Journal of Biotechnology 147 (2010) 169171

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Journal of Biotechnology
journal homepage: www.elsevier.com/locate/jbiotec

Short communication

Deep eutectic solvents (DESs) are viable cosolvents for enzyme-catalyzed

epoxide hydrolysis
Diana Lindberg a , Mario de la Fuente Revenga b , Mikael Widersten a,
a
b

Department of Biochemistry and Organic Chemistry, Uppsala University, Box 576, SE-751 23 Uppsala, Sweden
Instituto de Quimica Medica-CSIC Juan de la Cierva 3, E-28006 Madrid, Spain

a r t i c l e

i n f o

Article history:
Received 21 December 2009
Received in revised form 21 April 2010
Accepted 23 April 2010

Keywords:
Deep eutectic solvents
Epoxide hydrolase
Regioselectivity
Cosolvents

a b s t r a c t
A special group of ionic liquids, deep eutectic solvents (DESs) have been tested as cosolvents in enzymecatalyzed hydrolysis of a chiral (1,2)-trans-2-methylstyrene oxide. The choline chloride:ethane diol (ET),
choline chloride:glycerol (GLY) and choline:chloride:urea (REL) DESs were included in the reaction mixtures with epoxide and the potato epoxide hydrolase StEH1. The effect of the DESs on enzyme function
was primarily elevations of KM (up to 20-fold) and with lesser effects on turnover numbers (twofold
variation). The regioselectivity in hydrolysis of the (1R,2R)-2-trans-methylstyrene oxide was altered in
the presence of GLY or ET to favor epoxide ring opening at the benzylic carbon (R = 2.33), enhancing the
regioselectivity observed in buffer-only systems (R = 1.35). The DES solutions dissolved 1.5-fold higher
epoxide concentrations as compared to phosphate buffer. The total conversion of high concentration
(40 g/l) of (1S,2S)-MeSO was not negatively affected by addition of 40% GLY.
2010 Elsevier B.V. All rights reserved.

Advances in the area of biocatalysis have provided new ways

to perform enzyme catalysis in partly or fully nonaqueous environments. Such reaction conditions, however, often decrease the
stability and activity of enzymes (Hudson et al., 2005; Hobbs and
Thomas, 2007; Serdakowski and Dordick, 2008). Ionic liquids (ILs)
are considered as green solvents and have been extensively investigated in biocatalytic applications, with successful results (Park
and Kazlauskas, 2003; Yang and Pan, 2005; van Rantwijk and
Sheldon, 2007; de Maria, 2008; Roosen et al., 2008). Deep eutectic solvents (DESs) make up a group of IL-related solvents formed
from mixtures of polar components (liquids or solids) forming
complexes when mixed with drastically decreased melting temperatures. DESs are readily prepared in pure states from low-cost
starting materials, typically by mixing choline chloride (ChCl) with
an organic hydrogen donor, e.g. an amine, amide, alcohol, or carboxylic acid (Abbott et al., 2003, 2004). The resulting liquid consists
of both anions and cations, as well as neutral molecules, contributing both ionic and molecular solvent features to the DES (Zhang et
al., 2009). To date, only one previous investigation on DES as alternative solvents in enzyme-catalyzed conversions has been reported
(Gorke et al., 2008).
Due to their nontoxic nature, biodegradability and low cost,
we wanted to test the potential of DESs as alternative solvents in
enzyme-catalyzed reactions. As urea, one DES constituent, is also a
well known protein denaturant, while another constituent, glycerol

Corresponding author. Tel.: +46 018 471 4992; fax: +46 018 55 8431.
E-mail address: mikael.widersten@biorg.uu.se (M. Widersten).
0168-1656/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.jbiotec.2010.04.011

often acts as a protein stabilizer, an investigation on the effect of

DES on enzyme stability and activity was also deemed interesting.
Three different commercially available DESs were tested: ethaline
(ET) (ChCl:ethanediol), glyceline (GLY) (ChCl:glycerol), and reline
(REL) (ChCl:urea), all in 1:2 stoichiometric ratios.
Epoxide hydrolases (EH) catalyze hydrolysis of epoxides to the
corresponding vicinal diols. Both diols and epoxides are important prochiral and chiral intermediate molecules in chemical and
pharmaceutical industry (Archelas and Furstoss, 2001; Steinreiber
and Faber, 2001; de Vries and Janssen, 2003; Choi and Choi, 2005).
The stereochemistry of the product resulting from EH-catalyzed
hydrolysis is dependent on both enantio- and regioselectivity,
as EH:s can attack either carbon on the epoxide ring structure
(Fig. 1). To assess effects on enzyme stability, kinetic properties and
regioselectivity, the two trans-2-methylstyrene oxide (2-MeSO)
enantiomers were tested as substrates for the potato EH StEH1
(Elfstrm and Widersten, 2005) in the presence of DES cosolvents.
The (1S,2S)-enantiomer is a good substrate for StEH1 and was chosen for the activity and stability assays. The (1R,2R)-enantiomer, on
the other hand, is hydrolyzed by StEH1 with poor regioselectivity
(Lindberg et al., 2008), hence, providing a probe for DES effects on
selectivity.
To assess the effects on enzyme activity, StEH1-catalyzed
hydrolysis of (1S,2S)-2-MeSO was determined in the presence of
varying concentrations of the different DESs (Fig. 2). The enzyme
was least sensitive to GLY followed by ET and REL, with 50% relative activity retained in 60% GLY. When the enzyme was incubated
in DES for prolonged time periods, a DES-dependent inactivation was observed (Table 1). The half-lives of retained enzyme

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D. Lindberg et al. / Journal of Biotechnology 147 (2010) 169171

Table 2
Steady state kinetic parameters for StEH1 catalyzed hydrolysis of (1S,1S)-2-MeSO,
at different DES concentrationsa .
Solvent

Fig. 2. Effects on epoxide hydrolase activity in the presence of increasing concentrations of DES. Retained enzyme activity was measured in 0.1 M sodium phosphate,
pH 7.5, 0.3 mM (1S,2S)-2-MeSO and in the presence of DES (60% v/v).

activity decreased with DES concentration and, again, with the

urea-containing REL affecting the enzyme most negatively. In either
ET or GLY, StEH1 retained activity over several hours. GLY and
REL have similar solvent polarities of 0.84, and ET 0.80, according to Reichhardts normalized polarity scale (with water = 1 and
trimethylsilane = 0) (Gorke et al., 2008), which indicates that solvent polarity is not inuencing catalytic activity but that observed
effects were maybe due to chemical properties of the urea component of the DES.
The steady state kinetic parameters of StEH1-catalyzed hydrolysis of (1S,2S)-2-MeSO in different DES concentrations are presented
in Table 2. The catalytic efciencies determined in the different
Table 1
Half-lives of enzyme activity during prolonged incubation of StEH1 in DESa .
Solvent
Buffer
GLY

ET
REL

Conc. (%)
0
20
40
60
60
20
40

kinactive b (h1 )
<103
(2 4) 103
(3 2) 103
(5 0.5) 102
(2 0.2) 102
(3 0.7) 101
(7 3) 101

t1/2 (h)
>200
>200
>200
15
40
2
1

a
For probing StEH1 stability in DES, retained enzyme activity was measured after
incubation in DES (60%) in 0.1 M sodium phosphate and 0.3 mM (1S,2S)-2-MeSO.
b
kinactive was obtained from tting a single-exponential function to the data of
retained relative activity as a function of time; rel. activity (t) = A exp (kinactive t).

kcat (s1 )

KM (mM)

kcat /KM (s M)1

63 3

0.077 0.01

GLY

20
40
60

51 2
57 5
44 5

0.12 0.01
0.25 0.04
0.53 0.09

0.42 0.05
0.23 0.04
0.082 0.02

ET

20
40
60

63 2
61 6
31 6

0.24 0.02
0.49 0.08
1.2 0.3

0.26 0.03
0.12 0.02
0.026 0.008

REL

20
40

65 4
92 10

0.36 0.04
1.5 0.3

0.18 0.02
0.060 0.01

Buffer

Fig. 1. Stereoselective outcome of StEH1-catalyzed reaction of the two enantiomers

of 2-MeSO. The StEH1 regioselectivity towards 1 is >98% in preference of C-1, while
for enantiomer 2 the enzyme shows regioselective promiscuity and prefer C-1 to
C-2 with a factor of 2:1 at pH 7.5, 30 C.

Conc. (%)

0.82 0.1

a
2-MeSO enantiomers were purchased from Aldrich. The DESs, reline, glyceline
and ethaline, were purchased from Scionix (UK). DES solutions were prepared by
mixing 0.1 M sodium phosphate with DES after which pH was adjusted to 7.5. StEH1
was puried as described previously (Elfstrm and Widersten, 2005). Epoxides were
dissolved in acetonitrile and added to the reactions mixtures. Final acetonitrile concentrations were 13% (v/v). StEH1 activity was determined at 30 C in 080% (v/v)
DES with 0.20 mM (1S,2S)-2-MeSO and 0.010.04 M StEH1. Kinetic data for epoxide
hydrolysis were recorded at 225 nm,  = 4.3 (mM cm)1 . Steady state measurements were performed using serial dilutions of (1S,2S)-2-MeSO (0.020.4 mM) in 20,
40 and 60% of GLY or ET and 20 and 40% REL, in the presence of 0.020.04 M StEH1.
The steady state kinetic parameters were obtained after tting the Michaelis Menten
equation to the experimental data with SIMFIT (http://www.simt.man.ac.uk).

DESs showed that GLY affected StEH1 catalysis the least. Turnover
numbers remained relatively unaffected by cosolvent, whereas KM
increased with DES concentrations. The loss in activity at higher
DES concentrations (Fig. 2) should therefore not be attributed
to protein denaturation, but rather to destabilization of enzymesubstrate or reaction intermediate complexes.
The regioselectivity in StEH1-catalyzed hydrolysis of (1R,2R)2-MeSO was also affected by cosolvent. Hydrolysis in the dioland triol-containing ET and GLY, resulted to a larger degree in
the diols formed after epoxide ring opening at the benzylic carbons. This was not observed in the presence of the urea-containing
REL (Table 3). The underlying reason for this solvent-dependent
effect on regioselectivity is at present not known. It has, however,
been recently shown that regioselectivity in this enzyme is sensitive to changes in reaction conditions (Lindberg et al., 2010). The
observed improvement in regioselectivity with (1R,2R)-2-MeSO
caused by addition of GLY (or ET) is still insufcient for biocatalytic
usefulness.
All DES solutions dissolve approximately 1.5 times as much substrate as the buffer solution. In order to analyze the maximum
substrate tolerance in a DES, biocatalytically relevant amounts of
substrate, 20 and 40 g/l (1S,2S)-MeSO, was incubated with enzyme
at mass ratios of 1:250 and 1:500 (enzyme:substrate) in sodium-

Table 3
Enantiomeric excess of product diols (eep )a after StEH1-catalyzed hydrolysis of 2MeSO. Major diol enantiomers are (1R,2S) and (1S,2R), after hydrolysis of (1S,2S)and (1R,2R)-2-MeSO, respectivelyb .
Substrate

(1S,2S)-2-MeSO
(1R,2R)-2-MeSO

eep (%)
Buffer

GLY

ET

REL

96 0.002
15 0.06

97 0.005
38 0.02

98 0.007
40 0.03

96 0.007
18 0.01

eep = (cmajor diol cminor diol /cmajor diol + cminor diol ) 100.
15 mM (1S,2S)-2-MeSO was added to 0.1 mM sodium-phosphate (3% acetonitrile) or 40% GLY, ET or REL (v/v) fortied with 2 M StEH1. Reactions were run until
completion overnight at 30 C. Water was evaporated and the residues dissolved
in 80/20 hexane/isopropanol solution and analyzed by HPLC (Daicel Chiralpak ASH or Supelco Acentis C-18 columns) with mobile phases consisting of either 95/5
hexane/isopropanol for the chiral normal phase system or 35/65 methanol/0.1 M
sodium phosphate (pH 3.0) for the C-18 reverse phase system.
a

D. Lindberg et al. / Journal of Biotechnology 147 (2010) 169171

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References

Fig. 3. StEH1-catalyzed conversion (c) over time of 0.3 M (1S,2S)-2-MeSO to (1R,2S)phenylpropane diol in buffer (unlled circles) as compared to in 40% GLY (lled
circles). 0.3 mM (1S,2S)-2-MeSO was mixed with 2 M StEH1, dissolved in 0.1 mM
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