Enzymatic hydrogenation of diverse activated alkenes. Identification of two Bacillus old yellow enzymes with broad substrate profiles

Article abstract of DOI:10.1016/j.molcatb.2014.04.004

By whole cell transformation, 32 out of 71 strains showed OYEs activity toward maleimide in the first round screening. Among them, a Bacillus strain was identified to be active toward a selection of substrates with different electron-withdrawing groups. Two OYE homologous genes, bac-oye1 and bac-oye2 were cloned from this strain and overexpressed in Escherichia coli BL21(DE3). The recombinant enzyme Bac-OYE2 showed a broader pH range (6.0-10.5), while Bac-OYE1 was so sensitive to pH that it lost most of the enzyme activity below pH 6.0 or above pH 9.0. The reaction temperature exerted similar effects on the activities of both enzymes, but the stability of Bac-OYE2 was more sensitive to the temperature than Bac-OYE1. In addition to α,β-unsaturated aldehydes, ketones, nitroalkenes, and the double activated carboxylic acids, esters, nitriles and cyclic imides, Bac-OYE1 and Bac-OYE2 also exhibited activities toward the "borderline" substrates such as unsaturated lactones, mono carboxylic esters, showing their broader substrate scopes. These enzymes also had excellent enantioselectivity as evidenced by the reductions of several α,β-unsaturated cyclic ketones, α-substituted α,β-unsaturated carboxylic esters and 2-methyl maleimide. For example, methyl 2-acetamidoacrylate was reduced by Bac-OYE1 with >99% conversion and >99% ee.

Full text of DOI:10.1016/j.molcatb.2014.04.004

Journal of Molecular Catalysis B: Enzymatic 105 (2014) 118–125
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enzymatic hydrogenation of diverse activated alkenes. Identification
of two Bacillus old yellow enzymes with broad substrate profiles
Hailing Zhanga,b, Xiuzhen Gaob, Jie Renb, Jinhui Fengb, Tongcun Zhanga,
Qiaqing Wub,∗, Dunming Zhub,∗
a College of Bioengineering, Tianjin University of Science and Technology, Tianjin 300457, China
b National Engineering Laboratory for Industrial Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Institute of Industrial
Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China
a r t i c l e i n f o
a b s t r a c t
Article history:
By whole cell transformation, 32 out of 71 strains showed OYEs activity toward maleimide in the first
round screening. Among them, a Bacillus strain was identified to be active toward a selection of substrates
with different electron-withdrawing groups. Two OYE homologous genes, bac-oye1 and bac-oye2 were
cloned from this strain and overexpressed in Escherichia coli BL21(DE3). The recombinant enzyme Bac-
OYE2 showed a broader pH range (6.0–10.5), while Bac-OYE1 was so sensitive to pH that it lost most
of the enzyme activity below pH 6.0 or above pH 9.0. The reaction temperature exerted similar effects
on the activities of both enzymes, but the stability of Bac-OYE2 was more sensitive to the temperature
than Bac-OYE1. In addition to ␣,␤-unsaturated aldehydes, ketones, nitroalkenes, and the double activated
carboxylic acids, esters, nitriles and cyclic imides, Bac-OYE1 and Bac-OYE2 also exhibited activities toward
the “borderline” substrates such as unsaturated lactones, mono carboxylic esters, showing their broader
substrate scopes. These enzymes also had excellent enantioselectivity as evidenced by the reductions of
several ␣,␤-unsaturated cyclic ketones, ␣-substituted ␣,␤-unsaturated carboxylic esters and 2-methyl
maleimide. For example, methyl 2-acetamidoacrylate was reduced by Bac-OYE1 with >99% conversion
and >99% ee.
Received 11 December 2013
Received in revised form 7 April 2014
Accepted 8 April 2014
Available online 21 April 2014
Keywords:
Ene-reductase
Old yellow enzyme
Reduction of alkenes
Biocatalytic hydrogenation
Substrate specificity
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
It has been found that OYE-like enzymes could catalyze
the reduction of a variety of activated alkenes, including ␣,␤-
In recent years, enone/enoate-reductases (also called “Old Yel-
low Enzyme, OYE) have been attracting the interest of chemists for
its high potential in creating two stereogenic centers in asymmetric
reduction of C C bonds. The OYE-catalyzed asymmetric reduction
proceeds via hydride transfer from the N5 of reduced FMN onto C␤
of the substrate with a solvent-derived proton being transferred to
C␣, and finally results in a trans-fashion addition of the [2H] [1,2],
which is contrary to the cis-hydrogenaration by transition-metal-
based homogeneous catalysts. The trans-hydrogenation function
makes these enzymes important complementary tools for existing
chemical catalysts.
unsaturated aldehydes, ketones, nitroalkenes, carboxylic acids
and derivatives (esters, lactones, cyclic imides, cyclic acid anhy-
drides) [3–6] and are widely distributed in biological sources,
including plants [7,8], bacteria [9], fungi [10,11], and protozoa
[12]. However, there was no single conserved physiological role
has been attributed until now. It was suggested that a gen-
eral role of OYEs was the detoxification of a broad spectrum of
electrophilic compounds [13]. Studies suggested that the phys-
iological roles of OYE-containing organisms may resulted from
their specific metabolic requirements and environmental pressures
[14]. For example, OYE from Arabidopsis leaves metabolized ␣,␤-
unsaturated carbonyl compounds produced during insect attract
and bacterial pathogenesis [15].
Because of the exquisite stereoselectivity and broad acceptance
of various activating groups of OYE-like enzymes, some novel OYEs
have been discovered to expand the tool-box [16–19]. However,
many of the reported enzymes have either low activity or low stere-
oselectivity toward some specific substrates [20–22]. Therefore,
it is necessary to seek new OYEs with broad substrate profile. As
∗
Corresponding authors at: National Engineering Laboratory for Industrial
Enzymes and Tianjin Engineering Center for Biocatalytic Technology, Tianjin Insti-
tute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin
Airport Economic Area, Tianjin 300308, China. Tel.: +86 22 84861963;
fax: +86 22 84861996.
E-mail addresses: wu qq@tib.cas.cn (Q. Wu), zhu dm@tib.cas.cn (D. Zhu).
http://dx.doi.org/10.1016/j.molcatb.2014.04.004
1381-1177/© 2014 Elsevier B.V. All rights reserved.

H. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 118–125
119
such, the available strains stored in our laboratory were screened
in two rounds with maleimide and a set of ␣,␤-unsaturated com-
pounds with diverse structural features as substrate, respectively,
and a Bacillus strain was identified. Two OYE genes from this strain
were cloned and overexpressed. The recombinant enzymes showed
activity toward a diversity of substrates, especially for the less acti-
vated alkenes (or called “borderline substrates”) such as methyl
acrylate.
using universal primers 27F (5ꢀ-AGAGTTTGATCCTGGCTCA-3ꢀ) and
1492R (5ꢀ-GGTTACCTTGTTACGACTT-3ꢀ) and then sequenced at BGI
(China). The PCR was performed with the following conditions:
95 ◦C for 3 min, one cycle; 94 ◦C for 30 s/55 ◦C for 30 s/72 ◦C for 90 s,
30 cycles; 72 ◦C for 10 min, one cycle. The 16S rDNA sequence was
submitted to Genbank of NCBI for BLAST search. A phylogenetic
tree was constructed with the top 10 strains of the blast result by
using ClustalX (Version 1.83) and MEGA (Version 4.0) based on the
homologous 16S rDNA sequences.
2. Materials and methods
2.4. Cloning of ene-reductase genes
2.1. Chemicals
The Bacillus ene-reductase genes were amplified by two
rounds of PCRs without whole genomic sequence. Round 1:
The nucleotide sequence of the NADH: flavin oxidoreductase
from Bacillus vallismortis DV1-F-3 (NCBI reference sequence:
NZ JH600221.1) was chosen as template for primers design, which
were Bac-F: 5ꢀ-ATGGCCAGAACATTATTCACAC-3ꢀ and Bac-R: 5ꢀ-
TTACCAGCCTCTCTCATATTG-3ꢀ. With Genomic DNA of strain WX16
as template, PCR was performed with KOD Plus Polymerase under
the following conditions: 94 ◦C for 2 min, one cycle; 94 ◦C for
20 s/50 ◦C for 30 s/68 ◦C for 70 s, 30 cycles; 68 ◦C for 5 min, one cycle.
The products were performed a A-addition reaction before ligat-
ing onto pTZ57R/T vector by using Ex Taq Polymerase at 72 ◦C for
30 min. The constructed vector were transformed into E. coli Top10
for TA cloning and then sequenced.
Round 2: Nucleotide sequences of NADPH dehydrogenase
NamA (NCBI reference sequence: AGF27164) and putative NADH-
dependent flavin oxidoreductase (NCBI reference sequence:
AGF27122.1) from Bacillus amyloliquefaciens IT-45 were used
as template to redesign primers for the amplifications of
bac-oye1 and bac-oye2, respectively. The primers were as fol-
lows: for bac-oye1, F: 5ꢀ-ATGGCGAGAAAATTATTCACACCATGGAC-
3ꢀ and R: 5ꢀ-TCACCATGCTCTGTCGTATTGGACCG-3ꢀ; for bac-oye2
F: 5ꢀ-ATGAAGCAAACTTATAAACCGCTGTTTGAAC-3ꢀ and R: 5ꢀ-
CTACTTTTCAAACGGCACCCAGCCG-3ꢀ. PCRs were performed by
following the protocol described above except that the annealing
temperatures were 52 ◦C for both bac-oye1 and bac-oye2. The two
nucleotide sequences of bac-oye1 and bac-oye2 were deposited in
the GenBank database with accession no. KJ577134 and KJ577135,
respectively.
All of the chemicals were commercially available, except
for
racemic
(5S)-2-methyl-5-(prop-1-en-2-yl)
cyclohex-
anone (P3), dimethyl 2-methylsuccinate (P15), methyl
2-acetamidopropanoate (P16) and 3-methyl-2,5-Pyrrolidinedione
(P30), which were obtained by Pd/C-catalyzed hydrogena-
tion of (S)-carvone (S3), dimethyl itaconate (S15), methyl
2-acetamidoacrylate (S16) and 3-methyl maleimide (S30),
respectively [23].
NdeI, XhoI and NcoI were purchased from Fermentas (Germany).
Solution I ligase was purchased from Takara (Japan). Taq DNA poly-
merase was purchased from TIANGEN (Beijing, China). KOD DNA
polymerase was purchased from TOYOBO (Japan). InsTAclone PCR
cloning kit was from Thermo scientific (USA). Expression vectors,
pET32a(+) and pET28a(+), were purchased from Novagen (Merck,
Germany). Host cells, Escherichia coli Top10 and E. coli BL21(DE3)
were stored in our laboratory.
2.2. Screening of microorganisms for ene-reductase
Seventy-one strains, which were isolated from contaminated
soils, were stored in 15% glycerin in our laboratory. For cultivation,
the complete medium (pH 7.0) containing 1.5% glucose·H2O (w/v),
0.5% peptone (w/v), 0.5% yeast extract (w/v), 0.1% NaCl (w/v), 0.1%
KH2PO4 (w/v), 0.1% K2HPO4·3H2O (w/v) and 0.05% MgSO4·7H2O
(w/v) was used and 0.5% of the strains were inoculated into 50 mL
complete medium. All cultures were shaken at 30 ◦C for 24 to
48 h at 200 rpm and the cells were harvested by centrifugation at
12,000 × g for 10 min.
For the sake of heterologous expression, the encoding genes
of Bac-OYE1 and Bac-OYE2 were re-amplified from the recom-
bined pTZ57R/T vectors with the following primers: for Bac-OYE1
gene, the primers were bac-oye1-F, 5ꢀ-CATGCCATGGGGCATCAT-
CATCATCATCACGCGAGAAAATTATTCACACCGTGGAC-3ꢀ and bac-
oye1-R: 5ꢀ-CCGCTCGAGTCACCATGCTCTGTCGTATTGGACCG-3ꢀ with
NcoI and XhoI as restriction sites (italic) and N-terminal
His-tag (bold); for Bac-OYE2 gene, primers were bac-oye2-F: 5ꢀ-
CGCCATATGCACCATCATCATCATCATAAGCAAACTTATAAACCGCT-
GTTTGAAC-3ꢀ and bac-oye2-R: 5ꢀ-CCGCTCGAGCTACTTTTCAAAC-
GGCACCCAGCCG-3ꢀ with NdeI and XhoI as restriction sites (italic)
and N-terminal His-tag (bold) [9]. The 25th nucleotide A of bac-oye1
was changed into G (bold and italic) without amino acid replace-
ment in order to produce the recognition sequence of NcoI. The
open reading frames of bac-oye1 and bac-oye2 were ligated into
pET28a(+) and pET32a(+), respectively.
Maleimide was used in the first round screening, and
2-cyclohexen-1-one, trans-2-Hexen-1-al, crotonic acid, trans-␤-
nitrostyrene, methyl 2-acetamidoacrylate, dimethyl itaconate
and benzylidenemalononitrile were used in the second round
screening. For the first round screening, the resting cells
(200 mg mL−1 wet cell) and substrate maleimide (1 g L−1, final con-
centration) were mixed in 1 mL of phosphate buffer (50 mM, pH 7.0)
containing 0.5% (w/v) glucose [18,24]. The resulting mixtures were
incubated at 30 ◦C for 96 h at 200 rpm, 0.9 mL of ethyl acetate was
added to extract the products and unconverted substrates. Organic
extracts were dried with anhydrous Na2SO4, and analyzed by thin
layer chromatography (TLC) with ethyl acetate and petroleum ether
(1/2, v/v) as mobile phase and chiral GC analysis, which was per-
formed on an Agilent 7890 gas chromatography equipped with
FID detector, CP ChiraSil DEX Column (25 m × 0.25 mm × 0.25 ␮m,
Varian, USA) and He as carrier gas (2 mL min−1), and using the
following column temperature program: 10 ◦C min−1 from 100 to
180 ◦C, 180 ◦C for 5 min. Standard product succinimide was used as
reference.
2.5. Expression and purification of recombinant enzymes
The recombinant plasmids were transformed into E. coli
BL21(DE3) cells. Expressions of the genes were induced using
0.1 mM isopropyl-␤-d-1-thiogalactopyranoside (IPTG) at 37 ◦C
when the optical density at 600 nm (OD600) was 0.6–0.8. Cells har-
vested by centrifugation were suspended in 20 mM Tris–HCl (pH
7.4, containing 250 mM NaCl) and then broken with high pressure
2.3. 16S rDNA sequencing determination
Genomic DNA of functional strain WX16 was extracted accord-
ing to the protocol of TIANamp Bacteria DNA Kit (Tiangen Biotech,
China). The 16S rDNA was amplified by PCR with Taq polymerase

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H. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 118–125
homogenizer (APV, Holland). Cell debris was removed by centrifu-
gation at 25,000 × g for 25 min. Supernatant obtained was filtered
through a 0.22-␮m-pore-size filter, and then purified by Ni affinity
chromatography on ÄKTA purifier 10 (GE, USA) with His Trap HP
column (5 mL, GE, USA). The column was equilibrated with 20 mM
Tris–HCl buffer (pH 7.4) containing 250 mM NaCl and eluted with a
buffer containing 20 mM Tris–HCl, 250 mM NaCl and 500 mM imid-
azole (pH 7.4). The collected fractions with purified enzymes were
dialyzed in 20 mM phosphate buffer (pH 7.0) to remove imidazole.
Protein concentration was determined with the BCA Protein Assay
Kit (CWBIO, China).
enzymes, YqjM and morphinone reductase, which were performed
at the same time [5,25,26]. For other substrates, the product con-
figuration was determined as previously reported [16,27].
3. Results and discussion
3.1. Identification of a Bacillus strain with OYE activity
By function screening, 32 of 71 strains showed reduction activ-
ity toward maleimide which was a general active substrate of OYEs
[3,18,23]. In order to further identify strains with broad substrate
profile, a series of activated alkenes with diverse structural features
including 2-cyclohexen-1-one, trans-2-hexen-1-al, crotonic acid,
trans-␤-nitrostyrene, methyl 2-acetamidoacrylate, dimethyl ita-
conate and benzylidenemalononitrile, were selected as substrates
in the 2nd round screening, and strain WX16 showed the ene-
reductase activity toward most of the selected substrates (see Table
S1 in Supplemental materials). By 16S rDNA sequences analysis,
the strain shared 99% sequence identities with several strains from
the genus of Bacillus, which revealed that the strain WX16 belongs
to the Bacillus genus and the closest two strains were Bacillus sp.
CMJ2-5 and B. vallismortis (see Fig. S1 in Supplemental materials).
2.6. Effects of pH and temperature on enzyme activity
To determine the effect of pH on the activities of Bac-OYE1
and Bac-OYE2, enzyme activities toward 2-cyclohexen-1-one were
measured in the following buffers: citrate-phosphate buffer (pH
4.0–6.0), phosphate buffer (pH 6.0–8.0), Tris–HCl buffer (pH
8.0–9.0), NaOH–glycine buffer (pH 9.0–10.5) and NaOH–phosphate
buffer (pH 11.0–12.0).
To investigate the optimal temperatures of the two OYEs,
enzyme activity was assayed at various temperatures ranging
from 25 ◦C to 55 ◦C with 5 ◦C intervals using 2-cyclohexen-1-one
as substrate. Likewise, the thermal stabilities were evaluated by
measuring the residual activities after incubating the enzymes at
various temperatures for 100 min.
3.2. Cloning of OYE genes from Bacillus
According to the BLAST search and phylogenetic tree based on
16S rDNA, it was found that the closest two strains were Bacil-
lus sp. CMJ2-5 and B. vallismortis D20. By searching NCBI database
(http://www.ncbi.nlm.nih.gov/) using “B. vallismortis” and “old yel-
low enzyme” as key words led to a putative OYE gene (Protein
ID: WP 010328336.1) from B. vallismortis DV1-F-3 (NCBI refer-
ence sequence: NZ JH600221.1), which was chosen as template
for primers design. After PCR, a fragment of 1071 bp nucleotides
was obtained. To our surprise, by BLAST analysis, the fragment,
except the primer parts, showed 99% identity with a gene sequence
of B. amyloliquefaciens IT-45. Therefore, primers were redesigned
based on the nucleotide sequences of NADPH dehydrogenase NamA
gene (NCBI reference sequence: KSO 008335) and putative NADH-
dependent flavin oxidoreductase gene (NCBI reference sequence:
KSO 008125) from B. amyloliquefaciens IT-45. Two target genes
were obtained after PCR with the genomic DNA of WX16 as tem-
plate. These two genes (bac-oye1 and bac-oye2) showed 99.4% and
99.2% identities with NamA and the putative NADH-dependent
flavin oxidoreductase gene, respectively. The corresponding amino
acid sequences of the two enzymes, named as Bac-OYE1 and Bac-
OYE2, showed 100% and 99.7% identities with their templates,
respectively.
Enzyme activity assay method was the same as reported in Ref.
[16].
2.7. Substrate specificity
The substrate scopes of the two OYE enzymes were assayed by
screening a series of substrates. The activity assay was performed
by monitoring the oxidation of NADPH at 340 nm according to the
previously reported method [16], except the buffer was Tris–HCl
(pH 8.0, 50 mM). All of the substrate concentration was 5 mM,
except for S26, S27, S29, S31 and S28, which were difficult to dis-
solve and their final concentration was 1.25 mM (0.25 mM for S28).
The reaction was initiated by addition of NADPH.
2.8. Bioconversions
The bioconversion reactions for substrates S2, S3, S15, S16, S24
and S30 were performed in 1 mL of Tris–HCl buffe−r1(50 mM, pH
8.0). The reaction system contained d-glucose (11 g L ), d-glucose
dehydrogenase (1.5 g L−1), NADP+ (1 g L−1), OYE (2 g L−1) and sub-
strate (20 mM). The mixtures were incubated at 30 ◦C, 200 rpm for
24 h and then extracted with an equal volume of ethyl acetate. The
conversions and enantiomeric excess (ee)/diasteromeric excess
(de) values of the reactions were measured by chiral GC anal-
ysis on an Agilent 7890 gas chromatography using CP ChiraSil
DEX Column (25 m × 0.25 mm × 0.25 ␮m, Varian, USA) for S2,
S3, S16 and S24, and Gamma DEXTM 225 Capillary Column
(30 m × 0.25 mm × 0.25 ␮m, SUPELCO, Japan) for S15 and S30. Tem-
perature programs were as follows: For S2: 10 ◦C min−1 from 70
to 100 ◦C, 100 ◦C for 10 min, 5 ◦C min−1 to 120 ◦C, 60 ◦C min−1 to
180 ◦C, 180 ◦C for 3 min. For S3: 10 ◦C min−1 from 70 to 100 ◦C,
100 ◦C for 14 min, 60 ◦C min−1 to 180 ◦C, 180 ◦C for 3 min. For S24:
70 ◦C for 2 min, 5 ◦C min−1 from 70 to 140 ◦C, 140 ◦C for 3 min,
50 ◦C min−1 to 180 ◦C, 180 ◦C for 3 min. For S16: 95 ◦C for 10 min,
40 ◦C min−1 from 95 to 160 ◦C, 160 ◦C for 3 min. For S15: 10 ◦C min−1
from 70 to 100 ◦C, 100 ◦C for 7.2 min, 60 ◦C min−1 to 180 ◦C, 180 ◦C
for 3 min. For S30: 10 ◦C min−1 from 80 to 170 ◦C, 170 ◦C for 15 min,
10 ◦C min−1 to 180 ◦C, 180 ◦C for 3 min. In order to determine the
product configuration, the GC data were compared with those of
the reduction products of S15, S16 and S30 catalyzed by known
Phylogenetic analysis was performed for these two OYEs and
15 known OYE enzymes (Fig. 1) [18]. The results showed that the
two OYEs were distributed to different phylogenetical taxa, that
is to say, they were distantly related. Bac-OYE1 was most closely
related to the YqjM from Bacillus subtilis with 87.3% identity; Bac-
OYE2 was not clustered with other OYE enzymes but shares a 28.7%
identity with CrS from Thermus scotoductus. The identity between
Bac-OYE1 and Bac-OYE2 was only 27%.
3.3. Expression and purification of recombinant enzymes
Both bac-oye1 and bac-oye2 genes with a N-terminal His-tag
were expressed in E. coli BL21(DE3) after induced by 0.1 mM IPTG
at 37 ◦C to give soluble proteins. By Ni-NTA affinity chromatogra-
phy, the overall enzyme yields were 83.3% with 1.9-fold enrichment
for Bac-OYE1 and 91.0% with 3.4-fold enrichment for Bac-OYE2
(Table 1), respectively. The purified enzymes presented the typ-
ical yellow color of OYEs. SDS-PAGE (Fig. 2) showed that the
molecular mass of Bac-OYE1 and Bac-OYE2 were approximately

H. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 118–125
121
NEM Escherichia coli (P77258)
100
93
PETN reducate Enterobacter cloacae (Q6JL81)
morB Pseudomonas putida (Q51990)
62
NCR Zymomonas mobilis (Q5NLA1)
34
52
XenB Pseudomonas fluorescens (Q9RPM1)
OPR3 Lycopersicon esculentum (Q9FEW9)
OPR1 Arabidppsis thaliana (Q8LAH7)
100
28
100
OPR2 Arabidppsis thaliana (Q8GYB8)
KYE1 Kluyveromyces (P40952)
100
OYE3 Saccharomyces cerevisiae (P41816)
OYE1 Saccharomyces carlsbergensis (Q02899)
OYE2 Saccharomyces cerevisiae (Q03558)
SYE4 Shewanella oneidensis (Q8EBV3)
Bac-OYE2 Bacillus sp.
100
100
100
CrS Thermus scotoductus (YP 004203660)
YqjM Bacillus subtilis (P54550)
Bac-OYE1 Bacillus sp.
100
100
0.1
Fig. 1. Phylogenetic relationships of Bac-OYE1 and Bac-OYE2 (underlined) homologies to other OYEs with known function. Amino acid sequences were used to generate the
distance tree with ClustalX (Version 1.83) method with default settings. A distance neighbor-joining tree was then created using the MEGA (Version 4.0). The names of the
OYEs are indicated to the right of the phylogenetic tree with NCBI accession numbers given in parentheses.
Table 1
Purification of Bac-OYE1 and Bac-OYE2.
a
Enzyme
Specific activity (U mg−1
)
Total activity (U)a
Purification factor
Yield (%)
Bac-OYE1
Crude enzyme extract
Ni-NTA
Crude enzyme extract
Ni-NTA
1.4
2.7
0.9
3.1
240
200
191
175
1
1.9
1
100
83.3
100
91.0
Bac-OYE2
3.4
a
The activity unit was defined as the amount of enzyme consuming 1 ␮mol NADPH per minute (1 ␮mol min−1) using 2-cyclohexen-1-one as substrate.
41 kDa and 43 kDa, respectively. Only when NADPH was used as
the cofactor, the two enzymes showed activity, indicating that they
were NADPH-dependent, although Bac-OYE2 showed 99.7% iden-
tity with the putative NADH-dependent flavin oxidoreductase gene
from B. amyloliquefaciens IT-45, indicating that it might be wrongly
annotated in the database.
range with optimal pH at pH 8.0. The sensitivity toward pH of Bac-
OYE1 was similar to that of securinine reductase which exhibited
optimum activity in the range of pH 8.0–8.5 but dropped off sharply
between pH 7.0 and 8.0 [28]. As shown in Fig. 3a, the components
of the buffer had impact on enzyme activity as reported previously
[29], and both enzymes showed higher activities in Tris–HCl buffer.
The effects of temperature on the activities of the two enzymes
were tested between 25 ◦C and 55 ◦C. Bac-OYE1 and Bac-OYE2
showed the highest activities at 35 ◦C and 40 ◦C, respectively
(Fig. 3b). After incubation at different temperature for 100 min,
residual activities toward 2-cyclohexen-1-one were determined
to investigate their thermal stabilities. There was no activity loss
for Bac-OYE1 under 40 ◦C, whereas residual activity of Bac-OYE2
gradually decreased with the increment of incubation temperature
(Fig. 3c). It has been previously observed that temperature and pH
showed different effects on the OYE homologues from the same
genome [21,30].
3.4. Effects of pH and temperature on the activities of Bac-OYE1
and Bac-OYE2
In order to investigate the pH effects of the two enzymes, activ-
ities of Bac-OYE1 and Bac-OYE2 at different pH were measured. As
shown in Fig. 3a, Bac-OYE2 showed a broad pH range at pH 6.0–10.5,
which was more broader than Yers-ER from Yersinia bercovieri
whose pH range was 6.0–8.5 [3]. Notably, Bac-OYE2 had maxi-
mum activity around pH 9.0, more alkali than other known OYEs
[3,9,20,28]. In contrary to Bac-OYE2, Bac-OYE1 had a narrower pH

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H. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 118–125
(S12). They were different from morphinone reductase (MorR) from
Pseudomonas putida and estrogen binding protein (EBP1) from Can-
dida albicans which could not reduce maleimide [16]. Although
Bac-OYE1 had 87% identity with YqjM, they showed some dif-
ferences in substrate specificity. The activity of Bac-OYE1 toward
cinnamaldehyde (S6) and benzylideneacetone (S7) were 1.04 U/mg
and 0.65 U/mg, but no activity was detected for YqjM. In addition,
there was no report about the reduction of benzylidenemalononi-
trile (S27) catalyzed by YqjM, indicating that the substrate scope of
Bac-OYE1 was broader than YqjM.
Although the substrate scopes of Bac-OYE1 and Bac-OYE2
were similar, there were some differences between these two
enzymes. First, for most active substrates, Bac-OYE2 displayed
higher specific activities than Bac-OYE1. For example, both OYEs
were active toward trans-␤-nitrostyrene (S29), but the specific
activity of Bac-OYE2 (24.8 U mg−1) was 16 times over that of Bac-
OYE1 (1.54 U mg−1). Secondly, the substrate scope of Bac-OYE2 was
broader than that of Bac-OYE1. It was found that Bac-OYE1 showed
no activity toward trans-␤-methyl-␤-nitrostyrene (S31) while the
Fig. 2. SDS-PAGE analysis of purified enzymes. Lane M, protein marker; lane 1, crude
cell-free extract of Bac-OYE1; lane 2, purified Bac-OYE1 by Ni-NTA; lane 3, crude cell-
free extract of Bac-OYE2; lane 4, purified Bac-OYE2 by Ni-NTA. (For interpretation of
the references to color in this figure legend, the reader is referred to the web version
of this article.)
3.5. Substrate profiles
specific activity of Bac-OYE2 toward this substrate was 1.89 U mg−1
.
To investigate the substrate spectra of the two OYEs, substrates
with diverse electron withdrawing groups were tested in Tris–HCl
(pH 8.0). The activities of Bac-OYE1 and Bac-OYE2 toward all sub-
strates were summarized in Table 2. It was found that both of
the two enzymes were active toward ␣,␤-unsaturated aldehydes,
ketones, nitroalkenes, lactones and cyclic imides, and showed activ-
ities toward some carboxylic acid or esters and nitriles which were
not preferred by most of OYEs [31]. Especially, Bac-OYE2 showed
high activities toward dicarboxylic esters or nitriles (S27, S28). It
has been recognized that simple ␣,␤-unsaturated mono-carboxylic
acids or esters were not easily reduced by ene-reductases (ERs),
although they were good substrates for ERs from anaerobic orga-
nisms [31]. Monocarboxylates such as crotonic acid (S18), cinnamic
acid (S19) and cinnamonitrile (S26) were not reduced by these two
enzymes. However, Bac-OYE1 and Bac-OYE2 were active toward
␣,␤-unsaturated lactones and carboxylic acid esters such as 2(5H)-
furanone (S8), 5,6-dihydropyran-2-one (S10) and methyl acrylate
This is consistent with the fact that Bac-OYE1 and Bac-OYE2 had
only 27% identity with each other.
3.6. Enantioselectivity
In order to investigate the stereoselectivities of the two OYEs,
the biotransformations of several substrates were performed, and
the conversions and ee values of the products were determined.
Methyl 2-acetamidoacrylate (S16), the most simple dehydroamino
acid derivative, could be reduced by OYE1–3, YqjM and OPR1,
among these ene-reductases, YqjM was the best reported cata-
lyst with 41% conversion and 97% ee [25]. As shown in Table 3,
Bac-OYE1 could transform S16 with >99% conversion and 99% ee
value, indicating Bac-OYE1 was superior to other ene-reductases.
In addition to S16, Bac-OYE1 and Bac-OYE2 also effectively cat-
alyzed the reductions of substrates S2, S3, S15 and S30 with high
Fig. 3. Effects of pH and temperature on the activities of Bac-OYE1 (᭹) and Bac-OYE2 (ꢁ). (a) The pH profiles of Bac-OYE1 and Bac-OYE2. Citrate-phosphate (pH 4.0-6.0),
phosphate (pH 6.0–8.0), Tris–HCl (pH 8.0–9.0), NaOH–glycine (pH 9.0–10.5) and NaOH–K2HPO4 (pH 11.0–12.0) were used. (b) The temperature profiles of Bac-OYE1 and
Bac-OYE2. Phosphate buffer (pH 7.0 for Bac-OYE2 and pH 8.0 for Bac-OYE1) was used. (c) Thermal stabilities of Bac-OYE1 and Bac-OYE2. Phosphate buffer (pH 7.0 for Bac-OYE2
and pH 8.0 for Bac-OYE1) was used.

H. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 118–125
123
Table 2
Substrate specificities of Bac-OYE1 and Bac-OYE2.
Table 2 (Continued)
Entry
S18
Structure
Specific activity (U mg−1
)
Entry
Structure
Specific activity (U mg−1
)
Bac-OYE1
Bac-OYE2
Bac-OYE1
Bac-OYE2
O
O
0
0
HO
S1
3.25
6.92
O
O
S19
0
0
0
0
OH
OH
S2
S3
1.73
1.76
3.58
2.46
O
S20
HN
O
O
O
CHO
S4
S5
1.33
0
7.47
0.02
S21
S22
S23
0
0
0
0
0
0
O
CHO
CHO
O
S6
S7
1.04
0.65
4.82
0.02
O
O
O
HO
OH
O
O
O
O
S8
S9
0.08
0
0.04
0
O
O
S24
1.3
0.51
O
O
O
O
S10
S11
1.8
0
1.53
S25
S26
S27
2.65
0
0.1
0
OH
CN
O
O
O
0
OH
CN
CN
O
0.75
5.0
S12
S13
0.11
0
0.12
0
O
CN
O
S28
0.88
1.54
10.7
O
OH
O
Br
O
NO2
S14
S15
0
0
0
S29
S30
24.8
44
O
O
O
N
O
O
O
12.3
0.08
O
NO2
O
O
O
S31
0
1.89
S16
S17
0.09
0
0
N
H
O
OH
0
HO
O

124
H. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 118–125
Table 3
Bioreduction of ␣,␤-unsaturated alkenes by Bac-OYE1 and Bac-OYE2.
Entry
Structure
Bac-OYE1
Bac-OYE2
Conversion (%)
ee/de (%)
Conversion (%)
ee/de (%)
S2
S3
>99
99
97(R,R)
>99
97(R,R)
O
O
91(R,S)
>99(R)
99.8
93(R,S)
>99(R)
O
O
O
S15
S16
81
26
24
O
O
O
O
O
O
>99
99(S)
95(S)
N
H
S24
S30
>99
>99
76(R)
>99
>99
76(R)
N
O
O
>99(R)
>99(R)
enantioselectivity. For benzylidenemalononitrile (S27), which had
two nitrile groups as electron withdrawing groups, and trans-␤-
nitrostyrene (S29), Bac-OYE1 and Bac-OYE2 could reduce them to
the corresponding saturated compounds with 100% conversions.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.2014.
04.004.
4. Conclusion
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