Brassica napus
soluble epoxide hydrolase (BNSEH1)
Cloning and characterization of the recombinant enzyme expressed in
Pichia pastoris
Stefan Bellevik
1
, Jiaming Zhang
2
and Johan Meijer
1
1
Department of Plant Biology, Genetics Center, Swedish University of Agricultural Sciences, Uppsala, Sweden;
2
National
Biotechnology Laboratory of Tropical Crops, Chinese, Academy of Tropical Agricultural Sciences, Chengxi, Haikou, China
Epoxide hydrolase (EC 3.3.2.3) in plants is involved in the
metabolism of epoxy fatty acids and in mediating defence
responses. We report the cloning of a full-length epoxide
hydrolase cDNA (BNSEH1) from oilseed rape (Brassica
napus) obtained by screening of a cDNA library prepared
from methyl jasmonate induced leaf tissue, and the 5¢-RACE
technique. The cDNA encodes a soluble protein containing
318 amino acid residues. The identity on the protein level is
85% to an Arabidopsis soluble epoxide hydrolase (sEH) and
50–60% to sEHs cloned from other plants. A 5 · His tag
was added to the N-terminus of the BNSEH1 and the con-
struct was over-expressed in the yeast Pichia pastoris. The
recombinant protein was recovered at high levels after
Ni-agarose chromatography of lysed cell extracts, had a
molecular mass of 37 kDa on SDS/PAGE and cross-reacted
on Western blots with antibodies raised to a sEH from

ity to inhibitors, pH optimum, etc. [2]. Several geometrically
different but related epoxides such as cis-stilbene oxide
(CSO) and trans-stilbene oxide (TSO) have been found to be
useful substrates in order to distinguish soluble from
membrane bound epoxide hydrolases [3,4]. These substrate
pairs can be applied to crude extracts to assess the relative
contribution of membrane bound and soluble forms to the
total epoxide hydrolase activity in many species.
Based on sequence homology analysis epoxide hydrolase
was classified as a member of a super family of hydrolytic
enzymes including esterases and lipases, united by an a/b
hydrolase fold and a similar catalytic triad motif [5]. The
three-dimensional structure of epoxide hydrolase has been
resolved for mouse [6], fungal [7] and bacterial enzymes [8].
These studies have confirmed the predicted a/b fold struc-
ture, provided a detailed picture of the active site and
proposed a mechanism of the catalytic reaction supported by
site-directed mutagenesis. Epoxide hydrolases act through a
two-step mechanism in which an acidic nucleophile attacks
the epoxide ring, forming a covalent intermediate, which is
then hydrolysed by a polarized water molecule [9].
Epoxide hydrolases in mammals are essential in the
detoxification of epoxides that are toxic due to the
electrophilic and unstable nature of the epoxide ring [2].
The relevance of this enzyme for detoxification in plants is
uncertain, however. Certain plants store epoxy fatty acids in
seeds, e.g. Euphorbia lagascae contains up to 60% epoxy
fatty acids in the seed oil [10]. Epoxide hydrolase is probably
needed for complete b-oxidation of epoxy fatty acids, e.g.
during germination when seed stores are broken down prior

a host to generate high amounts of epoxy or hydroxy fatty
acids in the seed for production of technical oils or plastics
[19]. Successful engineering has already altered seed fatty acid
composition to create high laurate [20] and up-regulated
palmitate, stearate and c-linolenate (x-6) B. napus lines [21].
Several genes encoding sEH exist in potato, soybean and
Arabidopsis [11,13,22] and apparently also in oilseed rape
(S. Bellevik, J. Lin & J. Meier). Enzymatic characterization
is necessary to better understand the functional roles of the
isoforms and in this study we have used the yeast P. pastoris
as host for over-expression of the first sEH cloned from
B. napus (BNSEH1). We here present data concerning the
physico-chemical and biochemical properties of the recom-
binant enzyme.
EXPERIMENTAL PROCEDURES
Materials
Oligonucleotides were purchased from TAGC (Copenha-
gen, Denmark). All enzymes were purchased from MBI
Fermentas (Vilnius, Lithuania) unless stated otherwise.
CSO and TSO (Aldrich Chemical Co., Milwaukee, WI,
USA); b-naphtoflavone, sodium-parahydroxymercuri
benzoate, quercitin (3,3¢,4¢,5,7-pentahydroxyflanone) (Sigma
Chemical Co., St Louis, MO); chalcone oxide (Lancaster
Synthesis, Morecambe, UK); x-bromo-4-nitroacetophe-
none (Fluka AG; Buchs, Switzerland); 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide (Bio-Rad Laboratories, Hemel
Hempstead, UK); N,N¢-dicyclohexylcarbodiimide (kind gift
of A
˚
ke Engstro

. Hybridization was performed at high strin-
gency conditions using Hybond N
+
nylon filters (Amer-
sham Biosciences, Uppsala, Sweden) according to the
manufacturer’s instructions. Signals were detected after
36–56 h exposure on Biomax MS X-ray films (Amersham
Biosciences). One of the positive phage clones was excised
from Lambda Zap II using R408 helper phage coinfection
but was found to lack the initial bases of the 5¢-end
(including the initiating methionine codon).
5¢-Rapid amplification of cDNA ends (RACE)
ASMART
TM
RACE cDNA Amplification kit (Clontech)
was employed to amplify the missing 5¢-region of the
cDNA. Total RNA from young B. napus cv. Hanna
seedlings were isolated after grinding in liquid nitrogen
using phenol/chloroform extraction and precipitation with
lithium chloride. Several RACE PCR products correspond-
ing to the cDNA clone were obtained using one nested
primer pair after the first cDNA synthesis. In the first strand
synthesis Superscript II
TM
Rnase H
–
Reverse Transcriptase
(Life Technologies, Ta
¨
by, Sweden) was used with the

BamH1 site, a Kozak consensus sequence for a proper
translation initiation in P. pastoris and also the nine initial
bases missing in the isolated cDNA clone. The reverse
primer (5¢ AAG GTA GGA ATT CCT AGA ATT TGG
AGA TGA AGT C 3¢) contained an EcoR1 site after the
stop codon. The PCR product was amplified using Taq
DNA polymerase (Stratagene, La Jolla, CA, USA) and
blunt-end cloned into the pPCR-Script AMP SK(+)
cloning vector. After sequencing the chosen clone was
digested with BamH1 and EcoR1, transformed into E. coli
by electroporation and subcloned into the P. pastoris
expression vector pPIC3.5K (Invitrogen). JM106 electro-
competent cells were transformed, the selected clone
cultured and prepared by a Qiagen plasmid Midi kit,
linearized with Stu1 and purified with a PCR purification kit
(Qiagen, Hilden, Germany). Competent GS115 yeast cells
were electroporated with 15 lg of linearized plasmid before
plating onto selective media. The resulting colonies were
tested in liquid culture for epoxide hydrolase activity after
24 h of methanol induction (calculated as percentage
substrate turnover/OD cell suspension) using the [
3
H]TSO
assay [3]. Selected clones were grown in 2-L Fernbach flasks
under vigorous agitation to improve aeration and cells were
harvested after 4 days of methanol induction, centrifuged at
5296 S. Bellevik et al. (Eur. J. Biochem. 269) Ó FEBS 2002
3000 g for 5 min and stored at )80 °C if not processed at
once.
Purification of recombinant BNSEH1

concentrations needed to remove contaminating proteins
in the washing step and to elute the recombinant enzyme
in the elution step were determined in preliminary
experiments. Fractions were analysed for epoxide hydro-
lase activity and protein content based on the Peterson
procedure [24] using BSA as a standard, and also by SDS/
PAGE [25].
Western Blot analysis
Proteins were separated by SDS/PAGE, using 12.5%
polyacrylamide gels (Invitrogen). After completion of the
gel electrophoresis, separated proteins were detected by
Coomassie Brilliant Blue or by immunoblotting after wet
transfer to poly(vinylidene difluoride) filters (Schleicher &
Schull, Dassel, Germany). After blocking in milk/BSA,
filters were incubated with affinity purified rabbit polyclonal
anti-sEH Ig followed by HRP-conjugated swine anti-rabbit
Ig (Dako A/S, Glostrup, Denmark) and bands detected by
diaminobenzidine staining or ECL (Pierce, Rockford, IL,
USA). Antibodies were raised by immunization of rabbits
with recombinant sEH from A. thaliana (AtsEH1). The
AtsEH1 corresponds to the EST clone used as probe for the
library screening (see above). Preimmune serum served as
the negative control. The regional ethical committee
approved the animal experiments.
Oligomerization analysis of BNSEH1 by gel filtration
A HiPrep 26/60 Sephacryl S-100 High Resolution column
(Amersham Biosciences) was chosen for native gel filtration
analysis of BNSEH1 dissolved in 0.1
M
potassium phos-

a
values
were obtained from intersections of asymptotes to the curve
plot of log(k
cat
) vs. pH. Activation enthalpy was calculated
from Arrhenius plots of ln(k
cat
)vs.1/T based on values up
to the denaturation point (55 °C). For the kinetic param-
eters a range of approximately 10-fold higher and lower
substrate concentrations of the predicted K
m
was tested and
substrate conversions were kept below 5% to obtain
accurate estimates of initial velocities. The data were used
to draw a Lineweaver-Burk plot and the line equation was
used to calculate K
m
and V
max
values. When inhibitors were
tested the inhibitor was preincubated with enzyme for 5 min
at 30 °C before the substrate was added. The control
reactions received solvent alone. No effect of the solvents on
epoxide or diol partitioning between the organic and
aqueous phases was found.
RESULTS
Cloning of BNSEH1
An epoxide hydrolase clone was isolated from a cDNA

of the yeast cells through a French press the lysis efficiency
reached 90–95% as determined by phase-contrast micro-
scopy. The crude extract was filtered, centrifuged and
passed through a nickel resin with affinity for the histidine
tag. Fractions with more than 80% purity of BNSEH1 were
used for the biochemical analysis.
Ó FEBS 2002 Recombinant Brassica napus epoxide hydrolase (Eur. J. Biochem. 269) 5297
Physico-chemical properties of recombinant BNSEH1
The mass of BNSEH1 was estimated as 37 kDa based on
SDS/PAGE with Coomassie staining (Fig. 2A). The theor-
etical mass of BNSEH1 calculated from the predicted
amino acid sequence of the cDNA corresponds to
36 164 Da (37 096 Da with his-tag) assuming no post-
translational modifications. The BNSEH1 over-expressed
enzyme cross-reacted with antibodies raised against AtsEH1
and the sizes of the sEHs were indistinguishable upon
Western blot analysis (Fig. 2B). Twice the amount of
BNSEH1 protein relative to AtsEH1 was needed to reach
an equal signal intensity. No reaction was observed when
samples were incubated with preimmune serum or when a
P. pastoris extract without BNSEH1 was probed with the
specific antibodies.
Gel filtration analysis of recombinant BNSEH1
The BNSEH1 was subjected to size exclusion chromato-
graphy to determine its oligomeric state. BNSEH1 eluted as
a distinct single peak based on absorbance at 280 nm and
TSO activity (results not shown). The size of the native
BNSEH1 corresponded to 45 kDa.
Biochemical characterization of recombinant BNSEH1
The recombinant enzyme had low activity towards CSO

55 °C (Fig. 4A). At this temperature a threefold higher
activity was reached compared to standard conditions
chosen at 30 °C. The enzyme was almost completely
inactivated at 64 °C and at higher temperatures. The
enthalpy of activation of the reaction was estimated from
Arrhenius plots to  47 kJÆmol
)1
. The pH optimum for
enzymatic TSO hydrolysis was found to be broad and
around pH 6–7 (Fig. 4B). At pH 4.5 almost 50% of
maximum activity was obtained while activity dropped
rapidly at higher alkaline pH. The activity in potassium
Fig. 1. Protein sequence alignment
(
CLUSTALW
1.8) of cloned plant epoxide
hydrolases. The terminal asterisks illustrate
the suggested peroxisomal targeting signal.
The following sequences were retrieved for
analysis from the GenBank database;
B_napus (AJ459780), A_thaliana (D16628),
G_max (CAA55294), S_tuberosum
(U02497), E_lagascae 1 (AF482450),
N_tabacum (AAB02006). The predicted
catalytic residues are bolded and indicated
by * in the alignment. Identical and similar
residues are shown with black and grey
backgrounds, respectively.
5298 S. Bellevik et al. (Eur. J. Biochem. 269) Ó FEBS 2002
phosphate buffer at pH 6–6.5 was similar to Tris/HCl

concentration, respectively. b-Naphtoflavone had no effect
up to 100 l
M
but caused 50% inhibition at 1 m
M
. 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide had no effect on the
enzyme activity up to 1 m
M
. b-naphtoflavone, quercitin and
N,N¢-dicyclohexylcarbodiimide did not dissolve completely
at 1 m
M
final concentration so the free concentration is
actually lower. Addition of ethanol alone had only a minor
effect on the enzyme activity while addition of dimethyl-
sulfoxide caused almost 20% reduction in activity and the
solvent control was set to 100%.
Fig. 3. Kinetic analysis of recombinant BNSEH1. Enzyme activity was
determined using TSO at different concentrations from 0.98 to
187.5 l
M
in 0.1
M
potassium phosphate, pH 7, using 0.1 lgof
BNSEH1 enzyme. A Lineweaver-Burk plot used to calculate the kin-
etic parameters is shown based on data from one out of two repre-
sentative experiments.
Fig. 4. Effects of temperature and pH on recombinant BNSEH1 enzyme
activity. (A) Enzyme activity was measured using 100 l

The BNSEH1 was cloned and found to encode a protein of
318 amino acid residues. The recombinant enzyme was
functional and readily detected by assay of TSO hydrolysis
in yeast extracts upon over-expression in P. pastoris using a
methanol inducible promoter [27]. The his-tagged BNSEH1
was obtained at > 80% purity after one-step purification
on Ni-agarose and the subunit mass could be determined to
37 kDa. No misfolding seemed to occur since all protein
fractions recovered from the affinity chromatography
displayed activity and a symmetrical active peak was
obtained upon gel filtration analysis of the native enzyme.
Of the plant sEH sequences available in the database,
BNSEH1 is most closely related to the Arabidopsis AtsEH1
with 85% identity in the predicted amino acid sequence.
Identity to the other plant sEHs was in the range 50 to 60%
with a higher similarity to soybean (Glycine max)and
potato than to tobacco and E. lagascae sEH (Fig. 1). The
five catalytic residues of sEH in plants and mammals [9] are
conserved also in BNSEH1 suggesting that the properties
are similar to the potato and Arabidopsis enzymes [4].
Gel filtration analysis showed that the recombinant
BNSEH1 is a monomer. The apparent native mass of
45 kDa is slightly higher than expected for a monomeric
BNSEH1 but can be due to an altered diffusion in the gel
matrix because of the histidine tag. Potato and Arabidopsis
AtsEH1 were also shown to be monomers [4] while a
soybean sEH was reported to be a dimer [12]. An obvious
difference between the soybean and the other plant sEH is
an N-terminal extension of 25 amino acids (Fig. 1). The
function of these residues is not clear but it is tempting to

reported that carboxylate modifying agents such as
N,N¢-dicyclohexylcarbodiimide and its hydrolysis product,
N,N¢-dicyclohexylurea, showed strong inhibition of mam-
malian sEH using 4-nitrophenyl-trans-2,3-epoxy-3-phenyl-
propyl carbonate or 1,3-diphenyl-trans-propene oxide as
substrates. Also the B. napus sEH, using TSO as substrate,
was inhibited by these carboxylate modifying compounds
(Fig. 5), most probably through interference with the
activating tyrosine residues [29]. Chalcone oxides that
originally were reported as potent inhibitors for mammalian
sEH [30] does not seem to be very efficient on plant sEHs
including AtsEH1 [4] and BNSEH1. Recent experiments
with the soybean sEH, using the substrate 9,10-epoxystearic
acid, suggested that also the enantioselectivity differs
between plant and mammalian sEHs [31].
The BNSEH1 clone was isolated from a cDNA library
prepared from MeJa-treated leaves. sEH has been described
to be up-regulated at the transcript level by MeJa in potato
[11] but in Vicia sativa seedlings sEH activity remained
unaffected by MeJa treatment [32]. No MeJa induction of
sEH transcripts was observed in B. napus seedlings kept in
hydroponic cultures (S. Bellevik, F. Sitbon & J. Meier)
suggesting that BNSEH1 probably has a constitutive
expression. The only plant hormone besides MeJa reported
to induce sEH is auxin, which increased transcript levels of a
sEH in Arabidopsis within 1 h of treatment [14]. Analysis of
such experiments must keep in mind that sEHs recently
have been identified in multiple copies in several plants.
Preliminary Southern blot data suggest that at least four
epoxide hydrolase genes are present in B. napus (S. Bellevik,

Oilseed rape is the third largest oil crop in the world and
there are potential industrial applications for transgenic
plants modified for enzymes such as epoxide hydrolases.
For example, overproducing oxylipins in the vegetative
tissues resulting in a better endogenous biotic stress
protection would be useful for the agriculture and environ-
ment in terms of reduced pesticide spraying and increased
crop yields. A modified lipid composition in the seeds of
B. napus is also interesting as a renewable source for
technical oils [19]. Knowledge of the function and regulation
of epoxide hydrolase isoforms in plants open up possibilities
for future engineering to redirect fatty acid metabolism into
the desired products.
ACKNOWLEDGEMENTS
This work was supported by grants from the Foundation for Strategic
Research. We are grateful to Mikael Widersten, Uppsala University,
for discussions on enzyme kinetics.
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