Lipase-mediated epoxidation of the cyclic monoterpene limonene to limonene oxide and limonene dioxide

Article abstract of DOI:10.5560/ZNB.2012-0167

Limonene is an industrially interesting monoterpene that accumulates in bulk quantities as byproduct of the fruit juice industry. The corresponding epoxides are versatile synthetic intermediates and additives for the chemical industry. Due to a number of disadvantages of classical chemical epoxidation including serious safety issues and unwanted side-reactions, we here used a mild lipasecatalyzed chemo-enzymatic epoxidation system, with either free or different immobilized forms of Candida antarctica lipase B. Full limonene conversion (° 98%) was easily achieved at 40°C within less than 24 h. The enzymatic activities in the formation of limonene monoxide significantly varied from either 1-3.4 U/mg_biocatalyst at r.t. or from 1.7-4.9 U/mg_biocatalyst at 40°C. For the first time we showed that it is possible to generate high amounts of limonene dioxide (～ 30% with CalB on carrier 350 at 40°C) using this mild lipase-mediated epoxidation method. Enzyme activities and limonene dioxide yields strongly depend on the nature of the selected enzyme carrier, the immobilization method and the reaction temperature.

Full text of DOI:10.5560/ZNB.2012-0167

Lipase-mediated Epoxidation of the Cyclic Monoterpene Limonene to
Limonene Oxide and Limonene Dioxide
Lars O. Wiemanna, Christina Faltla and Volker Siebera,b
a
Fraunhofer Institut fu¨r Grenzfla¨chen- und Bioverfahrenstechnik IGB, Projektgruppe BioCat,
Schulgasse 11a, 94315 Straubing, Germany
Technische Universita¨t Mu¨nchen, Lehrstuhl Chemie biogener Rohstoffe, Schulgasse 16, 94315
b
Straubing, Germany
Reprint requests to Dr. Lars Wiemann. Phone: +49 9421 187-353. Fax: +49 9421 187-310.
E-mail: lars.wiemann@igb.fraunhofer.de
Z. Naturforsch. 2012, 67b, 1056 – 1060 / DOI: 10.5560/ZNB.2012-0167
Received June 18, 2012
Dedicated to Professor Heribert Offermanns on the occasion of his 75th birthday
Limonene is an industrially interesting monoterpene that accumulates in bulk quantities as by-
product of the fruit juice industry. The corresponding epoxides are versatile synthetic intermediates
and additives for the chemical industry. Due to a number of disadvantages of classical chemical
epoxidation including serious safety issues and unwanted side-reactions, we here used a mild lipase-
catalyzed chemo-enzymatic epoxidation system, with either free or different immobilized forms of
Candida antarctica lipase B. Full limonene conversion (> 98%) was easily achieved at 40 ◦C within
less than 24 h. The enzymatic activities in the formation of limonene monoxide significantly var-
ied from either 1 – 3.4 U/mgbiocatalyst at r. t. or from 1.7 – 4.9 U/mgbiocatalyst at 40 ◦C. For the first
time we showed that it is possible to generate high amounts of limonene dioxide (∼ 30% with CalB
on carrier 350 at 40 ◦C) using this mild lipase-mediated epoxidation method. Enzyme activities and
limonene dioxide yields strongly depend on the nature of the selected enzyme carrier, the immobi-
lization method and the reaction temperature.
Key words: Terpene, Biocatalysis, Chemo-enzymatic Epoxidation, Enzyme Immobilization,
Renewable Resources
Introduction
The ongoing “greening” of chemical production
both, are already in use in the polymer industry as reac-
tive diluents in cationic and UV-cure applications [6].
In classical chemical synthesis organic peroxides
processes currently enhances research activities that and metal catalysts or peroxy acids such as m-
try to open novel biocatalytic or combined chemo- chloroperbenzoic acid (m-CPBA) are added to gain
enzymatic routes for the conversion of sustainable re- the corresponding epoxides from alkenes [7]. m-CPBA
sources. In this context plant-derived monoterpenes are still is considered to be the work horse in synthetic
industrially interesting compounds as they accumu- chemistry, although it is expensive and difficult to han-
late in bulk amounts as extracts from biogenic waste dle, but most other peroxy acids are less stable and
streams. Common examples are pinene from technical more dangerous [8]. Moreover these techniques exhibit
forestry resins and wood pulp [1] and limonene as an a high potential to catalyze unwanted hydrolytic side
inexpensive by-product from the citrus fruit juice in- reactions. If possible the handling of the hazardous
dustry [2, 3]. The value of these bio-based compounds peroxy acids on an industrial scale generally is to be
can be increased via addition of further functionalities, avoided [7, 9, 10]. Therefore, on an industrial level,
e. g. by producing the corresponding epoxides as versa- epoxides are typically synthesized either by direct ox-
tile synthetic intermediates and chiral building blocks idation, by the chlorohydrin method or by the in situ-
for the chemical industry [4, 5]. Moreover, limonene generation of peroxy acids from formic or acetic acid
oxide and limonene dioxide, as well as mixtures of and hydrogen peroxide, and often with addition of min-
c
ꢀ 2012 Verlag der Zeitschrift fu¨r Naturforschung, Tu¨bingen · http://znaturforsch.com
Brought to you by | New York University Bobst Library Technical Services
Authenticated
Download Date | 5/20/15 1:35 PM

L. O. Wiemann et al. · Lipase-mediated Epoxidation of Limonene
1057
limonene to limonene monoxide, but also to gain sig-
nificant amounts of limonene dioxide (Scheme 1).
Results and Discussion
Chemo-enzymatic epoxidation of limonene
In our previous studies (unpublished data) we found
that the best results for the lipase-catalyzed epoxi-
dation of limonene (e. g. 25 mM) were obtained with
Candida antarctica lipase B (CalB) in reaction sys-
tems that use toluene as solvent with a double ex-
cess of caprylic acid (e. g. 50 mM) and H2O2 (e. g.
50 mM, from a 30% aqueous solution) as co-substrates
for the lipase-catalyzed in situ-generation of peroxy
acids, that in turn catalyze the Prileschajew epoxi-
dation of limonene to limonene monoxide. Thereby
we also observed a significant difference between
the free lyophilized form of Candida antarctica li-
pase B (CalB) and the carrier-bound lipase-preparation
Novozyme435, where CalB is adoptively bound to
a PMMA-carrier. Based on these results we now
used several different carrier systems exhibiting vary-
ing properties for the immobilization of CalB. These
enzyme carriers were used for the epoxidation of
limonene in the reaction system described above.
Scheme 1. Lipase-mediated epoxidation of limonene to
limonene oxide and limonene dioxide.
eral acids [7]. In consequence this leads to the accu-
mulation of large amounts of acidic and chlorinated
waste streams, not to mention the serious safety issues
and the problems with the formation of unwanted side
products.
Already two decades ago Bjo¨rkling et al. [11, 12]
showed that some lipases (EC 3.1.1.3) are able to form
peroxy acids from carboxylic acids in organic solvents
if H2O2 is present. Thus, hazardous peroxy acids can
be produced in situ in organic solvents under mild con-
ditions and used directly in the catalyzed epoxidation
of organic substrates such as alkenes via the Prilescha-
jew reaction. The first industrially important products
such as environment-friendly PVC stabilizers and sol-
ubilizers on the basis of epoxidized soybean oil have
successfully been produced via this chemo-enzymatic
method [13]. Further work has also proven the gen-
eral feasibility of this approach for the epoxidation of
cyclic monoterpenes [4, 5, 8, 14].
Enzyme immobilization
CalB was immobilized on 3 different carriers either
by adsorption (carrier C435 and carrier EC1) or by co-
In this work, we investigated different commer- valent binding (carrier 350). The immobilization pro-
cially available carrier systems for the immobilization cedure is described in the Experimental Section, and
of Candida antarctica lipase B and compared the ef- details on the applied carrier types and properties are
ficiency of the corresponding enzyme carriers in the shown in Table 1. All enzyme carriers were of spheri-
epoxidation of the cyclic monoterpene limonene. We cal shape with average sizes of 300 – 1000 µm for fa-
report for the first time on successfully shifting the cilitated handling in technical applications and were
product range to reach not only full conversion of made out of solid polyacrylic materials.
Table 1. Properties of enzyme
carriers.
Name
Binding
type
Carrier
material
Functional
group
Particle
size (µm)
Manufacturer
CalB
NZ435
EC1
–
–
–
lyophilized
enzyme powder
300 – 1000
c-LEcta
(Germany)
Novozymes
adsorption
PMMA
–
(Denmark)
adsorption
(non-ionic)
adsorption
(cationic)
covalent
polyacrylic
polyacrylic
polyacrylic
carboxylic
ester
carboxylic
acid
epoxide
(apolar)
350 – 700
350 – 700
300 – 700
Chiral Vision
(The Netherlands)
Chiral Vision
(The Netherlands)
Chiral Vision
(The Netherlands)
C435
350
Brought to you by | New York University Bobst Library Technical Services
Authenticated
Download Date | 5/20/15 1:35 PM

1058
L. O. Wiemann et al. · Lipase-mediated Epoxidation of Limonene
Biocatalyst Total activity per Specific activity at r. t. Total activity per Specific activity at 40 ◦C
Table 2. Biocatalytic activi-
ties for the epoxidation of
limonene to limonene monox-
ide calculated from the initial
product formation rates.
assay at r. t. (U)
(U/mgcarrier
)
assay at 40 ◦C
(U/mgcarrier)
CalB lyo
NZ435
EC1
C435
350
51.4
167.1
109.2
170.6
118.9
1.0
3.3
2.2
3.4
2.4
84.9
208.2
214.0
244.7
172.5
1.7
4.2
4.3
4.9
3.5
A determination of enzyme loadings via Brad- limonene dioxide were also generated. Figure 1 shows
ford [15] was not feasible, probably due to the dis- the 24 h kinetics of the chemo-enzymatic limonene
turbing influence of undefined additives and stabilizing conversion using biocatalyst 350 at 40 ◦C as an
agent in the lyophilized enzyme powder from c-LEcta example. The dashed line indicates total limonene
(Leipzig/Germany). The protein content given by the conversion by summing up the amount of both
manufacturer is 10 – 20%.
products (monoxide and dioxide). Thus full limonene
conversion was reached after approximately 8 h in
this case. In the first 1 to 1.5 h of the reaction more
than 60% of limonene monoxide is generated, but no
limonene dioxide yet. After 2 h, when about 70% of
Chemo-enzymatic limonene epoxidation (limonene
monoxide)
The lyophilized CalB powder showed the lowest the limonene was converted to limonene monoxide,
specific activity (1 U/mgenzyme) in the synthesis of the concentration of limonene dioxide slowly started to
limonene monoxide at r. t. All biocatalytic systems increase until it finally reached its maximum of 27.4%
where CalB was immobilized on a carrier (NZ435, after 24 h. On the other hand, limonene monoxide
EC1, C435 and 350) showed significantly higher spe- already reached its maximum of 85% after 6 – 7 h and
cific activities, ranging from 2.2 to 3.4 U/mgcarrier then started to decrease to 72.6% as it is converted
(Table 2). Assuming maximum enzyme loadings of to the dioxide. Although notable variations in the
1 – 10% on the carriers, the real specific activities in ratios of limonene monoxide/dioxide after 24 h were
U/mgprotein should be by far higher. Obviously the sur- observed for the different biocatalysts (Figs. 2 and 3),
face properties of the acrylic carriers have a positive the basic kinetics were rather similar to the one shown
impact on the micro environment of the biocatalyst in Fig. 1.
that significantly enhances the initial product forma-
Fig. 2 shows the results with the different biocat-
tion rates. Moreover we observed another considerable alysts for limonene conversion after 24 h at r. t. The
increase in the specific activity by simply elevating the highest amounts of limonene dioxide of 14.6 and
reaction temperatures to 40 ◦C. Thus we reached the 13.6% were detected for CalB lyo and 350, respec-
high specific activity of 4.9 U/mgcarrier in case of the tively, whereas C435 only reached 7.1%, but interest-
C435 carrier (Table 2). No conversion of limonene was ingly C435 showed the highest activity in the initial
observed in the absence of Candida antarctica lipase B
as a biocatalyst.
Yields of limonene conversion to limonene monoxide
and limonene dioxide
Nearly quantitative conversion (> 98%) of
limonene was achieved under the chosen reaction
conditions after 24 h with free CalB powder as well
as CalB-containing enzyme carriers as biocatalysts.
However, the conversion products were different mix-
tures of limonene monoxide and limonene dioxide.
To the best of our knowledge, this is the first report
Fig. 1 (color online). Kinetics of the chemo-enzymatic
limonene conversion to limonen monoxide and limonene
about lipase-mediated epoxidation of limonene where,
next to limonene monoxide, significant amounts of dioxide with biocatalyst 350 at 40 ◦C.
Brought to you by | New York University Bobst Library Technical Services
Authenticated
Download Date | 5/20/15 1:35 PM

L. O. Wiemann et al. · Lipase-mediated Epoxidation of Limonene
1059
served a strong dependency of the lipase activity in the
synthesis of both limonene monoxide and dioxide with
respect to the properties of the selected carrier mate-
rials and the reaction temperature. Thus it seems pos-
sible to predetermine certain desired product ratios of
limonene monoxide/dioxide mixtures by simply adopt-
ing the reaction parameters in terms of temperature,
enzyme carrier properties and immobilization method.
Currently, crucial parameters such as biocatalyst stabil-
ity (repetitive recycling), epoxidation yields at higher
substrate concentrations, as well as the possibility to
transfer this efficient, mild and bio-based epoxidation
system to other valuable terpenes are under investiga-
tion in our laboratories.
Fig. 2. Degree of limonene conversion into limonene monox-
ide and dioxide at r. t. after 24 h of reaction time.
Experimental Section
Materials
Novozyme 435 was obtained from Novozymes A/S
(Bagsvaerd/Denmark), lyophilized CalB (Candida Antarc-
tica lipase B) was a gift from c-LEcta (Leipzig/Germany).
Enzyme carriers (IB350, IB-C435 and IB-EC1) were re-
ceived from ChiralVision (Leiden/The Netherlands), D-
Limonene (96%) from Acros Organics (Geelen/Belgium).
Solvents and all other reagents were purchased from
Sigma-Aldrich (Steinheim/Germany) or Carl Roth (Karls-
Fig. 3. Degree of limonene conversion into limonene monox- ruhe/Germany) and used as obtained.
ide and dioxide at 40 ◦C after 24 h of reaction time.
Enzyme immobilization
1 g of each carrier (Table 1) was mixed with 4 mL of
formation of limonene monoxide. Increasing the reac-
tion temperature to 40 ◦C led to significantly increased
limonene dioxide yields (Fig. 3), except for biocatalyst
C435. The highest limonene dioxide yield of nearly
30% was observed for the biocatalyst 350. No side re-
actions or rearrangement of educts or products were
detected in any of the described reactions. In future
studies it will be tried to elucidate the effects of im-
mobilization and the carrier properties on the intrin-
sic kinetics of lipase-mediated epoxidation in more de-
tail in order to develop even more efficient epoxidation
systems.
pure ethanol (Merck) and incubated for 10 min. The sam-
ples were then filtered, thoroughly rinsed with 10 mL of
aqua dest. and dried at r. t. 0.25 g of a pre-treated carrier
was mixed with 1 mL of cooled enzyme solution containing
30 mg CalB (c-LEcta; Leipzig/Germany) in 100 mM acetate
buffer (pH = 6.9) and left standing on ice for 16 h. Finally
the samples were filtered, rinsed three times with 100 mM
ice-cold acetate buffer (pH = 5), dried at r. t., and stored at
4 ◦C.
Lipase activity for the chemo-enzymatic epoxidation of
limonene
Reactions of 50 mM caprylic acid, 25 mM limonene,
50 mM H2O2 (30% aqueous solution), and 50 mg of lipase-
immobilisates in toluene were carried out under permanent
magnetic stirring in sealed GC-glass vials at either r. t. (∼
23 ◦C) or 40 ◦C, all in triplicate. Samples of 10 µL were
withdrawn after 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 24 h, mixed
with 990 µL of ethyl acetate and then analyzed via GC-MS.
One enzyme unit (U) corresponds to the amount of immobi-
Conclusion
We have shown for the first time that the lipase-
mediated chemo-enzymatic conversion of limonene to
limonene dioxide with maximum yields of 30% is fea-
sible using immobilized Candida antarctica lipase B
under moderate reaction conditions (≤ 40 ◦C). We ob- lized lipase that catalyzes the formation of 1 µmol limonene
Brought to you by | New York University Bobst Library Technical Services
Authenticated
Download Date | 5/20/15 1:35 PM

1060
L. O. Wiemann et al. · Lipase-mediated Epoxidation of Limonene
oxide per minute and was calculated from the linear array of limonene monoxide: 7.21 – 7.32 min and limonene dioxide:
the initial product formation rates.
9.52 – 9.65 min.
Supporting information
GC-MS
Mass spectra of limonene oxide and dioxide after chemo-
enzymatic synthesis are given as Supporting Information
available online (DOI: 10.5560/ZNB.2012-1067).
Concentrations of limonene, limonene monoxide and
limonene dioxide were determined using a BPX5 column
(SGE, 30 m, I. D. 0.25 mm, film 0.25 µm) connected to
a QP2010 Plus gas chromatograph with a Single Quad
MS-detector (both Shimadzu, Japan) with helium as carrier
gas. 1 µL of the diluted sample was injected via SSL-injector
Acknowledgement
Financial support by BayernFIT – Forschung, Innovation,
(290 ◦C) starting at 60 ◦C for 1 min, then heated to 170 ◦C Technology (BayernFIT – Research, Innovation, Technol-
at 11 ◦C min−1 and finally to 270 ◦C at 70 ◦C min−1 and ogy) of the Free State of Bavaria (Germany) is gratefully
held for 3 min. Retention times, limonene: 5.72 – 5.78 min, acknowledged.
[1] R. P. Limberger, A. M. Aleixo, A. G. Fett-Neto, A. T.
Henriques, Electron. J. Biotechnol. 2007, 10, 500 – 507.
[2] W. A. Duetz, A. H. M. Fja¨llman, S. Ren, C. Jourdat,
B. Witholt, Appl. Environ. Microbiol. 2001, 67, 2829 –
2832.
[8] Y. Xu, N. R. B. J. Khaw, Z. Li, Green Chem. 2009, 11,
2047 – 2051.
[9] M. Ru¨sch, S. Warwel, Org. Lett. 1999, 1, 1025 –
1026.
[10] M. A. Moreira, T. B. Bitencourt, M. d. G. Nascimento,
[3] W. A. Duetz, A. H. M. Fja¨llmann, S. Ren, C. Jourdat,
Synth. Commun. 2005, 35, 2107 – 2114.
B. Witholt, Appl. Microbiol. Biotechnol. 2003, 61, [11] F. Bjo¨rkling, S. E. Godtfredsen, O. Kirk, J. Chem. Soc.,
269 – 277. Chem. Commun. 1990, 1301 – 1303.
[4] V. Skouridou, H. Stamatis, F. N. Kolisis, Biocatal. Bio- [12] F. Bjo¨rkling, H. Frykman, S. E. Godtfredsen, O. Kirk,
transform. 2003, 21, 285 – 290. Tetrahedron 1992, 48, 4587 – 4592.
[5] V. Skouridou, H. Stamatis, F. N. Kolisis, J. Mol. Cat. B [13] U. To¨rnvall, C. Orellana-Coca, R. Hatti-Kaul, D. Ad-
2003, 21, 67 – 69.
[6] F. P. Greenspan (FMC Corporation), US-Patent
lercreutz, Enzyme Microb. Technol. 2007, 40, 447 –
451.
3073792, 1963.
[7] C. Wiles, M. J. Hammond, P. Watts, Beilstein J. Org.
Chem. 2009, 5, 27; doi:10.3762/bjoc.5.27.
[14] E. G. Ankudey, H. F. Olivo, T. L. Peeples, Green Chem.
2006, 8, 923 – 926.
[15] M. M. Bradford, Anal. Biochem. 1976, 72, 248 – 254.
Brought to you by | New York University Bobst Library Technical Services
Authenticated
Download Date | 5/20/15 1:35 PM