A novel enantioselective epoxide hydrolase from Agromyces mediolanus ZJB120203: Cloning, characterization and application

Article abstract of DOI:10.1016/j.procbio.2014.01.003

A new strain Agromyces mediolanus ZJB120203, capable of enantioselective epoxide hydrolase (EH) activity was isolated employing a newly established colorimetric screening and chiral GC analysis method. The partial nucleotide sequence of an epoxide hydrolase (AmEH) gene from A. mediolanus ZJB120203 was obtained by PCR using degenerate primers designed based on the conserved domains of EHs. Subsequently, an open reading frame containing 1167 bp and encoding 388 amino acids polypeptide were identified. Expression of AmEH was carried out in Escherichia coli and purification was performed by Nickel-affinity chromatography. The purified AmEH had a molecular weight of 43 kDa and showed its optimum pH and temperature at 8.0 and 35 C, respectively. Moreover, this AmEH showed broad substrates specificity toward epoxides. In this study, it is demonstrated that the AmEH could unusually catalyze the hydrolysis of (R)-ECH to produce enantiopure (S)-ECH. Enantiopure (S)-ECH could be obtained with enantiomeric excess (ee) of >99% and yield of 21.5% from 64 mM (R,S)-ECH. It is indicated that AmEH from A. mediolanus is an attractive biocatalyst for the efficient preparation of optically active ECH.

Full text of DOI:10.1016/j.procbio.2014.01.003

Process Biochemistry 49 (2014) 409–417
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
A novel enantioselective epoxide hydrolase from Agromyces
mediolanus ZJB120203: Cloning, characterization and application
Feng Xuea,b, Zhi-Qiang Liua,b, Shu-Ping Zoua,b, Nan-Wei Wana,b, Wen-Yuan Zhua,b
,
Qing Zhua,b, Yu-Guo Zhenga,b,∗
a Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, PR China
b Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou 310014,
Zhejiang, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new strain Agromyces mediolanus ZJB120203, capable of enantioselective epoxide hydrolase (EH) activ-
ity was isolated employing a newly established colorimetric screening and chiral GC analysis method.
The partial nucleotide sequence of an epoxide hydrolase (AmEH) gene from A. mediolanus ZJB120203
was obtained by PCR using degenerate primers designed based on the conserved domains of EHs. Sub-
sequently, an open reading frame containing 1167 bp and encoding 388 amino acids polypeptide were
identified. Expression of AmEH was carried out in Escherichia coli and purification was performed by
Nickel-affinity chromatography. The purified AmEH had a molecular weight of 43 kDa and showed
its optimum pH and temperature at 8.0 and 35 ◦C, respectively. Moreover, this AmEH showed broad
substrates specificity toward epoxides. In this study, it is demonstrated that the AmEH could unusu-
ally catalyze the hydrolysis of (R)-ECH to produce enantiopure (S)-ECH. Enantiopure (S)-ECH could be
obtained with enantiomeric excess (ee) of >99% and yield of 21.5% from 64 mM (R,S)-ECH. It is indicated
that AmEH from A. mediolanus is an attractive biocatalyst for the efficient preparation of optically active
ECH.
Received 31 October 2013
Received in revised form
24 December 2013
Accepted 7 January 2014
Available online 18 January 2014
Keywords:
Cloning
Characterization
Epoxide hydrolase
Agromyces mediolanus ZJB120203
(S)-Epichlorohydrin
© 2014 Elsevier Ltd. All rights reserved.
1. Introduction
optically active pharmaceuticals such as ␤-blockers, l-carnitine,
and ferroelectric liquid crystals [16]. To develop the needs of high
Epoxide hydrolases (EHs, EC 3.3.2.3), ubiquitous in nature and
co-factor independent enzymes, catalyze the hydrolysis of epox-
ides to corresponding vicinal diols with the addition of a water
molecule [1,2]. EHs have been isolated or cloned from a wide range
of organisms including bacteria, yeast, fungi, plant, insect, and
mammals [3–8]. Most EHs are members of the ␣/␤ hydrolase fold
family base on their available sequences information [9–11]. These
EHs consist of a core domain that is composed of a central ␤-sheet
surrounded by ␣-helices and a variable cap domain positioned on
top of the substrate binding site. They use a catalytic mechanism
that involves an Asp–His–Asp/Glu (nucleophile–histidine-acid)
catalytic triad located on top loops of the main domain [12].
The observed intrinsic enantioselectivity of some EHs makes
them more useful in the preparation of enantiopure pharma-
ceuticals and fine chemicals [11–15]. In particular, enantiopure
epichlorohydrin (ECH) is a valuable intermediate for producing
performance enzymes, recent efforts have been focused on the
searching, cloning, expression, purification, characterization, cat-
alytic mechanism, modification of enantioselectivity and activity,
resolution of structure and homology modeling of various new EHs.
Some of EHs exhibit modest enantioseletivities toward ECH. How-
ever, only a few could preferentially hydrolyze (R)-ECH, retaining
the useful (S)-ECH for the synthesis of atorvastatin. For example,
Novosphingobium aromaticivorans EH has been cloned and applied
into resolution of (R,S)-ECH (50 mM) to provide (S)-ECH with 20.7%
yield and more than 99% enantiomeric excess (ee) [16]. Enantiopure
(S)-ECH could be obtained from its racemates (60 mM) with an opti-
cal purity of 100% ee and 20% yield in cyclohexane supplemented
with 2.0% (v/v) water using Aspergillus niger cells harboring EH [17].
18.5% of (S)-ECH with 98% ee from racemic ECH was obtained by the
dry cells of A. niger ZJB-09173 in cyclohexane supplemented with
4.0% (v/v) water [18]. Hence, exploring new enantioselective EHs is
necessary due to the limited number of EHs for the enantioselective
production of (S)-ECH.
In this study, a new strain, Agromyces mediolanus ZJB120203
producing a novel EH was isolated and identified. The gene encod-
ing the EH from A. mediolanus ZJB120203 was cloned and expressed
in Escherichia coli. Some characteristics of recombinant EH as well as
∗
Corresponding author at: Zhejiang University of Technology, Institute of Bio-
engineering, No. 18 Chaowang Road, Hangzhou 310014, Zhejiang Province, PR China.
Tel.: +86 571 88320630; fax: +86 571 88320630.
E-mail address: zhengyg@zjut.edu.cn (Y.-G. Zheng).
1359-5113/$ – see front matter © 2014 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.procbio.2014.01.003

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F. Xue et al. / Process Biochemistry 49 (2014) 409–417
its highly enantioselective hydrolysis of (R,S)-ECH were also stud-
2.4. Expression and purification of AmEH
ied.
For construction of expression vector of AmEH gene, the follow-
ing pair of primers, EHF3 and EHR3 (Table 1) were synthesized, and
the underlined parts were the Nde I and Not I sites. The amplified
DNA fragment was double-digested with Nde I and Not I, and then
ligated into pET28a expression vector treated the same restriction
enzymes to construct the recombinant plasmid pET28a-AmEH. This
recombinant plasmid was used for the expression of the AmEH gene
in E. coli BL21 (DE3).
2. Materials and methods
2.1. Strains and growth condition
E. coli JM 109 (Tiangen biotech Co., Ltd., Beijing, China) and
E. coli BL21 (DE3) (Invitrogen, Karlsruhe, Germany) were used for
gene cloning and gene expression, respectively. The E. coli strains
were cultured in Luria-Bertani (LB) medium at 37 ◦C, and supple-
mented with the appropriate antibiotic(s) [ampicillin (100 ␮g/mL)
or kanamycin (50 ␮g/mL)].
E. coli BL21 (DE3) cells harboring the pET28a-AmEH were grown
at 37 ◦C in LB medium containing Kan (50 ␮g/mL) until the optical
density reached 0.8 at 600 nm. Then, cells were induced by addition
of 0.1 mM isopropyl-␤-d-thiogalactopyranoside (IPTG) and growth
was carried out at 28 ◦C for extra 10 h. The cells were harvested
by centrifugation at 10,000 × g for 10 min, washed with 50 mM
phosphate buffer (pH 8.0). The cell pellet was re-suspended in
50 mM phosphate buffer (pH 8.0) and disrupted by sonication. Cell
debris was removed by centrifugation at 10,000 × g for 10 min. The
soluble fraction was applied to a Nickel–nitrilotriacetic (Ni–NTA)
column equilibrated with binding buffer [50 mM phosphate buffer
(pH 8.0)]. After being washed with washing buffer [500 mM NaCl,
20 mM phosphate (pH 8.0) and 20 mM imidazole], the bound
enzyme was eluted with elution buffer [500 mM NaCl, 20 mM phos-
phate (pH 8.0) and 500 mM imidazole], and then dialyzed against
50 mM phosphate buffer (pH 8.0) [23]. The collected pure enzyme
was used for characterization experiments. The purity of the pro-
tein was analyzed by 12% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) under denaturing conditions as
described by Laemmli [24]. The protein concentration was mea-
sured by the method of Bradford using the Bio-Rad protein assay
kit with bovine serum albumin as a standard.
2.2. Cloning of the EH gene
Genomic DNA was extracted from strain ZJB120203 using a
FastDNA® Spin Kit for Soil (MPBio, Santa Ana, CA) following the
manners provided by manufacturer. To obtain the partial EH gene
fragment, two degenerate primers EHF1 and EHR1 (Table 1) were
designed according to the sequences of two regions (HGWPG and
GHFAALE) that are highly conserved among five EHs gene (Gen-
Bank accession nos. ABF21120, AAO67343, AAF64646, CAA73331
and AFI98637). The PCR product was directly sequenced and the
resulting DNA sequence was used for alignment and homology
search. For cloning of the full-length EH gene, EHF2 and EHR2
primers (Table 1) were designed based on the EH gene sequence
of closest homologue from strain ZJB120203. The amplified DNA
fragments were cloned into the pGEM-T vector (Promega, Madi-
son, WI, USA), and then introduced into E. coli JM109. The resulting
plasmid was named pGEM-T-ameh. The successful clones were
identified by direct colony PCR and subjected to plasmid isolation,
then sequenced the whole length of EH gene.
2.5. Enzyme assay
2.3. Sequence analysis, homology modeling and docking
The standard assay was performed with racemic ECH and
the recombinant EH mixed in 10 mL phosphate buffer (pH 8.0).
The reaction mixture reaction was incubated at 30 ◦C, 150 rpm.
After 10 min, 1.0 mL biotransformation sample was taken and
centrifuged at 12,000 × g for 6 min and then 200 ␮L of biotrans-
formation sample was extracted with 600 ␮L of ethyl acetate and
centrifuged at 10,000 × g for 6 min. The organic layer was sep-
arated and dried with anhydrous sodium sulfate. The reaction
mixture was analyzed by GC and chiral GC to determine the enzyme
activity and ee value, respectively. The Agilent 6890N system (Agi-
lent, Santa Clara, CA) was equipped with a capillary HP-5 column
(0.35 mm × 30 m × 0.25 ␮L film thickness) with N2 as carrier gas.
Chiral separation was performed on a chiral capillary BGB-175 col-
umn (0.25 mm × 30 m × 0.25 ␮L film thickness). The initial column
temperature was set at 90 ◦C and the inlet and detector tempera-
tures were both 220 ◦C. The retention times of racemic ECH, (S)-ECH
and (R)-ECH were 2.8, 4.6 and 4.8 min, respectively [25]. The ee
was derived from the remaining epoxide of the two enantiomers
Sequence similarity searches were carried out using blastn and
blastp, respectively. Sequence analyses were inferred with ExPASy
[19]. A multiple alignment of EH amino acid sequences from various
organisms were constructed with software package DNAMAN and
the ESPript 2.2 network station [20].
The three-dimensional homology model of AmEH was gener-
ated using Build Homology Models (MODELER) in Discovery studio
(DS) 2.1 (Accelrys Software, San Diego, USA) using crystal struc-
tures of EHs (PDB accession no. 4i19) 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 Heath module
in DS 2.1. The geometry of loop regions was corrected using Refine
Loop/MODELER [21]. Finally, the best quality model was chosen for
further calculations, molecular modeling, and docking studies by
Autodock 4.0 [22].
Table 1
The primers for PCR used in the study.
Primer name
Sequence
Purpose
P1
P2
5ꢀ-AGAGTTTGATCCTGGCTCAG-3ꢀ
5ꢀ-AAGGAGGTGATCCAGCCGCA-3ꢀ
5ꢀ-YKSCAYGGYTGGCCMGG-3ꢀ
16S rDNA
16S rDNA
EHF1
EHR1
EHF2
EHR2
EHF3
EHR3
First partial fragment
First partial fragment
Full length amplification
Full length amplification
Expression primer
5ꢀ-SARYGCRGCAAAGTGTCC-3ꢀ
5ꢀ-ATGACCGCGGTGAGTCCCAC-3ꢀ
5ꢀ-TCATTGTTCATTTCCTCTCAATTG G-3ꢀ
5ꢀ-CGCCATATGACCGCGGTGAGTCC CAC-3ꢀ
5ꢀ-ATTTGCGGCCGCTCATTGTTCATTTCCTCTC AATTGG-3ꢀ
Expression primer
Note: Y represents identifies C or T; K represents G or T; S represents C or G; M represents A or C; R represents A or G. Restriction sites are underlined.

F. Xue et al. / Process Biochemistry 49 (2014) 409–417
411
[ee(%) = (S − R)/(S + R) × 100]. The extent of yield (y) was calculated
from [y = (AR + AS)/(A0R + A0S)] before the complete consumption of
epoxide, where the initial epoxide of (R) and (S) was denoted as A0,
and the remaining epoxide of (R) and (S) was as A. One unit (U) of
EH activity was defined as the amount of enzyme required to con-
vert 1 ␮mol ECH at 30 ◦C. Specific activity was defined as U/mg of
enzyme.
accession no. KC589436) of strain ZJB120203 showed high similar-
ity (99%) to the 16S rDNA sequence of A. mediolanus. Based on these
results, the strain ZJB120203 was identified as A. mediolanus, and
further named as A. mediolanus ZJB120203.
3.2. Cloning of the AmEH gene
The partial AmEH gene with length of 799 bp was amplified
using A. mediolanus ZJB120203 genome DNA as template with
the degenerate primers. Blastn searches in the GenBank databases
revealed that the PCR product (799 bp) was similar to a known EH
gene (GenBank accession no. JQ671543). Oligonucleotide primer
EHF2 and EHR2 were derived from the amino acid sequence of clos-
est homologue of strain. The full-length AmEH gene consisted of an
ORF of 1167 bp, was amplified with the primers EHF2 and EHR2. It
begins with ATG and terminating with TGA and encodes a polypep-
tide of 388 amino acid residue with a predicted molecular weight of
42,952 Da, and the theoretical isoelectric point was 4.8. The AmEH
nucleotide sequence has been deposited in the GenBank database
under accession no. JX467176.
2.6. Effects of pH and temperature on AmEH activity
The pH dependence of AmEH activity was investigated using
the following buffers with concentration of 0.1 M: sodium
acetate–acetic acid buffer (pH 4.0 to 7.0), boric acid-sodium borate
buffer (pH 7.5 to 8.5), sodium phosphate buffer (pH 6.5 to 8.0), and
glycine–sodium hydroxide buffer (pH 8.5 to 10.0). The enzyme sta-
bility at each pH was estimated by incubating the enzyme solution
with 0.1 M buffers at pH 4.0–10.0 at 4 ◦C for 30 min. AmEH activ-
ities were measured at pH 8.0 over a temperature range of 20 to
50 ◦C to determine optimum temperature. For determination of the
thermostability, the enzyme was pre-incubated in a water bath at
different temperatures for 1 h, and then the remaining activity was
assayed. All assays were performed in triplicate.
3.3. Sequence analysis, homology modeling and docking
2.7. Circular dichorism (CD) measurements
The amino acid sequence deduced from EH gene of A. mediolanus
ZJB120203 was compared with those of other organisms available
in the NCBI database (Fig. 1). The amino acid sequences align-
ment result showed that EH from A. mediolanus ZJB120203 showed
a high homology (99%) with Arthrobacter sp. JBH1 (GeneBank
accession no. AFI98637) [32]. But it was only 43%, 39%, 34%,
34%, 31% and 31% identical to other EHs from Sphingomonas sp.
KC8 (ZP 09141165), N. aromaticivorans DSM 12444 (YP 497537),
Rhodosporidium toruloides CBS 0349 (AAF64646), Rhodotorula glu-
tinis CIMW 147 (AAF64646), A. niger M200 (ABF21120), and
Agrobacterium radiobacter AD1 (CAA73331), respectively. Accord-
ing to protein sequences alignments (Fig. 1), this newly cloned
AmEH was shown to belong to the ␣/␤ hydrolase family, the
putative members of the catalytic triad were deduced to be
Asp181, His362 and Glu336 (highlighted in green) [33]. The two
conserved tyrosine residues (Tyr308 and Tyr239, highlighted in
pink) implicated in activation of the epoxide during catalysis
were also present in the EH from A. mediolanus ZJB120203. An
HGW[O]P motif which can form an oxyanion hole can be found
[9,34].
CD spectra were recorded using a J-815 spectropolarimeter
(Jasco Co., Tokyo, Japan). The EH were dissolved to a final con-
centration of 0.1 mg/mL in 20 mM sodium phosphate buffer (pH
8.0). The cell pathlength of 1.0 cm was used for the spectral range
190–350 nm. The scanning rate was 20 nm/min. Spectra manager
228 software was used for data acquisition [26,27].
2.8. Substrate specificity
Substrate specificity of AmEH was determined by measuring
the enzyme activity toward various epoxides. All reactions were
conducted in 0.1 M sodium phosphate buffer (pH 8.0) at 30 ◦C for
10 min with 50 mM substrate. The enzyme activity of AmEH toward
the epoxides was determined using the following methods: Epox-
ides 2 and 12 were analyzed by chiral GC-14C system equipped
with Chiraldex G-TA column [28,29]. Epoxides 8 to 10 were ana-
lyzed with a chiral capillary BGB-174 column. Epoxides 3 to 7 were
analyzed by chiral HPLC (Daicel CHIRALPAK AS-H column) using
hexane/2-propanol 4:1 v/v as mobile phase [30]. For the analysis
of the conversion of epoxide 11, a ␤-DEX 225 column was used
[31].
Homology modeling and molecular docking were performed to
gain insights into the binding mode of the substrates and the origin
of inverted enantioselectivity. The binding modes of (R)- and (S)-
ECH in the active site from molecular docking are shown in Fig. 2.
As shown in Fig. 2, both of (R)- and (S)-ECH form hydrogen bonds
with Tyr308 by the epoxide oxygen. We proposed that a sufficiently
small d (the distance between the Asp181 oxygen and the attacked
epoxide carbon) value could correspond to a near-attack conformer
as discussed by Bruice in other enzyme-catalyzed reaction [35], or
more generally to a productive position, and that consequently, this
distance should be shorter in the case of the preferred enantiomer
3. Results
3.1. Isolation and identification of strain ZJB120203
Five bacteria were isolated from the soil from coastal wet-
land of Yancheng city in Jiangsu province, China. Strain ZJB120203
was found to be the most suitable biocatalyst for further study,
as it showed the highest specific activity on racemic ECH.
It was deposited in China Center for Type Culture Collection
(CCTCC, http://www.cctcc.org/), and numbered CCTCC M2012299.
Strain ZJB120203 was taxonomically characterized and identi-
fied on the basis of morphological, physiological, and biochemical
tests. It was found to be an aerobic, Gram-negative, rod-shaped
(0.4–0.6 × 1.0–1.2 ␮m), motile by a single polar flagellum. Colonies
cultivated on nutrient agar for 3 days were white, soft, convex
and wet with smooth edges. The carbon source utilization by a
standardized micromethod employing the Biolog microstation was
determined (data not shown). The 16S rDNA sequence (GenBank
˚
[36]. There was a difference in d values between (R)-(3.5 A) and
˚
˚
(S)-ECH (3.8 A). The ꢀd value is expected to be 0.3 A, with the pre-
ferred (R)-ECH closer to the attacking Asp181 (Table 2). According
to the near attack conformations (NACs) postulated by Bruice [37],
the angle from the Asp181 oxygen via the attacked epoxide carbon
to the epoxide oxygen (˛1) and the Asp181 oxygen via the attacked
epoxide carbon to the other epoxide carbon (˛2) were also consid-
ered. The favored (R)-ECH has larger angles (˛1 and ˛2) than the
disfavored (S)-ECH (Table 2). These may be the reason that why the
hydrolysis of the (R)-ECH proceeds at a much higher rate than that
of the (S)-ECH.

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F. Xue et al. / Process Biochemistry 49 (2014) 409–417
Fig. 1. Multiple alignments of amino acid sequences of EHs. The protein accession numbers are: A. mediolanus ZJB120203 (AmEH, this paper), Arthrobacter sp. JBH1 (AsEH,
AFI98637), Sphingomonas sp. KC8 (SsEH, ZP 09141165), N. aromaticivorans DSM 12444 (NaEH, YP 497537), R. glutinis CIMW 147 (RgEH, AAF64646), R. toruloides CBS 0349
(RtEH, AAO67343), A. niger M200 (AnEH, ABF21120), and A. radiobacter AD1 (AD1EH, CAA73331). The amino acid residues that are conserved in all sequences are all labeled
in red. The catalytic triad (Asp181, His362 and Glu336) and the two tyrosines (Tyr308 and Tyr239) are marked in green and pink, respectively. The oxyanion hole of active site
motif is marked by blue triangles. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.4. Expression of the AmEH gene and its purification
competent cells. The recombinant cell was induced by 0.1 mM of
IPTG at 28 ◦C for about 10 h when the OD600 reached the value
of 0.8. Recombinant AmEH was purified from the supernatants of
cell lysates by single-step affinity chromatography on a Ni–NTA
In order to express the AmEH gene, the recombinant expression
vector pET28a-AmEH was transformed into the E. coli BL21 (DE3)

F. Xue et al. / Process Biochemistry 49 (2014) 409–417
413
Fig. 3. SDS-PAGE analysis of expression products by E. coli BL21 (DE3)
(pET28a-AmEH). Lane 1, E. coli BL21(DE3)/pET28a; lane 2, uninduced E. coli
BL21(DE3)/pET28a-AmEH; Lane 3, E. coli BL21(DE3)/pET28a-AmEH induced by IPTG;
Lane 4, soluble fraction of BL21(DE3)/pET28a-AmEH induced by IPTG; Lane 5, the
purified AmEH; Lane M, molecular weight mark.
3.5. Biochemical characterization of the AmEH
The pH optimum was detectable in the pH range from 4.0 to 10.0.
Optimum activity of AmEH toward ECH occurred at 8.0 (Fig. 4a).
The AmEH showed the high activity over a broad pH range from
6.0 to 9.0. However, the enzyme exhibited little activity below pH
5.0 or above pH 9.0. The AmEH was stable at pH 6.0–9.0, it retained
more than 79% residual activity after incubation at 30 ◦C for 30 min
(Fig. 4b).
The optimum temperature of the AmEH activity was deter-
mined by incubating the purified AmEH in the reaction mixture
for 10 min at temperatures from 20 to 50 ◦C, respectively, at pH 8.0.
The purified recombinant AmEH exhibited a maximum in activity
at 35 ◦C (Fig. 5a). Below 30 ◦C or above 40 ◦C, there was a signif-
icant decrease in the specific activity of AmEH. To determine the
stability against thermal denaturation, the recombinant enzymes
were pre-incubated at various temperatures (20–50 ◦C) for 1 h and
then cooled immediately to assay the residual activity at 30 ◦C. The
enzyme remained stable up to 35 ◦C (Fig. 5a), more than 80% activity
after incubation at 35 ◦C for 1 h. CD spectroscopy is an excellent tool
for rapid determination of protein secondary structures and inves-
tigation of enzyme thermostability [38]. To further investigate the
enzyme thermostability, we measured the CD spectra of AmEH at
different temperatures from 190 to 350 nm. The spectra showed
the effect of heat incubation on the structural change of AmEH. As
shown in the Fig. 5b, only little changes were observed in the CD
spectra and secondary structure content of the protein at tempera-
tures between 10 to 40 ◦C. But there were further changes in the CD
spectra at 50, 60 and 70 ◦C. This result is correspond exactly with
EH activity that decreased rapidly after incubation at temperatures
over 40 ◦C.
Fig. 2. Homology modeling and molecular docking. (a) Homology protein model of
AmEH; (b) (R)-ECH docking into the active site of the enzyme; (c) (S)-ECH docking
into the active site of the enzyme.
column. The SDS-PAGE result showed that a recombinant protein
about 43 kDa (Fig. 3), which was in accordance with the predicted
value, was expressed. The specific activity and amount of protein of
the recombinant EH were calculated to be 24.6 U/mg and 0.11 g/L,
respectively.
Table 2
Results of docking experiments.
Substrate
d [Å]a
˛
1
[◦]b
˛
[◦]b
2
R-ECH
S-ECH
3.5
3.8
125.0
94.3
118.8
88.3
The effects of metal ions and chemicals on the activity of the
AmEH were measured by incubating the enzyme in the presence
of reagents. The residual activities were assayed according to the
standard method. The enzyme activity was severely inhibited by
a
d: the distance between the Asp181 oxygen and the attacked epoxide carbon.
˛1: the angle from Asp181 oxygen via the attacked epoxide carbon to the epoxide
b
oxygen; ˛2: the angle Asp181 oxygen via the attacked epoxide carbon to the other
epoxide carbon.

414
F. Xue et al. / Process Biochemistry 49 (2014) 409–417
a
100
100
80
60
40
20
0
a
80
60
40
20
0
15
20
25
30
35
40
45
50
55
T(oC)
3
4
5
6
7
8
9
10
11
b
pH
b
20
0
100
80
60
40
20
0
10 oC
20 oC
30 oC
40 oC
50 oC
60 oC
70 oC
-20
-40
-60
-80
180 200 220 240 260 280 300 320 340 360
wavelength(nm)
4
5
6
7
8
9
10
pH
Fig. 5. Effects of temperature on the activity (ꢀ) and stability (♦) of the purified
AmEH (a). Assays were performed at various temperatures under the enzyme assay
condition. The remaining activity was assayed under the enzyme assay conditions
after the purified recombinant enzyme had been placed at indicated temperature
for 1 h with 0.1 M sodium phosphate buffer (pH 8.0). The results are means SD
from three independent determinations. The CD spectra of EH were collected in
triplicate at wavelengths from 190 to 350 nm with a scan speed of 20 nm/min (b).
(For interpretation of the references to color in this figure legend, the reader is
referred to the web version of this article.)
Fig. 4. Effects of pH on activity (a) and stability (b) of the purified AmEH. The activ-
ities were determined in standard conditions with 0.1 M in different buffers with
pH varying from 4.0 to 10.0. The remaining activity was assayed under the enzyme
assay condition after the purified recombinant enzyme had been incubated at 30 ◦
C
for 30 min in 0.1 M different buffer. The enzyme activity obtained in 0.1 M sodium
phosphate buffer (pH 8.0) was taken as 100%. The results are means SD from three
independent determinations.
the Hg2+, Ag2+, and Cu2+, partially inhibited by Co2+, Ni+, Fe3+, Ba2+
,
monosubstituted epoxides at C-1 position with bulky ring such as
styrene oxide and benzyl glycidyl ether and with aliphatic chains
such as ECH. In case of epoxides 4 to 6 which were derivative
of benzyl glycidyl ether, there were marked differences in enan-
tioselectivity with methyl position, a general trend can be found
that the ee value increases as the methyl on the phenyl ring is
shifted from the para- to the ortho-position. Thus, we further tested
some other epoxides 7 to 12, however, low activity and values
were observed for most of them. The activity and enantioselectiv-
ity toward glycidates-1,2-disubstitued epoxides were affected by
the side chain length at C-2 position. From the results, it is con-
cluded that the AmEH could be explored for the productions of
chiral epoxides with high value, besides enantiopure (S)-ECH.
Mn2+ and Zn2+, and slightly inhibited by Mn2+ and Fe3+. The addi-
tion of Ca2+, Mg2+, 2% (v/v) of Tween 80 and Triton-X100 induced
the AmEH activation slightly. The metal chelator EDTA also did not
inhibit the enzyme activity, indicating that this enzyme is not a
metalloenzyme (Table 3).
To determine enantioselective hydrolyzing activity of the puri-
fied AmEH, hydrolysis rate of EH toward (S) and (R)-ECH were
determined, and kinetic parameters were determined by non-
R
R
linear regression using a Sigma Plot program. Vmax and Km
of the purified AmEH toward (R)-ECH were 35.62 ␮mol/min/mg
S
S
and 56.6 mM, while Vmax and Km toward (S)-ECH were
7.90 ␮mol/min/mg and 161.4 mM respectively (Table 4), indicating
that (R)-ECH is hydrolyzed faster than (S)-ECH. The enantioselec-
tivity factor E was calculated as 12.9 for the hydrolysis of racemic
ECH. This value is slightly higher than the E value of 12.2 estab-
lished previously by the kinetic resolution of ECH with EH from N.
aromaticivorans in the same aqueous phase system [16].
3.6. Enantioselective resolution of racemic ECH by purified AmEH
Biotransformation of racemic ECH by the 2 mg purified AmEH at
the concentrations of 64 mM was performed under optimal reac-
tion conditions. The time-course changes in the concentration of
(R)- and (S)-ECH and the ee are shown in Fig. 6. The hydrolysis rate
of (R)-ECH showed to be much faster than that of (S)-ECH, and (R)-
ECH vanished completely after 35 min. The enantiopurity of the
remaining (S)-ECH increased from 0% to >99% after 35 min. Final
A number of potential substrates were tested to investigate sub-
strate specificity of the AmEH. As shown in Table 5, the AmEH has
a broad substrate specificity. Nevertheless, among these substrates
there are significant differences in activity and enantioselectivity.
The purified AmEH showed an enantioselective hydrolysis toward

F. Xue et al. / Process Biochemistry 49 (2014) 409–417
415
Table 3
paper, enrichment culture of soil samples supplemented with ECH
as sole carbon source, we also added the pH indicator (bromothy-
mol blue) in order to screen the microorganisms with the EH and
halohydrin dehalogenase activity, because the ECH may be trans-
form to 3-chloro-1,2-propanediol (MCP) by EH, and then the MCP
was degraded by halohydrin dehalogenase which can cause the
change of the pH. This is the first report to use this colorimet-
ric screening and chiral GC analysis method to screen the new
microorganism producing EH with enantioselectivity. Through this
method, microorganisms with EH activity were rapidly found. As
a result, a new strain, A. mediolanus ZJB120203 capable of the R-
stereospecific epoxide hydrolysis and halohydrin dehalogenation
(data not shown) was screened from soil sample.
Effects of metal ions and chemical agents on AmEH activity.
Reagent
Concentration
Relative activity (%)a
None
EDTA-Na2
CoCl2
NiCl2
FeCl3
BaCl2
CaCl2
MgCl2
MnSO4
ZnSO4
AgNO3
HgCl2
CuSO4
Triton-X100
Tween 80
DMSO
100
5 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
2% (v/v)
2% (v/v)
2% (v/v)
98.5 1.3
75.8 2.3
52.6 1.7
87.3 2.0
53.8 0.9
103.7 2.4
102.6 1.2
89.2 2.8
68.5 2.1
13.7 1.8
0
Further, an EH gene was isolated from strain ZJB120203. The
amino acid sequences alignment showed that EH from A. medi-
olanus ZJB120203 showed a high homology (99%) with Arthrobacter
sp. JBH1, but no information of biotransformation about the EH
from the Arthrobacter sp. JBH1 was available. In addition, it was
only 39% identical to EH from N. aromaticivorans DSM 12444
(YP 497537), which was previously used in the bioresolution of
enantiopure (S)-ECH. Until now, about six structures of EHs belong-
ing to ␣/␤ hydrolase family have been solved. Despite a generally
low sequence similarity, EHs posses a high conservation of the cat-
alytic triad composed of nucleophile–histidine-acid. These residues
located at the interface of the main domain and the cap domain
(Asp–His–Asp/Glu), while two tyrosines located in the cap domain
[1]. In our case, sequence alignment and homology modeling, indi-
cated that AmEH belongs to ␣/␤ hydrolase fold enzymes, which
shares the common features in that they have critical residues to
show EH activity such as the catalytic triad (Asp181, His362 and
Glu336), two tyrosines (Tyr308 and Tyr239) assisting in the ring open-
ing and HGXP motif. It indicated that the conservativeness at the
residues is the minimum requirement for EHs activity. The docking
study revealed the binding mode of the substrates and the origin of
inverted enantioselectivity. The Tyr308 make hydrogen bonds with
the oxygen of the ECH, while the carboxylate oxygen of Asp181 is
attacking one of the epoxide-ring carbons. This results in the for-
mation of a covalent enzyme-substrate ester intermediate, which is
hydrolyzed through the attack of water molecule in the second fast
step of reaction. The catalytic His362 together with Glu336 function
as a charge-relay pair, which is responsible for water activation. It
is reported that d and angles value are key factors influencing the
activity and enantioselectivity of EH [36]. In this study, we found
that there was a big difference in angle values between (R)-(125.0◦
and 118.8◦) and (S)-ECH (94.3◦ and 88.3◦), which leads to the AmEH
was more active on (R)-ECH. At the same time, the favored (R)-ECH
shows shorter distance than the disfavored (S)-ECH. Regulating the
d and angles value would improve the activity and enantioselectiv-
ity of the AmEH on (S)-ECH. Thus, structural changes in the binding
pocket imposed by the evolutionary process are predicted to make
the (R)-ECH to be positioned close to Asp181 for rapid nucleophilic
attack to proceed.
19.2 3.1
110.3 1.2
120.6 2.5
90.2 3.7
a
The enzyme activity was measured under the standard conditions using ECH as
substrate after incubating the enzyme with different metal ions and chemical agents
at 30 ◦C for 30 min. The activity in the absence of metal ions was recorded as 100%.
Table 4
Kinetic parameters of hydrolysis of (S)-and (R)-ECH with AmEH.
Substrate
Km (mM)
Vmax
(␮mol/min/mg)
kcat (1/s)
kcat/Km
[1/(mM s)]
(R)-ECH
(S)-ECH
56.6
161.4
35.62
7.90
25.74
5.71
0.453
0.035
yield of enantiopure (S)-ECH was 21.5% (theoretical yield = 50%) for
racemic ECH.
4. Discussion
EHs are known to be promising biocatalysts for the prepara-
tion of chiral epoxides and vicinal diols [15,39], because of their
excellent enantioselectivity on substrates. The already present
application of EHs in the production of chiral epoxides is antici-
pated to expand as new enzymes are being isolated, cloned, and
characterized.
Until now, several bacterial strains were isolated from soil sam-
ples, which can use DCP or ECH as a sole carbon source [40].
The pathway for the degradation of 1,3-dichloro-2-propanol (DCP)
or ECH was found in these microorganism (Fig. S2), the deha-
logenation steps are catalyzed by the haloalcohol dehalogenase,
whereas the epoxide hydrolysis step is catalyzed by EH. In this
35
30
25
20
15
10
5
120
100
80
60
40
20
0
(S)-ECH
(R)-ECH
ee(%)
The AmEH gene was successfully overexpressed in E. coli, and
the AmEH activity was confirmed. Characterization of the recombi-
nant AmEH showed that this enzyme has very excellent activities
toward ECH and other epoxides, especially under the conditions
with pH 8.0 and 35 ◦C. The AmEH is a potential biocatalyst for the
preparation of enantiopure styrene oxide and benzyl glycidyl ether
which are the important intermediate for the synthesis of pharma-
ceuticals and agrochemicals. The effect of selected metal ions and
chemical agents >on AmEH was similar to those from other sources.
Compared to the other known EHs, the racemic resolution of ECH
showed that this AmEH could unusually catalyze the hydrolysis of
(R)-ECH to produce enantiopure (S)-ECH [25,28,41–43]. This AmEH
has been applied into resolution of 64 mM (R,S)-ECH to provide
(S)-ECH with 21.5% yield, which is the highest yield compare to
0
0
5
10
15
20
25
30
35
40
Time(min)
Fig. 6. Enantioselective resolution of 64 mM racemic ECH by the purified AmEH.

416
F. Xue et al. / Process Biochemistry 49 (2014) 409–417
Table 5
Comparison of the enzyme activity and enantioselectivity of AmEH toward various epoxide substrates.
Serial number
1
Substrates
Relative activity (%)a
100
Absolute configurationb
ee (%)
Epichlorohydrin
Styrene oxide
S
>99
O
Cl
O
2
3
43.3
84.9
R
S
>99
>99
O
Benzyl glycidyl ether
O
O
O
4
Rac-m-benzyl glycidyl ether
54.9
S
88.4
O
5
6
Rac-p-benzyl glycidyl ether
Rac-o-benzyl glycidyl ether
41.2
35.8
S
S
47
O
O
91.1
O
O
7
8
3-Phenylglcidol
51.1
17.8
(R,R)
(R,S)
95.7
11.5
OH
O
COOMe
COOEt
Methyl-3-phenyl glycidate
O
9
Ethyl-3-phenyl glycidate
Cyclohexene oxide
8.6
(R,S)
9.4
O
10
15.8
–
meso
O
11
Allyl glycidyl ether
1,2-Epoxyoctane
89.6
14.5
n.d.c
n.d.
n.d.
n.d.
O
O
12
a
Value are expressed as percentage of the activity measure with ECH taken as 100%.
Absolute configuration, meaning the configuration of remaining epoxide after reaction.
Not determined.
b
c
previous reports [16–18]. Though the AmEH proved to have some
advantages to (S)-ECH production, the E-value was indeed low, and
the enzyme activity of AmEH was also required to enhance. To
improve the activity and selectivity, protein engineering methods
[44–48], including site-directed mutagenesis, saturation mutagen-
esis, error-prone polymerase chain reaction and DNA shuffling,
will be applied to enhance the activity and enantioselectivity of
AmEH.

F. Xue et al. / Process Biochemistry 49 (2014) 409–417
417
In conclusion, we successfully cloned a novel EH gene from a
newly isolated strain, A. mediolanus ZJB120203. The EH gene, with
the length of 1,167 bp encoding 388 amino acids, was actively
expressed in E. coli. The purified AmEH had a molecular mass of
43 kDa and showed its optimal activity under condition of temper-
ature 35 ◦C and pH 8.0. Enantiopure (S)-ECH with ee > 99% and a
yield of 21.5 were obtained from 64 mM racemates. Characteristics
investigation indicated that AmEH is an attractive biocatalyst for
the efficient preparation of optically active ECH.
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.procbio.2014.01.003.
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