Enzymatic epoxidation of β-caryophyllene using free or immobilized lipases or mycelia from the Amazon region

Article abstract of DOI:10.1016/j.molcatb.2013.05.021

The chemo-enzymatic epoxidation of the terpene β-caryophyllene is reported herein. This compound can form two products, the mono-epoxide 2 and the di-epoxide 3. Different experimental conditions, varying the source of the lipases (including mycelia from the Amazon region), the oxidizing agents (H ₂O₂ aq. (AHP) or urea-hydrogen peroxide (UHP)) and the substituted acyl donors on the alkyl chain (bromide and alkyl), along with the influence of organic medium, were evaluated. Depending on the experimental conditions the formation of a single product could be obtained. CAL-B was the most efficient catalyst (conv. >99%). When using the commercial lipases product 2 was obtained in conversions of 16-27%, and using the native lipases 2 was obtained in conversions of 20-23%. With the use of mycelia UEA 06 and UEA 53 the conversions were 16 and 21%, respectively. When the 2-bromo alkylated and 2-ethylhexanoic acids were used as acyl donors only the mono-epoxide 2 was obtained in conversions of 14-54% (24 h). AHP was found to be a better oxidizing agent than UHP, a shorter time and lower amount being required to obtain 2 or 3 as the sole product in good conversions (60 up to >99%). The organic solvents were also selective. When using n-hexane the preferred formation of 2 was observed with >99% conversion, and when ethyl acetate or toluene were used the conversion to 3 was also >99% (in 8 and 24 h, respectively).

Full text of DOI:10.1016/j.molcatb.2013.05.021

Journal of Molecular Catalysis B: Enzymatic 95 (2013) 48–54
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enzymatic epoxidation of ␤-caryophyllene using free or immobilized
lipases or mycelia from the Amazon region
Jaqueline Maria Ramos da Silvaa, Thiago Bergler Bitencourtb, Marcelo Alves Moreirac,
Maria da Grac¸ a Nascimentoa,∗
a Departamento de Química, Universidade Federal de Santa Catarina, Florianópolis, SC 88040-900, Brazil
b Departamento de Engenharia de Alimentos, Universidade Federal da Fronteira Sul, Laranjeiras do Sul, PR 85303-775, Brazil
c Departamento de Solos e Recursos Naturais, Universidade do Estado de Santa Catarina – UDESC, Lages, SC 88.520-000, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history:
The chemo-enzymatic epoxidation of the terpene ␤-caryophyllene is reported herein. This compound
can form two products, the mono-epoxide 2 and the di-epoxide 3. Different experimental conditions,
varying the source of the lipases (including mycelia from the Amazon region), the oxidizing agents (H2O2
aq. (AHP) or urea-hydrogen peroxide (UHP)) and the substituted acyl donors on the alkyl chain (bromide
and alkyl), along with the influence of organic medium, were evaluated. Depending on the experimental
conditions the formation of a single product could be obtained. CAL-B was the most efficient catalyst
(conv. >99%). When using the commercial lipases product 2 was obtained in conversions of 16–27%, and
using the native lipases 2 was obtained in conversions of 20–23%. With the use of mycelia UEA 06 and
UEA 53 the conversions were 16 and 21%, respectively. When the 2-bromo alkylated and 2-ethylhexanoic
acids were used as acyl donors only the mono-epoxide 2 was obtained in conversions of 14–54% (24 h).
AHP was found to be a better oxidizing agent than UHP, a shorter time and lower amount being required
to obtain 2 or 3 as the sole product in good conversions (60 up to >99%). The organic solvents were
also selective. When using n-hexane the preferred formation of 2 was observed with >99% conver-
sion, and when ethyl acetate or toluene were used the conversion to 3 was also >99% (in 8 and 24 h,
respectively).
Received 6 March 2013
Received in revised form 2 May 2013
Accepted 21 May 2013
Available online 29 May 2013
Keywords:
Lipases
Epoxidation
␤-Caryophyllene
Organic solvent
© 2013 Published by Elsevier B.V.
1. Introduction
esters [3]. Several methods for the oxidation of monoterpenes in
the presence of various metals as catalysts or MCPBA have been
The oxyfunctionalization of cyclic olefins is of industrial impor-
tance due to the possibility of transforming cheap and readily
available substrates into valuable intermediates for fine chemical
synthesis. Terpenes, which are widely distributed in nature, are of
particular importance due to the fact that their oxidation products –
terpenic aldehydes, alcohols and esters – are used as starting prod-
ucts for fragrances, flavors and therapeutic agents [1]. Epoxides
can undergo various chemical reactions with a variety of nucle-
ophiles and are readily converted into diols, amino-alcohols and
ethers. An important recent application is the use of chiral epox-
ides as intermediates for the production of chiral pharmaceuticals
[2].
reported. Nevertheless, most of these studies use bromide ions as
promoters and/or acetic acid media resulting in complex mixtures
of oxygenated derivatives with very low selectivity. In addition, the
commercial production of MCPBA is under threat owing to its haz-
ardous nature [4]. An alternative method for alkene epoxidation
mediated by an enzyme (especially lipases), in the presence of a
small amount of free fatty acids, was firstly described by Björkling
in the early 1990s [5].
Lipases are the most widely used class of enzymes in biotech-
nology and they show varied stability in the presence of organic
solvents, under extreme pH conditions and in ionic liquids (ILs).
Apart from hydrolysis, lipases can catalyze esterification and trans-
esterification reactions as well as the formation of peroxy-acids in
low-water media [6,7].
The use of immobilized enzymes, particularly lipase originat-
ing from Candida antarctica B (CAL-B), in the chemo-enzymatic
epoxidation of olefins, has been reported [8–12]. This lipase
has been highlighted due to its excellent efficiency in catalyz-
ing the perhydrolysis of octanoic acid. Skouridou et al. used this
lipase to catalyze the formation of peroxy octanoic acid from
Epoxides can be readily prepared from alkenes, through a num-
ber of methods including the use of m-chloroperoxybenzoic acid
(MCPBA), transition metal catalysts and even cyclic seleninate
∗
Corresponding author. Tel.: +55 48 37216844; fax: +55 48 37216850.
E-mail addresses: maria.nascimento@ufsc.br, graca@qmc.ufsc.br
(M.d.G. Nascimento).
1381-1177/$ – see front matter © 2013 Published by Elsevier B.V.
http://dx.doi.org/10.1016/j.molcatb.2013.05.021

J.M.R. da Silva et al. / Journal of Molecular Catalysis B: Enzymatic 95 (2013) 48–54
49
O
O
O
O
R
C
+
R C
O
OOH
OH
H2O2
lipases or mycelia
H2O
3
2
1
R=
CH3(CH2)n
CH3(CH2)3CH
CH2CH3
n = 2,3,9
CH3(CH2)nCH
Br
n = 1,3,5,7,9,11,13,15
Scheme 1. The chemo-enzymatic epoxidation of ␤-caryophyllene (1) by lipases.
the corresponding carboxylic acid and aqueous hydrogen perox-
ide (AHP) in toluene. They varied the amount of AHP over five
cycles of reaction. After five cycles the lipase activity decreased
by 60–90%, depending on the amount of hydrogen peroxide used
[11]. In addition to the use of aqueous hydrogen peroxide, a
complex with urea, called urea hydrogen peroxide (UHP), has
produced good results in the epoxidation of a range of alkenes
[12].
The use of H2O2 (AHP or UHP) as an olefin oxidant often requires
an efficient catalytic process in order to achieve high conversions
and selectivity, because of the poorer reactivity of H2O2 compared
to other classic epoxidizing reagents, such as organic peracids, per-
esters or persulfates [9].
fragrances, shampoos, toilet soaps and other toiletries as well as
in non-cosmetic products such as household cleaners and deter-
gents. Its use worldwide is in the region of <0.1 metric tons per
annum [26]. Park et al. investigated whether ␤-caryophyllene
oxide exerts its anti-cancer effects through the modulation of the
PI3K/AKT/mTOR/S6K1 and MAPK signaling. They observed that CPO
can significantly potentiate the apoptotic effects of various phar-
macological PI3K/AKT inhibitors when employed in combination
in tumor cells [27].
Herein, the study of some experimental parameters which affect
the chemo-enzymatic epoxidation of ␤-caryophyllene (1) to form
the mono- (2) and/or the di-epoxide (3) is reported. The influence
of using different biocatalysts (free lipases and mycelium-bound
lipases), the amount and type of oxidizing agent (UHP or AHP), the
acyl donor, and the use of different organic solvents were evaluated
in an attempt to optimize the process (Scheme 1).
The simplicity of the process and efficiency of the reaction
temperature and pressure allow the oxidation of double bonds of
compounds in situ. There is no need to isolate the peracid and the
reuse of the biocatalyst has shown significant advantages for all
process [8].
2. Experimental data
Silva et al. studied the chemo-enzymatic epoxidation of citronel-
lol catalyzed by CAL-B. The results showed that the organic medium
seemed to be one of the most important parameters in this reaction
[13].
Besides affecting the enzyme activity [14], the solvent is
important because it defines a major part of the environmental
performance of the processes in the chemical industry and also
impacts on cost, safety and health issues [15]. Laane et al. described
that the epoxidation activity is low in relatively polar solvents,
quite variable in solvents intermediate and high in apolar solvents
[14].
The solvent effect was also described by Svedendahl et al. In
this work, the direct epoxidation of but-2-enal and 3-phenylprop-
2-enal in aqueous and organic solution catalyzed by CAL-B was
realized, by activating both hydrogen peroxide and the ␣,␤-
unsaturated aldehyde, bringing them to a suitable position for
reaction. Substrate binding and binding of the reaction inter-
mediates were investigated both by molecular dynamics (MD)
simulations and experimental kinetics. This reaction was fasted in
aqueous solution [16].
The natural sesquiterpene ␤-caryophyllene (BCP) is an interest-
ing substrate due to its chiral centers, double bonds and availability,
since it is found in many essential oils of spices (e.g. cinna-
mon, origanum, black pepper, basil, and cloves) and in some
food/medicinal plants [17–19]. In addition, BCP is often used in
cosmetics, is an FDA-approved food additive and is a component
of Cannabis sativa extract (Sativex), an approved drug in Euro-
pean countries and in Canada [20]. BCP presents well-established
anti-inflammatory [17] and antioxidant effects [21,22] in addi-
tion to its antispasmodic [23], antiviral [24], and local anesthetic
effects [25]. According to Bhatia et al., ␤-caryophyllene alcohol
is the fragrance ingredient used in decorative cosmetics, fine
2.1. Materials
The terpene (␤-caryophyllene) (80%) was purchased from
Sigma–Aldrich. The solvents acetonitrile (99.5%), chloroform
(99.8%), ethanol (99.5%), cyclohexane (99%) and t-butanol (99%)
as well as octanoic (99.5%), dodecanoic (98%) and hexadecanoic
(C16) (98%) acids were acquired from Vetec. Dichloromethane
(99.5%), methanol (99.5%), and n-hexane (98.5%) were obtained
from Synth, t-butyl methyl ether (MTBE) (99.9%) was obtained from
Tedia, ethyl ether (98%) was purchased from Chemis, ethylacetate
(99.8%), N,N-dimethylformamide (DMF) (99%) and dimethylsulfox-
ide (DMSO) (99%) were obtained from Grupo Química and acetone
was obtained from Carlo Herba. Decanoic (98%) and tetradecanoic
acids (98%) were purchased from Fluka and 2-bromopentanoic
(99%), 2-bromohexanoic (97%), 2-bromohexadecanoic (97%) and
2-ethylhexanoic (99%) acids were obtained from Sigma–Aldrich.
Urea-hydrogen peroxide-UHP (percentage given as 30% in H2O2)
was supplied by Sigma–Aldrich and aqueous hydrogen peroxide
30% H2O2 (percentage given as wt% H2O2 in water) was pur-
chased from Vetec. Lipases originating from Candida antarctica B
(CAL-B, Novozym 435, 10,000 PLU/g immobilized on a polyacry-
late resin), Rhizomucor miehei (Lipozyme RM IM, 5-6 BAUN/g)
and Mucor miehei (Lipozyme IM, 5-6 BAUN/g) were donated by
Novozymes. Lipases originating from Burkholderia cepacia (PS-
C Amano I, 1638 U/g; PS Amano SD, 30,000 U/g; PS Amano –
30,000 U/g; PS Amano IM, 500 U/g; PS-C Amano II, 1000 U/g), Rhi-
zopus oryzae (F-AP15, 150 u/mg), Candida rugosa (AY Amano 30,
30,000 u/g), Pseudomonas fluorescens (AK, 25,000 U/g), Aspergillus
niger (A Amano 12, 120,000 u/g) and Mucor javanicus (M Amano
10, 10,000 u/g) were donated by Amano Pharmaceuticals Co. The
mycelium-bound lipases isolated from Amazonian plants (named

50
J.M.R. da Silva et al. / Journal of Molecular Catalysis B: Enzymatic 95 (2013) 48–54
Table 1
mycelia UEA 01, UEA 06, UEA 07, UEA 23, UEA 27, UEA 28, UEA 41,
and UEA 53 UEA 115) were isolated, characterized and kindly
provided by Dr. Sandra Patricia Zanotto of the State Univer-
sity of Amazonas (Universidade do Estado do Amazonas; UEA)
(Manaus-AM, Brasil) [28]. Three native lipases originating from
A. niger (LAN 18.2 U/mL), Rhizopus oligosporus (LRO 14.9 U/mL)
and Mucor hiemalis (lipase 32, 26.4 U/mL) were isolated from soil
micro-organisms in the region of Bueno Brandão (MG, Brazil),
characterized and kindly donated by Dr. Patricia O. Carvalho (USF-
Braganc¸ a Paulista, SP) [29,30].
Evaluation of different lipases in the chemo-enzymatic epoxidation of ␤-
caryophyllene.a
Entry
Lipases
Activity
Conversion (%)b
2
3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
CAL-B
PS-C Amano I
F-AP15
PS-C Amano SD
PS Amano
AY Amano 30
PS Amano IM
M Amano 10
AK Amano 20
PS-C Amano II
A Amano 12
Lipozyme RM IM
Lipozyme IM
LRO
10,000 PLU/g
1638 U/g
150 u/mg
23,000 U/g
30,000 U/g
30,000 u/g
500 U/g
10,000 u/g
25,000 U/g
1000 U/g
120,000 u/g
5–6 BAUN/g
5–6 BAUN/g
14.9 U/mL
18.2 U/mL
0
>99
0
17
16
16
16
23
17
23
25
25
27
24
26
23
20
0
0
0
0
0
0
0
0
0
0
0
0
2.2. General procedure for the chemo-enzymatic epoxidation of
ˇ-caryophyllene
In
a typical chemo-enzymatic epoxidation reaction of ␤-
caryophyllene, 0.6 mL (2.5 mmol) of the substrate, 1.2 mL of
aqueous hydrogen peroxide (30%), 0.16 mL (1.0 mmol) of octanoic
acid and 50 mg of lipase or 100 mg of mycelium, were added to
10 mL of dichloromethane (or other organic solvent when spec-
ified). The reaction was stirred at 150 rpm, at 25 ◦C, for 24 h.
Reaction samples were periodically collected and analyzed by gas
chromatography (GC) (Agilent Technology 7820 A) with flame ion-
ization detection. The separation was performed using a Shimadzu
CBP-5-M25-025 m column, with a column temperature program
of 80–250 ◦C (10 ◦C/min). The injector and detector temperatures
were set at 280 ◦C and 290 ◦C, respectively. The flow rate of the car-
rier hydrogen gas was of 7 mL/min, resulting in an analysis time of
15 min (1 Rt = 6.5 min, 2 Rt = 8.5 min, 3 Rt = 11 min). To obtain the
pure products, the reaction mixture was washed with saturated
Na2CO3 and purified by column chromatography using a mixture
of n-hexane:ethyl acetate (7:3, v/v) as the eluent. The products were
fully characterized by 1H- and 13C NMR, and the spectroscopic data
compared with those reported in literature [31].
LAN
0
a
Reaction conditions: ␤-caryophyllene (2.5 mmol), 30% H2O2 (5.0 mmol),
octanoic acid (1.0 mmol), lipase (50 mg), dichloromethane (10 mL), 150 rpm, r.t.
(∼25 ◦C).
b
Determined by GC.
However, when CAL-B (Table 1, entry 1) was used as the bio-
catalyst, di-epoxide 3 was obtained as a single product with >99%
conversion in 24 h of reaction.
The epoxidation of ␤-caryophyllene was then carried out using
nine mycelium-bound lipases from the Amazon region. The results
are presented in Table 2. In the reactions catalyzed by mycelia, the
mono-epoxide 2 was obtained as a single product. After 24 h of reac-
tion, the conversion degrees were 2–21%. In the case of the mycelia
UEA 06 and UEA 53, the conversions were 16 and 21%, respectively.
These results were also similar to those obtained with some com-
mercial lipases, such as PS Amano (16%) and AY Amano 30 (23%).
As observed in Table 2, the conversion degrees increased over time.
For example, after 168 h of reaction, the highest conversions were
achieved using the mycelium UEA 06 (64%), followed by UEA 23
(55%), UEA 01 (49%) and UEA 41 (48%).
These data show that both of the native lipases (LAN and
LRO) and the mycelia have great potential for use in epoxida-
tion reactions. This is the first report which uses these native
lipases and mycelia in epoxidation reactions in the preparation of
␤-caryophyllene epoxides. Also, these results are of interest consid-
ering the huge diversity of microorganisms which have not yet been
explored.
Mono-epoxide 2: (41%, 225 mg); m.p.:62–64 ◦C (Lit. [31]
62–63 ◦C) 1H NMR (400 MHz, CDCl3): ␦(ppm) 4.97 (1H, s), 4.85 (1H),
2.91(1H, q, J 10.4 Hz,), 2.58 (1H, d, J 9,2 Hz), 2.36 (1H, m), 2.15 (1H, q,
J 4,4 Hz), 2.11 (2H, m) 1.80–1.55 (5H, m), 1.34–1.29 (5H, m) 0.95 (6H,
m). 13C NMR (400 MHz, CDCl3): ␦ (ppm) 62.0, 59.0, 58.0, 56.0,49.0,
47.0, 39.0, 35.0, 33.0, 31.0, 30.0, 27.0, 25.0, 22.0, 16.0.
Di-epoxide 3: (38%, 225 mg); m.p.: 73–75 ◦C (Lit. [31] 72–73 ◦C)
1H NMR (400 MHz, CDCl3): ␦ (ppm) 3.08, (1H, q, J 4.8 Hz,), 2.99 (1H,
d, J 2.4 Hz), 2.89 (1H, q, J 4,8 Hz), 2.67 (2H, t, J 3,6 Hz), 2.57 (1H, d, J
5,2 Hz), 2.34 (1H, m), 2.22–2.06, (5H, m), 2.01 (1H, s), 1.81–1.41 (5H,
m), 1.29 (3H, s), 0.94 (3H, s). 13C NMR (400 MHz, CDCl3): ␦ (ppm)
63.0, 60.0, 50.0, 49.0, 40.0, 39.0, 34.0, 30.1, 29.8, 29.7, 27.2, 17.0.
The endocyclic epoxidation of ␤-caryophyllene with Nema-
nia aenea, Diplodia gossypina and Chaetomium cochliodes has been
3. Results and discussion
Some experimental parameters which may influence the
chemo-enzymatic epoxidation of ␤-caryophyllene to form mono-
2 and/or di-epoxide 3 will be discussed below.
Table 2
Conversions to mono-epoxide (2) obtained in the chemo-enzymatic epoxidation of
␤-caryophyllene using different mycelia.a
Conversion to 2 (%)b
3.1. Screening of lipases and mycelia
Entry
Mycelium
24 h
72 h
120 h
168 h
In a first approach, thirteen commercially available lipases and
three native lipases were used to obtain epoxides derived from ␤-
1
2
3
4
5
6
7
8
9
UEA 01
UEA 06
UEA 07
UEA 23
UEA 27
UEA 28
UEA 41
UEA 53
UEA 115
14
16
13
8
38
49
38
47
2
42
44
34
29
43
59
37
49
8
40
47
41
40
49
64
40
55
9
45
48
41
46
caryophyllene using H2O2
(30%) as an oxidant agent in 24 h of
aq
reaction (Table 1). Using the commercial lipases PS-C Amano I, F-
AP15, PS-C Amano SD, PS Amano, AY Amano 30, PS Amano IM, M
Amano 10, AK Amano 20, PS-C Amano II, A Amano 12, Lipozyme
RM IM, and Lipozyme IM (Table 1, entries 2–13) the mono-epoxide
2 was obtained in moderate conversions which ranged from 16 to
27%. Similar results were obtained with the use of non-commercial
lipases (Table 1, entries 14,15), such as LAN (20%) and LRO (23%). It
should be noted that these data are similar to those obtained with
commercial lipases and di-epoxide 3 was not detected.
2
11
10
21
15
a
Reaction conditions: ␤-caryophyllene (2.5 mmol), 30% H2O2 (5.0 mmol),
octanoic acid (1.0 mmol), mycelia (100 mg), dichloromethane (10 mL), 150 rpm, r.t.
(∼25 ◦C).
b
Determined by GC.

J.M.R. da Silva et al. / Journal of Molecular Catalysis B: Enzymatic 95 (2013) 48–54
51
Table 3
reported, but showed very low productivities because of the strong
Influence of time on the chemo-enzymatic epoxidation ␤-caryophyllene (1).
antifungal activity of ␤-caryophyllene and ␤-caryophyllene epox-
ide. It is unlikely that antifungal agents will be produced with fungi
because of the apparent strong product inhibition [32–34].
Considering the above results and in order to evaluate some
other experimental parameters which may influence the formation
of mono- or di-epoxide derived from ␤-caryophyllene, CAL-B was
selected as the biocatalyst.
Entry
Time (h)
AHP
UHP
2
3
2
3
1
2
3
4
5
2
4
6
8
24
60
32
34
13
nd
nd
31
58
83
99
16
50
60
82
14
nd
nd
nd
8
86
Reaction conditions: ␤-caryophyllene (2.5 mmol), UHP or AHP (5.0 mmol), octanoic
3.2. Influence of acyl donors
acid (1.0 mmol), CAL-B (50 mg), dichloromethane (10 mL), r.t., nd = not detected.
The stability of the enzyme may also be influenced by the size of
the acyl donor [35]. In the next part of this study, the influence of
the acyl donor chain length on the chemo-enzymatic epoxidation
of 1 using CAL-B as the biocatalyst and AHP as an oxidant agent was
investigated. As previously reported, the formation of peroxy-acid
is an important step in this reaction [5,9].
Thus, considering the above results, octanoic acid was selected
as the acyl donor for use in the subsequent experiments, which
included the influence of the type and amount of oxidizing agent
and organic solvent. The use of octanoic acid as an acyl donor has
been previously described in the literature. [8,11,13]
In this study, the following alkyl acids were used as acyl donors:
acetic (C2), butyric (C4), hexanoic (C6), octanoic (C8), decanoic
(C10), dodecanoic (C12), tetradecanoic (C14) and hexadecanoic
(C16). In 24 h of reaction only the di-epoxide 3 was formed in
conversions of >99% using the various alkyl acids (C4-C16), except
when acetic acid was the acyl donor where both the mono- (2)
and di-epoxide (3) were detected, in conversions of 80% and 20%,
respectively. It is well described in the literature that acetic acid is
not the best acyl donor in enzymatic reactions because it may be
aggressive toward the enzyme, forming strong bonds at the active
site [7].
Another important factor to be considered is the steric effect
of the acyl donor. Bulky chains, in general, might reduce the rate
of nucleophilic attack by H2O2 which consequently reduces the
reaction rate [36]. Thus, in order to expand the studies related to
the influence of acyl donors, 2-ethylhexanoic, 2-bromo-valeric, 2-
bromo-hexanoic and 2-bromo-hexadecanoic acids were used to
evaluate both steric and electronic effects on the epoxidation of
␤-caryophyllene (1).
With the use of 2-ethyl-hexanoic acid only the product 2 was
obtained, in 14% conversion in 24 h reaction. This result reinforces
the fact that the formation of peroxy-acid is an important step in
the formation of the corresponding epoxide. In this case, steric
hindrance was most likely the parameter which influenced the
low formation of 2. This data is also consistent with those pre-
sented by Hollmann et al., who evaluated the CAL-B activity in
esterification reactions with methyl valeric acid at positions ␣-, ␤-,
␥-with octan-1-ol. The enzyme activity showed a great dependence
on the position of the methyl group. When ␣- and ␤-substituted
acids were used, the product conversions were low (<25%), and it
was postulated that the substituents in these positions hinder the
proper connection of the substrate at the active site of the enzyme
[37].
When 2-bromo-valeric, 2-bromo-hexanoic and 2-bromo-
hexadecanoic acids were used, only mono-epoxide 2 was formed,
in conversions of 47, 54 and 36%, respectively, in 24 h of reaction.
These results probably reflect both the electron withdrawing effect
of the halogen atom close to the carboxyl group and the steric effect
of the alkyl chain. Recently, it was described that the CAL-B activ-
ity may be strongly influenced by the presence of strong acids. In
general, acids with pKa values lower than 4.8 decreased the lipase
activity. These acids can protonate the aspartate or glutamate at the
active site of CAL-B. If one of these residues is protonated, they can-
not act effectively in the catalytic triad of CAL-B to activate Ser105
(via His224) for the nucleophile attack and thus the lipase activity
is decreased in the presence of strong acids. This also explains the
lower conversion (80%) when acetic acid (pKa 4.75) was used as the
acyl donor rather than octanoic acid (99%), in 24 h of reaction [37].
3.3. The influence of the type and amount of oxidizing agent
The type and concentration of peroxide donor used in chemo-
enzymatic epoxidation are important factors. A high concentration
of peroxide donor can cause inhibition of the enzyme by changing
its tertiary and/or quaternary structure. In addition, an excess of
peroxides can cause changes in the substrate reactivity through
direct oxidation and the peroxide, depending on its strength
(expressed as the % of free oxygen), can also change the course
of the enzymatic reactions through the excessive production of
peroxy-acids [12,36].
Thus, the chemo-enzymatic formation of 2 and 3 using two dif-
ferent peroxide donors, AHP (aqueous hydrogen peroxide – 30%)
and UHP (urea hydrogen peroxide – 30%), was investigated as a
function of the reaction time. Aliquots were withdrawn at 2, 4, 6, 8
and 24 h and analyzed by GC. The results are presented in Table 3.
With the use of AHP or UPH, mono-epoxide 2 was obtained as
a single product in conversions of 60 and 16%, respectively, in 2 h
of reaction (Table 3, entry 1). Also, in the case of UPH, only mono-
epoxide 2 was obtained in 4 and 6 h of reaction forming the product
in conversions of 50 and 60%, respectively (Table 3, entries 2 and
3). However, after 4 and 6 h using APH as the oxidizing agent, both
products 2 (32 and 31%, respectively) and 3 (31 and 58%, respec-
tively) were formed (see also Table 3, entries 2 and 3).
In relation to the results obtained after 8 h of reaction, the con-
versions into mono-epoxide 2 were 13% and 82% and di-epoxide 3
were 83% and 8% for AHP and UHP, respectively (Table 3, entry 4).
However, in 24 h of reaction, 3 was the main product, which was
formed in conversions of 99% and 86%, respectively (Table 3, entry
5).
The above results show that the formation of 2 and 3 was time
dependent. The data also revealed that, in general, the formation
of mono-epoxide 2 was favored in the presence of UHP, since this
reagent releases the oxidant agent in a more controlled manner
[12]. On using AHP, the formation of di-epoxide 3 was favored. As
recently described, the H2O2 concentration used determines the
rate and amount of peracid formed, which, in turn, influences the
efficiency of the chemo-enzymatic reaction [36].
In the next part of this study, the influence of each oxidant
amount was evaluated in the epoxidation of ␤-caryophyllene. Fig. 1
presents the conversions into products 2 and 3 as a function of UPH
(in mmol).
It was observed that with an increase in the amount of UHP
from 1 to 3 mmol the conversion to mono-epoxide also increased,
from 20 to 96%, respectively. For this amount of UHP the two prod-
ucts were detected, and from 5.0 to 16 mmol of oxidizing agent the

52
J.M.R. da Silva et al. / Journal of Molecular Catalysis B: Enzymatic 95 (2013) 48–54
Table 4
Effect of organic solvent on the chemo-enzymatic epoxidation of ␤-caryophyllene.a
100
80
60
40
20
0
Entry
Solvent (Log P)c
Conversion (%)b
8 h
24 h
2
3
2
3
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
n-Hexane (3.50)
Cyclohexane (3.20)
Toluene (2.50)
Chloroform (2.00)
t-Butanol (1.45)
MTBE (1.43)
Dichloromethane (0.93)
Ethyl ether (0.85)
Ethyl acetate (0.68)
THF (0.49)
>99
>99
0
62
59
>99
19
99
0
82
>99
98
>99
0
0
0
>99
38
0
0
81
0
>99
0
0
0
0
>99
76
0
0
24
>99
>99
0
0
64
93
0
90
0
>99
92
>99
29
0
7
>99
10
>99
0
8
0
71
0
0
Acetone (−0.23)
Ethanol (−0.24)
Acetonitrile (−0.33)
Methanol (−0.76)
DMF (−1.00)
0
4
8
12
16
UHP (mmol)
0
0
0
7
0
22
0
Fig. 1. Influence of the amount of UHP on the chemo-enzymatic epoxidation of
␤-caryophyllene to form the mono-epoxide 2 (ꢀ) and di-epoxide 3 (᭹). Reaction
conditions: ␤-caryophyllene 2.5 mmol, UHP 1.0–15.0 mmol, octanoic acid 1.0 mmol,
CAL-B 50 mg, dichloromethane 10 mL, r.t. 24 h.
DMSO (−1.30)
0
a
Reaction conditions: substrate 2.5 mmol, H2O2 5 mmol, octanoic acid 1 mmol,
CAL-B 50 mg, solvent 10 mL, 150 rpm, 8 and 24 h, r.t. (∼25 ◦C).
b
Conversions were determined by GC.
Ref. [14].
c
di-epoxide 3 was obtained as the main product in conversions of
87–96%.
The results obtained using AHP as the peroxide donor are pre-
sented in Fig. 2.
of the corresponding peroxy-acid was also favored and thus the
formation of 3 increased (87–99%), in 24 h of reaction.
Thus, the choice of peroxide type and amount proved to be
important parameters in this reaction, in which products 2 or 3
can be selectively obtained in good conversion degrees.
As can be observed, with 2.0 mmol of AHP the mono-epoxide
was obtained with a conversion degree of 93%. With the use of
larger amounts of AHP, ranging from 2.5 to 15 mmol, both products
were formed. The mono-epoxide 2 was obtained in 81–26% and the
di-epoxide 3 in 19–74% conversions. In the case of 4.0 mmol of AHP
the di-epoxide was obtained as the main product, in conversion
degrees of 94 to 99%. These results were to be expected considering
that AHP is considered to be a more energetic oxidant agent than
UHP.
The results show that the conversion into 2 and/or 3 was
dependent on the amount and type of peroxide donor employed.
In both cases, in reactions in which the amount of peroxide was
< 2.5 mmol, mono-epoxide was the main product. With the use of
between 3–4 mmol of AHP and UHP the regioselectivity decreased
and a mixture of products was detected (Figs. 1 and 2). As previ-
ously noted, on using more than 5 mmol of peroxide the formation
3.4. Effects of the organic medium
The influence of the organic medium was then evaluated. It
is well established that enzyme activity is strongly affected by
the choice of organic solvent [13,14,16]. Log P (logarithm of the
partition coefficient of the solvent for the standard octanol/water
two-phase system) is the most useful parameter to classify the
biocatalytic reaction of a solvent [14].
Thus, in order to evaluate this effect in the epoxidation of
␤-caryophyllene, sixteen solvents with different polarities were
selected and the conversions to epoxides 2 and 3 in 8 and 24 h are
presented in Table 4.
When dichloromethane (log P = 0.93) and chloroform
(log P = 2.00) were used both products were obtained, with
conversions of 19–62% for 2 and 38–81% for di-epoxide 3, in 8 h
of reaction. With the use of n-hexane (log P = 3.50), cyclohexane
(log P = 3.20), t-butanol (log P = 1.45), MTBE (log P = 1.43), ethyl
ether (log P = 0.85), THF (log P = 0.49), acetone (log P = −0.23),
ethanol (log P = −0.24), acetonitrile (log P = −0.33) and DMF
(log P = −1.00), the mono-epoxide 2 was obtained as a single
product. With the use of t-butanol, THF, DMF and ethanol, the con-
versions were of 59, 82, 98, and 7%, respectively (Table 4, entries
5, 10, 12 and 15). On using the other solvents, the conversions
were all >99%. It is interesting to note that with the use of both
toluene and ethyl acetate, the di-epoxide 3 was obtained as a
single product with conversions of >99% (Table 4, entries 3 and 9).
Within 24 h of reaction using toluene, chloroform,
dichloromethane or ethyl acetate the di-epoxide 3 was obtained in
>99% conversion (Table 4, entries 3, 4, 7 and 9). In the same reaction
time and using n-hexane, THF or ethanol only mono-epoxide 2
was formed with conversions of >99% (Table 4, entries 1, 10 and
12).
100
80
60
40
20
0
0
4
8
12
16
H2O2(mmol)
When the epoxidation reaction was carried out in the presence
of cyclohexane, MTBE, diethyl ether, acetone or acetonitrile, both
products were obtained but, in most cases, with the predominant
Fig. 2. Effect of the amount of H2O2 (30%) in the chemo-enzymatic epoxidation of
␤-caryophyllene to form the mono-expoxide 2 (ꢀ) and di-epoxide 3 (᭹). Reaction
conditions: as in Fig. 1, except for AHP (1.0–15.0 mmol).

J.M.R. da Silva et al. / Journal of Molecular Catalysis B: Enzymatic 95 (2013) 48–54
53
formation of mono-epoxide 2 (Table 4, entries 2, 6, 8, 11 and 13).
Interestingly, with the use of acetonitrile the main product was the
di-epoxide 3 with 71% conversion.
organic medium. Depending on the experimental conditions, only
one epoxide (2 or 3) was obtained, this being an advantage associ-
ated with the use of biocatalysts in these reactions.
When a more polar solvent, such as DMF, was used the degrees of
conversion into 2 were 7 and 22% at 8 and 24 h, respectively (Table 4,
entry 15). In the presence of methanol and DMSO no product was
detected (Table 3, entries 14 and 16).
These interesting results indicate that the appropriate choice of
organic solvent and reaction time may promote the selective for-
mation of only one product. However, no direct relationship with
the log P values was detected. In other studies reported in the liter-
ature the solvent was found to affect the formation of products in
enzyme-catalyzed reactions, but with no direct correlation to log P
[38–40].
In addition to the polarity of the organic solvents, problems
related to the reagents and or product solubility must be con-
sidered and obtaining an ideal combination of these parameters
can be a difficult task. For example, as presented in Table 4, good
results were also achieved when polar solvents such as MTBE,
dichloromethane, acetone, ethyl acetate, ethanol and acetonitrile
were used. Using these solvents it is expected a higher solubility of
the oxidizing agent (PHA), thus a higher production of the octanoic
peroxy acid and consequently an increase in the production of cor-
responding epoxides 2 and 3.
However, besides the selectivity, the solvent must be as “green”
as possible. The idea of “green” solvents expresses the goal to min-
imize the environmental impact resulting from the use of solvents
in chemical production [15].
In this regard, according to Hellweg et al., dichloromethane
and chloroform cannot be considered as good solvents. The
use of chlorinated solvents should be avoided because, along
with nitrobenzene, they have the potential for long-term and
widespread exposure [41]. In this study, with the use of the
above cited solvents product 3 was obtained with a conversion of
>99%.
As reported, to obtain mono-epoxide 2, hexane was the most
appropriate solvent, the product being formed in a conversion
of >99% in 8 h. Hexane can be considered as an environmentally
favorable solvent. To produce only the di-epoxide 3 in conver-
sion degrees of >99%, toluene can be selected, also considering the
assessment of environmental parameters [15,41,42].
Finally, the results of this study demonstrate that the organic
medium is an important parameter in chemo-epoxidation reac-
tions catalyzed by lipases. Bearing in mind the goal of minimizing
the environmental impact, depending on the medium, only one
product (2 or 3) could be obtained with high selectivity and con-
version degrees.
Acknowledgements
This study was supported by Universidade Federal de Santa
Catarina (UFSC-Brazil), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq-Brazil), CAPES and INCT-Catalysis
which provided financial support and scholarships. We also are
grateful to Amano Pharmaceutical Co. (Japan), Novozymes (Brazil)
for the donation of lipases, Dr. Patrícia O. Carvalho of Universidade
de São Francisco (USF-Brazil) for the donation of lipases and Dr. San-
dra P. Zanotto of Universidade Estadual do Amazonas (UEA-Brazil)
for the donation of mycelia.
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