Enzyme-mediated epoxidation of methyl oleate supported by imidazolium-based ionic liquids

Article abstract of DOI:10.1016/j.molcatb.2010.09.019

In the present manuscript we describe and discuss the use of hydrophobic and hydrophilic ionic liquids (ILs) as efficient supports to the enzyme-catalyzed epoxidation of biodiesel. The use of nine different lipases in three different ILs (BMI.PF₆, BMI.NTf₂ and BMI.BF ₄) gave high biodiesel conversion rates in short reaction times using hydrogen peroxide (30%, v/v) as the epoxidation agent. A drastic behavior change is observed by altering the media from a hydrophobic IL to a hydrophilic IL. For instance, the use of Amano A. lipase (from Aspergillus niger) in hydrophilic BMI.BF₄ yielded the epoxidized compound in 89% in the first reaction hour, and in the mean time, hydrophobic BMI.PF₆ yielded the same product in 67%. The use of other lipases resulted in the desired epoxidized derivative and also in the 1,2-diol as a result of a reversible epoxy ring-opening promoted in the reaction media. Conversions and selectivities depended on the nature of the IL, on reaction time and on the selection of the lipase enzyme.

Full text of DOI:10.1016/j.molcatb.2010.09.019

Journal of Molecular Catalysis B: Enzymatic 68 (2011) 98–103
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enzyme-mediated epoxidation of methyl oleate supported by
imidazolium-based ionic liquids
Wendylene S.D. Silvaa,b, Alexande A.M. Lapisc, Paulo A.Z. Suareza,b,∗, Brenno A.D. Netoa,b,∗
a Laboratory of Medicinal and Technological Chemistry, University of Brasília (IQ-UnB), Brasilia, DF, Brazil
b National Institute of Catalysis (INCT), Brazil
c Universidade Federal do Pampa, Unipampa, Bagé, RS, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 July 2010
Received in revised form
27 September 2010
Accepted 29 September 2010
Available online 7 October 2010
In the present manuscript we describe and discuss the use of hydrophobic and hydrophilic ionic liquids
(ILs) as efficient supports to the enzyme-catalyzed epoxidation of biodiesel. The use of nine different
lipases in three different ILs (BMI.PF6, BMI.NTf2 and BMI.BF4) gave high biodiesel conversion rates in
short reaction times using hydrogen peroxide (30%, v/v) as the epoxidation agent. A drastic behavior
change is observed by altering the media from a hydrophobic IL to a hydrophilic IL. For instance, the use
of Amano A. lipase (from Aspergillus niger) in hydrophilic BMI.BF4 yielded the epoxidized compound in
89% in the first reaction hour, and in the mean time, hydrophobic BMI.PF6 yielded the same product in
67%. The use of other lipases resulted in the desired epoxidized derivative and also in the 1,2-diol as a
result of a reversible epoxy ring-opening promoted in the reaction media. Conversions and selectivities
depended on the nature of the IL, on reaction time and on the selection of the lipase enzyme.
© 2010 Elsevier B.V. All rights reserved.
Keywords:
Epoxidation
Catalysis
Ionic liquids
Enzyme
Lipase
1. Introduction
and stabilizers, reactive diluents for paints, intermediates in the
production of polyurethane-polyols, polyester polymers and alkyd
Due to increasing environmental concerns and petroleum sup-
ply security, the use of biomass as raw material for polymers,
chemicals, fine chemicals and fuel applications has become an
imperative issue in recent years [1]. In this context, one observes
widespread interest in the chemistry of fats and oils as well as
their derivatives [2], which are now renowned as a wealth of
bio-based products and viable alternatives to petroleum-based
fuels. As a consequence, demands for renewable feedstocks to
obtain industrial products have been granted increasing impor-
tance. For example, it has been shown that epoxidation of fats, oils
and fatty acid mono-esters is an important way to improve their
physical–chemical properties and also to obtain starting materi-
als for a large number of products. For instance, the epoxidation
of biodiesel increased its overall oxidative stability and lowered
its overall friction coefficient, even when done in the presence
of additives, improving its quality [3,4]. Moreover, epoxy deriva-
tives of fats and oils are also applied as lubricants, PVC-plasticizers
resins, as well as components of blended lubricants and adhesives
[5,6]. Methyl oleate epoxide, for example, is a key intermediate used
in the synthesis of other derivatives such as carbonates [7] and
azides [8]. It is worth mentioning that nowadays different prod-
ucts based on epoxidized derivatives of fats and oils are already
commercially available.
Besides, the use of environmentally friendly reaction media is
highly interesting in the current world’s scenery and demands.
In this sense, the use of ionic liquids (ILs) as reaction
media has become a viable and attractive industrial alterna-
tive to organic solvents [9]. Additionally, it has been shown
by some of us that imidazolium-based ILs (Fig. 1 1-n-butyl-3-
methylimidazolium hexafluorophosphate, BMI.PF6; 1-n-butyl-3-
methylimidazolium bis(trifluoromethylsulfonyl)imide, BMI.NTf2;
1-n-butyl-3-methylimidazolium tetrafluoroborate, BMI.BF4) are
excellent media that support different enzyme-catalyzed reactions
[10]. Enzymes are usually active in ILs that comprise the species
BF4−, PF6− and NTf2− [11], possibly due to the lower hydrogen bond
basicity of the enzyme-compatible anions [12]. In this sense, the
use of enzymes supported in an appropriate IL would be a very
attractive system to promote epoxidation reactions of biodiesel
(methyl oleate). In addition, we can envisage the use of hydrogen
peroxide as the oxidant agent in the reaction, which is an attrac-
tive idea from both environmental and economic standpoints [6].
∗
Corresponding authors at: Laboratory of Medicinal and Technological Chem-
istry, University of Brasília (IQ-UnB), Brasilia, DF, Brazil. Tel.: +55 61 31073867;
fax: +55 61 32734149.
E-mail addresses: psuarez@unb.br (P.A.Z. Suarez),
brenno.ipi@gmail.com (B.A.D. Neto).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.09.019

W.S.D. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 98–103
99
Furthermore, vegetable oils, especially soybean oil, are relatively
inexpensive [13].
+
+
N
+
N
N
_
N
N
N
The current industrial process for obtaining these epoxides is
based on the Prileschajew reaction [14], which uses short chain
percarboxylic acids. For all reasons mentioned above, it is neces-
sary to develop novel, cheap, efficient and environmentally friendly
methodologies to perform the epoxidation of fats and oils and their
derivatives, such us methyl oleate [15]. Recently, different research
groups have studied heterogeneous catalytic systems based on alu-
minum oxide and bi-metal oxides and hydrogen peroxide, which
have lower activity compared to the industrial process but are easy
to recover and reuse [6,16]. It is worth mentioning that several
deactivation issues were described referring to the presence of both
Brönsted [17] and Lewis [16] acid sites, which are probably related
to the acid-catalyzed decomposition of hydrogen peroxide. On the
other hand, it was recently shown that CALB enzyme catalyzes
the epoxidation of methyl oleate in homogeneous media in the
presence or absence of acyl donors, achieving good activities and
selectivities [18a]. In fact, the use of enzymes to perform epoxida-
tion reaction is not new and some articles are found in the literature
[18b–d]. However, to the best of our knowledge, the potential
of lipase enzymes to promote epoxidation reactions of bio-based
compounds in ILs was not exploited. Based on our interest in bio-
based fuels [19] and polymers [20] and in our experience using
enzyme-catalyzed reactions in ILs [10,21a], we disclose herein that
the use of some lipases, from different microorganisms, supported
in hydrophobic (BMI.PF6, BMI.NTf2) or hydrophilic (BMI.BF4) ILs
_
_
NTf2
PF6
BF4
BMI.NTf2
BMI.PF6
BMI.BF4
(hydrophilic)
(hydrophobic)
(hydrophobic)
Fig. 1. Hydrophobic and hydrophilic imidazolium-based ionic liquids (ILs).
results in a heterogeneous catalytic system for the synthesis of
epoxidized methyl oleate derivatives with high yield and selectivity
in short reaction times under an eco-friendly process.
2. Results and discussion
An initial screening was performed using nine different com-
mercially available lipases to promote the epoxide formation
(EMO) and epoxy ring-opening (DIOL) reactions (Scheme 1) in a
hydrophobic IL (BMI.NTf2). The results are summarized in Table 1.
It is interesting to highlight that all reactions carried out in the
presence of acyl donor (acetic acid 10 mol%) gave worse results,
probably due to enzyme instabilization. For this reason we decided
not to use any acyl donor in further reactions under the tested
conditions.
As shown in Table 1, despite the hydrophobicity of the tested
IL, all lipases were successfully supported in the ionic media and
O
Lipase enzyme
O
H O (30%
)
v/v
2
2
+
N
N
_
Methyl oleate
X
O
OH
O
O
HO
O
O
+
DIOL
1,2-diol (
)
EMO
Epoxidized methyl oleate (
)
Scheme 1. Enzyme- (lipase-) catalyzed epoxidation (EMO) and epoxy ring-opening (DIOL) reaction using methyl oleate as the substrate and hydrogen peroxide (30%, v/v)
in different imidazolium-based ILs at 30 ◦C.
Table 1
Epoxidation and epoxy ring-opening using hydrophobic BMI.NTf2 and hydrogen peroxide (30%, v/v) at 30 ◦C.
Ent.
Enzyme
Time (h) and yield (%)
Prod.
1 h
2 h
3 h
4 h
5 h
64
18
62
14
69
14
54
12
67
11
62
12
54
12
53
12
78
9
45
27
62
18
55
20
45
23
46
27
58
15
55
15
50
25
58
14
48
26
54
15
58
13
34
20
47
28
49
32
49
24
33
45
61
20
56
12
58
13
52
21
66
16
47
23
45
25
52
22
47
26
68
10
58
14
34
48
45
29
47
25
48
25
59
16
56
15
57
24
47
23
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
From Aspergillus niger (Amano A.)
1
2
3
4
5
6
7
8
9
From Candida antarctica B (Novozyme 435)
From Candida rugosa (Amano AY)
From Penicillium camembertii (Amano G 50)
From Penicillium roqueforti (R Amano K)
From Pseudomonas cepacia (PS-D Amano I)
From Pseudomonas fluorescens (Amano lipase AK)
From Porcine pancreas (type II, Aldrich)
From Lipase type II (Calf tongue roof)

100
W.S.D. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 98–103
80
70
60
50
40
30
20
10
Lipase type II
EMO
DIOL
1
2
3
4
5
Time (h)
Fig. 2. Observed behavior of EMO and DIOL formation catalyzed by Lipase Type II
(Calf tongue roof) enzyme in BMI.NTf2 at 30 ◦C. Note that the observed behavior is
similar for all tested lipase enzymes (see Table 1).
showed good activity. This means that all enzymes showed a com-
parable behavior. Indeed, the use of this ‘wet’ hydrophobic ionic
liquid gave very interesting results. In the first reaction hour, in all
cases, a high yield of EMO formation and low yield of DIOL are
observed. The results obtained for EMO conversion in the reac-
tion performed using Lipase type II (Table 1, Entry 9) are even
more expressive. It was possible to close the oxirane ring in 78%,
whilst the DIOL formation was accomplished in mere 9% yield.
Additionally, the methyl oleate conversion (EMO + DIOL) ranged
from good to excellent in all cases. Moreover, the requested reac-
tions time are very short. In Fig. 2 (for Lipase type II) we see
a representative graphic of the reaction during a period of five
hours.
It is interesting to point out that the reaction conducted in the
absence of hydrogen peroxide does not result in DIOL formation
independently of the IL tested. This showed that closing the ring is
required prior to the DIOL formation from the oxirane ring (EMO)
catalyzed in the ionic media. Somehow, it is expected and logical.
As can be depicted from Fig. 2 and the data in Table 1, we could
observe that the epoxy ring opening is a consequence of the EMO
formation, as shown in Scheme 2.
Fig. 3. Obtained yields of EMO and DIOL in the first reaction hour under the studied
conditions using ‘wet’ hydrophobic IL BMI.NTf2 at 30 ◦C.
liquid effect. Indeed, the use of pure EMO and H2O2 in BMI.NTf2
results in DIOL formation in poor yields.
Based on an elegant study of the diffusion coefficients of metal
compounds in “wet” and “dry” ILs [22], it was pointed out that
“wet” ILs (as the current case) are nanostructured materials which
allow polar neutral molecules to undergo fast diffusion in polar
or ‘wet regions’ [23]. Additionally, it was explained that ILs must
be understood as nanostructured media when supporting enzymes
[23]. Moreover, some researchers have very recently described that
in the present context, enzymes in water-immiscible ILs should
be also considered as included into hydrophilic gaps of the net-
work [24]. Therefore, the observed stabilization of enzymes could
be attributed to the maintenance of this strong network around the
protein, displaying exceptional synthetic activity and operational
stability [25a].
It is worth noting that the obtained epoxide yield is among the
highest ever published for methyl oleates. Moreover, the reaction
time is very short, rendering this methodology one of the fastest
ever described. Note that higher yields (almost quantitative) but
requiring longer reaction times have already been published ear-
It has earlier been stated that lipases do not perform epoxide
ring opening and that these reactions can be mediated by Epoxide
hydrolases [21b]. As a consequence, it can be attributed to the ionic
Table 2
Epoxidation and epoxy ring opening using ‘wet’ BMI.PF6 and hydrogen peroxide (30%, v/v) at 30 ◦C.
Ent
Enzyme
Time (h) and yield (%)
Prod.
1 h
2 h
3 h
4 h
5 h
67
19
66
15
69
20
60
29
73
22
77
15
67
20
80
11
18
32
72
11
72
15
65
19
65
22
78
16
66
19
68
21
76
16
57
30
68
19
68
8
70
25
67
14
68
18
61
31
70
21
67
20
72
11
74
18
77
15
77
16
70
18
69
20
64
27
82
14
73
19
64
21
81
16
79
14
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
From Aspergillus niger (Amano A.)
1
2
3
4
5
6
7
8
9
From Candida antarctica B (Novozyme 435)
From Candida rugosa (Amano AY)
62
19
73
15
75
18
63
22
66
20
71
12
73
16
From Penicillium camembertii (Amano G 50)
From Penicillium roqueforti (R Amano K)
From Pseudomonas cepacia (PS-D Amano I)
From Pseudomonas fluorescens (Amano lipase AK)
From Porcine pancreas (type II, Aldrich)
From Lipase type II (Calf tongue roof)

W.S.D. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 98–103
101
O
OH
O
O
HO
O
O
reaction
media
H2O
1,2-diol (DIOL)
Epoxidized methyl oleato (EMO)
Scheme 2. Lipase-catalyzed ring opening (DIOL) from EMO in ILs.
lier [25b]. Since we could not avoid the epoxy ring opening under
the studied conditions, it is crucial for our main goal that EMO
be obtained within the first reaction hour, since the best results
were acquired in this period of time. In Fig. 3 we visualize the best
yields of EMO and DIOL in the first reaction hour for all tested
enzymes.
Enzymes such as Candida rugosa (Table 1, Entry 3) and from Peni-
cillium roqueforti (Table 1, Entry 5) afforded EMO in good yields as
well (69% and 67%, respectively). The best DIOL yield was achieved
using the lipase from Candida antarctica B (Table 1, Entry 2) in five
hours (48%). The obtained results indicate that, under the tested
conditions, all studied lipases preferentially promote methyl oleate
conversion into EMO instead of oxirane ring opening to give the
DIOL product.
90
80
70
60
50
40
30
20
10
Penicillium roqueforti
EMO
DIOL
To verify the efficiency of hydrophobic ILs in the epoxidation
reaction and epoxy ring opening, we decided to perform the same
reaction using ‘wet’ hydrophobic BMI.PF6 as the reaction medium.
All results are summarized in Table 2.
1
2
3
4
5
It is very clear from the data in Table 2 that the anion effect
plays a role in the enzyme-catalyzed epoxidation of methyl oleate.
As expected, the anion effect over enzymatic reactions can be very
drastic [26]. Considering the first hour of reaction in BMI.PF6, the
best EMO yield (80%) was obtained when a lipase from Porcine pan-
creas was used (Table 2, Entry 8), whilst in the mean time the DIOL
yield was only 11%. Selecting Lipase type II (Table 2, Entry 9), only
18% of EMO and 32% of the DIOL were produced using the same
media. It is worth mentioning that, when utilizing similar reaction
conditions, however with BMI.NTf2 as reaction media, the yield was
78% and 9% (for Lipase type II), respectively, as shown in Table 1
(Entry 9). The present case exemplifies the importance of anion
selection and screening when using different enzymes.
Another important issue to be considered is the reaction time.
We noted that in BMI.PF6 at the settled temperature (30 ◦C), Lipase
type II (Table 2, Entry 9), lipase from Penicillium roqueforti (Table 2,
Entry 5) and from Porcine pancreas (Table 2, Entry 8) gave the best
EMO yields (79%, 82% and 81%, respectively). Nevertheless, the
required reaction time was 5 h. During the course of the reaction,
the yield increased for Lipase type II (Table 2, Entry 9) and oscillated
by using the lipase from Penicillium roqueforti (Table 2, Entry 5) as
well as that from Porcine pancreas (Table 2, Entry 8), as seen in Fig. 4.
As may clearly be depicted from Fig. 4, Lipase Type II displays a
different behavior. Nevertheless, it also shows a high EMO yield and
low DIOL yield. Moreover, all enzymes tested in BMI.PF6 showed
good conversion rates, affording EMO in very good yields either
within the first or fifth reaction hours, depending on the enzyme.
A better visualization is provided by Fig. 5 for the first and for the
fifth reaction hours.
Time (h)
90
80
70
60
50
40
30
20
10
Porcine pancreas
EMO
DIOL
1
2
3
4
5
Time (h)
80
70
60
50
40
30
20
10
Lipase type II
EMO
DIOL
In the case of the lipase from Candida antarctica B in BMI.PF6
(Table 2, Entry 2), the highest yield was detected in the second
hour (72%). Lipase from Penicillium camembertii (Table 2, Entry 4)
afforded EMO in 73% in the third reaction hour and from Pseu-
domonas fluorescens (Table 2, Entry 7) displayed the highest yield
in the fourth hour (72%). For all other lipases tested in BMI.PF6 as
the reaction media, the maximum yield was reached either in the
first or in the last hour of the established reaction time. The best
1
2
3
4
5
Time (h)
Fig. 4. Observed behavior of EMO and DIOL formation catalyzed by lipases from
Penicillium roqueforti, Porcine pancreas and Lipase type II in BMI.PF6 at 30 ◦C.

102
W.S.D. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 98–103
Table 3
Epoxidation and epoxy ring opening using hydrophilic BMI.BF4 and hydrogen peroxide (30%, v/v) at 30 ◦C.
Ent
Enzyme
Time (h) and yield (%)
Prod.
1 h
2 h
3 h
4 h
5 h
89
–
69
25
31
47
33
36
27
39
28
33
46
31
39
39
32
32
28
40
24
60
29
27
52
44
29
52
52
20
57
3
37
38
29
42
35
41
53
39
34
37
25
28
33
25
42
26
55
2
31
27
26
21
47
22
21
54
59
39
27
22
37
22
30
18
49
17
35
24
45
28
31
30
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
EMO
DIOL
From Aspergillus niger (Amano A.)
1
2
3
4
5
6
7
8
9
27
19
41
20
67
18
26
19
45
47
41
22
34
28
31
37
From Candida antarctica B (Novozyme 435)
From Candida rugosa (Amano AY)
From Penicillium camembertii (Amano G 50)
From Penicillium roqueforti (R Amano K)
From Pseudomonas cepacia (PS-D Amano I)
From Pseudomonas fluorescens (Amano lipase AK)
From Porcine pancreas (type II, Aldrich)
From Lipase type II (Calf tongue roof)
DIOL yield was achieved using lipase from Penicillium camembertii
(Table 2, Entry 4) in the fourth hour (31%).
best result is obtained when the lipase from Aspergillus niger is
employed (Table 3, Entry 1). In the first reaction hour, using BMI.BF4
as reaction medium, we noted the highest yield of EMO in all cases
(89%, Table 3, Entry 1), even considering the results obtained with
hydrophobic BMI.NTf2 or BMI.PF6. The reaction was highly selec-
tive and very fast. Additionally, no DIOL formation could be noted
during this period, indicating the high selectivity of the reaction
under the tested condition. Curiously, the best yield of DIOL for-
mation (among all) was equally obtained using the same enzyme
(Table 3, Entry 1), but in the third reaction hour (60%).
It is worth noting that some authors have previously reported
that the lipase from Aspergillus niger is not efficient at all to perform
transesterification reactions in ILs (no biodiesel formation noted)
[10]. However, it is the best lipase among all tested herein to per-
form the epoxidation reaction in a hydrophilic IL. Since BMI.BF4
is a hydrophilic IL, water is readily available in the presence of the
enzyme to promote other side reaction, that means ester hydrolyze.
However, the ester hydrolysis is very slow when compared to
the epoxidation reaction. After 6 h of reaction, the only enzyme
that promoted the presence of a significant content of free fatty
acid in the reaction bulk is that from Pseudomonas fluorescens,
which displays a 33% of acid content. Less than 10% of free fatty
acid was noted after 6 h of reaction in the presence of all other
enzymes.
Despite the quality of the obtained results in the two ‘wet’
hydrophobic ILs, it was necessary to conduct the same study in a
hydrophilic IL. Generally, considering imidazolium-based ILs, both
cations and anions play a major role in stabilizing enzymes in the
presence of water [27]. Since it is nowadays well accepted, we could
expect a very different behavior using BMI.BF4 as a medium to
support the lipases. All reactions were carried out under the same
conditions (at 30 ◦C and hydrogen peroxide 30%, v/v). The results
are summarized in Table 3.
All reactions performed in BMI.BF4 afforded EMO and DIOL in
distinctive proportions. Nonetheless, it is important to highlight
that reactions carried out in this hydrophilic medium significantly
increased the DIOL yields for all tested lipases. Somehow, this
should be expected, especially because water is now available
in much higher concentrations than those detected when using
hydrophobic ILs. For instance, we observed 52% of DIOL in the third
reaction hour using lipase from Penicillium camembertii (Table 3,
Entry 4).
In a general way, EMO formation ranged from reasonable to
good yields in all cases described in Table 3. Overall, the use of
hydrophilic IL gave poor results when compared to those obtained
using hydrophobic ILs (compare Tables 1–3). Nevertheless, the
Fig. 5. Obtained yield of EMO and DIOL in the first and in the fifth reaction hours under the studied conditions using BMI.PF6 at 30 ◦C.

W.S.D. Silva et al. / Journal of Molecular Catalysis B: Enzymatic 68 (2011) 98–103
103
Finally, for all tested cases, enzymes and ILs could be recov-
ered and reused without any loss of activity. Enzyme was filtered,
washed with acetone and reused without any loss of activity. The
ILs were easily recovered after enzyme filtration and reused several
times after removal of solvent.
was employed: 30% A + 70% B in 0 min, 100% B in 15 min, 50%
B + 50% C in 30 min, followed by isocratic elution with 50% B + 50% C
for the last 20 min. Epoxidation reaction. In a thermo-controlled
bath (30 0.1 ◦C) enzyme lipase (100 mg), IL (1 mL), methyl oleate
(2 mL) and hydrogen peroxide (30%, v/v, 1 mL) were added to a
Schelenk flask. The mixture was stirred for five hours and the prod-
uct removed and directed analyzed by HPLC. Enzyme and IL reuse.
All enzymes could be efficiently recovered and reused. Enzyme can
be filtered, washed with acetone and reused without loss of activ-
ity. The ILs can be easily recovered after filtered the enzyme and
could be reused several times after solvent remove (vacuum).
3. Conclusions
Overall, the use of hydrophobic ILs (BMI.NTf2 and BMI.PF6) allow
the EMO formation in high yields and within a few hours and, in
many cases, in just one hour of reaction. It is preferred to carry out
the EMO formation under the tested conditions, with low yields of
DIOL. The use of a hydrophilic IL (BMI.BF4) has shown that DIOL
formation is viable and competes directly with the EMO formation.
However, the highest yield of EMO (89%) was obtained using the
enzyme from Aspergillus niger in this medium. The reaction takes
place in just one hour. Moreover, the DIOL yield can be increased
in hydrophilic ILs. This result is one of the best ever published
considering both selectivity and reaction time. Moreover, the use
of a hydrophobic IL favors the competition reaction of epoxida-
tion and hydrolysis, but epoxidation is much faster. And free acid
content is less than 10% after 6 h of reaction. It is also notewor-
thy that no other product could be detected during the reaction,
indicating the high selectivity of the catalytic system described
herein.
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General: Enzymes and all other chemicals were purchased from
commercial sources (Amano, Acros or Aldrich) and used without
further purification. We used standard and commonly used pro-
cedures to the synthesis and purification of ILs. Authentic samples
of EMO, DIOL and methyl oleate were used to determine the HPLC
method [28]. HPLC analyses were performed on a Shimadzu LC-20A
Prominence liquid chromatograph equipped with an SPD-M20A
diode-array detector and a four-solvent delivery system. The sol-
vents were filtered through a 0.45-mm Millipore filter prior to
use and degassed by continuous stripping with nitrogen. Injection
volumes of 1 mL and a flow rate of 1 mL min−1 were used in all
analyses. All samples were dissolved in propan-2-ol-n-hexane (5:4,
v/v). All solvents were HPLC-grade and were used as purchased,
without further purification. A Shim-pack VP-ODS (particle size
4.60 mm, 250P 4.6 mm I.D.) column was obtained from Shimadzu.
HPLC method: reservoir A contained water, reservoir B contained
acetonitrile, and reservoir C contained propan-2-ol-n-hexane (5:4,
v/v). A 50-min ternary gradient with two linear gradient steps
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