Identification of a marine NADPH-dependent aldehyde reductase for chemoselective reduction of aldehydes

Article abstract of DOI:10.1016/j.molcatb.2013.01.010

A putative aldehyde reductase gene from Oceanospirillum sp. MED92 was overexpressed in Escherichia coli. The recombinant protein (OsAR) was characterized as a monomeric NADPH-dependent aldehyde reductase. The kinetic parameters K_m and k_cat of OsAR were 0.89 ± 0.08 mM and 11.07 ± 0.99 s^-1 for benzaldehyde, 0.04 ± 0.01 mM and 6.05 ± 1.56 s^-1 for NADPH, respectively. This enzyme exhibited high activity toward a variety of aromatic and aliphatic aldehydes, but no activity toward ketones. As such, it catalyzed the chemoselective reduction of aldehydes in the presence of ketones, as demonstrated by the reduction of 4-acetylbenzaldehyde or the mixture of hexanal and 2-nonanone, showing the application potential of this marine enzyme in such selective reduction of synthetic importance.

Full text of DOI:10.1016/j.molcatb.2013.01.010

Journal of Molecular Catalysis B: Enzymatic 90 (2013) 17–22
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Identification of a marine NADPH-dependent aldehyde reductase for
chemoselective reduction of aldehydes
Guangyue Li, Jie Ren, Qiaqing Wu∗∗, Jinhui Feng, Dunming Zhu∗, Yanhe Ma
National Engineering Laboratory for Industrial Enzymes, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xi Qi Dao, Tianjin Airport Economic Area,
Tianjin 300308, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A putative aldehyde reductase gene from Oceanospirillum sp. MED92 was overexpressed in Escherichia coli.
The recombinant protein (OsAR) was characterized as a monomeric NADPH-dependent aldehyde
reductase. The kinetic parameters Km and kcat of OsAR were 0.89 0.08 mM and 11.07 0.99 s−1 for
benzaldehyde, 0.04 0.01 mM and 6.05 1.56 s−1 for NADPH, respectively. This enzyme exhibited high
activity toward a variety of aromatic and aliphatic aldehydes, but no activity toward ketones. As such, it
catalyzed the chemoselective reduction of aldehydes in the presence of ketones, as demonstrated by the
reduction of 4-acetylbenzaldehyde or the mixture of hexanal and 2-nonanone, showing the application
potential of this marine enzyme in such selective reduction of synthetic importance.
Received 20 November 2012
Received in revised form
31 December 2012
Accepted 12 January 2013
Available online 23 January 2013
Keywords:
Oceanospirillum sp.
Aldehyde reductase
Bioreduction
© 2013 Elsevier B.V. All rights reserved.
Chemoselectivity
Marine enzyme
1. Introduction
catalytic mechanism, although there is considerable variation in
the substrate-binding pocket [8,9]. These enzymes metabolize
The reduction of carbonyl compounds to alcohols is one of the
most important reactions in synthetic organic chemistry. Although
many methods are available for the reaction, few are chemoselec-
tive for the reduction of aldehydes in the presence of ketones and
such methods are still in demand [1–4]. Enzymatic reduction offers
an attractive approach because the enzymes usually display a better
selectivity and the biocatalytic reaction conditions are considered
to be more environmentally benign. However, there are only a few
examples where biocatalysts have been exploited to the chemose-
lective reduction of aldehydes in the presence of ketones and other
functional groups [5–7]. On the other hand, the benzylic alcohols
are key starting materials in the synthesis of scented substances
for cosmetics, fragrances and flavor industry, which in general are
produced from the less expensive aldehydes. As such, aldehyde
reductases have been searched with aim to develop biocatalytic
methods for chemoselective reduction of aldehydes.
a diverse range of substrates, including aliphatic and aromatic
aldehydes, monosaccharides, steroids, prostaglandins [8,10–12]. A
NADPH-dependent human liver aldehyde reductase (HlAR) was
extracted from human liver and the enzyme catalyzed the reduc-
tion of various aldehydes [13,14]. HlAR was thus used as the
template in genome mining to seek for effective aldehyde reductase
in AKR superfamily. Since the enzymes originated from the highly
diverse marine microorganisms offered a great source of biocata-
lysts for organic synthesis [15–17], a putative aldehyde reductase
(OsAR) from Oceanospirillum sp. MED92 [18], which had 41.64%,
35.82%, 15.34% and 13.69% identity with HlAR [14], Sporobolomyces
salmonicolor aldehyde reductase (SsAR) [19], Vigna radiata alde-
hyde reductase (VrAR) [20] and aflatoxin-metabolizing aldehyde
reductase (AfAR) [21], was selected as the target enzyme. Herein
we report the biochemical characterization and substrate profile of
the recombinant OsAR enzyme. The results showed that OsAR was
an aldehyde specific reductase, and catalyzed the chemoselective
reduction of aldehydes in the presence of ketones.
The aldo–keto reductases (AKR) are a superfamily of enzymes
that includes a number of related NAD(P)H-dependent oxidore-
ductases, such as aldehyde reductase, aldose reductase. The
AKR enzymes share a common (␣/␤)8 structure, and conserved
2. Materials and methods
2.1. Materials
∗
Corresponding author. Tel.: +86 22 84861962; fax: +86 22 84861996.
Corresponding author. 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).
∗∗
FastDigest Restriction Enzymes were purchased from Fer-
mentas, St. Leon Roth, Germany. Vector pET21a(+) was purchased
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.01.010

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G. Li et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 17–22
Table 1
Purification parameters of QsAR.
Specific activity (U mg−1
)
Total activity (U)
Purification factor
Yield (%)
Cell-free extract
Ni column
1.8
16
200
172
1
8.9
100
86
from Novagen (Merck, Germany). NADPH was from Codexis (USA);
all aldehydes and ketones were purchased from Alfa Aesar or
Sigma–Aldrich.
2.2. Cloning, expression and purification of OsAR
The OsAR gene from Oceanospirillum sp. MED92 with six His-
tag at N-terminal and recognition sites for the restriction enzymes
NdeI and SalI was synthesized by Shanghai Xuguan Biotechno-
logical Development Co. LTD. The gene product was digested
with NdeI/SalI, and cloned into the NdeI/SalI sites of pET21a (+)
(Novagen). After confirmation of the sequence, the plasmid PET-
21a-QsAR was transformed into E. coli BL21 (DE3).
E. coli BL21 (DE3) cells carrying the recombinant plasmid were
cultivated in 5 ml LB medium containing ampicillin (100 ␮g/ml)
overnight at 37 ◦C. The culture was inoculated into 1 l LB medium
containing ampicillin (100 ␮g/ml) and grown at 37 ◦C. The culture
was induced by addition of isopropyl ˇ-d-1-thiogalactopyranoside
(IPTG) with a final concentration of 1 mM when OD600 reached 1.2,
and then allowed to grow for additional 12 h at 20 ◦C. After cen-
trifugation at 6000 × g for 15 min at 4 ◦C, the bacterial pellet was
washed with phosphate buffer (50 mM, pH 7.0), and resuspended
in a phosphate buffer (20 mM pH 7.4) containing 0.5 M of NaCl and
40 mM of imidazole.
The cells were lysed by sonication and the supernatant was
collected by centrifugation at 10,000 × g for 30 min at 4 ◦C. Pro-
tein purification was performed at 23 ◦C using an AKTA purifier
10 system with UNICORN 5 software (GE Healthcare). The OsAR
was purified using affinity chromatography with a HisTrapTM
FF crude column (GE, USA). The column was pre-equilibrated
with 50 ml equilibrium buffer (20 mM phosphate buffer, 0.5 M
NaCl, 40 mM imidazole, pH 7.4). The sample (30 ml) was loaded
with a flow rate of 1.0 ml/min. After washing with 50 ml of the
equilibrium buffer, the bounded target protein was eluted with
elution buffer (20 mM PB, 0.5 M NaCl, 0.5 M imidazole, pH 7.4).
The target protein fraction was desalted through dialyzed against
20 mM phosphate buffer (pH 6.5). The protein was collected and
stored at −20 ◦C. Protein concentration was measured by Bradford
method [22].
Fig. 1. SDS-PAGE analysis of OsAR. M: protein marker. (1) Control with empty vec-
tor. (2) Cell-free extract. (3) Purified QsAR.
in absorbance at 340 nm using a molar absorption coefficient of
6.22 mM−1 cm−1. One unit of enzyme activity was defined as the
amount of enzyme catalyzing the conversion of 1 ␮mol NADPH per
minute.
2.5. Effects of pH, temperature and metal ions on enzyme activity
The pH dependence of OsAR activity was studied using the
following buffer system (100 mM): sodium acetate (pH 5.0–6.0),
sodium phosphate (pH 6.0–8.0), and Tris–HCl (pH 8.0–9.0). For
determining pH stability, the enzyme was pre-incubated in buffers
2.3. Molecular weight determination
The molecular mass of OsAR was determined by gel filtration
chromatography using a Superdex 200 10/300 GL column and the
low molecular weight gel filtration calibration kit (GE). The sodium
phosphate buffer (50 mM, pH 7.2) containing 150 mM NaCl was
used as the eluent. The flow rate was 0.4 ml/min and the absorbance
at 280 nm was monitored.
2.4. Activity assay
Enzyme activity was determined in a 0.2 ml of sodium phos-
phate buffer (0.1 M, pH 6.5) containing substrate (2 mM), NADPH
(0.3 mM), and 20 ␮L (20 ␮g/ml) of the purified enzyme OsAR. The
substrate was added as solution in dimethyl sulfoxide (DMSO),
which did not exceed 1% of the total volume [23]. The reaction
was initiated by the addition of the enzyme, and monitored for
1–3 min with SPECTRAMAX M2e (MD, USA) at 30 ◦C. The activity
was determined by measuring NADPH oxidation from the decrease
Fig. 2. Effects of pH on the enzyme activity (square) and stability (circle). The
enzyme activity (16 U/mg) measured in sodium phosphate buffer (100 mM, pH 6.5)
was defined as 100%.

G. Li et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 17–22
19
benzaldehyde and NADPH in the range of 0.01–0.2 mM were used
for the activity assay. All experiments were conducted in triplicate.
2.7. Selective reduction of aldehydes
The bioreduction of 4-acetylbenzaldehyde was carried out
as follows: d-Glucose (280 mg), d-glucose dehydrogenase (11 U),
NADPH (10 mg), QsAR (40 U) and 4-acetylbenzaldehyde (50 mg)
were mixed in sodium phosphate buffer (25 ml, 100 mM, pH 6.5).
The mixture was stirred at 25 ◦C for 18 h. The mixture was extracted
with methyl tert-butyl ether. The organic extract was dried over
anhydrous sodium sulfate and removal of the solvent gave prod-
uct, 4-acetylbenzyl alcohol (38.2 mg, 76.4% yield). 1H NMR (CDCl3),
ı: 2.58 (d, 3H), 4.76 (s, 2H,), 7.44(d, 2H, 2JH H = 7.2 Hz,), 7.93 (d,
2H, 2JH H = 7.2 Hz)). The bioreductions of hexanal and 2-nonanone
were as follows: d-Glucose (36 mg), d-glucose dehydrogenase (2 U),
NADPH (1.0 mg), QsAR (4 U), hexanal (10 mM) and 2-nonanone
(10 mM) were mixed in sodium phosphate buffer (2 ml, 100 mM,
pH 6.5). The mixture was stirred at 25 ◦C for 12 h. The mixture was
extracted with methyl tert-butyl ether. The products were iden-
tified by comparison with authentic samples in an Agilent 7890
gas chromatography with Gamma DEXTM 225 capillary column
(30 m × 0.25 mm × 0.25 mm, SUPELCO, Japan). The column temper-
ature was controlled as follows: 50 ◦C for 5 min; 30 ◦C/min to 80 ◦C;
80 ◦C for 5 min; 20 ◦C/min to 100 ◦C; 100 ◦C for 8 min. The retention
times for hexanal and hexanol were 6.99 and 8.66 min; 16.08 and
16.44 min for 2-nonanone and 2-nonanol, respectively.
Fig. 3. Effects of temperature on enzyme activity (circle) and stability (square). The
enzyme activity measured at 30 ◦C was defined as 100%.
Table 2
Effects of metal ions, EDTA, detergent and reducing agent on OsAR.
Chemicals
Concentration
Relative activity (100%)
MnCl2
(NH4)2SO4
MgSO4
ZnCl2
NiSO4
CuSO4
FeCl3
EDTA
SDS
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
1 mM
5 mM
0.1%
103
103
110
98
94
11
100
99
43
3. Results and discussion
TritonX-100
␤-Mercaptoethanol
Control
1%
5 mM
11
75
100
3.1. Purification and biochemical characterization of OsAR
A putative aldehyde reductase (OsAR) from Oceanospirillum sp.
MED92 (NCBI accession number: ZP 01167697.1) was found by
NCBI BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) using
a human liver aldehyde reductase (HlAR) as template [13], and
expressed in E. coli BL21 (DE3) at 20 ◦C as a soluble protein. The
recombinant protein (OsAR) was purified in one chromatographic
step using a HisTrapTM FF crude column and the purification
parameters are shown in Table 1. SDS-PAGE analysis at each stage
of the purification is shown in Fig. 1. The purified enzyme gave a sin-
gle band of about 36 kDa, which is consistent with the calculated
value (36.3 kDa). Its molecular mass determined by gel filtration
chromatography was about 36 kDa, indicating that OsAR existed as
monomer. This aggregation states was consistent with other known
aldehyde reductases, such as SsAR [19], HlAR [14], VrAR [20] and
Candida magnoliae aldehyde reductase (CmAR) [26].
OsAR was highly specific for NADPH as a coenzyme, and no activ-
ity was observed in the reduction of benzaldehyde when NADPH
was replaced by NADH. This is in agreement with aldehyde reduc-
tases VrAR [20], SsAR [19] and CmAR [26]. In contrast, the two
other known aldehyde reductases AfAR [21,27] and HlAR could use
both NADH and NADPH as cofactor, but the affinity for NADH was
significantly lower than that observed for NADPH [14,28].
of different pH (5.0–9.0) for 24 h at 4 ◦C. The residual activity was
measured by following the standard assay procedure. The opti-
mum temperature of the enzyme was determined by measuring
the enzyme activity in the range of 20–45 ◦C. For thermostability,
the enzyme was incubated at different temperatures (15–45 ◦C) for
30 min followed by measuring the residual activity by following the
standard assay procedure.
Effect of metal ions, EDTA, detergent and reducing agent to
the enzyme activity of QsAR were tested by pre-incubating with
the enzyme at different working concentrations. Various chemi-
cals were added to the enzymatic preparation and pre-incubated
for 30 min at 4 ◦C, and the residual activity of the enzyme was
measured under standard conditions [24,25]. All experiments were
conducted in triplicate.
2.6. Determination of kinetic parameters
The kinetic parameters were obtained by measuring the initial
velocities of the enzymic reaction and curve-fitting according to
the Michaelis–Menten equation using GraphPad Prism 5 software
(GraphPad Software Inc). For benzaldehyde, the activity assay was
performed in a mixture containing a varying concentration of benz-
aldehyde (0.1–8 mM) and 0.6 mM of NADPH. For NADPH, 10 mM of
As shown in Fig. 2, OsAR exhibited maximum activity at pH
6.5, and more than 90% of the activity was retained in the pH
range of 6.0–7.0. The optimum pH of QsAR was similar with known
Table 3
Kinetic parameters of aldehyde reductases.
Enzyme
Km (mM)
kcat (s−1
)
kcat/Km (mM−1 s−1
)
Reference
OsAR
HlAR
ARII
0.89 0.08
3.4
25.5
11.07 0.99
0.47
24.67
12.44
0.14
0.97
2.57
This work
[14]
[30]
MbAR
0.033
0.085
[29]

20
G. Li et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 17–22
Table 4
Substrate specificity of aldehyde reductase.
No.
Substract
Structure
Relative activity (100%)
Aldehydes
O
1
Benzaldehyde
100a
146
O
2
4-Bromobenzaldehyde
4-Chlorobenzaldehyde
4-Fluorobenzaldehyde
Br
Cl
F
O
3
4
141
80
O
O
5
6
4-(Trifluoromethyl)benzaldehyde
Pentafluorobenzaldehyde
171
110
F3C
F
F
F
F
O
F
O
7
4-Cyanobenzaldehyde
165
NC
O
8
9
4-Formylbenzoic acid
4-Acetoxybenzaldehyde
p-Tolualdehyde
65
HOOC
O
O
O
110
96
O
10
O
11
4-Isopropylbenzaldehyde
79
O
12
13
4-tert-Butylbenzaldehyde
p-Methoxybenzaldehyde
NDb
80
(H3C)3C
O
O
O
14
4-(Methylthio)benzaldehyde
61
S
15
16
Hexanal
Octanal
115
72
17
2-Methylbutyraldehyde
65
18
19
trans-2-Hexen-1-al
Cinnamaldehyde
35
69
20
Mannose
ND

G. Li et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 17–22
21
Table 4 (Continued)
No.
Substract
Glucose
Structure
Relative activity (100%)
21
ND
Ketones
22
Acetophenone
ND
ND
23
24
chloro-acetophenone
a-bromoacetophenone
O
Br
ND
O
25
26
2-Indanone
2-Nonanone
ND
ND
O
27
3-Bromoheptan-4-one
ND
Br
28
29
Cyclohexanone
ND
ND
Tetrahydrothiophen-3-one
30
31
Benzylideneacetone
ND
2-Cyclohexen-1-one
trans-3-Nonen-2-one
ND
ND
32
a
The specific activity for benzaldehyde was 16 U/mg and defined as 100%.
ND, the activity was not detected.
b
Effects of Cu2+, Fe3+, NH4+, Mg2+, Mn2+, Ni2+, Zn2+, EDTA,
TritonX-100, ␤-mercaptoethanol and SDS on enzyme activity were
also examined (Table 2). The activity of OsAR was strongly inhibited
aldehyde reductases AfAR, HlAR and Mung Bean aldehyde reduc-
tases (MbAR), which displayed a pH optimum of 6.5 and high
activity nearby neutral pH [14,28–30]. Similar to a known S.
salmonicolor aldehyde reductases (ARII) [30], QsAR was stable over
a broad range of pH 6.0–9.0 at 4 ◦C for 24 h, but the activity
decreased significantly in more acid conditions (Fig. 2).
The optimal reaction temperature of aldehyde reductase OsAR
for benzaldehyde reduction was 40 ◦C (Fig. 3). This enzyme was
stable at 20 ◦C for 30 min, and its thermal stability rapidly decreased
above 25 ◦C (Fig. 3). This might be due to the origin of QsAR, a marine
bacterium Oceanospirillum sp. MED92 isolated from a surface water
sample from the eastern Mediterranean Sea, a low temperature
marine environment [18].
by Cu2+ (about 90% inhibition), but Fe3+, NH4+, Mg2+, Mn2+, Ni2+
,
and Zn2+ showed no obvious effects on the enzyme activity.
Similar strong inhibition by Cu2+ was observed for the alde-
hyde reductase CmAR, but it was also inhibited completely by
Zn2+ [26]. EDTA had little effect on the activity of QsAR, sug-
gesting that metals were not required for the activity of OsAR,
which was consistent with catalytic mechanism of AKR superfa-
mily [30]. The activity of OsAR was inhibited by Triton X-100, SDS
and ␤-mercaptoethanol. This may be due to the combined effect
of factors such as reduction in hydrophobic interactions which

22
G. Li et al. / Journal of Molecular Catalysis B: Enzymatic 90 (2013) 17–22
normally play a crucial role in maintaining tertiary structure, and
their direct interactions with the protein molecule [25]. The inhi-
bition of OsAR activity by ␤-mercaptoethanol suggests that the
four cysteine residues in the enzyme may be involved in the for-
mation of disulfide bond, which is necessary for OsAR’s enzyme
activity.
The Michaelis–Menten constant Km for benzaldehyde was
0.89 0.08 mM, and 0.04 0.01 mM for NADPH. The kcat was
11.07 0.99 s−1 for benzaldehyde and 6.05 1.56 s−1 for NADPH,
respectively. The Km of QsAR for benzaldehyde is in the range
(0.033–25.5 mM) of known aldehyde reductases [14,20,29,30].
Compare to the aldehyde reductases HlAR, ARII and MbAR
[14,29,30], QsAR displayed higher kcat/Km (Table 3), suggest-
4. Conclusion
In conclusion, a gene from Oceanospirillum sp. MED92, which
was annotated as a putative oxidoreductase included in the
aldo–keto reductase family, was successfully cloned and over-
expressed in E. coli. The purified OsAR exhibited high activity
toward a variety of substituted benzaldehydes and aliphatic alde-
hydes, but no activity for ketone functional group. As such, OsAR
is a NADPH-dependent, highly specific aldehyde reductase. This
enzyme represents the first aldehyde reductase of marine origin,
which could catalyze the chemo-specific reduction of aldehydes in
the presence of ketones, a synthetically useful transformation.
ing the enzyme QsAR possesses
efficiency.
a relative higher catalytic
Acknowledgments
This work was financially supported by the Chinese Academy of
Sciences (KGCX2-YW-203 and KSCX2-EW-G-14) and National Key
Basic Research and Development Program (2011CB710801).
3.2. Substrate specificity
The catalytic activity of OsAR was measured toward a wide
range of aldehydes and ketones, and the results are summarized
in Table 4. From Table 4, it can be seen that OsAR catalyzed the
reduction of various aromatic and aliphatic aldehydes. For benz-
aldehyde derivatives with butyl, methylthio, methoxy, isopropyl
and methyl substituents, the activity was generally lower than the
analogs with electron-withdrawing substituents such as chloro,
bromo, trifluoromethyl and cyano groups, suggesting the elec-
tronic property of the substituent exert some effects on the enzyme
activity. This is consistent with the results for aldehyde reductase
from mung bean [29] and a microbial 7␣-hydroxysteroid dehydro-
genase [31]. Regarding aliphatic aldehydes, OsAR was active for
both saturated and unsaturated ones and showed higher activity
toward medium-chain saturated aliphatic aldehydes than long-
chain and short-chain counterparts. QsAR displayed no activity
toward mannose and glucose. This is different from the known
aldehyde reductases HlAR and CmAR, which showed activity
toward glucose [14,26]. No activity was detected for ketones,
suggesting that OsAR is highly specific to aldehydes. This speci-
ficity is different from the known aldehyde reductases HlAR
[14], ARII [30] and AfAR [28], which displayed activity toward
ketones.
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3.3. Selective reduction of aldehydes
The high activity toward aldehydes and the lack of activity
for ketones suggest that OsAR should be an attractive biocata-
lyst for the chemoselective reduction of aldehydes in the presence
of ketones. Indeed, 4-acetylbenzaldehyde was reduced to give 4-
acetylbenzyl alcohol as the sole product. For the reduction of a
mixture of hexanal and 2-nonanone by OsAR, hexanal was reduced
to hexanol in 100% conversion, and no 2-nonanol was detected.
There are only a few examples of biocatalytic chemoselective
reduction of aldehydes [5–7]. In these studies the chemoselec-
tive reduction of aldehydes were carried out by using an E. coli
JM109 whole cell or plant cells as the biocatalyst. OsAR offers the
first example of employing an isolated aldehyde reductase for such
chemoselective aldehyde reduction, which may be applied in the
synthesis of benzylic alcohols, the key starting materials in the pro-
duction of scented substances for cosmetics, fragrances and the
flavor industry.
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Physiol. 119 (1999) 621.
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