Synthesis of eugenyl acetate by enzymatic reactions in supercritical carbon dioxide

Article abstract of DOI:10.1016/j.bej.2016.06.018

Supercritical carbon dioxide (SC-CO₂) as reaction medium has gained attention in the production of terpenic esters catalyzed by lipases. Therefore, this work investigated the production of eugenyl acetate by esterification of eugenol and acetic anhydride in SC-CO₂ using two commercial lipases (Lipozyme 435 and Novozym 435) as catalysts. The influence of enzyme concentration (1–10% weight/weight), substrates’ molar ratio (1:1 to 5:1), temperature (40–60?°C) and pressure of SC-CO₂ (10–30?MPa) on the esterification rate (X; %) and specific productivity (SP; kg of product/kg of catalyst x hour) were evaluated. A home-made high-pressure stirred-batch reactor (100?ml) was used in the experiments. The use of Novozym 435 achieved higher conversion and specific productivity of eugenyl acetate than Lipozyme 435. An excess of acetic anhydride (5:1?M/M) and high enzyme concentration (10%) achieved higher esterification rates than the lowest conditions (1% and 1:1?M/M). The optimal temperatures and pressure for the synthesis of eugenyl acetate in SC-CO₂ were 50 and 60?°C at 10?MPa, respectively. The phase behavior of the reaction system and the synthesis in organic medium were also studied. Kinetic experiments performed at 40, 50 and 60?°C indicated that the reaction follows the simple Ping-Pong Bi-Bi mechanism and the affinity of acetic anhydride to enzyme was larger than that of eugenol.

Full text of DOI:10.1016/j.bej.2016.06.018

Biochemical Engineering Journal 114 (2016) 1–9
Contents lists available at ScienceDirect
Biochemical Engineering Journal
journal homepage: www.elsevier.com/locate/bej
Regular article
Synthesis of eugenyl acetate by enzymatic reactions in supercritical
carbon dioxide
Philipe dos Santosa, Giovani L. Zabotb, M. Angela A. Meirelesa, Marcio A. Mazuttic,
Julian Martíneza,∗
a College of Food Engineering, Food Engineering Department, University of Campinas, UNICAMP, 13083-862, Campinas, SP, Brazil
b Academic Center, Federal University of Santa Maria, UFSM, 96506- 302, Cachoeira do Sul, RS, Brazil
c Chemical Engineering Department, Federal University of Santa Maria, UFSM, 97105-900, Santa Maria, RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Supercritical carbon dioxide (SC-CO2) as reaction medium has gained attention in the production of
terpenic esters catalyzed by lipases. Therefore, this work investigated the production of eugenyl acetate by
esterification of eugenol and acetic anhydride in SC-CO2 using two commercial lipases (Lipozyme 435 and
Novozym 435) as catalysts. The influence of enzyme concentration (1–10% weight/weight), substrates’
molar ratio (1:1 to 5:1), temperature (40–60 ◦C) and pressure of SC-CO2 (10–30 MPa) on the esterification
rate (X; %) and specific productivity (SP; kg of product/kg of catalyst x hour) were evaluated. A home-
made high-pressure stirred-batch reactor (100 ml) was used in the experiments. The use of Novozym 435
achieved higher conversion and specific productivity of eugenyl acetate than Lipozyme 435. An excess
of acetic anhydride (5:1 M/M) and high enzyme concentration (10%) achieved higher esterification rates
than the lowest conditions (1% and 1:1 M/M). The optimal temperatures and pressure for the synthesis of
eugenyl acetate in SC-CO2 were 50 and 60 ◦C at 10 MPa, respectively. The phase behavior of the reaction
system and the synthesis in organic medium were also studied. Kinetic experiments performed at 40, 50
and 60 ◦C indicated that the reaction follows the simple Ping-Pong Bi-Bi mechanism and the affinity of
acetic anhydride to enzyme was larger than that of eugenol.
Received 12 February 2016
Received in revised form 23 May 2016
Accepted 14 June 2016
Available online 16 June 2016
Keywords:
Eugenol
Lipozyme 435
Novozym 435
Esterification
Supercritical
© 2016 Elsevier B.V. All rights reserved.
1. Introduction
physical or biotechnological processes must be employed for the
isolation and purification of the formed products [5].
Low molecular weight esters represent an important class of
aroma, consisting of compounds derived from short chain acids
such as acetates, propionates and butyrates, which are often
responsible for fruity aroma [1–3]. These esters, known for their
flavoring properties, are present in essential oils of natural matter,
which are technically difficult to extract, isolate and purify. Fur-
thermore, conventional chemical synthesis leads to the formation
of undesirable products to the food and pharmaceutical industries.
Therefore, biocatalyzed chemical synthesis becomes of great inter-
est due to the high chemo-, regio- and stereo-selectivity of the
enzymes, which provide possible in vitro synthesis of naturally
existing single enantiomers of specific compounds [2]. Moreover,
the esters can be considered as natural [2–4], since the process
meets the required conditions by the legislation. For example, the
substrates or raw materials used in process are natural and only
Lipases (glycerol ester hydrolases, EC 3.1.1.3) belong to the
hydrolase group and are responsible for catalyzing the hydrolysis
of glycerol esters and long-chain fatty acids, producing alcohol and
acid [6]. In many research works, lipases have been employed as
catalysts for the synthesis of esters, such as isoamyl acetate (banana
flavor) [3,7], isoamyl butyrate (pear flavor) [8,9] and cinamyl
acetate (a compound of cinnamon essential oil) [10]. Besides, a
recent research showed that lipases are stable in pressurized fluids,
which increased their potential use in esterification reactions [11].
Among the supercritical fluids (SC) used in industrial processes, car-
bon dioxide (CO2) is the most common due to its advantages, such
as low cost, nontoxicity, non-flammability, inertness, full recovery
and moderate critical properties (Pc = 7.38 MPa, Tc = 304.2 K) when
compared to other green solvents. Therefore, reactions in supercrit-
ical CO2 can be carried out with low energy cost for pressurization,
and at temperatures that do not damage the enzymes [12,13].
Moreover, if SC-CO2 cannot improve reaction rate, the adjustable
solvent power of the fluid allows the design of a production process
∗
Corresponding author.
E-mail address: julian@unicamp.br (J. Martínez).
http://dx.doi.org/10.1016/j.bej.2016.06.018
1369-703X/© 2016 Elsevier B.V. All rights reserved.

2
P. dos Santos et al. / Biochemical Engineering Journal 114 (2016) 1–9
with integrated downstream separation of products and unreacted
substrates [11].
natant and washings were collected for protein analysis by the
Lowry method [18], described as follows.
Eugenyl acetate is an aroma ester generally found in the essen-
tial oil of clove buds (Syzygium aromaticum). Besides eugenyl
acetate, clove oil is rich in eugenol, beta-carophyllene, alfa-
humulene and other minor compounds. The European Food Safety
Authority (EFSA) evaluated and considered the application of
eugenyl acetate safe as aromatic substance in food products. Actu-
ally, eugenyl acetate is listed on the database of the European
Union as authorized substance to be used by the food industry [14].
Besides the flavoring property of eugenyl acetate, several works
have reported other properties of industrial interest, such as antiox-
idant capacity [15], antimicrobial [16] and anticancer properties
[17].
In this context, the aim of the present work was to investi-
gate the synthesis of eugenyl acetate through enzymatic reactions
in SC-CO2 media. The effects of enzyme and substrates’ concen-
tration, temperature, pressure and number of reuse cycles of the
enzymes were evaluated for two commercial immobilized lipases.
The differences between two commercial immobilized lipases from
C. antartica were shown. Moreover, experiments were performed
to determine the kinetic parameters, and finally the phase behavior
of the reaction system and the synthesis in organic medium were
studied.
A standard curve was prepared with bovine serum albumin
(BSA) powder (Sigma Aldrich). Samples, supernatant and washings
were diluted in order to fit within the BSA standard curve range
(0.02–0.6 mg/ml). 50 ␮l of sample and 450 ␮l of distilled water were
placed in each tube. Next, 5 ml of biuret reagent was added to each
tube and mixed thoroughly with a vortex. The biuret reagent was
prepared by mixing three solutions: solution A: cupric sulfate at 1%;
solution B: sodium potassium tartrate at 1%; solution C: 2% sodium
carbonate in 0.1 M of NaOH with ratio of 1:1:50 (A:B:C). The mixture
was then let incubating for 10 min prior to the addition of 500 ␮l per
tube of 1.0 N Folin & Ciocalteu’s reagent (Dinâmica, Diadema/SP),
and the samples were mixed immediately. Color was developed for
30 min in the dark at room temperature and the absorbance was
measured at 650 nm. All absorbance determinations were made
using a UV–vis spectrophotometer (Hach, DR/4000U, Colorado,
USA). All experiments were replicated at least three times.
The water content of immobilized lipases was determined
by Karl Fischer titration using a model 701 Metrohm apparatus
(Herisau, Switzerland) equipped with a 5 ml burette and an extrac-
tor, which was operated at 120 ◦C and a nitrogen (White Martins
S.A., Campinas-SP/Brazil) flow rate of 50 × 10−9 ml/min. The Karl
Fischer reagent used in the titration was from Merck (Darmstadt,
Germany).
The mean particle size distributions of Lipozyme 435 and
Novozym 435 were determined based on the static light scatter-
ing method using a Multi-Angle Static Light-Scattering Mastersizer
(Malvern Instruments, Worcestershire, UK). The real densities of
immobilized enzymes were measured by helium pycnometry,
whereas bulk density was measured by weighing a known volume
of solid material. Finally, the ratio between real and bulk density
determined the porosity of the packed enzyme bed.
2. Materials and methods
2.1. Materials
Two commercial lipases from Candida antarctica (Lipozyme 435
and Novozym 435), both immobilized on a macroporous anionic
resin, were kindly supplied by Novozymes Brazil (Araucária-
PR/Brazil). The reagents eugenol and eugenyl acetate were obtained
from Sigma Aldrich. Acetic anhydride, n-hexane and ethyl acetate
were supplied by Synth (Diadema-SP/Brazil). All chemicals were
analytical grade. Carbon dioxide (99.9%) was purchased from White
Martins S.A. (Campinas-SP/Brazil).
2.3. Synthesis of eugenyl acetate in SC-CO2
The experimental homemade apparatus used in all reac-
tion experiments consists in a CO2 booster (Maximator M-111,
Zorge/Germany), a solvent reservoir, a cooling (Solab SL152/18,
2.2. Characterization of the enzymes
Êxodo Científica, Hortolândia/SP, Brazil) and
a heating ther-
The activity of the immobilized enzymes was determined as the
initial rate of the esterification reaction of oleic acid (Sigma Aldrich)
with propanol (Sigma Aldrich) at a molar ratio of 3:1, with hex-
ane (Synth) as reaction medium and enzyme concentration of 5%
(w/w) in the substrates. The mixture was kept at 50 ◦C in a shaker
incubator (TE-421, Tecnal, Piracicaba-SP/Brazil) at 150 orbitals per
minute (OPM) for 30 min. Then, the oleic acid content was deter-
mined by titration with KOH 0.1 M in ethanol (Synth). A unit of
activity (U) was defined as the amount of enzyme needed to con-
sume 1 ␮mol of oleic acid per minute. All determinations of lipase
activity were replicated at least three times. The residual activity
(%) of the lipase was defined as the ratio between the activity of the
untreated enzyme (U0) and that of the lipase treated with SC-CO2
(U), as stated in Eq. (1).
mostatic (Marconi S.A., Campinas-SP/Brazil) baths. Manometers
(Zurich, São Paulo-SP/Brazil), a magnetic stirrer (IKA, RCT Basic,
Staufen/Germany), thermocouples, control valves (Autoclave Engi-
neers), a micrometric valve (Autoclave Engineers, Erie/PA, USA) and
a stainless steel vessel of 100 ml. Fig. 1 shows the schematic flow
diagram of the high-pressure stirred-bath reactor unit.
First, an amount of immobilized lipase was placed inside the
high-pressure stirred-bath reactor. After 30 min of thermal stabi-
lization and to remove the residual superficial water in the catalyst
and the wall reactor, the reaction mixture formed by eugenol and
acetic anhydride was introduced in the stirred-batch reactor. Next,
the reactor was pressurized with CO2 at a rate of 10 MPa min−1
.
After the end of the established reaction time (1 h), the system was
depressurized at 1 MPa min−1. In all experiments the stirring rate
was fixed at 600 rpm. The evaluated process variables were pres-
sure (10–30 MPa), temperature (40–60 ◦C), enzyme concentration
(1–10%), concentration of substrates (molar ratio from 1:1 to 5:1
of acetic anhydride: eugenol) and the reuse of the enzyme (1, 2
and 3 times). In the kinetic experiments each point of the curve at
the same process condition represents the mean of the performed
experiments at the established reaction time. All experiments were
replicated at least three times.
ꢀ
ꢁ
U
Residual Activity % =
( )
x100
(1)
U0
The protein content of the enzymes was determined by the
Lowry method [18], but the original method is not suitable for
immobilized lipase forms, so a preliminary desorption step was
executed as according to the method proposed by Petry et al. [19].
A known amount of immobilized lipase (0.1–0.2 g) was stirred in
2–4 ml of the extraction buffer/solvent, 10% formic acid in 45% ace-
tonitrile (in water), for 2 h, at room temperature (22–25 ◦C). The
material was washed three times with the same buffer/solvent for
10 min in each step, and finally with distillated water. The super-
From the amount of eugenol and eugenyl acetate after the
reaction, the mass balance and the reaction stoichiometry, it was
possible to determine the esterification rate or conversion (X, %),

P. dos Santos et al. / Biochemical Engineering Journal 114 (2016) 1–9
3
Fig. 1. Diagram of the homemade unit for high-pressure stirred-batch reactor containing: V-1, V-2, V-3, V-4 and V-5– Control valves; VM – Micrometer valves; VS – Safety
valve (Pmax = 30 MPa); C- Compressor; F-Compressed air filter; FC – CO2 Filter; BR – Cooling bath; BP- Pump (Booster); BA – Heating bath; I-1 and I-2– Pressure indicators;
I-3 – Temperature indicators; IC-1–Indicators and controllers of magnetic stirrer; IC-2–Indicators and controllers of temperature of micrometer valve; AM – Magnetic stirrer.
which was obtained by the molar ratio between the products (np)
and substrates (ns), using Eq. (2):
was coupled to a cooling ultra-thermostatic bath (Marconi S.A.,
Model MA-184, Campinas-SP/Brazil) and a metallic jacket on the
cell coupled with a heating thermostatic bath (Polyscience, Illi-
nois/USA). The equilibrium cell includes a movable piston, which
allows pressure control inside the cell.
Phase behavior was visually observed through the pressure
manipulation using the pump with CO2 as pneumatic fluid. First,
the amount of substrates and enzyme were introduced inside the
view cell. The cell was closed and loaded with a determined amount
of CO2. After the desired temperature was reached, the pressure
was increased until the observation of a single phase. From this
point, the pressure was slowly decreased until the apparition of a
new phase. Three temperatures were evaluated (40, 50 and 60 ◦C)
with enzyme concentration and substrates’ molar ratio of 1% and
5:1 (acetic anhydride/eugenol), respectively, which are the same
used in the reaction experiments.
ꢀ
ꢁ
np
ns
X % =
( )
x100
(2)
The specific productivity of eugenyl acetate (SP) was expressed
as the mass ratio between product (mp) and catalyst (mc) per unit
time (t), as shown in Eq. (3):
ꢂ
ꢃ
mp
SP kg/kg × h
=
(3)
mc × t
The esterification of eugenol was also performed in n-hexane
for comparison with SC-CO2. The reaction was carried out at atmo-
spheric pressure and temperature, substrate molar ratio (5:1 acetic
anhydride/eugenol) and concentration of enzyme (1%) equal to
those used in the reactions in SC-CO2 at the best condition of
conversion and specific productivity. The reaction volume was
completed to 100 ml of hexane and the mixture was incubated in a
shaker incubator (TE-421, Tecnal, Piracicaba-SP/Brazil) under agi-
tation of 150 orbitals per minute (OPM). After one hour an aliquot
of 1.5 ml was filtered (Chormafil Xtra PA-20/25, Macherey-Nagel,
Düren/Germany) and analyzed by gas chromatography (see Sec-
tion 2.5.) to determine the concentrations of eugenol and eugenyl
acetate. The experiments were replicated three times.
2.5. Analytical methods
After the reaction the reactant (eugenol) and product (eugenyl
acetate) contents were determined using a gas chromatograph
with a flame ionization detector (GC-FID). The method was based
according to Zabot et al. [21]. A GC–FID chromatograph (Shimadzu,
CG17A, Kyoto/Japan) equipped with a capillary column of fused sil-
ica DB-5 (J&W Scientific, 30 m × 0.25 mm × 0.25 ␮m, Folsom,/USA)
was used. Each sample was filtered (Chormafil Xtra PA-20/25,
Macherey-Nagel, Düren/Germany) and diluted to 5 mg/ml in ethyl
acetate, and 1 ␮l was injected into the chromatograph. The sample
split ratio was 1:20. The carrier gas (Helium, 99.9% purity, White
Martins, Campinas,/Brazil) flowing at 1.1 ml/min. The injector and
the detector temperatures were 220 and 240 ◦C, respectively. The
column was heated from 60 ◦C to 246 ◦C at 3 ◦C/min. At these con-
ditions the retention times of eugenol and eugenyl acetate peaks
were 24 and 32 min, respectively. Quantification was performed
using external standard calibration curves (R2 = 0.999 to eugenol
and R2 = 0.996 to eugenyl acetate).
2.4. Phase behavior experiments
The solubility of substrates in SC-CO2 depends on CO2 density,
which can be tuned by changes in temperature and/or pressure,
and an increase in solubility of substrates in SC-CO2 can lead to
an increase in the reaction rate. On the other side, a decrease on
reaction rate can occur due to formation of two phases (liquid and
gas) [20]. Therefore, phase behavior experiments were performed
through a static method without sampling in a high-pressure
variable-volume view cell. Basically, the apparatus consists of a
view cell with two sapphire windows for visual observations, a
magnetic stirrer to promote the agitation in the mixture, an abso-
lute pressure transducer (Novus, TP-Model 520, Brazil), a “PT 100”
type thermocouple inside the cell, a CO2 pump (Thermo Separa-
tion Products, Model Constametric 3200 P/F, Waltham/USA) that

4
P. dos Santos et al. / Biochemical Engineering Journal 114 (2016) 1–9
2.6. Kinetic model
cost, specifically in biocatalyzed processes where high esterifica-
tion and low enzyme concentrations should be achieved. Therefore,
the effects of enzyme concentration and substrates’ molar ratio
on the conversion and specific productivity of eugenyl acetate
using Lipozyme 435 and Novozym 435 were evaluated. In these
experiments the temperature and pressure were fixed at 40 ◦C and
10 MPa, respectively. In these conditions the immobilized lipases
have shown their highest residual activities [24–26]. Moreover, the
stirring rate was fixed in a high value to ensure that the reaction
would be kinetically controlled and the inter-particle diffusion and
external mass transfer effects were negligible [27,28]. The results
are shown in Table 1.
The experimental data of conversion versus time were adjusted
with a second-order model, as shown in Eq. (4). The quality of this
adjustment was evaluated from the determination coefficient (R2)
using the MATLAB software (2014Ra, MathWorks, Natick, MA, USA)
X % = at2 + bt + c
(4)
( )
where Xis the conversion (%), t is time (h) and a,b and c are
adjustable parameters.
Through the analytical derivative of the adjusted model for
eugenol conversion, the eugenol conversion rate at each time t was
calculated, as shown in Eq. (5).
The ANOVA results show that the effect of enzyme concentra-
tion on the conversion was statistically significant (p < 0.05 and
ꢀ
ꢁ
dX
dt
Fcal = 37.97) using immobilized lipase Lipozyme 435, and not signif-
v = Ce,0
(5)
icant for Novozym 435 (p = 0.362 and Fcal = 1.14). It can be observed
that, for Lipozyme 435, the highest conversion of eugenyl acetate
was obtained with high concentration of the enzyme (10%) at the
three evaluated substrate molar ratios, but for Novozym 435 this
behavior was not observed in any condition. Moreover, at the
highest substrate molar ratio 5:1 (acetic anhydride/eugenol) for
Lipozyme 435, the concentration of lipase did not affect the con-
version. The same behavior was found to the specific productivity
of eugenyl acetate, but the magnitude of the specific productiv-
ity decreases with the amount of enzyme increase, even with an
increase of reaction rate.
The increase of enzyme loading results in higher reaction rate, as
exposed on Table 1. Although the reaction time was kept constant
at one hour, the highest rate and specific productivity for both com-
mercial lipases were obtained with 1% enzyme and 5:1 molar ratio.
Eventually, with longer reaction time some experimental runs
would overlap for larger enzyme content. However, in the indus-
trial point of view it is interesting to use lower amount of catalyst
(enzyme), which results in reduced cost. Other works using SC-
CO2 as reaction medium obtained similar results [29,30]. Chiaradia
et al. [16], studying the eugenyl acetate esterification in a solvent-
free system found a significant effect of the enzyme (Novozym 435)
concentration on the conversion, and concluded that lower concen-
trations can be used without affecting the reaction conversion. It
must be emphasized that the conversion increased with the sub-
strates’ concentration, but at the same molar ratio, 5:1, the increase
of enzyme concentration from 5.5 to 10% did not affect the reaction
rate (conversion). This probably happened because in these con-
ditions the amount of substrates was the controlling factor of the
reaction rates instead of the enzyme concentration. Furthermore,
the reaction rates achieved in these experiments were not large
enough to compensate the amount of used immobilized lipase, thus
the lowest specific productivities were obtained.
where v is the reaction rate at the time t (mol/l s), Ce,0 is the initial
(t = 0) concentration of eugenol (mol/l) and dX/dt is the analytical
derivative of the conversion calculated with Eq. (4).
Next, the reaction rate was adjusted with the Ping-Pong Bi-Bi
model without inhibition, as described by Eq. (6).
Vmax
A B
[ ] [ ]
v =
(6)
KmB A + KA B + A
B
m [ ] [ ] [ ]
[ ]
where Vmax is the maximum reaction rate (mol/l g s), A and B are
[ ] [ ]
the acetic anhydride and eugenol concentrations (mol/l), respec-
tively, and KmA and KmB are the Michaelis-Mentem constants for
acetic anhydride and eugenol (mol/l).
The Ping-Pong Bi-Bi model was fitted to the calculated reaction
rate (v) using a minimum of constrained nonlinear multivariable
function algorithm from the MATLAB software. The objective func-
tion was defined as sum of the squares, as shown in Eq. (7).
n
ꢄ
ꢅ
ꢆ
2
fobj
=
vexp − vsim
(7)
i=1
where vexp is the experimental reaction rate (mol/l s), vsim is the
simulated reaction rate (mol/l s) and n is the total of experimental
points.
The activation energies were obtained from the slope of the
ꢀ
ꢁ
k
hK
T
B
straight line of ln
vs reciprocal of absolute temperature (1/T),
m
as described by the Arrhenius Eq. (8) and the Eyring absolute rate
equation [22,23].
ꢀ
ꢁ
kBT
Ea
ln
= −
+ ln A
( )
(8)
hKm
RT
where Km is the Michaelis-Menten constant,h is the Planck constant
(6.6256 × 10−34 J s), kB is Boltzman constant (1.3805 × 10−24 J/K), A
is the frequency factor, Ea is the activation energy and R is the molar
gas constant (8.314 J/K mol).
3.2. Effect of acetic anhydride and eugenol molar ratio
Acetic acid has been traditionally used as acyl donor to flavor
ester reactions (direct esterification) [31]. Moreover, several woks
have reported an inhibition of enzyme by high concentration of acid
[3], probably due to the decrease of pH in the reaction system. In
addition, acetic acid can be a lipase inhibitor, reacting with serine
in the active site of the enzyme [32]. Alternatively, the synthesis of
esters can be performed by transesterification with acetates and by
acylation with acetic anhydride [7]. Table 1 shows the conversions
and specific productivities obtained with three acetic anhydride
concentrations and three different enzyme loadings.
2.7. Statistical analysis
The results of conversion and specific productivity of eugenyl
acetate were statistically evaluated by ANOVA, using the software
Statistica for Windows 6.0 (Statsoft Inc., USA) in order to detect
significant differences. The significant differences at level of 5%
(p < 0.05) were analyzed by the Tukey test.
3. Results and discussion
A positive effect of molar ratio on the conversion and spe-
cific productivity of eugenyl acetate was observed, and the ANOVA
results showed that the acetic anhydride/eugenol molar ratio was
statistically significant in the conversion using both immobilized
lipases, Lipozyme (p < 0.05 and Fcal = 104.20) and Novozym 435
3.1. Effect of the enzyme concentration
In industrial applications the process variables should be con-
trolled to obtain the maximum specific productivity with minimum

P. dos Santos et al. / Biochemical Engineering Journal 114 (2016) 1–9
5
Table 1
Effect of enzyme concentration and substrates’ molar ratio on the conversion and specific productivity of eugenyl acetate using two different lipases in SC-CO2.
Enzyme (%)1
Molar Ratio2
Conversion (%)
Lipozyme 435
Specific productivity (kg/kg h)
Lipozyme 435
Novozym 435
Novozym 435
1
1
1
5
5
5
10
10
10
1:1
3:1
5:1
1:1
3:1
5:1
1:1
3:1
5:1
4.42 0.18eA
1.63 0.02eB
15.60 0.69cdA
33.23 0.26aA
5.11 0.54eB
19.61 0.85cA
26.58 0.36bA
14.45 2.92dA
19.37 0.99cA
19.51 0.49cA
3.29 0.02abA
2.59 0.10bcB
6.22 0.41aB
1.89 0.39cdA
1.37 0.02deB
1.13 0.12deB
0.80 0.07eA
0.72 0.01eB
0.64 0.03eA
1.21 0.04cdB
6.73 0.45bA
10.09 0.07aA
0.78 0.08dB
1.71 0.07cA
1.62 0.02cA
1.11 0.21cdA
0.84 0.04dA
0.60 0.01dA
6.50 0.25deB
19.34 1.34aB
12.27 2.53bcA
15.70 0.27abB
18.50 1.20aB
10.34 0.95cdB
16.30 0.01abB
20.27 1.00aA
Experiments were carried out at fixed conditions of time (1 h), stirring (600 rpm), pressure (10 MPa) and temperature (40 ◦C). Different indexes (a, b, c, d) in the same column
(lowercase) or in the same line (uppercase) indicate that the means differ significantly by Tukey’s test (p ≤ 0.05). For example, lowercases compares the means at different
experimental conditions with the same enzyme and uppercases compares the means of different enzymes at the same experimental condition.
1
Enzyme concentration expressed by weight of substrates, w/w (%).
Acetic anhydride/eugenol molar ratio (M/M).
2
Table 2
may also have been immobilized during the immobilization pro-
cess, increasing the real density of the particles. Moreover, the mean
Characteristics of two different immobilized lipases from Candida antarctica.
particle diameter of Lipozyme is higher than that of Novozym 435.
According to Yadav and Devi [34] the bead size range of Novozym
435 is 0.3–0.9 mm, and the same particle distribution was observed
for both immobilized lipases. Despite the higher specific enzymatic
activity of Novozym 435, the study of the effects of SC-CO2 pres-
sure and temperature on the conversion and specific productivity
of eugenyl acetate was performed with Lipozyme 435 because few
papers dealing with enzymatic reactions using a food grade immo-
bilized lipase (Lipozyme 435) are found in literature.
Characteristic
Novozym 435
Lipozyme 435
Enzymatic Activity (U/g)
Protein Content (mg/g of particle)
Specific Enzymatic Activity (U/mg)
Water Content (g/g, %)
Bulk Density (g/cm3)
167.1 0.39
85.4 5.41
1.95 0.07
1.62 0.04
0.35 0.01
0.96 0.01
0.64 0.01
350.3 2.5
173.9 0.36
108.2 6.38
1.60 0.01
2.71 0.06
0.35 0.01
1.21 0.06
0.71 0.01
452.4 12.6
Real Density (g/cm3)
Porosity (ad.)
Mean Diameter (␮m)
(p < 0.05 and Fcal = 447.40). In fact, the major effect on the conver-
sion was that of acetic anhydride/eugenol molar ratio. The same
behavior was observed by Chiaradia et al. [16], who concluded that
the molar ratio of acetic anhydride/eugenol presented a positive
effect, followed by the effect of temperature on eugenyl acetate
production in a solvent-free system using Novozym 435. It is also
observed on Table 1 that the conversion and specific productiv-
ity of eugenyl acetate using Novozym 435 was higher than that of
Lipozyme 435 at all experimental conditions, except under the low-
est molar ratio with 1 and 5% of enzyme, which is probably due to
experimental errors. In order to explain the reasons why Novozym
435 outperformed Lipozyme 435 in the eugenol conversion, the
characterization of both lipases was performed and the results are
exposed on Table 2.
According to the enzyme supplier, Lipozyme 435 is food grade
while Novozym 435 is analytic or technical grade, information also
reported by Melgosa et al. [24]. This can be confirmed by the spe-
cific enzymatic activity (SA), which represents the ratio between
the enzymatic activity and protein content. The SA of Lipozyme 435
was 1.6 U/mg while Novozym 435 achieved SA about 18% higher,
1.9 U/mg. The water content of Novozym 435 was similar to that
found by Habulin et al. [33] (1.44%) and lower than the obtained
by Yadav and Devi [34] (3%), but the titrable water content for
Lipozyme 435 was about four times higher than that reported by
Melgosa et al. [24] (0.7 0.2%).
Particle size, porosity and density are important characteristics
of catalysts, which can influence mass transfer and intraparticle
diffusion of substrates and products, and consequently the reac-
tion rate and specific productivity. The measured bulk density for
both immobilized lipases was the same as reported by the enzyme
supplier, and closer to that reported by Yadav and Devi [34] for
Novozym 435 (0.45 g/cm3). However, the real density of Lipozyme
was higher than that of Novozym 435. In fact, the immobiliza-
tion material is the same for both lipases (macroporous resin of
poly-methyl methacrylate (Lewatit VP OC 1600)) [24,35], but since
Lipozyme 435 contains more protein with lower SA, these proteins
3.3. Effect of pressure and temperature of SC-CO2
Based on the results shown on Table 1, the effects of tempera-
ture and pressure were evaluated at the conditions that achieved
high conversion and specific productivity. Thus, the enzyme con-
centration and acetic anhydride/eugenol molar ratio were kept
constant at 1% and 5:1, respectively. According to Oliveira et al.
[25] for Novozym 435 and Melgosa et al. [24] for Lipozyme 435
and Lipozyme RM IM, the increase of temperature in a supercrit-
ical reaction process causes a decrease of the residual activity of
enzymes. The same behavior was reported to pressure by several
works [24,25,36]. Indeed, according to Knez, Habulin and Primozˇicˇ
[37] the pressure-induced deactivation of enzymes occurs mostly
above 150 MPa. Based on the literature survey, the experiments
were performed at temperatures from 40 to 60 ◦C and pressures
from 10 to 30 MPa. Table 3 shows the effects of temperature and
pressure on the conversion and specific productivity of eugenyl
acetate using the Lipozyme 435 as catalyst.
The ANOVA analyses showed that the effects of pressure and
temperature were statistically significant (p < 0.05 and Fcal > Ftab
)
on the conversion and specific productivity of eugenyl acetate. A
positive effect of pressure at 40 ◦C and a negative effect at 50 and
60 ◦C can be observed on Table 3. The positive effect can be related
to the increase of CO2 density with pressure, which improves its
solvation power in the reaction medium [20]. In turn, the nega-
tive effect on the conversion can be connected with the dilution of
the substrates caused by the greater amount of CO2 pumped into
the reactor, causing an increase in the molar ratio between SC-CO2
and substrates [20,38]. Knez et al. [38] studied the effect of pres-
sure on the conversion of free fatty acid into fatty acid ester using
lipase from Rhizomucor miehei (Lipozyme RM IM) in dense CO2.
According to the authors, at pressures above 10 MPa the conversion
decreased due to the dilution effect caused by larger CO2 amount,
which reduced the substrates’ molar fraction in the reaction bulk,
slowing down the esterification.

6
P. dos Santos et al. / Biochemical Engineering Journal 114 (2016) 1–9
Table 3
Effect of temperature and pressure on the conversion and specific productivity of eugenyl acetate using Lipozyme 435.
Temperature (◦C)1
Pressure (MPa)2
Conversion (%)
Specific productivity (kg/kg h)
40
40
40
50
50
50
60
60
60
10
20
30
10
20
30
10
20
30
19.34 1.34c
20.76 0.25c
22.80 0.08c
32.26 1.25a
21.17 0.85c
24.77 3.80bc
30.75 0.74ab
20.17 1.85c
24.70 0.26bc
6.22 0.41b
6.38 0.08b
7.01 0.02b
9.77 0.36a
6.23 0.26 b
7.53 1.15 b
9.47 0.23a
6.05 0.35 b
7.51 0.19 b
Experiments were carried out at fixed conditions of time (1 h), agitation (600 rpm), enzyme concentration (1%) and acetic anhydride/eugenol molar ratio (5:1). Different
indexes (a, b, c, d) in the same column (lowercase) indicate that the means differ significantly by Tukey’s test (p < 0.05).
1
Temperature.
Pressure of SC-CO2.
2
The effect of temperature from 40 to 50 ◦C was positive for the
Table 4
Influence of reuse cycles of Lipozyme 435 on the conversion and specific productivity
conversion, from 19 to 32% at 10 MPa, from 20 to 21% at 20 MPa
and from 22 to 24% at 30 MPa. Besides, the increase of temperature
of eugenyl acetate in SC-CO2 at 10 MPa and 40 ◦C.
from 50 to 60 ◦C caused a decrease on the conversion, which can be
related to the changes in the physical properties of the solvent, such
as limitations in mass transfer, viscosity, surface tension and due to
the decrease of the solvation power of the substrates at lower sol-
vent density. The esterification of eugenol was also carried out in
n-hexane for comparison with the results obtained at supercritical
conditions. The conversion in n-hexane was 15.3 1.8%, whereas
in SC-CO2 (10 MPa and 40 ◦C) it was near 19%. According to Laudani
et al. [39], the higher diffusivity and lower viscosity and surface
tension of SC-CO2 are responsible for the reduction of interphase
transport limitations, thus increasing the reaction rate and esteri-
fication of eugenol.
Number of Cycles
Conversion (%)
Specific productivity (kg/kg h)
1
2
3
19.34 1.34
17.60 0.29
13.10 0.06
6.22 0.41
5.45 0.60
4.43 0.02
Experiments were carried out at fixed conditions of time (1 h), agitation (600 rpm),
enzyme concentration (1%) and acetic anhydride/eugenol molar ratio (5:1).
Researches dealing with esterification reactions in supercritical
media have shown that the conversion can be influenced by the
solubility of substrates in SC-CO2, which depends on the system’s
temperature and pressure. The solubilities of eugenyl acetate and
eugenol in SC-CO2 were reported by Cheng et al. [40], Souza et al.
[41] and Guan et al. [42], who showed that the solubility of these
compounds increases with CO2 density. However, few publications
dealing with phase equilibrium of the whole enzymatic esterifica-
tion system have been found in the literature. Phase equilibrium
experiments of the system eugenol/acetic anhydride/Lipozyme
435/SC-CO2 were performed to observe the behavior at each exper-
imental condition exposed on Table 3, and the results are shown in
Appendix A (Supplementary data, Fig. S1).
Fig. 2. Effect of the number of depressurization/pressurization cycles on the residual
activity (bar chart, %) and water content (᭿, %) of Lipozyme 435 treated with SC-CO2
at 10 MPa and 40 ◦C.
At 10 MPa and 60 ◦C the reaction mixture contained solid
(enzyme), liquid and gas phases, and at the other conditions the
equilibrium between solid and supercritical phase was observed.
The transition from three-phase (solid-liquid-gas) to two-phase
(solid-supercritical) system at 40, 50 and 60 ◦C was observed at
pressures around 8.0–8.2, 9.1–9.3 and 11–13 MPa, respectively. At
the end of all phase equilibrium experiments a white and opaque
solid material was observed on the view cell. This material can be
observed in Fig. S1 (Appendix A: Supplementary data) at condi-
tions of 50–60 ◦C and 20–28 MPa, where the reaction bulk became
more opaque. This solid material may be a small particle displaced
from the support of the immobilized lipase. Moreover, this can help
elucidating the decrease of conversion at high temperature and
pressure observed in the reactions using Lipozyme 435.
10 MPa and 40 ◦C, as shown on Table 4. The substrates and prod-
ucts were extracted from the high-pressure vessel at 2 h with a
CO2 flow rate of 2.84 × 10−4 kg/s, which was enough to remove
all the compounds from the reactor, as established by previous
experiments. The data on Table 4 show that the conversion and
specific productivity of eugenyl acetate decreased continuously
with the increasing number of cycles. The catalytic ability of the
lipase CALB (Candida antarctica lipase B) in an immobilized form
decreases with the increase of the utilization cycles [20] or depres-
surization/pressurization cycles [24,25]. The reduction of enzyme
activity after SC-CO2 exposure has been related to several reasons.
One of them is the water content needed to preserve the confor-
mation of enzyme, and thus its optimal activity [24,43]. Therefore,
the effect of the number of depressurization/pressurization cycles
on the residual activity (%) and water content (%) of Lipozyme 435
treated with SC-CO2 was evaluated. Each cycle was carried out at
40 ◦C, 10 MPa, 1 h of exposure time, and the depressurization rate
was fixed at 1 MPa min−1. The results are presented in Fig. 2.
It can be noted that the residual enzyme activity and the water
content decreased with the increase of the number of depres-
3.4. Reuse of Lipozyme 435
The main advantage of immobilized lipases over the free form
is that the enzyme can be reused many times in the process, thus
reducing the cost of catalyst. Several works have shown that the
enzyme activity decreases continuously with the increasing num-
ber of cycles, so the reusability of Lipozyme 435 was analyzed at

P. dos Santos et al. / Biochemical Engineering Journal 114 (2016) 1–9
7
Fig. 3. Kinetics of eugenyl acetate conversion (A) and specific productivity (B) in SC-CO2 at 40, 50 and 60 ◦C. Experiments were carried out at fixed conditions of pressure
(10 MPa), agitation (600 rpm), enzyme concentration (1%) and acetic anhydride/eugenol molar ratio (5:1).
Fig. 4. (A) Lineweaver-Burk plot of reciprocal of the reaction rates versus reciprocal acetic anhydride [A] concentration at different eugenol [B] concentrations. (B) Effect
of acetic anhydride [A] concentration on the reaction rate at different eugenol [B] concentrations. Experiments were carried out at fixed conditions of agitation (600 rpm),
enzyme concentration (1%), time (1 h), temperature (40 ◦C) and pressure (10 MPa).
surization/pressurization cycles. The initial titrable water content
of Lipozyme 435 was 2.71 0.06%, and after exposure to SC-
CO2 it decreased to 2.54 0.20, 2.27 0.22 and 1.66 0.19%, for
1, 2 and 3 cycles respectively. These results of residual activity
are in accordance with those reported by Melgosa et al. [24] for
Lipozyme RM IM and Lipozyme 435, in which several depressur-
ization/pressurization steps led to activity loss of both immobilized
lipases. Moreover, the authors did not report the water content of
the Lipozyme 435 after treatment with SC-CO2, because of the low
values found. Habulin et al. [33] related the decrease of residual
activity to the extraction of essential water from the immobilized
lipase Novozym 435, in which the water content fell from 1.44% for
the untreated enzyme to 0.88% for lipase treated with SC-CO2.
for all reaction times the conversion should be twice the obtained
at the previous point. Based on the kinetic experiments and the
phase behavior, the optimal conditions for the synthesis of eugenyl
acetate were 50 and 60 ◦C at 10 MPa. Although a two-phase region
(liquid and gas) appears at 60 ◦C and 10 MPa, the reaction rate was
almost equal to those of the experiments at 50 ◦C and 10 MPa.
The kinetics of many reactions have been successfully described
by the Ping-Pong Bi-Bi model, such as hydrolysis, esterification
and transesterification, using organic solvents [22,44] or supercrit-
ical fluids [29,30] as reaction media. In the kinetic study it is first
necessary to determine possible inhibitions caused by any sub-
stance involved in the reaction [44]. In this work, the substrates
are eugenol and acetic anhydride, and the products are eugenyl
acetate and acetic acid. A second route of eugenol esterification
can occur between acetic acid and eugenol, resulting in eugenyl
acetate and water. This second route and the effect of acetic acid
on the kinetics were not considered. The reaction routes are shown
in Appendix A (Supplementary data, Fig. S2). However, acetic acid is
an important inactivating compound, causing an indirect inhibition
by the decrease of pH in the reaction medium, changing the isoelec-
tric point of proteins and thus inactivating the enzyme. Moreover,
acetic acid can also act as a direct inhibitor of the enzyme, forming
a dead-end complex. According to Romero et al. [44], this effect has
little importance when the initial concentration of substrates is low.
Fig. 4 shows the Lineweaver-Burk plot of reciprocal of the reaction
rates versus reciprocal concentrations of acetic anhydride [A] and
3.5. Kinetic experiments
The kinetics of the production of eugenyl acetate was investi-
gated at 10 MPa, with 1% (w/w) of enzyme, 5:1 molar ratio and
stirring at 600 rpm. The results are shown in Fig. 3(A) for conver-
sion (%) and (B) for specific productivity (kg/kg h). It can be observed
that, at longer times, the conversion increased for the reaction at
the three tested temperatures, 40, 50 and 60 ◦C. However, the spe-
cific productivity of eugenyl acetate decreased with time for all
temperatures. This behavior can be explained observing Eq. (3),
which suggests that the increase of reaction time decreases its spe-
cific productivity. To keep the specific productivity at similar values

8
P. dos Santos et al. / Biochemical Engineering Journal 114 (2016) 1–9
Table 5
Differences were also observed in the particle size distribution and
water content of the immobilized lipases. Novozym 435 achieved
higher conversion and specific productivity of eugenyl acetate than
Lipozyme 435.
Kinetic parameters for the production of eugenyl acetate in SC-CO2 at 40, 50 and
60 ◦C using Lipozyme 435.
Parameter
40 ◦
3.6
17.0
260.2
C
50 ◦
4.2
17.5
190.0
C
60 ◦
4.5
14.0
147.5
C
The influence of temperature and pressure was verified in reac-
tions conducted with 1% of enzyme and substrate molar ratio of
5:1, and the optimal condition for the synthesis of eugenyl acetate
in SC- CO2 was determined at 50 and 60 ◦C at 10 MPa, in which
the study of phase behavior identified that the reaction mixture
contained solid (enzyme), liquid and gas phases. The conversion
and specific productivity of eugenyl acetate decreased with the
increasing number of cycles, and the residual enzyme activity and
the water content decreased with the increase of the number of
depressurization/pressurization cycles. The results suggested that
the reaction mechanism could be described to follow the simple
Ping-Pong Bi-Bi mechanism within the range of studied concen-
trations. The affinity of acetic anhydride to the enzyme was larger
than that of eugenol, and the affinity of both substrates to the lipase
and the maximum reaction rate increased with temperature. The
second step of the reaction kinetics required more energy that the
first step, so it was identified as the rate-limiting reaction.
Vmax(␮mol/g s)
Kma
Kmb
(␮M)
(␮M)
R2 (%)a
97.1
97.5
94.9
b
fobj
8.0 × 10−14
0.7 × 10−14
3.0 × 10−14
a
Lack of fit to Eq. (4).
Values for adjustment of Ping-Pong Bi-Bi model (Eq. (6)).
b
eugenol [B] at 40 ◦C and 10 MPa (A) and the effect of substrates on
the reaction rate (B).
The variation of the concentrations of the substrates acetic anhy-
dride [A] and eugenol [B] caused an increase on the reaction rate,
as shown in Fig. 4(B). Moreover, in the plot of Fig. 4(A) the esterifi-
cation of eugenol followed the classical Michaelis-Menten kinetics.
However, the three fitting straight lines in Fig. 4(A) were not parallel
to each other, suggesting that the enzymatic reaction did not fol-
low a simple Ping-Pong Bi-Bi mechanism within the range of tested
concentrations [22,45]. The kinetic parameters were obtained by
fitting the model to the experimental data using a subroutine (fmin-
search) of the software MATLAB [29]. The values calculated by the
Lineweaver-Burk plot of reciprocal of the reaction rates versus
reciprocal concentrations were used as initial guess. Table 5 shows
the kinetic parameters adjusted with the model for the production
of eugenyl acetate in SC-CO2.
Acknowledgements
The authors wish to thank CAPES (Project 2952/2011),
CNPq (Ph.D. fellowship: 142373/2013-3) and FAPESP (Projects
2013/02203-6, 2015/11932-7 and 2009/54137-1) for the financial
support.
The affinity of the enzyme to the substrates can be determined
by the value of 1/Km, which indicates that the affinity of the enzyme
to acetic anhydride is larger than to eugenol. This suggests that
acetic anhydride is the acyl donor that binds to the active sites
of lipase. Similar behavior also was observed by Romero et al.
[41], investigating the esterification of isoamyl alcohol and acetic
anhydride using immobilized lipase in n-hexane. According to the
authors, the Ping-Pong Bi-Bi mechanism can be divided in two
steps: a) First, the acyl donor binds the enzyme, forming an enzyme-
anhydride complex (EA). Through a unimolecular isomerization
reaction, the EA complex is transformed to an enzyme-acyl com-
plex (EC) and the first product (acetic acid) is released; b) The
second substrate (eugenol) binds to the enzyme-acyl complex (EC)
forming a ternary complex, enzyme-acyl-eugenol. Again, through a
unimolecular isomerization reaction the ternary complex is trans-
formed to an enzyme-ester complex, finally releasing the second
product (eugenyl acetate) and the enzyme in its initial conforma-
tion.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bej.2016.06.018.
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