Continuous-Flow Chemo and Enzymatic Synthesis of Monoterpenic Esters with Integrated Purification

Article abstract of DOI:10.1016/j.mcat.2018.04.007

Monoterpenic esters are very important flavor and fragrance compounds due to their organoleptic properties. Despite their importance, many drawbacks are found for the production of monoterpenic esters. Here in we report two different approach's (chemo and enzymatic) for the continuous production of monoterpenic esters with integrated purification arriving on the desired molecules with high yields (>95%) and short reaction times.

Full text of DOI:10.1016/j.mcat.2018.04.007

MolecularCatalysis453(2018)39–46
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
Continuous-Flow Chemo and Enzymatic Synthesis of Monoterpenic Esters
with Integrated Purification
Carlos A.A. Adarmea, Raquel A.C. Leãob,c, Stefania P. de Souzab, Ivaldo Itabaiana Jrd,
Rodrigo O.M.A. de Souzab, Claudia M. Rezendea
a
Aroma Analysis Laboratory, Chemistry Institute, Federal University of Rio de Janeiro, Rio de Janeiro, CEP 21941909, Brazil
Biocatalysis and Organic Synthesis Group, Chemistry Institute, Federal University of Rio de Janeiro, CEP 21941909, Brazil
Faculdade de Farmácia, Universidade Federal do Rio de Janeiro, Rio de Janeiro, CEP 21941170, Brazil
Departamento de Engenharia Bioquímica, Escola de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, CEP 21941909, Brazil
b
c
d
A R T I C L E I N F O
A B S T R A C T
Keywords:
continuous-flow
biocatalysis
monoterpenic esters
geranyl acetate
Monoterpenic esters are very important flavor and fragrance compounds due to their organoleptic properties.
Despite their importance, many drawbacks are found for the production of monoterpenic esters. Here in we
report two different approach’s (chemo and enzymatic) for the continuous production of monoterpenic esters
with integrated purification arriving on the desired molecules with high yields (> 95%) and short reaction
times.
1. Introduction
(triacylglycerol acylhydrolases E.C. 3.1.1.3.) have gained greater pro-
minence over the years [11]. They have been used for the production of
Short-chain esters are commonly flavor and fragrance compounds
used in the food, cosmetic and pharmaceutical industries [1,2]. In
particular, natural monoterpenic esters receive special attention for
their organoleptic properties like rose (e.g. citronellol and geraniol es-
ters), fruit (e.g. linalyl acetate) or mint (menthyl acetate) notes [3], for
being natural flavors [4] and generally have higher prices than those
produced synthetically. 5 According to the European [6] and US [7]
legislations, a “natural flavor” substance should be obtained only by
physical procedures (e.g. distillation) or by enzymatic or micro-
biological processes from natural sources (animal or vegetable).
Nevertheless, some drawbacks are found for the production of mono-
terpenic esters such as low concentration of the desired compounds,
variability in the composition and yield (seasonality) and others [8].
These factors make it difficult to transpose to an industrial scale,
making the process more costly and expensive.
A cheaper and alternative method to obtain monoterpenic esters is
by chemical synthesis that traditionally employs strong acid or basic
catalysis [9] in a process environmentally unfriendly. In this way, there
is an increasing interest in the development of new clean strategies for
their production for industrial scale approaches.
Nowadays, the flavor and fragrance industries are interested in the
biocatalysis for the production of flavor esters, because is a greener way
to obtain this natural products derivatives due to the selectivity, reu-
sability and softer reaction conditions required for biocatalysts [10].
From all enzymes that can be used for such transformations, lipases
flavor compounds by direct esterification or transesterification in or-
ganic solvents or under solvent free reactions, being able to accept a
wide range of substrates and do not need cofactors [12–19]. However,
the cost of the biocatalyst make sometimes unviable their use for in-
dustrial proposes [20]. Continuous flow systems are an alternative to
the traditionally batch process, with the advantage to improve the reuse
of the biocatalyst and decrease the damage of the immobilized enzyme
compared to batch reactions, due to the use of packed-bed reactors
[21,22]. With the advent of continuous-flow technology researchers are
able to improve important strategic parameters for industrial manu-
facturing such as energy efficiency, purification steps and reaction time
[23–27].
Following our interest on the development of new methodologies
for practical continuous-flow protocols [28–31], in this work we re-
ported the continuous flow synthesis of monoterpenic esters comparing
chemical and enzymatic catalysis for the acylation of five commercial
monoterpenic alcohols (geraniol, citronellol, myrtenol, menthol and
linalool) and two essential oils (Cymbopogon martinii and Mentha ar-
vensis). We started with the biocatalized transesterification of the
monoterpenic alcohols using ethyl acetate as acylating agent and for the
chemical approach we have used acetic anhydride in a batch reactor
were various parameters were studied such as temperature, con-
centration of the alcohol, concentration of enzyme and reaction time.
The best reaction condition obtained was translated to a continuous
flow system. Finally, we have integrated synthesis and purification in
E-mail addresses: souzarod21@gmail.com, souzarod21@yahoo.com.br (R.O.M.A. de Souza).
https://doi.org/10.1016/j.mcat.2018.04.007
Received 6 February 2018; Received in revised form 12 March 2018; Accepted 7 April 2018
2468-8231/©2018ElsevierB.V.Allrightsreserved.

C.A.A. Adarme et al.
MolecularCatalysis453(2018)39–46
order to arrive on the desired product in a single step.
2. Materials and Methods
Table 1
Immobilization efficiency of new biocatalysts compared with N435.
Biocatalyst
Immobilization Amount of
Initial rate
Specific
Activity (U/
g)c
efficiency (%)a
protein
(mM min−1
)
(mg g−1 of
support)
2.1. Enzyme and chemicals
The kit of hydrolases was purchased from Selectazyme®. The im-
mobilized enzymes Novozym 435®,Lipozyme RM IM® and the reagents
p-nitrophenyl palmitate (pNPP), p-nitrophenol (pNP), ethyl acetate,
acetic anhydride, sodium carbonate, geraniol, citronelol, mirtenol,
mentol and linalol were purchased from Sigma-Aldrich Co. (St. Louis,
USA). Lipase B from Candida antarctica (Lipozyme CaLB, soluble form)
was purchased from Novozymes (Brazil). Purolite® ECR8205F and
Purolite® ECR8214F (crosslinked copolymers of metacrylate containing
oxirane groups (ECR8205F: 35-34% of oxirane; (ECR8214F 55-65% of
oxirane)) were purchased from Purolite International Limited (Wales,
UK). The essential oil of Cymbopogon martinii (Poaceae) was purchased
from Ferquima® and the essential oil of Mentha arvensis, (Lamiaceae)
was purchased from Mentol de Campinas S.A.
N435
CALBEPO_A 55.1
CALBEPO_B 57.1
–
30b
4.3
4.5
6.4
3.6
2.9
620
800
2500
a
measured by desorption with Triton X-100 and Lowry assay.
obtained after desorption assay with Triton X-100.
obtained by hydrolytic activity assay. One unit of lipase activity was ex-
b
c
pressed as the release of 1 μmol pNP per minute under the assay conditions.
Immobilization yield
(total starting activity − total residual activity)
=
× 100
Total starting activity
(1)
(2)
Immobilization Eficiency
Observed activity
2.2. GC-MS
=
× 100
(total starting activity − total residual activity)
Analyses were performed on an Agilent 7820A gas chromatograph
(GC) (Palo Alto, CA, USA) equipped with a G2613A auto-sampler
coupled to a quadrupole mass spectrometer (MS) Agilent (MS 5977E).
The identities of the terpenic esters were characterized by comparison
with NIST 14 and ADAMS (2007) libraries. Helium carrier gas was at
1 mL min−1 with constant flow mode in a HP-5MS capillary column
(30 m × 0.25 mm × 0.25 μm). The oven was set at 60 °C to 150 °C in
3 °C min−1, injector at 250 °C set in split mode (1:20) and injection the
volume of 1.0 μ L. Mass spectrometer includes ion source at 230 °C,
quadrupole at 150 °C, ionization voltage of 70 eV and mass spectra were
obtained in full scan mode (40–350 m/z).
Purolite® ECR8205F and Purolite® ECR8214F polymers were applied
as supports for lipase B from Candida antarctica immobilization, named
respectively CALBEPO_A and CALBEPO_B. The difference between the
two supports used is only the number of epoxy groups available for
immobilization. For this purpose, 1 mL of the enzyme solution
(0.62 U. mL−1 of specific activity) was diluted in 3 mL of phosphate
buffer (25 mM, pH 7.0) and added to the appropriate support (1 g). The
mixture was stirred during 4 h at 40 °C using a flask shaker, followed by
vacuum filtering. The final biocatalysts were dried over night at am-
bient temperature. To check the covalent binding, supports were sub-
mitted to desorption with Triton X-100 according to Cabrera [33].
Soluble protein was determined by the Lowry method [34] using bovine
serum albumin (BSA) as protein standard (Table 1).
2.3. GC-FID
Analyses were performed on an Agilent 7890N gas chromatograph
(GC) (Palo Alto, CA, USA) equipped with a G2613A auto-sampler
coupled to flame ionization detector (FID). Hydrogen carrier gas was at
1,3 mL min−1 with constant flow mode in a DB-WAX capillary column
(30 m × 0.25 mm × 0.25 μm). Oven was set at 90 °C (1 min) to 220 °C
in 12 °C min−1. Detector and injector were set at 250 °C. Injection was
in split mode 1:20 and injection the volume 1.0 μ L.
2.5. Hydrolytic activity assay
The hydrolysis reaction was carried out using p-nitrophenyl palmi-
tate (pNPP). Substrate solution was prepared by mixing one volume of
10 mM solution of pNPP in 2-propanol with nine volumes of 10 mM
phosphate buffer solution pH 8.0 containing 0.44% (mass fraction) of
Triton X-100 and 0.11% (mass fraction) of arabic gum. The lipase ac-
tivity was measured using 100 μL of lipase solution or suspension and
900 μL of the substrate solution (10 mM pNPP) at 55 °C for 2 min. The
absorbance of pNP released was spectrophotometrically monitored at
410 nm. One unit of lipase activity was expressed as the release of
1 μmol pNP per minute under assay conditions. The calibration curve
was prepared using pNP as standard. Values are given as mean
standard deviation in triplicate for each point.
2.4. Lipase selection
All experiments were carried out in batch conditions according to
the section 2.6. One milliliter of a solution of 150 mg mL−1 of geraniol
in ethyl acetate and 50 mg of each enzyme for the selection test were
used. Reactions were performed at 50 °C for 48 h with magnetic stirring.
Samples at 24 and 48 h were analyzed by GC-MS.
2.6. Experimental design
2.5. Lipase immobilization
For batch reactions, we first performed a full factorial design 23 with
three central points to obtain the experimental error. For these ex-
periments, we used the enzyme N435 that were carried out according to
Table 1. In the case of chemical catalyzed reactions, we made a full
factorial design 24 with three central points. The matrixes of experi-
ment are shown in Tables 2 and 3.
In all experiments, immobilization efficiency and yields were de-
termined by measuring the hydrolytic activities and the protein con-
centrations in the supernatant solution. Immobilization yields (Eq. (1))
were calculated after determining the amount of protein and enzyme
units that disappeared from the supernatant and comparing with the
initial protein and enzyme concentrations offered to the reaction (units
per gram of support). Efficiency (Eq. (2)) was calculated after de-
termining the activity of the immobilized enzyme and comparing with
the number of enzyme units that disappeared from the supernatant
(theoretically immobilized). Soluble protein was determined by the
Bradford method using bovine serum albumin (BSA) as protein stan-
dard. 32
2.7. Batch reactions
Reagents were added to 4 mL vials on silicon carbide plates under
controlled temperature (40–60 °C for the biocatalyzed reaction and
70–110 °C for the chemical reaction) and magnetic stirring. The amount
40

C.A.A. Adarme et al.
MolecularCatalysis453(2018)39–46
Table 2
Table 4
Real and code values (-1: lower level; 0: intermediate level; +1: high level) for
Selected results for the evaluation of different free enzyme from the ester-
ification reaction between geraniol and ethyl acetate.
the full factorial design 23 of the biocatalyzed reaction.
Variable
-1
0
+1
Entry
Lipase Source
Conversion (%)
24 h
Temperature (°C)
40
1
50
50
2
150
60
3
250
48 h
Time (h)
Substrate concentration (mg mL−1
)
1
2
3
4
5
6
Lipase B − Candida antarctica
Lipase E − Alcaligenes sp
Lipase F- Alcaligenes sp
Pseudomonas stutzeri
Lipase C- Alcaligenes sp
Lipase D- Alcaligenes sp
88
87
85
70
72
68
96
90
90
81
85
84
Table 3
Real and Code values (-1: lower level; 0: intermediate level; +1: high level) for
the full factorial design 24 of the chemical reaction.
Reaction conditions: 1 mL solution containing 150 mg of geraniol in ethyl
acetate and 50 mg of each enzyme. at 50 °C for 48 h under magnetic stirring.
Conversions were determined by GC-MS.
Variable
-1
0
+1
Temperature (°C)
Time (h)
70
0,5
90
1
110
1,5
Sodium acetate concentration (%)
Acetic acid/acetic anhydride (v/v)
2
6
10
0.5000
Supporting Information).
0.0250
0.2625
As shown on Tables 4 and 5, both free and immobilized enzymes
could lead to desired products with good conversions after 24 and 48 h.
The best conversion after 48 h was obtained for the free enzymes
Candida antarctica B (96%), Alcaligenes sp E (90%) and F (90%), and for
the immobilized enzyme N435 (90%). Another interesting performance
was observed with the immobilized Candida antarctica B on epoxy re-
sins (Entries 4 and 5, Table 5) where a similar conversion was obtained
when compared to N435. But a closer look into the amount of protein
present in CALBEPO_A and CALBEPO_B (Table 1) revels that the reac-
tion catalyzed by these enzymes was much more efficient then N435,
since we have only 4.5 mg of protein loaded in each gram of support
against 30 mg. g of support−1 presented by commercial preparation.
This difference in the amount of protein between the home-made en-
zymes and the commercial biocatalyst is crucial for increasing the
productivity of the reaction. In these biocatalysts, lipase B from Candida
antarctica was immobilized on two epoxy resins through covalent
bonds. This type of interaction restricts lipase to a more active and
stable conformation, turning the final biocatalyst more robust, since the
conformational restriction turns the enzyme more resistant to deleter-
ious effects of solvent, as well high temperatures. Previous work by our
research group [35] has demonstrated that these biocatalysts have ex-
cellent performance in reactions of kinetic dynamic resolution of
amines and in esterifications when compared to the commercial en-
zyme Novozyme 435. Since there was not a big difference on conver-
sion between free and immobilized enzymes, we decided to move for-
ward using the immobilized enzyme N435. It is important to note that
industrial application of free enzymes can be limited by a poor long-
term operational stability, difficulty of recovering and re-use of the
enzyme.
of enzyme and reagents was added according to the experimental de-
sign. At the end, the reaction mixture was analyzed by GC-FID.
2.8. Continuous flow reaction
The reaction mixtures were pumped using the Asia flow system
equipped with a stainless steel column filled with the enzyme (packed
bed reactor). For each experiment, ethyl acetate was initially pumped
through the system during 3 min. The same procedure was made after
the experiment in order to clean the system from any remaining re-
actant or starting material. For the biocatalyzed continuous flow reac-
tion, we used a column with an inner volume of 2.4 mL (i.d. = 8.0 mm,
h = 4.9 cm) and for the chemical esterification reaction, a coil with
inner volume of 1,0 mL (i.d. = 1.5 mm, h = 57.0 cm) was employed.
2.9. Continuous flow reaction and purification
This system consisted in 3 steps: reaction, continuous work-up and
liquid-liquid separation. Different flow rates between the organic and
the aqueous phase have been studied to determinate the best rate (1:1,
1:3, 1:4). Different concentrations of sodium carbonate solutions were
tested (3, 5 and 10%), as well as several approaches towards a better
mixing efficiency between the two phases (Scheme 1).
3. Results and Discussion
3.1. Lipase selection
3.2. Biocatalyzed acetylation of geraniol with ethyl acetate
We began our studies evaluating different free (Table S1) and im-
mobilized enzymes (Table S2) on the transesterification of geraniol
with ethyl acetate at 50 °C for 48 h. The main results are shown in
Tables 4 and 5 (for further details with all results obtained see
3.2.1. Experimental design
In order to find the best reaction conditions, we performed a 23 full
factorial design in batch conditions, using as model the enzyme N435
Scheme 1. Representation of the continuous-flow reaction followed by in-line purification with liquid-liquid phase separator.
41

C.A.A. Adarme et al.
MolecularCatalysis453(2018)39–46
Table 5
Table 7
Selected results for the evaluation of different immobilized enzyme for the es-
terification reaction between geraniol and ethyl acetate.
Continuous flow biocatalyzed transesterification reaction between geraniol and
ethyl acetate catalyzed by different immobilized enzymes.
Entry
Lipase Source
Conversion (%)
Entry
Res. Time (min)
Conversion (%)a
24 h
48 h
N435
LipoRMIM
CALBEPO_A
CALBEPO_B
1
2
3
4
Amano IM, Burkholderia cepacia
Lipozyme RM IM® (Rhizomucor miehei)
N435® (Candida antarctica B)
Candida antarctica B immobilized on epoxy resin
(CALBEPO_A)
69
67
89
89
83
67
90
91
1
2
3
4
5
6
7
8
1
2
4
6
66
73
84
88
90
89
90
90
39
46
54
56
57
60
66
74
64
65
78
82
85
87
90
90
42
51
70
77
81
83
89
89
8
5
Candida antarctica B immobilized on epoxy resin
(CALBEPO_B)
90
91
10
30
60
Reaction conditions: 1 mL solution containing 150 mg of geraniol in ethyl
acetate and 50 mg of each enzyme at 50 °C for 48 h under magnetic stirring.
Conversions were determined by GC-MS.
Reaction Conditions: A solution containing 150 mg of geraniol in ethyl acetate
was pumped through a packed-bed reactor containing 1 g of immobilized en-
zyme. The column dimension were: i.d = 8.0 mm, h = 4.9 cm. Reactions were
carried out at 50 °C.
for the biocatalyzed acetylation of geraniol with ethyl acetate. The in-
dependent variables tested were temperature, time and substrate con-
centration. The respective levels of the variables for the 23 full factorial
design are present on Table 2. The results obtained based on the ex-
perimental design can be found on Table 6.
a
Conversions were determined by GC-FID.
CALBEPO_B) and experiments were performed at different residence
times (from 1 min to 60 min) in order to investigate the best conversion
towards the desired product. The maximum residence time was estab-
lished at 1 h based in our batch optimization. The results are shown in
Table 7.
As shown in Table 6, the best conversions were obtained when
running the reaction at the lower substrate concentration at 40 °C or
60 °C for at least 1 h (Entries 1, 2, 8 and 9, Table 6). Nevertheless, for
the substrate concentration of 150 mg mL−1 at 50 °C (Entries 5, 6 and 7,
Table 6), the results were also high (87%), which seems that the use of a
higher concentration of starting material can maximize product for-
mation leading to a better productivity of the process developed. Ac-
cording to the estimate effects for the full factorial design 23, the in-
crease in substrate concentration let to a negative effect, but the
interaction of temperature and substrate concentration have a positive
one (See Supporting information for further details, Table S3). In this
way, we have chosen 50 °C and 150 mg mL−1 of substrate as the best
condition for our biocatalyzed reaction.
Table 7 shows that similar results were obtained for 8 min − 60 min
residence time for N435 and CALEPO_A. In the case of RM IM, the best
result was obtained for 60 min of residence time, being far away from
the excellent results achieved by the other immobilized enzymes. For
the immobilized enzyme CALEPO_B, similar results were obtained for
30 and 60 min of residence time. A brief look to the space-time-yield of
N435 and CALEPO_A can show us that both enzymes reach high values,
2.4 g h−1 of product and 2.26 g h−1 respectively. Taking into account
the amount of protein present in each support, CALEPO_A is 6 times
more productive then N435, delivering 680 mg of product h−1.mg
ptn−1. Unfortunately, the long term stability of CALEPO_A is still low
which compromises its industrial application. In this way, we mon-
itored the continuous-flow esterification reaction of geraniol at the best
conditions found on Table 7 (Entry 5) a period of 8 h, when aliquots
were taken every 30 min in order to determine the stability of N435.
Fig. 1 shows the long-term stability results for N435.
3.2.2. Biocatalyzed transesterification reaction under continuous flow
conditions
With these results, we started to study the lipase-catalyzed reaction
under continuous flow conditions. The temperature was set at 50 °C and
the concentration of substrate was fixed at 150 mg mL−1. Besides the
fact that we have optimized these conditions to N435, we decided to
evaluate also the performance of other immobilized enzymes under
continuous flow conditions. The column was filled with 1 g of im-
mobilized enzyme (N453, Lipozyme RM IM, CALBEPO_A or
As we can see in Fig. 1, the immobilized enzyme N435 retained its
activity after a period of 8 h and the conversion remained constant at
approximately 90%. These results show that the immobilized enzyme
was stable for the experimental condition tested, allowing continuous
operation for prolonged times an important feature for a biocatalyst
aiming to be applied on production scale.
With these results in hands, we decided to extend the scope of the
protocol developed and decided to use the immobilized enzyme N435
as a catalyst for the esterification of other monoterpenic alcohols
(myrtenol, citronellol, menthol, linalool and the essential oil of
Cymbopogon martini), at the best condition obtained previously. Table 8
shows the results for these experiments at 50 °C and 8 min of residence
time.
As expected, citronellol and myrtenol show high conversions, si-
milar to those obtained with geraniol, since they are all primary alco-
hols. Unfortunately, the residence time applied for menthol and linalool
was not enough in order to achieve a significant conversion and this is
probably due to the fact that this monoterpenic alcohols are more
sterically hindered.
Table 6
Results for the N435 biocatalyzed acetylation of geraniol with ethyl acetate (full
factorial design 23).
Entry
Temperature
(°C)
Time
(h)
Substrate concentration (mg/mL)
Conversion**
(%)
1
2
3
4
40
40
40
40
50
50
50
60
60
60
60
1
3
1
3
2
2
2
1
3
1
3
50
50
87
89
59
73
87
87
87
89
89
71
82
250
250
150
150
150
50
50
250
250
5(C)*
6(C)*
7(C)*
8
9
10
11
3.3. Chemical esterification reaction
Reaction conditions: Temperature, time and concentration of geraniol were
used according to the experimental design using a 1 mL solution of geraniol and
ethyl acetate at appropriate concentration and 50 mg of N435. *Central point.
**Conversions were determined by GC-FID.
Additionally to the biocatalyzed esterification reaction, we studied
the esterification reaction between geraniol and acetic anhydride
without the addition of a catalyst, which would need laborious
42

C.A.A. Adarme et al.
MolecularCatalysis453(2018)39–46
Fig. 1. (a)Long-term stability test for N435 immobilized enzyme. (b) Chromatographic region of GC-FID analysis, for each type of reactor, at different sodium
carbonate concentration under continuous flow purification.
separation by the end of the reaction time. Traditionally, esters are
prepared from an alcohol and a carboxylic acid in a reaction catalyzed
by a base or an acid. To avoid the use of the catalyst, we choose acetic
anhydride that is more reactive than carboxylic acids. To control the pH
of the reaction media, we use sodium acetate that acts as an acid sca-
venger. The addition of sodium acetate makes the reaction media het-
erogeneous, hindering the translation of batch conditions to con-
tinuous-flow conditions. In a previously solubility test, we found that
the addition of acetic acid make the reaction media homogenous at
60 °C. Since acetic acid can act as acyl donor and also as a catalyst, we
evaluated its addition through the variable acetic acid/acetic anhydride
ratio in the experimental design.
acetic anhydride ratio under batch conditions. It was observed that salt
concentration is statistically insignificant to the reaction conversion
and the addition of acetic acid, evaluated through the variable acetic
acid/acetic anhydride ratio, has a negative effect on the reaction out-
come (for further details see Supporting Information, Tables S4 and S5).
3.3.2. Chemical esterification reaction under continuous flow process
Aiming at improving the esterification process to obtain geranyl
acetate, we performed the reaction under continuous flow. We eval-
uated temperature (90 °C − 150 °C) and the residence time (2 min −
10 min). Reagents were pumped separately at a molar ratio of 1:1.35
(geraniol: acetic anhydride) and results are summarized on Table 9.
Table 9 shows that conversions around 90% could be obtained at
110 °C with only 2 minutes of residence time. Nevertheless, the best
conversions (> 99%) were obtained in the range of 4 to 10 min of re-
sidence time at 150 °C. For 2 min at 150 °C, the conversion was about
95%, with an isolated yield of 93%. Definitely, temperature plays an
3.3.1. Experimental design
As usual, we started our optimization performing a full factorial
design 24 with three central points, and studied the influence of the
variables temperature, time, concentration of salt and the acetic acid/
43

C.A.A. Adarme et al.
MolecularCatalysis453(2018)39–46
associated to the high temperature (150 °C) used during the continuous
Table 8
Continuous-flow esterification reaction catalyzed by N435 for different
monoterpenic alcohols.
flow reaction.
Conversiona
(%)
3.4. Continuous flow purification
Monoterpenic alcohol
Taking these results into account, we decided to integrate synthesis
and purification in order to arrive at the end of our continuous flow
process with a product which would not need any further step of pur-
ification. The best reaction condition found for the continuous flow
chemical esterification reaction was used to investigate the continuous
flow purification of geranyl acetate. A continuous flow liquid-liquid
extraction was envisioned in order to extract remaining impurities from
the chemical synthesis (acetic acid and unreacted acetic anhydride), by
a sodium carbonate work-up at different concentrations (3, 5 and 10%
p/v).
First, we decided to determine the best proportion between organic
and aqueous phase (1:1; 1:3; 1:4) at a fixed sodium carbonate con-
centration (3%). In order to explore these changes, we needed to keep
the total flow rate at 0.5 mL min−1 to maximize the efficiency of our
continuous flow liquid-liquid separator (see further details on
Supporting Information). The criterion chosen to define the relationship
between the organic and aqueous phase used in the purification process
was the reduction of the percentage area of the acetic acid.
After the heating zone, where the acetylation reaction was taking
place, we added a second T-junction in order to deliver the sodium
carbonate solution for the work-up process. The extraction process went
through a mixing zone (2.2 mL coil, i.d = 1.0 mm, h = 280 cm) and
then through the liquid-liquid separator in order to arrive on the de-
sired purified product. The best proportion between organic and aqu-
eous phase to remove impurities was 1:4, as shown in Scheme 2. For
this proportion, was observed the less intense chromatographic peaks,
indicating that a major quantity of impurities were separated from the
geranyl acetate.
Based on the best proportion found between organic and aqueous
phases, we decided to investigate increasing sodium carbonate con-
centrations in order to evaluate the effect on the purification step of our
process. Another variable also investigated by us was the type of reactor
used as mixing zone. A packed-bed reactor filled with glass pieces, in
order to enhance mass transfer and mixing, was used instead of the coil
reactor and results are presented on Fig. 1. The efficiency of the pur-
ification process at different sodium carbonate concentrations were
evaluated based on the peaks observed in GC-FID for acetic anhydride
and acetic acid, the two major impurities found in this process.
The best result was obtained using a fixed-bed column reactor and
an aqueous solution of 10% sodium carbonate. With these conditions,
were did not observe the peaks corresponding to acetic acid and acetic
anhydride, indicating the removal of the impurities during the con-
tinuous purification. Nevertheless, we observed that for these experi-
mental condition there is a slight hydrolysis (≈ 4%, based on relative
areas) of the geranyl acetate. This was concluded based on the presence
of a chromatographic peak corresponding to geraniol on the final pro-
duct.
Citronellol
Myrtenol
Menthol
Linalool
91
86
0.2
0.1
87b
Essential oil of Cymbopogon martinii
Reaction Condition: A solution containing 150 mg of alcohol or essential
oil in ethyl acetate was pumped through a packed-bed reactor containing
1 g of immobilized enzyme. The column dimension were: i.d = 8.0 mm,
h = 4.9 cm. Reactions were carried out at 50 °C and 8 min of residence
time.
a
Conversions were determined by GC-FID.
Conversion is based on total geraniol content (75%) of the essential
b
oil.
Table 9
Continuous-flow esterification reaction between geraniol and acetic anhydride
at different reaction conditions.
Entry
Res. Time (min)
Conversion (%)a
90 °C
110 °C
130 °C
150 °C
1
2
3
4
5
2
4
6
8
61
79
83
86
88
90
93
95
96
96
91
95
97
98
99
95
99
99
99
99
10
Reaction conditions: reagents were pumped separately at different flow rates in
order to achieve a molar ratio of 1:1.35 (geraniol: acetic anhydride). Coil di-
mensions: i.d. = 1.5 mm, h = 57.0 cm.
a
Measured by GC-FID.
Table 10
Continuous flow chemical esterification of different monoterpenic alcohols
with acetic anhydride.
Monoterpenic alcohol
Conversion (%)a
Citronellol
Myrtenol
Menthol
Linalool
Essential oil of Cymbopogon martinii
Essential oil of Mentha arvensis
99
99
94
–
99b
89b
Reaction conditions: Reagents were pumped separately at different flow rates
in order to achieve a molar ratio of 1:1.35 (geraniol: acetic anhydride) Coil
dimensions: i.d. = 1.5 mm, h = 57.0 cm. Reaction were carried out at 150 °C.
Measured by GC-FID.
Conversion is based on total geraniol content (75%) of the essential oil
from Cymbopogon martini and total menthol content (45%) of the essential oil
from Mentha arvensis.
a
b
Finally, we performed an experiment with the continuous synthesis
and purification based on the best conditions established for the pro-
duction of geranyl acetate. The optimized process could lead us to the
production of geranyl acetate with a final chromatographic purity of
94.1% (Scheme 3).
important role and continuous-flow process can lead this chemical es-
terification to outstanding results with very short residence times.
Again, these results prompted us to perform the chemical esterification
of other monoterpenic alcohols (citronellol, myrtenol, menthol, lina-
lool, and the essential oils of Cymbopogon martini and Mentha arvensis).
Temperature of 150 °C and a residence time of 4 min were used in order
to evaluated the related reactions and results are shown on Table 10.
The results presented on Table 10 shows that primary alcohols
(citronellol and myrtenol) have a high conversion, similar to the results
obtained for geraniol in the previous test. In the case of menthol, very
good conversions were obtained (94%), something not observed for the
biocatalyzed reaction. For linalool, we observed degradation probably
4. Conclusion
In conclusion, we have developed a continuous flow approach for
monoterpenic esters production through biocatalysis or chemical cat-
alysis under continuous-flow conditions. For the biocatalyzed approach,
we found that the Novozyme 435 was the best choice for the continuous
flow environment due to the long-term stability, leading to high con-
version in short residence time and low temperatures. For the chemical
44

C.A.A. Adarme et al.
MolecularCatalysis453(2018)39–46
Scheme 2. GC-FID analysis for different proportions tested under continuous flow purification protocol. Proportions of organic to aqueous phase: a: 1:1; b: 1:3 and c
1:4.
Scheme 3. Schematic representation of the continuous-flow synthesis and characterization of geranyl acetate and GC-FID chromatogram of the final product.
45

C.A.A. Adarme et al.
MolecularCatalysis453(2018)39–46
approach, we found that acetic anhydride is a good acyl donor, that
lead to the desired geranyl acetate with high conversion (> 99%) in
only 4 min at 150 °C. The best experimental conditions for the chemical
esterification reaction was applied with success for the esterification of
citronellol, myrtenol, menthol and the essential oils of Cymbopogon
martinii and Mentha arvensis, which also presented high conversions.
Finally, we were able to establish a continuous flow synthesis and
purification of geranyl acetate with a chromatographic purity of 94.1%
of the final product, being possible to apply the process developed to
biocatalyzed or chemical catalyzed reactions.
[12] V. Athawale, N. Manjrekar, M. Athawale, Lipase-catalyzed synthesis of geranyl
methacrylate by transesterification: study of reaction parameters, Tetrahedron Lett.
43 (2002) 4797–4800.
[13] S. Gryglewicz, E. Jadownicka, A. Czerniak, Lipase catalysed synthesis of aliphatic,
terpene and aromatic esters by alcoholysis in solvent-free medium, Biotechnol. Lett.
22 (2000) 1379–1382.
[14] G.B. Oguntimein, W.A. Anderson, M. Moo-Young, Synthesis of geraniol esters in a
solvent-free system catalysed by Candida antarctica lipase, Biotechnol. Lett. 17
(1995) 77–82.
[15] H. Ghamgui, M. Karra-Chaâbouni, S. Bezzine, N. Miled, Y. Gargouri, Production of
isoamyl acetate with immobilized Staphylococcus simulans lipase in a solvent-free
system, Enzyme Microb. Technol. 38 (2006) 788–794.
[16] N.N. Ghandi, K.D. Mukherjee, Specificity of papaya lipase in esterification with
respect to the chemical structure of substrates, J. Agric. Food Chem. 48 (2000)
566–570.
[17] M. Zanetti, T.K. Carniel, A. Valério, J.V. Oliveira, D. Oliveira, P.H.H. Araújo,
H.G. Riella, M.A. Fiori, Synthesis of geranyl cinnamate by lipase-catalyzed reaction
and its evaluation as an antimicrobial agent, J. Chem. Technol. Biotechnol. 92
(2017) 115–121.
[18] E.T. Liaw, K.J. Liu, Synthesis of terpinyl acetate by lipase-catalyzed esterification in
supercritical carbon dioxide, Bioresour. Technol. 101 (2010) 3320–3324.
[19] P. Claon, C.C. Akoh, Lipase-catalized synthesis of terpene esters by transester-
ification in n-hexane, Biotechnol. Lett. 16 (1994) 235–240.
Acknowledgments
Authors thank CAPES, FAPERJ, CNPq and FINEP for financial sup-
port.
Appendix A. Supplementary data
[20] R.A. Sheldon, S. Pelt, Enzyme immobilization in biocatalysis: why, what and how,
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.mcat.2018.04.007.
Chem. Soc. Rev. 42 (2013) 6223–6235.
[21] I. Itabaiana, L.S.M. Miranda, R.M.O.A. Souza, Towards a continuous flow en-
vironment for lipase-catalyzed reactions, J. Mol. Catal. B: Enzym. 85-86 (1-9)
(2013).
[22] M.B. Plutschack, B. Pieber, K. Gilmore, P.H. Seeberger, The Hitchhiker’s Guide to
Flow Chemistry, Chem. Rev. (2017), http://dx.doi.org/10.1021/acs.chemrev.
7b00183.
[23] F.J.e. Agostino, S.N. Krylov, Advances in steady-state continuous-flow purification
by small-scale free-flow electrophoresis, Trends Anal.Chem. 72 (2015) 68–79.
[24] C. Wiles, P. Watts, Continuous flow reactors: a perspective, Green Chem. 14 (2012)
38–54.
[25] D.T. Mcquade, P.H. Seeberger, Applying flow chemistry: methods, materials and
multistep synthesis, J. Org. Chem. 78 (2013) 6384–6389.
References
[1] K. p. Dhake, D.D. Thakare, B.M. Bhanage, Lipase: A potential biocatalyst for the
synthesis of valuable flavour and fragrance ester compounds, Flavour Fragr. J. 28
(2013) 71–83.
[2] A. Larios, H.S. García, R.M. Oliart, G. Valerio-Alfaro, Synthesis of flavor and fra-
grance esters using Candida antarctica lipase, Appl. Microbol. Technol 65 (2004)
373–376.
[3] The Good Scents Company. Flavor and fragrance information catalog Accessed in
07/01/17.
[4] R. Teranishi, E.L. Hornstein, I. Wick, Flavor Chemistry, Thirty years of progress, 1
Ed., Springer, 112-113 1999, 2018.
[26] G. Jas, A. Kishchning, Continuous flow techniques in organic synthesis, Chem. Eur.
J. 9 (2003) 5708–5723.
[27] B. Gutmann, D. Cantillo, C.O. Kappe, Continuous-Flow Technology—A Tool for the
Safe Manufacturing of Active Pharmaceutical Ingredients, Angew. Chem. Int. Ed. 54
(2015) 6688–6728.
[5] S. Serra, C. Fuganti, E. Brenna, Biocatalytic preparation of natural flavours and
fragrances, Trends Biotechnol. 23 (2005) 193–198.
[28] R.O.M.A. Souza, L.S.M. Miranda, U.T. Bornscheuer, A Retrosynthesis Approach for
[6] Council directive, The Council of the European Communities, 88/388, EEC, 1988.
[7] Food and Drug Administration, US Code of Federal Regulations, 21 CFR, 101.22,
1985.
[8] P. Molina, M.R. Pimentel, G.M. Pastore, Pseudomonas: a promising biocatalyst for
the bioconversion of terpenes, Appl. Microbiol Biotechnol 97 (2013) 1851–1864.
[9] C. Sell, The chemistry of fragrance. From perfumer to consumer, 2 Ed., RSC
Publishing, 52-81, 2006.
[10] (a) P. Lozano, J.M. Bernal, A. Navarro, A clean enzymatic process for producing
flavour esters by direct esterification in switchable ionic liquid/solid phases, Green
Chem. 14 (2012) 3026–3033;
Biocatalysis in Organic Synthesis, Chem. Eur. J. (2017).
[29] A.S. Miranda, M.V.M. Silva, F.C. Dias, S.P. Souza, R.A.C. Leão, R.O.M.A. Souza,
Continuous flow dynamic kinetic resolution of rac-1-phenylethanol using a single
packed-bed containing immobilized CAL-B lipase and VOSO4 as racemization cat-
alysts, React. Chem. Eng. 2 (2017) 375–381.
[30] A.S. Miranda, L.S.M. Miranda, R.O.M.A. Souza, Lipases Valuable catalysts for dy-
namic kinetic resolutions, Biotechnol. Adv. 33 (2015) 372–393.
[31] R.A.C. Leão, S.P. Souza, D.O. Nogueira, G.M.A. Silva, M.V.M. Silva, M.L.E. Gutarra,
L.S.M. Miranda, I.I. Junior, R.O.M.A. Souza, Consecutive lipase immobilization and
glycerol carbonate production under continuous-flow conditions, Catal. Sci.
Technol. 6 (2016) 4743–4748.
[32] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding, Anal. Chem. 72
(1976) 248–254.
[33] Z. Cabrera, G. Fernandez-Lorente, R. Fernandez-Lafuente, J.M. Palomo,
J.M. Guisan, Novozym 435 displays very different selectivity compared to lipase
from Candida antarctica B adsorbed on other hydrophobic supports, J. Mol. Catal. B:
Enzym. 57 (2009) 171–176.
(b) K.P. Dhake, D.D. Thakare, B.M. Bhanage, Lipase: A potential biocatalyst for the
synthesis of valuable flavour and fragrance ester compounds, Flavour Fragr, J. 28
(2013) 71–83;
(c) J. Sun, L.W. Lee, S.Q. Liu, Biosynthesis of Flavour-Active Esters via Lipase-
Mediated Reactions and Mechanisms, Aust. J. Chem. 67 (2014) 1373–1381;
(d) R. Kirdi, N. Ben Akacha, M. Gargouri, Biosynthesis Coupled to the Extraction of
Geranyl Acetate in a Liquid-Gas System: Optimization of the Transesterification
Reaction and Modeling of the Transfer, Chemical & Biochemical Engineering
Quarterly 30 (1) (2016) 117–125;
(e) L.M. Todero, J.J. Bassi, F.A.P. Lage, M.C.A.C. Corradini, J.C.S. Barboza,
D.B. Hirata, A.A. Mendes, Enzymatic synthesis of isoamyl butyrate catalyzed by
immobilized lipase on poly-methacrylate particles: optimization, reusability and
mass transfer studies, Bioprocess and Biosystems Engineering 38 (8) (2015)
1601–1613.
[34] O.H. Lowry, N.J. Rosebrough, A.L. Farr, R. Randall, Protein Measurement with the
folin phenol reagent, J. Biol. Chem. 193 (1951) 265–275.
[35] S.P. Souza, R.A.D. Almeida, G.G. Garcia, R.A.C. Leão, J. Bassut, R.O.M.A. Souza,
I.I. Itabaiana, Immobilization of lipase B from Candida antarctica on epoxy-func-
tionalized silica: characterization and improving biocatalytic parameters, J. Chem.
Technol. Biotechnol. (2017), http://dx.doi.org/10.1002/jctb.5327.
[11] R.C. Rodrigues, R. Fernandez-Lafuente, Lipase from Rhizomucor miehei as an in-
dustrial biocatalyst in chemical process, J. Mol. Catal. B: Enzym. 64 (2010) 1–22.
46