Styrene monooxygenase from Pseudomonas sp. LQ26 catalyzes the asymmetric epoxidation of both conjugated and unconjugated alkenes

Article abstract of DOI:10.1016/j.molcatb.2010.08.012

A novel styrene monooxygenase (SMO) was isolated from Pseudomonas sp. LQ26, a styrene degrader from activated sludge. Sequence alignment demonstrated that it was the most distant member of all SMOs originating from the genus of Pseudomonas. The substrate spectrum of this enzyme extended beyond typical SMO substrates to 1-allylbenzene analogues, previously reported as non-substrates for the SMO from Pseudomonas fluorescens ST. The results demonstrate for the first time the asymmetric epoxidation of both conjugated and unconjugated alkenes catalyzed by SMO and suggest that a much broader substrate spectrum is expected for SMOs.

Full text of DOI:10.1016/j.molcatb.2010.08.012

Journal of Molecular Catalysis B: Enzymatic 67 (2010) 236–241
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Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Styrene monooxygenase from Pseudomonas sp. LQ26 catalyzes the asymmetric
epoxidation of both conjugated and unconjugated alkenes
Hui Lina,b,1, Jing Qiaoa,b,1, Yan Liua, Zhong-Liu Wua,∗
a Chengdu Institute of Biology, Chinese Academy of Sciences, P.O. Box 416, Chengdu 610041, China
b Graduate University of Chinese Academy of Sciences, Beijing 100039, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 May 2010
Received in revised form 24 August 2010
Accepted 24 August 2010
Available online 18 September 2010
A novel styrene monooxygenase (SMO) was isolated from Pseudomonas sp. LQ26, a styrene degrader from
activated sludge. Sequence alignment demonstrated that it was the most distant member of all SMOs
originating from the genus of Pseudomonas. The substrate spectrum of this enzyme extended beyond
typical SMO substrates to 1-allylbenzene analogues, previously reported as non-substrates for the SMO
from Pseudomonas fluorescens ST. The results demonstrate for the first time the asymmetric epoxidation of
both conjugated and unconjugated alkenes catalyzed by SMO and suggest that a much broader substrate
spectrum is expected for SMOs.
Keywords:
Epoxidation
Pseudomonas
© 2010 Elsevier B.V. All rights reserved.
Styrene monooxygenase
Unconjugated alkene
Biocatalysis
1. Introduction
Here, we present a novel SMO (designated as StyAB2) that
can perform the asymmetric epoxidation of 1-allylbenzene and
As a widely-used starting material for synthetic polymers,
styrene is present in many industrial and domestic effluents. Over
the last few decades, a large number of styrene-degrading microor-
ganisms have been isolated [1,2]. The majority of reports indicate
that the predominant catabolic pathway for styrene degradation
is through the oxidation of vinyl side chain catalyzed by styrene
monooxygenase (SMO). It is a two-component monooxygenase
encoded by styA and styB, consisting of an oxygenase (StyA) and
a NADH-flavin oxidoreductase (StyB) [3].
other unconjugated styrenes in addition to conventional conju-
gated styrene derivatives. StyAB2 was isolated from Pseudomonas
sp. LQ26, a styrene degrader from activated sludge. Compared with
all reported StyAB sequences from the genus of Pseudomonas at
DNA level, SMO from Pseudomonas sp. LQ26 shows a maximum
identity of 79%.
2. Materials and methods
SMOs display excellent enantiomeric selectivity in styrene
epoxidation, which has attracted many efforts to the synthesis
of chiral epoxides [4–9], a group of extremely important building
blocks in organic synthesis. SMOs from Pseudomonas fluorescens ST
[10,11] and Pseudomonas sp. VLB120 [6], have been used to con-
vert conjugated styrene derivatives as well as aromatic sulfides. A
recent isolated SmoA from metagenome [12] and a self-sufficient
SMO from Rhodococcus opacus [13] display similar substrate spec-
trum. Only one report has described the attempt to apply SMO in
the epoxidation of unconjugated alkenes and the results suggest
that 1-allylbenzene does not serve as a substrate [11].
2.1. Chemicals
Styrene, 4-bromostyrene, 3-chlorostyrene, 1,2-dihydrona-
phthalene, 2-vinylpyridine, 1H-indene and 1-allylbenzene were
purchased from Alfa-Aesar (Tianjin, China) or Acros Organics (Geel,
Belgium). 1-(2-Methylallyl)benzene, 1-allyl-2-hydroxybenzene,
1-allyl-3-methylbenzene and 1-allyl-4-methoxybenzene were
synthesized using Wittig reaction [14]. Racemic and (S)-styrene
epoxides were from Sigma–Aldrich (St. Louis, MO, USA). Other
racemic mixtures of epoxides were synthesized according to the
literatures [15,16].
2.2. Isolation of styAB2 from Pseudomonas sp. LQ26
∗
Corresponding author at: Chengdu Institute of Biology, Chinese Academy of Sci-
ences, 9 South Renmin Road, 4th Section, Chengdu, Sichuan 610041, China.
Tel.: +86 28 85238385; fax: +86 28 85238385.
The styrene degrading strain Pseudomonas sp. LQ26 was iso-
lated from activated sludge collected from a municipal sewage
treatment plant in Chengdu, China, using a modified indigo for-
E-mail address: wuzhl@cib.ac.cn (Z.-L. Wu).
1
These authors contributed equally to the present work.
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.08.012

H. Lin et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 236–241
237
mation assay [17] by conventional enrichment approach. The
2.6. Whole-cell bioconversion and product analysis
strain was deposited at China Center for Type Culture Collec-
tion (Wuhan, China) under the acquisition number of CCTCC M
2010188. Partial segment of 16 s rDNA was amplified by PCR with
two universal primers 5ꢀ-AGAGTTTGATCCTGGCTCAG-3ꢀ (27f) and
5ꢀ-GGTTACCTTGTTACGACTT-3ꢀ (1492r), cloned into pMD18-T vec-
tor (Takara, Dalian, China) and sequenced. The sequence has been
deposited at GenBank under accession no. GU731675. Gene styAB2
was amplified with primers 5ꢀ-ATGAAAAAGCGTATCGGTATTG-
3ꢀ and 5ꢀ-TTCGAAGGCATGAAGCATG-3ꢀ, designed according to
the reported consensus sequence for Pseudomonas species and
deposited at GenBank under accession no. GU593979.
Recombinant E. coli BL21 cells with wet weight of 0.5–2.0 g
were resuspended in 10 ml 100 mM potassium phosphate buffer
(pH 6.5) containing 20% (v/v) bis-(2-ethylhexyl) phthalate (BEHP)
and 10 mg substrate. The reaction was carried out at 30 ◦C for
24 h with gyratory shaking at 220 rpm and terminated by extrac-
tion with ether. The combined organic extracts were dried with
anhydrous sodium sulfate, concentrated under reduced pressure
and subjected to high performance liquid chromatography (HPLC)
analysis performed on a Shimadzu Prominence LC-20AD system
connected to a PDA-detector. Chemical yields were analyzed by
reverse-phase HPLC on a Luna C18 column (4.6 mm × 150 mm, Phe-
nomenex, Torrance, CA, USA). Optical purities were determined by
chiral HPLC using Daicel Chiralcel OD-H (compound 4) and OJ-H
(compound 3), or Chiralpak AD-H (compounds 1 and 2) and AS-H
(compounds 5–11) columns.
4-Bromostyrene was used as the substrate to study the
effects of reaction temperature and pH. Eighteen milligram of
4-bromostyrene was used as the substrate in 20 ml 100 mM potas-
sium phosphate buffer containing 10% (v/v) cyclohexane with of
0.5 g wet weight of recombinant E. coli BL21 cells. The reactions
were performed in duplicate assays at 30 ◦C with varied pH values
for 18 h or at pH 6.5 with varied temperatures for 24 h. Substrate
conversion was measured by reverse-phase HPLC on a Luna C18
column. The reactions were performed at 30 ◦C in potassium phos-
phate buffer with pH values ranging from 6.0 to 8.0, or in potassium
phosphate buffer with a pH value of 6.5 at temperatures varying
from 25 to 45 ◦C.
4-Chloroindole was used as the substrate to investigate the func-
tional expression of SMO. The pelleted cells from 1 ml culture were
resuspended in 1 ml 100 mM potassium phosphate buffer (pH 6.5)
containing 0.5 mM 4-chloroindole in a 24-well plate and incubated
at 37 ◦C with shaking at 220 rpm for 10 or 20 min. The formation of
colored product was measured by determining the absorbance at
620 nm and the estimated concentrations were calculated based
on the extinction coefficient of 4,4ꢀ-dichloroindigo [18]. 1U was
defined as the activity which converts 1 ␮mol of 4-chloroindole per
minute. The OD600 of one was corresponding to a dry cell weight
(DCW) of 2.97 mg/ml.
2.3. Expression of StyAB2, StyA and StyB in E. coli
The DNA fragments encoding either styAB2, styA or styB were
amplified with primers 5ꢀ-GAGGAGGTCATATGAAAAAGCGTATC-
GGTATTGTTG-3ꢀ (AB1F) and 5ꢀ-TGACAAGCTTTTAATTCAGGGGCA-
GCGGATTG-3ꢀ (AB2R), AB1F and 5ꢀ-TGACAAGCTTCAGGCTGCAA-
TGGTCGGC-3ꢀ, 5ꢀ-GAGGAGGTCATATGACGCTAAAGACAGATGCGG-
3ꢀ and AB2R, respectively. They were each digested with Nde I and
Hind III (New England Biolabs, Beverly, MA, USA), and inserted into
pET-28a(+) vector (Novagen, Madison, WI). The resulting expres-
sion vectors, pETAB, pETA or pETB were transformed into E. coli
BL21(DE3). Single colonies were grown overnight at 37 ◦C in LB
media containing 50 ␮g kanamycin/ml. Two milliliter of overnight
culture was then inoculated into 200 ml of Terrific Broth (TB
medium) containing 50 ␮g kanamycin/ml in a 500 ml flask. For the
expression of StyAB2, the cultures were incubated at 37 ◦C for 3 h
before induction and the incubation continued at 20 ◦C for another
18 h with gyratory shaking at 220 rpm. For the expression of StyA
or StyB, the induction was initiated with the addition of 0.05 mM
IPTG at 20 ◦C. The incubation continued at 20 ◦C for another 24 h.
2.4. Purification of recombinant StyA and StyB
The entire procedure was performed at 4 ◦C. To prepare the sol-
uble cell extract, 3 g recombinant E. coli cells (wet weight) treated
with lysozyme were lysed by sonication in buffer A, which consisted
of 0.1 M potassium phosphate (pH 7.0), 20% glycerol (v/v), 1 mM
phenylmethyl sulfonylfluoride (PMSF), 0.5 M potassium chloride,
0.1 mM dithiothreitol and 5 mM imidazole. Insoluble cell debris
was removed by centrifugation. The resulting supernatant was
loaded onto a Ni2+-NTA agarose column (Bio-Rad), which has been
equilibrated with buffer A at a flow rate of 1 ml/min. The recombi-
nant protein StyA or StyB was eluted at a flow rate of 1 ml/min with
buffer A containing 250 mM imidazole (no PMSF). Fractions con-
taining StyA or StyB were verified by SDS-PAGE, pooled, dialyzed
and used freshly.
2.7. Spectral data for epoxidation products
(S)-Styrene oxide (1): 1H NMR (600 MHz, CDCl3): ı 7.27–7.35
(m, 5H, Ar–H), 3.85 (m, 1H, CH), 3.13 (m, 1H, CH2), 2.79 (m, 1H,
CH2). [˛]D25 = +32.1 (c 1.02, CHCl3) {lit. [19] [˛] D25 = +21.1 (83 mM,
CHCl3) for 99% ee, (S)}; >99% ee; retention times: tR (R) 10.27 min,
tR (S) 10.67 min.
(S)-2-(4-bromophenyl)oxirane (2): 1H NMR (600 MHz, CDCl3):
ı 7.47 (d, 2H, Ar–H, J = 8.22 Hz), 7.15 (d, 2H, Ar–H, J = 8.22 Hz), 3.82
(m, 1H, CH), 3.14 (t, 1H, CH2, J = 4.8 Hz), 2.74 (dd, 1H, CH2, J = 2.4 Hz,
2.5. Determination of StyA activity in reconstituted system
J = 5.4 Hz). [˛]D25 = +14.8 (c 0.98, CHCl3) {lit. [20] [˛] 24 = +20.5
D
(c 1.27, CHCl3) for 99% ee, (S)}; >99% ee; retention times: tR (R)
11.06 min, tR (S) 11.64 min.
The activity of StyA was determined by measuring the formation
of (S)-styrene oxide by HPLC. The reaction mixture contained 8 ␮M
of purified StyA, 20 ␮M of purified StyB, 20 ␮M formate dehydroge-
nase (from Pichia pastoris KM71), 0.16 M sodium formate, 0.2 mM
NADH, 0.8 mM NAD+, 0.03 mM FAD, and varying concentrations
of styrene (from a 50-fold stock in DMSO), in 100 mM potassium
phosphate buffer (pH 6.5). Reaction mixtures were incubated at
30 ◦C on a shaker at 280 rpm for 1 h. The organic phase was ana-
lyzed by reverse-phase HPLC on a Luna C18 (4.6 mm × 150 mm)
column at a flow rate of 1 ml/min. The mobile phase consisted of a
methanol–water mixture at a ratio of 80:20. Estimates of kcat and
Km were obtained by non-linear regression with GradPad Prism
program (Graphpad, San Diego, CA, USA).
(S)-2-(3-chlorophenyl)oxirane (3): 1H NMR (600 MHz, CDCl3):
ı 7.09–7.19 (m, 4H, Ar–H), 3.75 (m, 1H, CH), 3.05 (m, 1H, CH2), 2.67
(m, 1H, CH2). [˛]D25 = +52 (c 0.52, CHCl3) {lit. [19] [˛] 25 = +10.8
D
(65 mM, CHCl3) for 99% ee, (S)}; >99% ee; retention times: tR (S)
11.46 min, tR (R) 11.70 min.
(S)-2-(oxiran-2-yl)pyridine (4): 1H NMR (600 MHz, CDCl3): ı
8.57 (d, 1H, Ar–H, J = 4.86 Hz), 7.66–7.69 (m, 1H, Ar–H), 7.21–7.24
(m, 2H, Ar–H), 4.01 (dd, 1H, CH, J = 2.34 Hz, J = 4.26 Hz), 3.18 (dd,
1H, CH2, J = 4.26 Hz, J = 5.88 Hz), 2.93 (dd, 1H, CH2, J = 2.34 Hz,
J = 5.88 Hz). [˛]D25 = +12 (c 0.2, CHCl3) {lit. [21] [˛]D19 = +14 (c 0.56,
CHCl3) for 99% ee, (S)}; >99% ee; retention times: tR (R) 15.05 min,
tR (S) 15.83 min.

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H. Lin et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 236–241
(1S, 2R)-1,2-dihydronaphthalene oxide (5): 1H NMR (600 MHz,
CDCl3): ı 7.09–7.41 (m, 4H, Ar–H), 3.85 (m, 1H, CH), 3.73 (m, 1H,
CH), 2.76–2.82 (m, 1H, CH2), 2.53–2.57 (m, 1H, CH2), 2.40–2.43 (m,
1H, CH2), 1.74–1.80 (m, 1H, CH2). [˛]D25 = −39 (c 0.21, CHCl3) {lit.
[19] [˛]D20 = −134.5 (68 mM, CHCl3) for 99% ee, (1S, 2R)}; >99% ee;
retention times: tR (1R, 2S) 11.37 min, tR (1S, 2R) 12.87 min.
(1S, 2R)-indene oxide (6): 1H NMR (600 MHz, CDCl3):
ı
7.20–7.52 (m, 4H, Ar–H), 4.28 (m, 1H, CH), 4.15 (m, 1H, CH), 3.24
(m, 1H, CH2), 3.01 (m, 1H, CH2). >99% ee; retention times: tR (1R,
2S) 12.58 min, tR (1S, 2R) 13.95 min.
Fig. 1. Phylogenetic relationship of DNA sequences between styAB2 from Pseu-
domonas sp. LQ26 and from other Pseudomonas strains retrieved from databases.
Phylogenetic tree was generated with ClustalW method.
(S)-2-benzyloxirane (7): 1H NMR (600 MHz, CDCl3): ı 7.22–7.32
(m, 5H, Ar–H), 3.13–3.15 (m, 1H, CH), 2.91 (dd, 1H, CH2, J = 5.64,
J = 14.52), 2.91 (dd, 1H, CH2, J = 5.22, J = 14.52), 2.81 (dd, 1H, CH2,
J = 4.44, J = 8.94), 2.91 (dd, 1H, CH2, J = 5.64, J = 14.52). 36% ee; reten-
tion times: tR (R) 6.12 min, tR (S) 6.54 min.
proved a better substrate than indole for library screening because
of enhanced color development [17]. Varied expression tempera-
ture (Fig. 2) and IPTG concentration were attempted. When 1 mM
IPTG was used, expressions at 20 and 15 ◦C yielded the best results
regarding both activity and specific activity per unit of dry cell
weight. Enzymatic activity reached a maximum of ∼5 U/mg DCW
at around 21 h (Fig. 2). Lower incubation temperature (10 ◦C) sig-
nificantly affected cell growth.
The functional expression of StyAB2 was further enhanced
by reducing IPTG concentration to zero. The addition of IPTG
had little effect on cell growth, but appeared unfavorable to
StyAB2 expression. The maximum whole-cell specific activity was
∼8 U/mg DCW in the absence of IPTG. Lower activities of 5–6 U/mg
DCW was observed for cells induced with IPTG. The unintended
induction of protein expression was most probably due to the
leaky nature of the system, which has been reported previously
[23].
(S)-2-benzyl-2-methyloxirane (8): 1H NMR (600 MHz, CDCl3):
ı 7.21–7.31 (m, 5H, Ar–H), 2.89 (d, 1H, CH2, J = 14.16), 2.82 (d, 1H,
CH2, J = 14.16), 2.65 (d, 1H, CH2, J = 4.98), 2.61 (d, 1H, CH2, J = 4.98),
1.28 (s, 3H, CH3). 13% ee; retention times: tR (R) 9.41 min, tR (S)
10.36 min.
(S)-2-(4-methoxybenzyl)oxirane (9): 1H NMR (600 MHz,
CDCl3): ı 7.16 (d, 2H, Ar–H, J = 8.58), ı 6.85 (d, 2H, Ar–H, J = 8.58),
3.79 (s, 3H, CH3), 3.09–3.12 (m, 1H, CH), 2.86 (dd, 1H, CH2, J = 5.52,
J = 14.52), 2.74–2.78 (m, 2H, CH2), 2.81 (dd, 1H, CH2, J = 2.64,
J = 4.94). 86% ee; retention times: tR (R) 9.56 min, tR (S) 9.90 min.
(S)-2-(oxiran-2-ylmethyl)phenol (10): 1H NMR (600 MHz,
CDCl3): ı 7.10–7.17 (m, 2H, Ar–H), 6.78–6.86 (m, 2H, Ar–H),
4.88–4.93 (m, 1H, CH), 3.85 (dd, 1H, CH2, J = 3.36 Hz, J = 12.18 Hz),
3.75 (dd, 1H, CH2, J = 6.36 Hz, J = 12.18 Hz), 3.25 (dd, 1H, CH2,
J = 9.54 Hz, J = 15.54 Hz), J = 12.18 Hz, 3.02 (dd, 1H, CH2, J = 7.5 Hz,
J = 15.54 Hz). 80% ee; retention times: tR (R) 11.43 min, tR (S)
10.47 min.
(S)-2-(3-methylbenzyl) oxirane (11): 1H NMR (600 MHz,
CDCl3): ı 7.19–7.21 (m, 1H, Ar–H), 7.04–7.06 (m, 3H, Ar–H),
3.12–3.15 (m, 1H, CH), 2.88 (dd, 1H, CH2, J = 5.76 Hz, J = 14.46 Hz),
2.75–2.80 (m 2H, CH2), 2.55 (dd, 1H, CH2, J = 2.7 Hz, J = 5.04 Hz), 2.34
(s, 3H, CH3). [˛]D25 = +18 (c 0.05, CHCl3); 29% ee; retention times:
tR (R) 5.6 min, tR (S) 6.1 min.
3. Results and discussion
3.1. Isolation of styAB2 from Pseudomonas sp. LQ26
It is well-known that most of the experimentally character-
ized SMOs are from the genus of Pseudomonas. All the SMOs are
two-component monooxygenases consisting of a FAD-dependent
hydroxylase and a NADH-flavin oxidoreductase encoded by styA
and styB. They are highly conserved with an overall identity of
>94% for styAB at DNA level. Accordingly, we amplified the puta-
tive styAB2 gene in Pseudomonas sp. LQ26 simply using primers
complementary to the consensus sequence. The isolated styAB2
gene showed a maximum of 79% sequence identity with other
styABs. It appeared as the most distant member of all SMOs orig-
inating from the genus of Pseudomonas, as shown in a rooted
phylogenetic tree with all identified homological styABs in Pseu-
domonas species aligned using the ClustalW method of DNAStar
(Fig. 1).
3.2. Overexpression of StyAB2 in recombinant E. coli
Functional expression of StyAB2 was achieved in the Novagen
pET system and verified by both SDS-PAGE and whole-cell activ-
ity towards 4-chloroindole based on the known indigo-forming
capacity of SMOs [12,22]. The substrate had been used in our pre-
vious work in the directed evolution of cytochrome P450 2A6, and
Fig. 2. Effect of temperature on the functional expression of StyAB2. Total activity
(A) and specific activity (B) were measured in triplicate using whole E. coli cells
expressing StyAB2. The expression was carried out at 10 ◦C (ꢀ), 15 ◦C (ꢁ), 20 ◦C (ꢁ),
25 ◦C (ꢂ) and 30 ◦C (᭹) with the induction of 1 mM IPTG.

H. Lin et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 236–241
239
Fig. 3. pH (A) and temperature (B) dependence of activities of StyAB2. Whole E. coli cells expressing StyAB2 was used to convert 4-bromostyrene, and the yield of product
was measured by reverse-phase HPLC.
3.3. Reaction conditions and kinetic parameters of SMO
4-bromostyrene, 3-chlorostyrene, 1,2-dihydronaphthalene, 2-
vinylpyridine, 1H-indene or 1,2-dihydronaphthalene (Fig. 5A)
afforded the corresponding (S)-epoxides with excellent enan-
tiomeric excesses (>99%) at yields of up to 100%. Electron-
withdrawing group appeared unfavorable for the reaction. Bromo-
and chloro-substituted substrates afforded lower product yields.
Compounds with electron-withdrawing group directly connected
to the terminal alkene, such as (E)-4-phenylbut-3-en-2-one, (E)-
1-(3-chloroprop-1-enyl)benzene and (E)-1-(2-nitrovinyl)benzene
did not serve as substrates for StyAB2.
The whole-cell biotransformation reactions using 4-
bromostyrene as substrate were performed in a bi-phase system
containing 20% (v/v) bis-(2-ethylhexyl) phthalate (BEHP) or 10%
(v/v) cyclohexane, which had been demonstrated valuable to
reduce the cytotoxicity effect of epoxide product [24]. The whole-
cell bioconversion system performed fairly well at 25–32 ◦C with
the maximum yield achieved at 30 ◦C. The yields dropped signif-
icantly when higher reaction temperature was applied (Fig. 3B).
Fig. 3A showed that the optimum reaction pH was around 6.5.
The product yield dropped from 54% to 41% and 31%, respectively,
when the reaction pH was set to 7.0 and 6.0.
Based on these results, we measured the steady-state kinetics of
styrene epoxidation at 30 ◦C, pH 6.5. The reactions were carried out
in a reconstituted system with purified enzymes. Kinetic proper-
ties of StyA were investigated at varying concentrations of styrene
(Fig. 4). The apparent Km value determined by non-linear regres-
sion was 0.41 0.14 mM, similar to that of StyAB for Pseudomonas
sp. VLB120. Its turnover frequency kcat was 1.5 0.1 min−1, sig-
nificantly lower than that of StyAB from Pseudomonas sp. VLB120
(1.6 s−1) [3].
The attempt to apply SMO in the epoxidation of unconjugated
alkenes has been described in one report, and the results sug-
gest that 1-allylbenzene does not serve as a substrate [11]. For the
newly isolated StyAB2, we found that it was able to catalyze the
epoxidation of 1-allylbenzene (Fig. 5B). The enantiomeric excess
of the epoxide product was 36%, similar to a previous report on
the same reaction catalyzed with chloroperoxidase (37% ee) [25].
Several unconjugated alkenes served as substrates as well, yielding
the corresponding (S)-epoxides with low to moderate enantiomeric
excesses (Fig. 5B). The highest enantioselectivity was found for
the substrates 1-allyl-4-methoxybenzene (86%) and 1-allyl-2-
hydroxybenzene (80%), which might be due to the advantage of
electron-donating nature of the methoxy and hydroxy group.
We compared the protein sequences of StyAB2 and the SMO
from P. fluorescens ST. They share 89% identity and 91% similarity,
and none of the discrepant residues are apparently critical, either
based on a model structure [26] or a recently released X-ray struc-
ture of SMO [27]. The alignment of the SMO from P. fluorescens
ST with StyAB2 was shown in Fig. S1 of the Supplementary mate-
rial. Consequently, no obvious differences have been observed for
their active cavities modeled according to the X-ray structure (data
not shown). It is hard to explain the different substrate spectrum
between these two SMOs based on present information. Elucida-
tion of the molecular basis for the novel activity of StyAB2 awaits
further work on mutational analysis and sophisticated structure-
relationship studies.
3.4. Biotransformation of conjugated and unconjugated alkenes
by StyAB2
The biotransformation of several alkenes with structure simi-
lar to styrene was attempted. Conjugated alkenes, such as styrene,
The novel activity presented here provides new insights into
the substrate specificity of SMOs. The products from unconjugated
alkenes represent a group of important synthetic building blocks
and intermediates. It is also quite promising to harness the stere-
oselectivity of StyAB2 for this group of substrates with carefully
designed structures and optimized reaction parameters. This work
is currently under way in this laboratory.
Insofar as we know, this is the first report on the epoxidation of
unconjugated alkenes catalyzed by SMO. Recently isolated SMOs,
Fig. 4. Steady-state kinetics of styrene epoxidation catalyzed by StyAB2. Each point
presented is the mean of triplicate assays in a reconstituted system.

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H. Lin et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 236–241
Fig. 5. Production of chiral epoxides by whole-cell catalyzed bioconversion of styrene derivatives (A) and unconjugated alkenes (B).
such as the self-sufficient SMO from R. opacus [13] and SmoA from
metagenome [12], although both more diverse in their sequence
spaces, have not been tested for unconjugated alkenes.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.molcatb.2010.08.012.
4. Conclusions
We have isolated a novel styrene monooxygenase StyAB2, and
functionally expressed it in E. coli. The enzyme is able to catalyze
the epoxidation of both unconjugated 1-allylbenzene analogues
and conventional substrates of conjugated styrene analogues. Since
only a very few SMOs have been experimentally assigned so far, the
catalytic specificity of most SMO sequences from genomes are yet
to be elucidated. Our results suggest that a much broader substrate
spectrum is expected for SMOs and although known for decades,
their applications in the generation of molecular diversity are yet
to be explored.
References
[1] A. Mooney, P.G. Ward, K.E. O’Connor, Appl. Microbiol. Biotechnol. 72 (2006)
1–10.
[2] N.D. O’Leary, K.E. O’Connor, A.D. Dobson, FEMS Microbiol. Rev. 26 (2002)
403–417.
[3] K. Otto, K. Hofstetter, M. Rothlisberger, B. Witholt, A. Schmid, J. Bacteriol. 186
(2004) 5292–5302.
[4] J.B. Park, B. Buhler, T. Habicher, B. Hauer, S. Panke, B. Witholt, A. Schmid,
Biotechnol. Bioeng. 95 (2006) 501–512.
[5] S. Panke, M. Held, M.G. Wubbolts, B. Witholt, A. Schmid, Biotechnol. Bioeng. 80
(2002) 33–41.
[6] F. Hollmann, P.C. Lin, B. Witholt, A. Schmid, J. Am. Chem. Soc. 125 (2003)
8209–8217.
[7] K. Hofstetter, J. Lutz, I. Lang, B. Witholt, A. Schmid, Angew. Chem. Int. Ed. 43
(2004) 2163–2166.
Acknowledgments
[8] G. Sello, F. Orsini, S. Bernasconi, P. Di Gennaro, Tetrahedron: Asymmetry 17
(2006) 372–376.
[9] K.L. Tee, O. Dmytrenko, K. Otto, A. Schmid, U. Schwaneberg, J. Mol. Catal. B:
Enzyme 50 (2008) 121–127.
[10] G. Bestetti, P. Di Gennaro, A. Colmegna, I. Ronco, E. Galli, G. Sello, Int. Biodeterior.
Biodegrad. 54 (2004) 183–187.
This work was supported by National Natural Science Foun-
dation of China (20802073/B020104), 100 Talents Program of the
Chinese Academy of Sciences and Sichuan Province Science Foun-
dation for Young Scholars 08ZQ026-023.

H. Lin et al. / Journal of Molecular Catalysis B: Enzymatic 67 (2010) 236–241
241
[11] S. Bernasconi, F. Orsini, G. Sello, A. Colmegna, E. Galli, G. Bestetti, Tetrahedron
Lett. 41 (2000) 9157–9161.
[12] E.W. van Hellemond, D.B. Janssen, M.W. Fraaije, Appl. Environ. Microbiol. 73
(2007) 5832–5839.
[13] D. Tischler, D. Eulberg, S. Lakner, S.R. Kaschabek, W.J.H. van Berkel, M. Schlo-
mann, J. Bacteriol. 191 (2009) 4996–5009.
[14] G. Wittig, U. Schoellkopf, Org. Syn. Coll. 5 (1973) 751.
[15] R.P. Hanzlik, M. Edelman, W.J. Michaely, G. Scott, J. Am. Chem. Soc. 98 (1976)
1952–1955.
[16] M. Fieser, L.F. Fieser, Reagents for Organic Synthesis, Wiley-Interscience, New
York, 1967.
[17] Z.-G. Zhang, Y. Liu, F.P. Guengerich, J.H. Matse, J. Chen, Z.-L. Wu, J. Biotechnol.
139 (2009) 12–18.
[18] Z.-L. Wu, L.M. Podust, F.P. Guengerich, J. Biol. Chem. 280 (2005) 41090–41100.
[19] A. Schmid, K. Hofstetter, H.-J. Feiten, F. Hollmann, B. Witholt, Adv. Synth. Catal.
343 (2001) 732–737.
[20] K. Matsumoto, T. Kubo, T. Katsuki, Chem. Eur. J. 15 (2009) 6573–6575.
[21] Y. Genzel, A. Archelas, Q.B. Broxterman, B. Schulze, R. Furstoss, J. Org. Chem. 66
(2000) 538–543.
[22] S. Panke, B. Witholt, A. Schmid, M.G. Wubbolts, Appl. Environ. Microbiol. 64
(1998) 2032–2043.
[23] F.W. Studier, Protein Expr. Purif. 41 (2005) 207–234.
[24] S. Panke, V. de Lorenzo, A. Kaiser, B. Witholt, M.G. Wubbolts, Appl. Environ.
Microbiol. 65 (1999) 5619–5623.
[25] A.F. Dexter, F.J. Lakner, R.A. Campbell, L.P. Hager, J. Am. Chem. Soc. 117 (1995)
6412–6413.
[26] K.A. Feenstra, K. Hofstetter, R. Bosch, A. Schmid, J.N.M. Commandeur, N.P.E.
Vermeulen, Biophys. J. 91 (2006) 3206–3216.
[27] U.E. Ukaegbu, A. Kantz, M. Beaton, G.T. Gassner, A.C. Rosenzweig, Biochemistry
49 (2010) 1678–1688.