Characterization of a newly synthesized epoxide hydrolase and its application in racemic resolution of (R,S)-epichlorohydrin

Article abstract of DOI:10.1016/j.catcom.2011.09.010

In the current study, an epoxide hydrolase (EH) gene from Rhodosporidium toruloides was synthesized and expressed in Escherichia coli. After purification, we found that the optimal pH and temperature of this enzyme were 7.5 and 35°C, respectively. The recombinant EH obtained in this study was temperature-sensitive and the activity decreased significantly above 45°C. The values of apparent K_m and V_max were 0.5953 mol/l and 0.0105 mol/(L?min). In addition, enantiomeric excesses value of (R)-epichlorohydrin could reach 100% after 40-min reaction. Moreover, this EH showed a broad substrates specificity toward epoxides. To the best of our knowledge, this is the first time to report the application of R. toruloides EH in racemic resolution of (R,S)-epichlorohydrin to produce (R)-epichlorohydrin.

Full text of DOI:10.1016/j.catcom.2011.09.010

Catalysis Communications 16 (2011) 133–139
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Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom
Short Communication
Characterization of a newly synthesized epoxide hydrolase and its application in
racemic resolution of (R,S)-epichlorohydrin
⁎
Zhi-Qiang Liu, Li-Ping Zhang, Feng Cheng, Li-Tao Ruan, Zhong-Ce Hu, Yu-Guo Zheng , Yin-Chu Shen
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China
Engineering Research Center of Bioconversion and Biopurification, Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, People's Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 17 July 2011
Received in revised form 1 September 2011
Accepted 12 September 2011
Available online 20 September 2011
In the current study, an epoxide hydrolase (EH) gene from Rhodosporidium toruloides was synthesized and
expressed in Escherichia coli. After purification, we found that the optimal pH and temperature of this enzyme
were 7.5 and 35 °C, respectively. The recombinant EH obtained in this study was temperature-sensitive and
the activity decreased significantly above 45 °C. The values of apparent Km and Vmax were 0.5953 mol/l and
0.0105 mol/(L·min). In addition, enantiomeric excesses value of (R)-epichlorohydrin could reach 100%
after 40-min reaction. Moreover, this EH showed a broad substrates specificity toward epoxides. To the
best of our knowledge, this is the first time to report the application of R. toruloides EH in racemic resolution
of (R,S)-epichlorohydrin to produce (R)-epichlorohydrin.
Keywords:
Epoxide hydrolase
Characterization
Racemic resolution
Epichlorohydrin
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
development in bioinformatics and gene engineering, it is possible
to obtain novel EHs by analysis of putative EHs sequence and activity
Epoxide hydrolases (EHs, EC 3.3.2.x) catalyze the hydrolysis of
epoxides to corresponding vicinal diols with the addition of a H2O
molecule, without any cofactors and metal ions [1,2]. EHs have been
found in various sources [3–6] and most of the EHs belong to the α/β
hydrolase fold family [7,8]. Previous studies demonstrated that microbi-
al EHs had a broad substrate specificity, high enantioselectivities, and
reaction rates, which has received more attention due to its promising
potential for the enantioselective resolution of racemic epoxides [3,9–
11]. Recently, the discovery and production of EHs in microorganisms
allowed applying EHs to a large scale.
information [18,19].
To explore the production of (R)-epichlorohydrin by biological
method, we synthesized an EH gene from Rhodosporidium toruloides
CBS14 and then successfully expressed it in Escherichia coli. After purifi-
cation, the catalytic properties of the recombinant EH and its application
in racemic resolution of (R,S)-epichlorohydrin were investigated in this
study.
2. Materials and methods
Enantiopure epoxides are key intermediates of many bioactive
2.1. Strains and plasmid
compounds [12,13]. As
a promising enantiopure epoxide, (R)-
epichlorohydrin was widely used for synthesis of β-blockers, L-car-
tine, and ferroelectric liquid crystals [14,15]. Choi et al. have
reported the preparation of chiral epichlorohydrin in organic sol-
vents using Aspergillus niger cells harboring EH, the enantiomeric
excesses (e.e.) value of (R)-epichlorohydrin could reach 100% and
yield was around 18% [16]. Rhodotorula glutinis EH has been cloned
by Kim's group and applied into resolution of (R,S)-epichlorohydrin
to provide (R)-epichlorohydrin with 25% yield [17]. However, the
yield and e.e. with existed EHs are low and not desirable for indus-
trial production in large scale by far [12]. Therefore, finding new EHs
or employing engineering enzymes is getting more and more attrac-
tion in the production of (R)-epichlorohydrin [18]. With the recent
E. coli BL21(DE3) was used as a host to express recombinant EH.
pET28b(+) was used as vector for the expression of EH gene in E.
coli BL21 (DE3). E. coli transformants were grown in Luria–Bertani
(LB) medium. All the chemicals used were of analytical grade and
commercially available.
2.2. Expression and purification of EH
A nucleotide sequence of EH from R. toruloides CBS14 (Genbank acces-
sion No. AAN32662) [20] with a length of 1227 bp was synthesized using
PCR assembly method [21] after optimization of the codons by a gene de-
signer software against E. coli as host [22]. The synthesized gene with 6×
His-tag was inserted into expression vector pET28b(+) between NcoI
and XhoI restriction endonuclease sites. The ligated plasmid pET28b-EH
was transformed into E. coli BL21 (DE3).
⁎
Corresponding author. Tel./fax: +86 571 88320630.
E-mail address: zhengyg@zjut.edu.cn (Y.-G. Zheng).
1566-7367/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.catcom.2011.09.010

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Z.-Q. Liu et al. / Catalysis Communications 16 (2011) 133–139
Permanent frozen stock of E. coli cells BL21(DE3) harboring the plas-
with a capillary HP-5 column (0.35 mm ID×30 m, 0.25 μl film thick-
ness) and a FID detector with N2 as carrier gas. And then the 1 μl organic
layer was subjected to chiral GC analysis to calculate the e.e. value. The
chiral GC system was equipped with a chiral capillary BGB-175 column
(0.25 mm ID×30 m, 0.25 μl film thickness) fitted with a FID detector
and He was used as carrier gas. The temperatures of the oven, injector,
and detector for analysis of racemic epichlorohydrin were 90, 220, and
220 °C, respectively. The retention times of racemic epichlorohydrin,
(R)-epichlorohydrin and (S)-epichlorohydrin were 2.70, 5.69, 5.50 min,
respectively.
mid pET28b-EH was used to inoculate into 50 ml LB broth medium con-
taining 50 μg ml−1 Kanamycin (Kan), and cultures were grown at
250 rpm and 37 °C. When the cultures reached optical densities of
0.6–0.8 at 600 nm, the temperature was lower to 28 °C, and isopropyl-
1-thio-β-D-galactoside (IPTG) was added to a final concentration of
0.1 mM. After 7 h induction, the cells were harvested by centrifugation
at 9,000 rpm for 20 min.
All purification steps were carried out at 4 °C. The wet cell paste
was disrupted by sonication in buffer (20 mM NaH2PO4, pH 8.0;
300 mM NaCl). And then the cell debris was removed by centrifuga-
tion at 12,000 rpm for 20 min. Subsequently, the soluble fraction
was loaded onto a Nickel-NTA superflow column (10 ml) equilibrated
with a binding buffer (20 mM NaH2PO4, pH 8.0; 300 mM NaCl). The
column was eluted with 2 volumes of washing buffer (20 mM
NaH2PO4, pH 8.0; 300 mM NaCl; 50 mM imidazole) and unbounded
proteins were washed out from the column. Later, the expected pro-
tein was eluted from the column with an elution buffer (20 mM
NaH2PO4, pH 8.0; 300 mM NaCl; 500 mM imidazole). These proteins
were analyzed using a 12% SDS-polyacrylamide gel electrophoresis
(SDS-PAGE).
One unit of EH activity was defined as the amount of enzyme required
to convert 1 μmol (S)-epichlorohydrin at 30 °C. Specific enzyme activities
were defined as units per mg enzyme.
2.6. Determinations of kinetics parameters
The values of Km and Vmax for the EH were determined by assaying
purified enzyme at increasing substrate concentrations ranging from
0.2% to 2.0% (v/v). The temperature, pH and quantity of the enzyme
were kept the same as the standard enzyme activity assay described
above. Apparent Km and Vmax were calculated according to Lineweaver–
Burk plots.
2.3. SDS-PAGE
2.7. Homology modeling and docking
Enzyme fractions and the molecular mass under denaturing condi-
tions were determined by SDS-PAGE performed using a Mini-gel system
(Bio-Rad, Hercules, USA). SDS-PAGE was performed according to
Laemmli's discontinuous Tris–glycine buffer system [23]. Proteins in
the gel were stained with Coomassie brilliant blue R-250 for 1 h, and
then destained with 0.5 M NaCl aqueous solution.
The three-dimensional homology model of EH was generated using
Build Homology Models (MODELER) in Discovery Studio (DS) 2.1
(Accelrys Software, San Diego, USA) using crystal structures of EH
from A. niger at 1.8 Å resolution (PDB accession code 1QO7) and crystal
structure of EH from Agrobacterium radiobacter AD1 at 2.1 Å resolution
(PDB accession code 1EHY) as templates. The generated structures
were improved by subsequent refinement of the loop conformations
by assessing the compatibility of an amino acid sequence to known
PDB structures using the Protein Health module in DS 2.1. The geometry
of loop regions was corrected using Refine Loop/MODELER. Finally, the
best quality model was chosen for further calculations, molecular
modeling, and docking studies by Autodock 4.0 [24].
2.4. Influence of factors on EH and its substrate specificity
The effects of pH, temperature and chemicals on the EHs were sys-
tematically investigated. The optimal pH for the reaction was deter-
mined with 100 μl epichlorohydrin in a 10 mL reaction system at 30 °C,
in a pH range of 3.0–9.0 using citric acid–sodium citrate buffer (pH
3.0–6.0), K2HPO4–KH2PO4 buffer (pH 6.0–8.0) and Tris–HCl buffer (pH
7.0–9.0). The optimal temperature of the enzyme was determined by
assaying enzyme activities at temperatures ranging from 25 to 55 °C.
Thermal stability was investigated by incubating enzymes at tempera-
tures ranging from 35 to 65 °C in 10 mL of 100 mM phosphate buffer
at pH 7.5 in water bath. The enzyme activities were assayed at 30 °C
after incubation at regular time intervals (30–600 min). The residual ac-
tivity was calculated by comparing final activity to initial activity of EHs
before incubation. The half-life of EH at each temperature was calculated
by plotting the natural logarithm of residual activity (ln RA) at each tem-
perature against time. The effects of different chemicals were assessed
by suspending 0.01 g of purified EH in 9 mL of 100 mM phosphate
buffer at 30 °C with series of metal ions (final concentration of
5 mM) and EDTA (final concentration of 10 mM) in 10 ml of pH 7.5
buffers containing 100 μl epichlorohydrin. The substrate specificity
of the EH was evaluated using different epoxides, each presented at
a concentration of 20 mM.
3. Results
3.1. Expression of EH gene
Based on the reported nucleotide sequence, the R. toruloides EH gene
was synthesized after optimization of the codons. Subsequently, the EH
gene and expression plasmid pET28b were treated with restriction
2.5. Enzyme assay
0.01 g recombinant EH was added into 10 mL phosphate buffer (pH
7.5) containing 100 μl epichlorohydrin. The reaction was carried out at
30 °C for 10 min in a thermomixer (Eppendorf, Hamburg, Germany).
After reaction, 1.0 ml biotransformation sample was taken and centri-
fuged at 12,000 rpm for 5 min and then 200 μl of supernatant was
extracted with 800 μl of ethyl acetate and centrifuged at 10,000 rpm
for 5 min. The organic layer was taken and dried by anhydrous sodium
sulfate. 1 μl of the reaction mixture was analyzed by GC and chiral GC for
enzyme activity and e.e., respectively. The GC system was equipped
Fig. 1. SDS-PAGE analysis of recombinant EH. Lane M, the protein size standard; Lane 1,
E. coli BL21(DE3); Lane 2, E. coli BL21(DE3)/pET28b; Lane 3, uninduced E. coli BL21
(DE3)/pET28b-EH; Lane 4, E. coli BL21(DE3)/pET28b-EH induced by IPTG; Lane 5,
crude extract; Lane 6, the purified EH.

Z.-Q. Liu et al. / Catalysis Communications 16 (2011) 133–139
135
a
b
citric acid-sodium citrate buffer
100
80
60
40
20
0
KH2PO 4-K2HPO 4 buffer
Tris-HCl
100
80
60
40
20
0
4
5
6
7
8
9
4
5
6
7
8
9
pH
pH
c
d
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
100
80
60
40
20
0
20
25
30
35
40
45
50
55
60
65
0
100
200
300
400
500
600
Temperture (
0
C)
Time (min)
e
280
240
200
160
120
80
0
2
4
6
8
10
Concentration of Tween-20 (%)
Fig. 2. a) Effect of pH on epoxide hydrolase activity. b) pH stability of epoxide hydrolase. The purified EH was pretreated in different buffers with pH ranging from 4.5 to 9.0 at 30 °C
for 1 h, and then the EH activities were determined according to the standard enzyme assay method. The activities of enzymes that assayed under the standard reaction were taken
as 100%. c) Effect of temperature on epoxide hydrolase activity. d) The thermo-stability of epoxide hydrolase activity. The purified enzymes were incubated at different tempera-
tures for 30–600 min, and then the EH activities were determined according to the standard enzyme assay method. The activities of enzymes that assayed under the standard re-
action were taken as 100%. ■ the liner of 25 °C; ● the liner of 35 °C; ◆ the liner of 45 °C; ▼ the liner of 55 °C. e) Effect of Tween-20 on the activity of epoxide hydrolase. Tween-20
with concentration from 0 to 9% was added to the reaction mixture, and then the EH activities were determined according to the standard enzyme assay method. The activities of
enzymes that assayed under the standard reaction without addition of Tween-20 were taken as 100%.
endonucleases of NcoI and XhoI, and ligated by T4 DNA ligase to con-
struct recombinant plasmid. And then the recombinant plasmid
was transformed into E. coli BL21 (DE3) and several clones were se-
lected. The recombinant plasmids were extracted and further se-
quenced to verify the existence of the EH gene. The positive
transformant harboring recombinant pET28b-EH was cultivated
and induced by 0.1 mM of IPTG. The cells were harvested and
disrupted by sonication, after which the recombinant EH was puri-
fied by His-tag-affinity chromatography. The SDS-PAGE analysis
(Fig. 1) clearly shows that a band with molecular mass of around
46 kDa on Lane 6 is obtained, which is in agreement with the pre-
dicted value based on the amino acid sequence of EH. The specific ac-
tivity and amount of protein of the recombinant EH were calculated
to be 113.1 U/mg and 0.79 g/l, respectively.

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Z.-Q. Liu et al. / Catalysis Communications 16 (2011) 133–139
3.2. Influence of factors on EH and its substrate specificity
on the EH activity were also investigated, the results showed that the
addition of Tween-20 has a positive effect on EH activity for the produc-
tion of (R)-epichlorohydrin because of a correlation between the effects
of the Tween-20 and the substrate used. Further investigation illustrated
that enzyme with 5% of Tween-20 exhibited the highest hydrolysis ac-
tivity. However, further increase in the concentration of Tween-20 did
not improve the enzyme activity.
3.2.1. Effect of pH on EH activity
Fig. 2a shows that the maximum activity of the EH is around pH 7.5,
but the lowest enzyme activity was found at pH 9.0. Moreover, a rapid
decrease in the enzyme activity was observed under acidic and basic
conditions, while neutral pH condition seemed to favor the enzyme ac-
tivity. To study the pH stability, EH was pretreated in different buffers
with pH ranging from 4.5 to 9.0 at 30 °C for 1 h and the results showed
that the EH was stable at pH 7.5. In addition, pH higher or lower than 7.5
resulted in the significant decrease in the relative activity of EH, and
only 10% of the residual enzyme activity was left at pH 9.0 (Fig. 2b),
which showed that the pH stability of this enzyme is not satisfied and
needs further improvements.
3.2.5. Substrate specificity
Table 2 shows the hydrolytic activity of the recombinant EH to-
wards a set of substrates. The purified enzyme had no activity to-
wards 1,3-chlorophenyl glycidol ether, 2-[(4-chlorophenoxy)methyl]
oxirane and oxirane-2,3-dicarboxylic acid, and very low activity
with 2-[(4-methylphenoxy)methyl]oxirane, but showed high activity
with 2-(chloromethyl)oxirane, 2-[(benzyloxy)methyl]oxirane, and 2-
[(2-methylphenoxy)methyl]oxirane. The substrate specificity study
demonstrated that this recombinant enzyme could be explored for
the productions of multiple diols with high values, besides enantio-
pure (R)-epichlorohydrin.
3.2.2. Effect of temperature on EH activity
Fig. 2c shows that the activity gradually increased with the increas-
ing of temperature from 20 to 35 °C, and reached its maximum at 35 °C,
which indicates that the optimal temperature of the EH is around 35 °C.
After this apex point, the activity sharply decreased. Only negligible EH
activity was observed at 60 °C. In order to test the thermo-stability of
EH, the purified enzyme was incubated at various temperatures (25–
55 °C) for various times and then residual activity was determined
according to the standard enzyme assay method. The residual activity
was obtained in accordance with the activity at the 0th h and its natural
logarithm value (Ln E0/E) was plotted against time. Fig. 2d shows the
results of the thermo-stability of EH. The Kd of the enzyme for temper-
atures at 25, 35, 45, and 55 °C were 0.0041, 0.0126, 0.0195, and
0.0362 min−1, respectively. Accordingly, the half-life (t1/2) of the puri-
fied enzyme at 25, 35, 45, and 55 °C were calculated to be 2.820, 0.917,
0.592, and 0.319 h, respectively, which demonstrates that this enzyme
was not thermo-stable.
3.3. Determination of kinetic parameters
The catalytic efficiency on (S)-epichlorohydrin hydrolysis and as-
sociated kinetic parameters of the recombinant EH were measured
using Lineweaver–Burk plots. The (R)-epichlorohydrin productivity
was determined at concentrations of (R,S)-epichlorohydrin from
0.2% to 2% and the Michaelis constant Km and Vmax values were esti-
mated as well. As illustrated in Fig. 3, the values of apparent Km and
Vmax are 0.5953 mol/L and 0.0105 mol/(L min) for the EH, respective-
ly. In addition, the e.e. value of (R)-epichlorohydrin reached 100%,
which suggests that the EH in this study had a relatively satisfied sub-
strate specificity on (S)-epichlorohydrin.
3.2.3. Effect of metal ions and other reagents on EH activity
3.4. The time course of racemic resolution of (R,S)-epichlorohydrin
To investigate the effects of the metal ions and chemicals on EH ac-
tivity, 0.01 g of the purified EH was preincubated according to the meth-
od described in Materials and methods section 2.4. The results showed
that EH activity was slightly inhibited by Zn2+ and strongly inhibited
by Hg2+, Fe2+, Ag+, Ba2+, Mg2+, Al3+, Cu2+ and Mn2+. Moreover,
EDTA, Co2+, Ca2+ and Ni2+ did not show any significant effects on the
EH activity (Table 1). However, it appears that there are no metal ions
tested in this study that could activate EH activity, which illustrated
that the EH obtained in the current study was not metal ion-dependent
in terms of performing its catalysis.
The time course of the racemic resolution of epichlorohydrin was
investigated using 1% (v/v) of (R,S)-epichlorohydrin as substrate
under the optimal condition. After 40 min resolution, the e.e. value
reached 100%. The enantiopure (R)-epichlorohydrin was remained
and the yield could be 18% (Fig. 4).
3.5. Homology modeling and molecular docking
The secondary structure and tertiary structure analysis showed
that EH obtained in this study consisted of 19 α-helix and 8 β-sheet.
In addition, one lid-like structure was formed by 7 α-helix. Crystal
structures of A. niger EH (PDB accession code 1QO7, resolution
1.80 Å) [26] and A. radiobacter EH (PDB accession code 1EHY, resolu-
tion 2.10 Å) [27] were used as templates for homology modeling of
R. toruloides EH. The best quality model as shown in Fig. 5a was cho-
sen for further calculations, molecular modeling, and docking studies.
The modeling results showed that the EH belongs to α/β enzyme
family containing a core motif and lid-like motif. The NC-loop com-
bined core motif and hat-like motif are different with the one in the
lid-like motif. The conserved catalytic residues Asp 190, Glu 359 and
His 385 from EH protein have similar orientations and locations in the
EH model.
The molecular docking studies of the EH could show substrate in-
teractions in the active site (Fig. 5b and c), which showed that both of
(R)- and (S)-epichlorohydrin could dock into the catalytic pocket of
EH, however the distances of hydrogen bonds with Tyr 261 were dif-
ferent. The mechanism of EH to catalyze (S)-epichlorohydrin can be
proposed as follows: two tyrosines, 261 and 262, bind and concomi-
tantly activate the substrate by forming hydrogen bonds (both
2.8 Å) at the epoxide O-atom, and the Asp 190 then initiates the nu-
cleophilic attack at the sterically less hindered C-atom of substrate,
3.2.4. The effect of lyoprotectants and detergents on EH activity
It is reported that lyoprotectants and detergents have a very impor-
tant effect on the EH activity [25]. In this study, the effects of lyoprotec-
tants and detergents including SDS, Tween-20, Tween-80 and D-sorbitol
Table 1
Effect of metal ions and other reagents on epoxide hydrolase activity.
Chemicals
Concentration (mM)
Relative activity (%)
Control
EDTA
HgCl2
AlCl3
AgNO3
ZnCl2
BaCl2
MgCl2
CaCl2
NiCl2
100
106.0 2.1
0
10
5
5
5
5
5
5
5
5
5
5
5
5
55.0 3.3
51.5 1.7
83.1 2.6
50.7 1.3
42.9 2.9
101.7 3.1
100.2 2.0
104.5 2.7
31.3 2.1
45.9 1.3
63.8 1.8
CoCl2
CuCl2
FeCl2
MnCl2

Z.-Q. Liu et al. / Catalysis Communications 16 (2011) 133–139
137
Table 2
Substrate specificity of the recombinant EH.
Substrates
Products
Relative activity (%)
100
Conversion rate (%)
2-(chloromethyl)oxirane
3-chloropropane-1,2-diol
46.7
2-[(benzyloxy)methyl]oxirane
3-(benzyloxy)propane-1,2-diol
164
129
76.6
2-[(2-methylphenoxy)methyl]oxirane
3-(2-methylphenoxy)propane-1,2-diol
60.3
27.7
0
2-[(4-methylphenoxy)methyl]oxirane
3-(4-methylphenoxy)propane-1,2-diol
3-(4-chlorophenoxy)propane-1,2-diol
3-(4-chlorophenoxy)propane-1,2-diol
59.3
1,3-Chlorophenyl glycidol ether
0
2-[(4-chlorophenoxy)methyl]oxirane
0
0
oxirane-2,3-dicarboxylic acid
2,3-dihydroxybutanedioic acid
0
0
resulting in ring-opening and the formation of a covalently bound
ester intermediate. Subsequently, the Glu 359 located in low position
would help His 385 to activate the H2O molecules which makes the
intermediate reacts with water and formed ester hydrolyzed, and
then 3-chloro-1,2-diol released.
4. Discussion
As a promising biocatalyst, EH has been widely put into industrial
applications for the preparation of enantiopure pharmaceuticals and
other fine chemicals because of its high enantioselectivity [3]. Many

138
Z.-Q. Liu et al. / Catalysis Communications 16 (2011) 133–139
12000
10000
8000
6000
4000
2000
0
-20
0
20 40 60 80 100 120 140 160 180 200
1/S(L/mol)
Fig. 3. Lineweaver–Burk double reciprocal plots of epoxide hydrolase.
EHs from microbial origins had been researched and some of them
were promising for preparative scale biotransformation [2]. This
work focused on cloning the EH gene and investigating its character-
istics for exploring industrial applications.
Though the R. toruloides CBS14 EH has been well studied [28,29], its
application in production of (R)-epichlorohydrin is still not investigated
and reported yet. In the current work, an EH gene from R. toruloides
CBS14 was synthesized and expressed in E. coli. The recombinant EH
showed higher activity in neutral or basic condition than in acidic con-
dition and it was sensitive to temperature, which is similar to the previ-
ous reports [1]. The racemic resolution of epichlorohydrin showed that
this EH could catalyze the hydrolysis of (S)-epichlorohydrin to produce
enantiopure (R)-epichlorohydrin, however its productivity is lower as
compared to EH from R. glutinis [17]. In addition, the substrate specific-
ity study suggested that this recombinant EH has potential use for the
production of diols with high values.
It is reported that not only e.e. value could increase, but also the
spontaneous hydrolysis could be inhibited, and the substrate solubil-
ity would be improved as well in the organic media [30]. In this study,
series of organic solvents were tested to investigate the influence of
organic solvents on EH and its catalytic process using DEAE-
cellulose immobilized enzyme as catalyst [16]. It is found the EH func-
tioned very well and the e.e. of product could reach 100%. In addition,
the spontaneous hydrolysis was avoided when hexane was used as
organic medium, which is similar to previous reports [31]. Further
studies are undergoing in our lab to investigate the detailed function
of EH in the organic media.
100
80
60
40
20
0
100
80
60
40
20
0
yield (%)
e.e. (%)
Fig. 5. Homology modeling and molecular docking. a. Homology protein model of ep-
oxide hydrolase. b. (S)-epichlorohydrin docking into the active site of epoxide hydro-
lase. c. (R)-epichlorohydrin docking into the active site of epoxide hydrolase.
Analysis of catalytic characteristics based on structures modeling
and molecular docking provides a new insight to this synthesized EH
and paves the fundamental for its further application [32]. The energies
of binding were expected to depend on several factors including hydro-
gen bonds with the two tyrosines (Tyr 261 and Tyr 262, activate the
substrate), the docked energy and the distance (d) between the attack-
ing O-atom of Asp 190 and the epoxide C-atom. It is reported that d
value is a key factor influencing the activity and enantioselectivity of
EH [33]. In this study, we found that though the docked energies for
0
10
20
30
40
Reaction Time (min)
Fig. 4. Time course of racemic resolution of epichlorohydrin.

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139
[8] R. Rink, J.H.L. Spelberg, R.J. Pieters, J. Kingma, M. Nardini, R.M. Kellogg, B.W. Dijkstra,
D.B. Janssen, Journal of the American Chemical Society 121 (1999) 7417–7418.
[9] K. Faber, M. Mischitz, W. Kroutil, Acta Chemica Scandinavica 50 (1996) 249–258.
[10] S. Hwang, C.Y. Choi, E.Y. Lee, Biotechnology and Bioprocess Engineering 13
(2008) 453–457.
[11] K.E. Jaeger, T. Eggert, A. Eipper, M.T. Reetz, Applied Microbiology and Biotechnol-
ogy 55 (2001) 519–530.
[12] W.J. Choi, Applied Microbiology and Biotechnology 84 (2009) 239–247.
[13] M. Kotik, V. Stepanek, M. Grulich, P. Kyslik, A. Archelas, Journal of Molecular Ca-
talysis B: Enzymatic 65 (2010) 41–48.
[14] Y.C. Jiang, J.Y. Wu, C. Liu, M.C. Hu, S.N. Li, Q.G. Zhai, Catalysis Communications 11
(2010) 727–731.
[15] I.K. Song, S.H. Lee, S.H. Song, D.R. Park, J.C. Jung, J.H. Song, S.Y. Woo, W.S. Song,
M.S. Kwon, Catalysis Communications 10 (2008) 160–164.
[16] W.J. Choi, E.Y. Lee, S.J. Yoon, S.T. Yang, C.Y. Choi, Journal of Bioscience and Bioen-
gineering 88 (1999) 339–341.
(R)-(−2.944 kcal/mol) and (S)-epichlorohydrin (−2.928 kcal/mol)
were close, there was still a big difference in d values between (R)-
(5.7 Å) and (S)-epichlorohydrin (4.3 Å), which leads to the EH obtained
in this study was more active on (S)-epichlorohydrin. Regulating the d
value and making the distance closer between (S)-epichlorohydrin
and Asp 190 would help to improve the activity and enantioselectivity
of this enzyme on (S)-epichlorohydrin. In addition, to improve the
thermo-stability, enantioselectivity, productivity and activity of EH,
other strategies such as rational design or by directed molecular evolu-
tion of proteins are also powerful tools [34] to combine advantages of
EH from different origins [35] based on the investigation of the structure
modeling and docking.
[17] H.S. Kim, J.H. Lee, S. Park, E.Y. Lee, Biotechnology and Bioprocess Engineering 9
(2004) 62–64.
[18] H.S. Kim, S.J. Lee, E.J. Lee, J.W. Hwang, S. Park, S.J. Kim, E.Y. Lee, Journal of Molec-
ular Catalysis B: Enzymatic 37 (2005) 30–35.
[19] H.B. Zheng, M.T. Reetz, Journal of the American Chemical Society 132 (2010)
15744–15751.
[20] H. Visser, C.A.G.M. Weijers, A.J.J. van Ooyen, J.C. Verdoes, Biotechnology Letters 24
(2002) 1687–1694.
[21] R. Rydzanicz, X.S. Zhao, P.E. Johnson, Nucleic Acids Research 33 (2005)
W521–W525.
[22] A. Villalobos, J.E. Ness, C. Gustafsson, J. Minshull, S. Govindarajan, BMC Bioinfor-
matics 7 (2006) 285–292.
In conclusion, R. toruloides CBS14 EH gene was successfully syn-
thesized, cloned and expressed in E. coli. The optimal pH and temper-
ature of purified EH were found to be 7.5 and 35 °C, respectively. The
recombinant EH was sensitive to temperature, and the activity de-
creased significantly at 45 °C. The values of apparent Km and Vmax
were calculated to be 0.5953 mol/l and 0.0105 mol/(L min) with the
epichlorohydrin as substrate. The application of recombinant EH in
racemic resolution of (R,S)-epichlorohydrin showed that the e.e.
value and productivity of (R)-epichlorohydrin reached around 100%
and 18%. After further modification by rational or irrational protein
design methods, this recombinant EH could be a potential candidate
for upscale production of (R)-epichlorohydrin.
[23] U.K. Laemmli, Nature 227 (1970) 680–685.
[24] G.M. Morris, D.S. Goodsell, R.S. Halliday, R. Huey, W.E. Hart, R.K. Belew, A.J. Olson,
Journal of Computational Chemistry 19 (1998) 1639–1662.
[25] N.A.E. Kronenburg, J.A.M. de Bont, Enzyme and Microbial Technology 28 (2001)
210–217.
[26] N. Sakai, Y. Tajika, M. Yao, N. Watanabe, I. Tanaka, Proteins 57 (2004) 869–873.
[27] C.L. Hung, J.H. Liu, W.C. Chiu, S.W. Huang, J.K. Hwang, W.C. Wang, The Journal of
Biological Chemistry 282 (2007) 12220–12229.
[28] J. Maritz, H.M. Krieg, C.A. Yeates, A.L. Botes, J.C. Breytenbach, Biotechnology Let-
ters 25 (2003) 1775–1781.
[29] M.S. Smit, Trends in Biotechnology 22 (2004) 123–129.
[30] S. Karboune, A. Archelas, R. Furstoss, J. Baratti, Journal of Molecular Catalysis
B: Enzymatic 32 (2005) 175–183.
[31] S. Karboune, A. Archelas, J. Baratti, Enzyme and Microbial Technology 39 (2006)
318–324.
Acknowledgements
The support of this work by the National Basic Research Program of
China (973 Program) (No. 2011CB710806), National Natural Science
Foundation of China (No. 21176224) and the Research Project of
Natural Science Foundation of Zhejiang Province (Nos. Z4080032
and R3110155) is gratefully acknowledged.
[32] Z.Q. Liu, Y. Gosser, P.J. Baker, Y. Ravee, Z.Y. Lu, G. Alemu, H.G. Li, G.L. Butterfoss,
X.P. Kong, R. Gross, J.K. Montclare, Journal of the American Chemical Society 131
(2009) 15711–15716.
[33] M.T. Reetz, M. Bocola, L.W. Wang, J. Sanchis, A. Cronin, M. Arand, J.Y. Zou, A.
Archelas, A.L. Bottalla, A. Naworyta, S.L. Mowbray, Journal of the American Chem-
ical Society 131 (2009) 7334–7343.
[34] L.Y. Rui, L. Cao, W. Chen, K.F. Reardon, T.K. Wood, The Journal of Biological Chem-
istry 279 (2004) 46810–46817.
[35] J.H. Woo, Y.O. Hwang, J.H. Kang, H.S. Lee, S.J. Kim, S.G. Kang, Journal of Bioscience
and Bioengineering 110 (2010) 295–297.
References
[1] Z.Q. Liu, Y. Li, Y.Y. Xu, L.F. Ping, Y.G. Zheng, Applied Microbiology and Biotechnol-
ogy 74 (2007) 99–106.
[2] A. Steinreiber, K. Faber, Current Opinion in Biotechnology 12 (2001) 552–558.
[3] E. Blee, F. Schuber, European Journal of Biochemistry 230 (1995) 229–234.
[4] R.J. Linderman, E.A. Walker, C. Haney, R.M. Roe, Tetrahedron 51 (1995) 10845–10856.
[5] I. Osprian, W. Kroutil, M. Mischitz, K. Faber, Tetrahedron: Asymmetry 8 (1997) 65–71.
[6] C.A.G.M. Weijers, J.A.M. de Bont, Journal of Molecular Catalysis B: Enzymatic 6
(1999) 199–214.
[7] M. Nardini, I.S. Ridder, H.J. Rozeboom, K.H. Kalk, R. Rink, D.B. Janssen, B.W. Dijkstra,
The Journal of Biological Chemistry 274 (1999) 14579–14586.