. Nevertheless, since the alcohol molecules react
with the acyl−enzyme intermediate rather than the enzyme in
its native form, a high concentration of alcohol creates Figure 1. S/N responses for each independent parameter at each
competition for the enzyme’s active site during the acylation level. The MR is shown in blue, the biocatalyst load (Cat) is shown in
step. Such interactions between the enzyme’s active site and green, the temperature (T) is shown in purple, and the reaction time
the alcohol molecules create complexes that are not catalytic (t) is shown in red.
competent, which negatively impacts the reaction rate. A
similar pattern of inhibition by alcohols was observed in similar reaction time and the temperature showed significance within
biochemical reactions performed by Pseudomonas cepacian,32,33 the best reliability range, as they presented p-values equal to
Candida rugosa,34 and Candida antarctica.35 0.0004 and 0.0037, respectively, whereas the MR and the
A similar behavior was observed for the catalyst load. catalyst load presented p-values much higher than 0.05. It is
Although a high enzyme concentration contributes to the rapid important to note that the contribution from the reaction time
formation of enzyme−substrate complexes, which increases the is about three times more significant than that of the
reaction rate, excessive enzyme amounts can lead to temperature, representing 74.64 versus 23.49% of the total
aggregation, preventing substrate binding to the active site. It conversion.
is noteworthy to emphasize that the optimal MR and catalyst Since the statistical analysis indicated that the reaction time
load levels are below the maximum level. This fact holds (t) was the most influential parameter affecting the conversion,
economic significance since the quantities of raw materials 2D contour surface graphs were generated to study the
employed in a chemical process directly influence the ultimate correlation effect of time with the other variables. The contour
cost of the final product. plots shown in Figure 2 connect points of equal values of
3.2. Statistical Analysis. The Taguchi method utilizes conversion, which can unveil interactions between two distinct
signal-to-noise (S/N) ratio values to determine the significance factors.
of each parameter analyzed. The present study applied the For instance, the temperature and reaction time are
“bigger is better” function to identify these ratios from the positively correlated. That indicates that a concurrent incre-
conversion values. The information on the average S/N of all ment of both the temperature and reaction time produces an
levels of each factor, in addition to the delta values, is shown in increment in conversion, although, as can be noted by the
Table 3 and Figure 1. The delta value results are obtained by inclination of the contour lines toward the reaction time, an
increment in the reaction time is more prevalent than an
Table 3. Response to Mean S/N Ratios and Order of increment in the temperature. The relationship between the
Variables for the Molar Ratio (MR), the Biocatalyst Load MR and the time, on the other hand, exhibits a different
(Cat), the Temperature (T), and the Reaction Time (t) behavior. The high conversion contour lines are displayed at
factor levels MR Cat T t reaction time levels above two and MR levels below two. At
1 32.58 32.53 31.47 29.38
MR levels higher than two, a decrease in conversion of at least
2 35.38 35.07 34.40 35.35
20% is expected due to the inhibition of the enzyme. Finally,
3 34.39 34.76 36.48 37.63
the contour lines for the catalyst load displayed the lowest level
delta 2.80 2.53 5.01 8.25
of interaction with the reaction time. At reaction times close to
level 3 (6 h), a conversion of at least 75% is predicted despite
the level of catalyst load.
subtracting the factor with the highest S/N value from the Based on the data obtained from the Taguchi method, it was
factor with the lowest S/N value. This type of analysis utilizes possible to derive an equation that expresses the conversion as
the delta value to rank the factors and define their significance a function of each of the independent parameters, as shown in
for the process. As already expected, the analysis of Table 3 eq 7
shows that the reaction time and temperature stood out among
the other analyzed parameters, with delta values of 8.25 and X (%) = 0.11A + 0.691B + 1.209C + 10.97D 43.5
5.01, respectively. (7)
The analysis of the variance provided an analogous outcome. In eq 7, X is the conversion for the esterification reaction,
The values obtained in this analysis are presented in Table 4, and A, B, C, and D are the encoded values of the MR, the
where the main highlight is given to the p-value, which is the biocatalyst load, the temperature, and the reaction time,
value that determines the significance of the factors during the respectively. The statistical analysis also concluded that the
studied reaction. The significance of a specific parameter is best time was presented in level 3, as 6 h (L3), the best
linked to its p-value, which can guarantee up to 95% temperature was 50 °C (L3), an adequate biocatalyst load was
confidence, provided that this value is less than 0.05. The defined as 15% (L3), and finally, the MR stood out in level 2,
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Table 4. Results of Analysis of Variance (ANOVA) for Parameters That Affect the Production of the Flavor Ester
factors SS DF IN F value p-value contribution (%)
molar ratio 60,620 2 30,310 0.05 0.8351 0.03
biocatalyst 73,607 2 36,803 3.01 0.1575 1.84
temperature 879,167 2 439,583 36.95 0.0037 23.49
time 2916,847 2 1,458,423 127.71 0.0004 74.64
total 3,930,241 6 100.00

3.3. Characterization of Geranyl Butyrate. The peak

observed in the chromatogram shown in Figure S1 is related to
the reaction product, geranyl butyrate. The maximum
absorption for geranyl butyrate was observed at 220 nm, and
the intensity of the peak indicates that the flavor ester was
produced in a significant amount, with the concentration of the
sample being equivalent to 40 mg/mL.
The 1H NMR spectrum, shown in Figure S2, displays
characteristic peaks assigned to the hydrogen atoms of the
ester structure. The doublet observed at δ 4.1 ppm (Figure
S2a) is related to the methylene hydrogen atoms directly
linked to the oxygen atom of the ester functional group. The
triplet peak observed at δ 2.3 ppm (Figure S2b) is assigned to
the methylene hydrogen atoms bonded to the ester carbonyl
carbon atom. The peaks referring to chemical shifts at δ 5.1
and δ 5.4 ppm are attributed to hydrogen atoms linked to sp2
carbons.
The 13C NMR spectrum, shown in Figure S3, was also used
to track the conversion of the alcohol into the flavor ester by
the appearance of the carbonyl carbon atom, which resonates
at δ 177 ppm. The peaks in the range of δ 130−140 ppm refer
to dehydrogenated sp2 carbons linked to the methyl branch.
Vinyl carbons at δ 123 and 124 ppm have very close chemical
shifts due to the similarity of the structural profiles of the two
carbon atoms (both have chemically similar neighborhoods).
The peak with a chemical shift close to δ 60 ppm is attributed
to the carbon linked directly to the oxygen of the ester group.
3.4. Computational Modeling and the Mechanism of
Figure 2. Contour surfaces for the production of geranyl butyrate.
the Reaction. The active site of ET2 is composed of a
The temperature (T), the MR, and the catalyst load (Cat) are
evaluated against the reaction time (t), the most influential parameter catalytic triad formed by residues Ser146, His258, and Asp201, as
affecting the conversion. shown in Figure 4. Substrates with appropriate conformations
and chemical affinity with the residues present in the vicinity of
the active site can bind and undergo catalysis. In the enzyme−
substrate complex (ET2-BA) obtained by molecular docking
in a ratio of 1:5 (L2). Equation 7 predicts a conversion of 96% and MD simulations, the second coordination shell residue
at the optimal parameter conditions. Figure 3 shows the parity Tyr21 stabilizes and orients the BA molecule toward the
plot comparing the predicted response from eq 7 to the nucleophilic residue Ser146 through hydrogen bonding (HT−
response values for the conversion obtained in the experi- OC = 1.53 Å). The hydrophobic carbon chain of BA interacts
ments. with the side chain of residues Ile94, Leu154, and Phe265 through
weaker nonpolar interactions. These interactions are respon-
sible for retaining the ET2-BA complex together, as shown by
the low rmsd displayed during the course of 100 ns of
simulation.
The most representative structure of the catalytic competent
ET2-BA complex (RA), obtained by cluster analysis of MD
simulations and optimized by hybrid QM/MM calculations, is
shown in Figure 4 inset (a) and in Figure 5. Key bond lengths
are shown in Table S1. In RA, the electrophile−nucleophile
(OS−CB) distance is 2.78 Å. The hydrogen atom HS of the
nucleophilic residue Ser146 is participating in a hydrogen bond
network that connects it with the residues His258 and Asp201.
The first and rate-limiting step of the acylation reaction
Figure 3. Parity graph evaluating the observed responses versus involves the nucleophilic attack of the OSHS side chain of
expected responses provided by eq 7. Ser146 onto the electrophilic carbon atom CB of BA. This
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Figure 4. Computational model of ET2 showing explicit TIP3P water molecules (left). The active sites of ET2 in complex with butyric acid (BA)
and acyl-ET2 in complex with geraniol (GE) are shown as insets (a,b).

Figure 5. Mechanism of the Acrylation of BA Catalyzed by ET2. The carbon atoms of the substrate (BA) are shown in purple, while the enzyme
atoms are shown in green. All bond lengths are displayed in angstroms, and free energies are in kcal/mol (B3LYP), relative to RA.

Figure 6. Mechanism of the deacylation of BA and geraniol catalyzed by acyl-ET2. The carbon atoms of BA, geraniol, and the enzyme are shown in
purple, yellow, and green, respectively. Bond lengths are displayed in angstroms and free energies in kcal/mol (B3LYP), relative to RD.

process takes place with a free energy barrier of 13.0 kcal/mol T1A, the electrophile-nucleophile ratio is decreased to 2.23 Å.
and is associated with T1A. In the structure of transition state This step also involves two proton transfer events (OS−HS to
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HS−Nε and Nδ−HH to HH−OA) which invert the protonation enzyme and in the reaction rate. This work is now underway
state of His258. The gem-diol intermediate IA generated after and will be reported in due course.
this step is exergonic by 4.9 kcal/mol. This unstable
intermediate collapses with a very low energy barrier (6.4
kcal/mol), which is associated with T2A. The collapse of IA
■
*
ASSOCIATED CONTENT
sı Supporting Information
takes place with the same proton transfer events as in T1A but The Supporting Information is available free of charge at
in the opposite direction. The product of the acylation reaction https://pubs.acs.org/doi/10.1021/acsomega.3c08483.
(PA) is the acyl enzyme (acyl-ET2), where the butyric group
(butyrate) is added to the Ser146 side chain and a free water Chromatogram showing the peak at 220 nm, 1H NMR
molecule. spectrum of geranyl butyrate,13C NMR spectrum of
The second process involved in the production of the flavor geranyl butyrate (highlighting the main peak), compar-
ester geranyl butyrate is deacylation. In this process, the acyl- ison of B3LYP and M06-2X DFT functionals, key bond
distances in Ångströms for acylation and deacylation
ET2 enzyme binds the second substrate, a molecule of geraniol
reaction mechanisms, and (x, y, z) coordinates of the
(GE), to form the second enzyme−substrate complex, acyl-
QM region for each discussed structure in the
ET2-GE, shown as RD in Figure 6.
manuscript (PDF)
In the first step of the deacylation mechanism, acyl-ET2
undergoes a nucleophilic attack from the oxygen atom OG of
GE at the carbon atom CB of the butyrate group previously
inserted at the Ser146 side chain. This process takes place with a
■ AUTHOR INFORMATION
Corresponding Author
free energy barrier of 14.0 kcal/mol, associated with transition José Cleiton Sousa dos Santos − Engineering and Sustainable
state T1D. A more sterically crowded gem-diol (ID) is formed Development Institute, University of International Integration
in this process and is endergonic by 4.0 kcal/mol. It is of Afro-Brazilian Lusophony, Redenção, Ceará 62790-970,
important to note that the nucleophilic attack can also be Brazil; orcid.org/0000-0002-1511-5180;
performed by an adventurous water molecule. Such an event Phone: +55(85) 9975-23838; Email: [email protected]
would take the enzyme back to IA instead of ID, and it is a
known disadvantage of performing esterification reactions in Authors
water. Although intermediate ID is thermodynamically less Francisco Simão Neto − Department of Chemical
stable than IA (with respect to its respective reactants), its Engineering, Federal University of Ceara, Fortaleza, Ceará
collapse occurs with a higher free energy barrier of 8.5 kcal/ 60455-760, Brazil
mol. The higher energy barrier associated with T2D is due to Paulo Gonçalves de Sousa Junior − Department of Organic
the presence of the bulky alkyl group, which stabilizes the gem- and Inorganic Chemistry, Federal University of Ceara,
diol through the electron-donating effect. The collapse of ID Fortaleza, Ceará 60440-900, Brazil
leads to the production of geranyl butyrate and regenerates the Carlos José Alves da Silva Filho − Department of Organic
free enzyme. The nucleophilic attack step is the rate-limiting and Inorganic Chemistry, Federal University of Ceara,
step of the deacylation mechanism, which makes the Fortaleza, Ceará 60440-900, Brazil
deacylation mechanism the slowest of the two reactions Lucas Pinheiro Coutinho − Department of Analytical
involved in flavor ester production. Chemistry and Physical Chemistry, Federal University of
In summary, our integrated experimental, statistical, and Ceara, Fortaleza, Ceará 60020-181, Brazil
computational approach evaluated the parameters that most Rafael Leandro Fernandes Melo − Department of
affected the production of geranyl butyrate by ET2 through an Metallurgic Engineering, Federal University of Ceara,
esterification reaction. The reaction time was indicated as the Fortaleza, Ceará 60440-554, Brazil
most relevant parameter affecting the conversion, followed by Javier Rocha-Martin − Department of Biochemistry and
the temperature. An increase in the concentration of the acyl Molecular Biology, Faculty of Biological Sciences,
acceptor as well as the catalyst load did not enhance the Complutense University of Madrid, Madrid 28040, Spain
production of the flavor ester significantly. However, at higher Maria Alexsandra de Sousa Rios − Department of
levels, an inhibition pattern was observed for these two factors, Mechanical Engineering, Federal University of Ceara,
which negatively affected the conversion. The final experiment Fortaleza, Ceará 60455-760, Brazil; orcid.org/0000-
performed at the optimum conditions (t = 6 h, T = 50 °C, MR 0002-3145-0456
= 1:5, and Cat = 15%) exhibited a conversion of about 93%. Ada Amélia Sanders Lopes − Engineering and Sustainable
Theoretical studies unveiled the binding modes of each Development Institute, University of International Integration
substrate in the active site of ET2. A balance of hydrophobic of Afro-Brazilian Lusophony, Redenção, Ceará 62790-970,
and hydrophilic forces exerted by resides Ile94, Leu154, and Brazil
Phe265 through weak nonpolar interactions and hydrogen Norberto de K. V. Monteiro − Department of Analytical
bonding provided by Tyr21 are responsible for the stabilization Chemistry and Physical Chemistry, Federal University of
of the enzyme−substrate complex. The nucleophilic attack step Ceara, Fortaleza, Ceará 60020-181, Brazil; orcid.org/
of the deacylation reaction was identified as the rate-limiting 0000-0002-5847-5733
step of the entire process. That step is associated with a free Marcos Carlos de Mattos − Department of Analytical
energy barrier of 14.0 kcal/mol, which is equivalent to a rate Chemistry and Physical Chemistry, Federal University of
constant (kcat) in the order of 103 s−1. Ceara, Fortaleza, Ceará 60020-181, Brazil; orcid.org/
Our team is developing a technology to immobilize ET2 for 0000-0003-4291-5199
the production of geranyl butyrate as well as other flavor esters, Leonardo Farias Serafim − Department of Chemistry, Georgia
without the use of toxic organic solvents. Preliminary State University, Atlanta, Georgia 30302, United States
experiments showed an enhancement in the stability of the Complete contact information is available at:
16999 https://doi.org/10.1021/acsomega.3c08483
ACS Omega 2024, 9, 16992−17001
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https://pubs.acs.org/10.1021/acsomega.3c08483 (15) Chang, M. Y.; Chan, E.-S.; Song, C. P. Biodiesel production

catalysed by low-cost liquid enzyme Eversa® Transform 2.0: Effect of
Notes free fatty acid content on lipase methanol tolerance and kinetic model.
The authors declare no competing financial interest. Fuel 2021, 283, 119266.
(16) Monteiro, R. R. C.; Arana-Peña, S.; da Rocha, T. N.; Miranda,

■ ACKNOWLEDGMENTS
We gratefully acknowledge the following Brazilian Agencies for
L. P.; Berenguer-Murcia, Á .; Tardioli, P. W.; dos Santos, J. C. S.;
Fernandez-Lafuente, R. Liquid Lipase Preparations Designed for
Industrial Production of Biodiesel. Is It Really an Optimal Solution.
Scientific and Technological Development: Fundação Cear- Renew. Energy 2021, 164, 1566−1587.
ense de Apoio ao Desenvolvimento Científico e Tecnológico (17) de Sousa Junior, P. G.; do Nascimento Camara, A. G.; Oliveira,
(FUNCAP) (PS1-0186-00216.01.00/21; UNI-0210- A. R. T.; de Castro Lima, G.; Lima, G. V.; Coutinho, L. P.; Nunes
00537.01.00/23), Conselho Nacional de Desenvolvimento Holanda Alexandre, J. Y.; Serafim, L. F.; de Mattos, M. C.; de Kássio
Científico e Tecnológico (CNPq) (311062/2019-9; 440891/ Monteiro, N. V.; Sousa dos Santos, J. C. Optimization and
2020-5; 307454/2022-3), and Coordenação de Aperfeiçoa- Theoretical Analysis of Lipase-Catalyzed Enzymatic Esterification of
mento de Ensino Superior (CAPES) (finance code 001). The Glycerol for Efficient Glycerides Synthesis. J. Biochem. Eng. 2023, 198,
authors thank the Northeastern Center for Application and 109033.
Use of NMR (CENAUREMN) for NMR spectroscopy. (18) Cavalcante, F. T. T.; da Fonseca, A. M.; Holanda Alexandre, J.
Y. N.; dos Santos, J. C. S. A stepwise docking and molecular dynamics

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