Biocatalytic properties of a recombinant aldo keto reductase with broad substrate spectrum and excellent stereoselectivity

1. BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS
Biocatalytic properties of a recombinant aldo-keto reductase
with broad substrate spectrum and excellent stereoselectivity
Yan Ni & Chun-Xiu Li & Hong-Min Ma & Jie Zhang &
Jian-He Xu
Received: 11 August 2010 /Revised: 7 October 2010 /Accepted: 9 October 2010 /Published online: 28 October 2010
# Springer-Verlag 2010
Abstract In the screening of 11 E. coli strains overexpress-
ing recombinant oxidoreductases from Bacillus sp.
ECU0013, an NADPH-dependent aldo-keto reductase
(YtbE) was identified with capability of producing chiral
alcohols. The protein (YtbE) was overexpressed, purified to
homogeneity, and characterized of biocatalytic properties.
The purified enzyme exhibited the highest activity at 50°C
and optimal pH at 6.5. YtbE served as a versatile reductase
showing a broad substrate spectrum towards different
aromatic ketones and keto esters. Furthermore, a variety of
carbonyl substrates were asymmetrically reduced by the
purified enzyme with an additionally coupled NADPH
regeneration system. The reduction system exhibited excel-
lent enantioselectivity (>99% ee) in the reduction of all the
aromatic ketones and high to moderate enantioselectivity in
the reduction of α- and β-keto esters. Among the ketones
tested, ethyl 4,4,4-trifluoroacetoacetate was found to be
reduced to ethyl (R)-4,4,4-trifluoro-3-hydroxy butanoate, an
important pharmaceutical intermediate, in excellent optical
purity. To the best of our knowledge, this is the first report of
ytbE gene-encoding recombinant aldo-keto reductase from
Bacillus sp. used as biocatalyst for stereoselective reduction
of carbonyl compounds. This study provides a useful
guidance for further application of this enzyme in the
asymmetric synthesis of chiral alcohol enantiomers.
Keywords YtbE . Recombinant aldo-keto reductase .
Bacillus sp. . Asymmetric reduction . Chiral alcohols
Introduction
Aldo-keto reductases (AKRs) are a growing superfamily of
more than 140 widely distributed individual members
belonging to 15 families, which are widely distributed in
plants, animals, and microorganisms (Jez and Penning
2001; Ellis 2002; Hyndman et al. 2003; www.med.upenn.
edu/akr/). They catalyze the NADPH-dependent reduction
of a variety of substrates including aliphatic and aromatic
aldehydes/ketones, monosaccharides, steroids, prostaglan-
dins, isoflavinoids, polyketides, and so forth (Bohren et al.
1989; Jez et al. 1997; Petrash 2007).
Chiral alcohols are frequently required as important
and valuable intermediates in the synthesis of numerous
pharmaceuticals and other fine chemicals. Asymmetric
reduction of prochiral ketones is an effective and
promising route for producing chiral alcohols, which
has an inherent advantage of achieving up to 100%
theoretical yield (Kataoka et al. 2003; Woodley 2008).
Biocatalytic transformation using isolated enzymes or
whole cell systems offers some advantages in comparison
with chemical methods, such as mild and environmentally
benign reaction conditions and remarkable chemo-, regio-,
and stereoselectivity (Nakamura et al. 2003; Kroutil et al.
2004; Goldberg et al. 2007). In addition to the conven-
tional biocatalyst screening, bioinformatics seems to
increase the possibility of discovering useful biocatalysts.
Screening of a library of overexpressed enzymes from
microorganisms with known wealth of data that has
accrued from sequencing whole genomes is a potential
tool to search a catalyst for desired reactions (Yamamoto
Y. Ni :C.-X. Li (*) :H.-M. Ma :J. Zhang :J.-H. Xu (*)
Laboratory of Biocatalysis and Bioprocessing,
State Key Laboratory of Bioreactor Engineering,
East China University of Science and Technology,
Shanghai 200237, People’s Republic of China
e-mail: chunxiuli@ecust.edu.cn
J.-H. Xu
e-mail: jianhexu@ecust.edu.cn
Appl Microbiol Biotechnol (2011) 89:1111–1118
DOI 10.1007/s00253-010-2941-4

2. et al. 2003; Moore et al. 2007; Matsuda et al. 2009). In
fact, 18 key reductases from bakers’ yeast have been
overexpressed in Escherichia coli and tested for the ability
of reduction (Kaluzna et al. 2004; Kaluzna et al. 2005;
Hammond et al. 2007).
Recently, we isolated a ketone reductase-producing
Bacillus sp. ECU0013 with excellent stereoselectivity and
high substrate concentration tolerance (Xie et al. 2010). In
this study, in the search for oxidoreductases with activity
and high stereoselectivity towards prochiral ketones, we
cloned 11 genes encoding oxidoreductases from the
strain and expressed them heterologously in E. coli
BL21(DE3). The identification of an aldo-keto reductase
(YtbE) with capability of producing alcohols was
achieved by screening of these recombinant reductases.
Purification of YtbE was performed, and the purified
enzyme was characterized of enzymatic properties,
especially with respect to its substrate specificity and
stereospecificity.
Materials and methods
Bacterial strains and vectors
E. coli DH5α and E. coli BL21 (DE3) were routinely
grown in Luria–Bertani (LB) medium and were used as
the cloning and expression hosts, respectively. Ampicillin
(100 μgml−1
) and kanamycin (50 μgml−1
) were used for
the selection of recombinant strains in E. coli. Bacillus sp.
ECU0013 was isolated and identified in our laboratory
(Xie et al. 2010). The strain was deposited in China
General Microbiological Cultures Center, with an acces-
sion number of CGMCC No. 2549. Cells were cultured as
previously described. Plasmid pMD18-T for the direct
cloning of PCR products was from TaKaRa (Dalian,
China) and plasmid pET28a (+) for heterogeneous
expression studies was obtained from Novagen (Shanghai,
China).
Cloning and construction of expression plasmids
The genomic DNA of Bacillus sp. ECU0013 was extracted
and purified using the TIANamp Bacteria DNA Kit from
Tiangen (Shanghai, China). DNA fragments containing the
different oxidoreductases-encoding genes were amplified
by polymerase chain reaction (PCR) using primers with the
BamHI and XhoI restriction sites. The amplified DNAs
were purified and directly cloned into the TA cloning site of
pMD-18T and then introduced into DH5α. The desired
genes were subcloned into expression plasmid pET28a (+)
using standard methods and then transformed into E. coli
BL21 (DE3) cells.
Expression and purification of recombinant carbonyl
reductases
Recombinant protein was expressed in E. coli BL21 cells
transformed with pET28a. Cultures were grown at 37°C in
LB medium containing 50 μg/ml kanamycin. When the
OD600 of the culture reached 0.6, IPTG was added to a final
concentration of 0.5 mM. The culture was incubated at
25°C for a further period of 12 h. Cells were harvested by
centrifugation, washed twice and resuspended in buffer A
(20 mM sodium phosphate buffer, pH 7.4, 500 mM NaCl,
10 mM imidazole). Cell suspension was disrupted by
sonication and the cell lysate was centrifuged at 10,000×g
for 20 min. The clear supernatant was loaded onto a Ni–
NTA column equilibrated with buffer A. The column was
washed with buffer A, and the retained proteins were eluted
with an increasing gradient from 10 to 500 mM of
imidazole in buffer A at a flow rate of 1 ml/min. The
purity of fractions was assessed by SDS–PAGE. The
fractions containing the pure protein were pooled and
dialyzed against sodium phosphate buffer for desalting.
Protein analysis
Gel electrophoresis was performed on 15% SDS–
polyacrylamide gel with Tris–glycine buffer system. Protein
bands were visualized by staining the gel with silver stain.
The apparent molecular masses were determined by gel
filtration chromatography using a TSK gel 2000SWxl
column (Tosoh, Japan) connected to an HPLC system
equilibrated with 100 mM sodium phosphate buffer
(pH 6.7) containing 0.1 M Na2SO4 at a flow rate of
0.4 ml/min. Protein molecular weight standards were horse
heart cytochrome c (12.4 kDa), bovine carbonic anhydrase
(29 kDa), bovine serum albumin (66 kDa), yeast alcohol
dehydrogenase (150 kDa), and sweet potato β-amylase
(200 kDa) from Sigma (Shanghai, China).
Enzyme assays
The reductase activity was assayed spectrophotometrically
at 30°C by monitoring the decrease in the absorbance of
NADPH at 340 nm. The standard assay mixture (1 ml) was
composed of 50 mM sodium phosphate buffer (pH 7.0),
2.0 mM ethyl pyruvate, 0.05 mM NADPH, and an
appropriate amount of enzyme. One unit of enzyme activity
was defined as the amount of enzyme catalyzing the
oxidation of 1 μmol NADPH per minute. The kinetic
parameters of the purified reductase were determined by
assaying (in triplicate) the activity on the substrate ethyl
pyruvate of different concentrations (1–30 mM) at a fixed
NADPH concentration. The maximal reaction rate (Vmax)
and apparent Michaelis–Menten constant (Km) of the
1112 Appl Microbiol Biotechnol (2011) 89:1111–1118

3. purified reductase were calculated from Lineweaver–Burk
plot.
pH and temperature optima and thermostability
The optimum pH was determined by standard activity assay
at different pH (4.0–10.0), with sodium citrate for a pH
range from 4.0 to 6.0, sodium phosphate for pH range from
6.0 to 8.5, and Gly–NaOH for pH range from 8.5 to 10.0.
The optimum temperature was determined under standard
conditions at different temperatures in the range of 20–
65°C. The stability was studied by incubating the purified
enzymes (0.1 mg·ml−1
) in the same buffer at 30°C, 40°C,
and 50°C, and the residual activity was assayed as
described above.
Effect of metal ions and additives on activity
Influence of various metal ions and additives on enzyme
activity was investigated by pre-incubating the enzyme with
different compounds in 50 mM sodium phosphate buffer
(pH 7.0) for 20 min at 30°C. The enzyme activity was
estimated using standard assay protocol. Relative activity
was expressed as a percentage of the activity in the absence
of any test compound.
Enantioselectivity
The enantioselectivity was determined by examining the
reduction of aromatic ketones and keto esters using an
NADPH regeneration system consisting of the purified
reductase and glucose dehydrogenase (GDH). The reaction
mixture containing 0.5 mM NADP+
, 2 mM aromatic
ketones or 10 mM keto esters, 0.8 U of the purified
reductase, 0.4 U of GDH, and 5% glucose in 0.4 ml of
50 mM potassium phosphate buffer (pH 7.0) was incubated
at 30°C with shaking for 12 h. After the reaction, each
reaction mixture was extracted twice with ethyl acetate. The
enantiomeric excess (ee) of the product and the level of
conversion were determined by GC or HPLC analysis. GC
analyses were performed using a CP-Chirasil-DEX CB
(Varian, USA) or Beta-DEX 120 column (Supelco, USA).
HPLC analyses were performed using Chiralcel OD-H
columns (Daicel Co., Japan; 250 mm×Φ4.6 mm).
Results
Screening of the recombinant reductases
Our approach was to find new reductases with capability of
reducing prochiral ketones to corresponding chiral alcohols.
Analysis of the Bacillus subtilis 168 genome (Kunst et al.
1997) for genes encoding oxidoreductases resulted in 11
open reading frames as candidates to design specific primers
for discovering synthetically useful biocatalysts. With stan-
dard cloning techniques and the genomic DNA of Bacillus
sp. ECU0013 as template, E. coli overexpression plasmids
for the 11 proteins of interest were created. During the
screening process, a ytbE gene-encoding reductase (YtbE)
was found to be able to reduce 2-chloroacetophenone to (R)-
2-chloro-1-phenylethanol, and was chosen as a potential
biocatalyst for further studies. The ytbE nucleotide sequences
identified in this study have been submitted to GenBank
(accession no. HM590486). A BLAST search revealed that
the deduced amino acid sequence of ytbE gene is practically
identical to that of a putative aldo-keto reductase from B.
subtilis 168 (Genbank accession no. CAB14865).
Protein purification
The N-terminal His-tagged recombinant enzyme, which
was fully soluble, was purified to electrophoretic homoge-
neity by immobilized metal affinity chromatography. The
specific activity of enzyme after purification was 1.62 U
mg−1
, corresponding to a 1.3-fold improvement in purity
compared to the crude extract. Samples of the crude extract
and target fraction after purification were analyzed by
SDS–PAGE (Fig. 1), which revealed a single band with an
apparent molecular size of 35.7 kDa, corresponding to the
theoretical value of the enzyme (Fig. 1, lane 3). Addition-
ally, the purified protein eluted from the TSK gel column as
a single peak with an elution volume corresponds to an
apparent molecular mass of 31.5 kDa, suggesting a
monomeric structure.
Substrate specificity
Substrate specificity of the enzyme was assessed rapidly by
using spectrophotometric assays. The ability of the enzyme
to catalyze the reduction of different aromatic ketones and
keto esters was determined (Table 1). Acetophenone
Fig. 1 SDS–PAGE analysis
of the purified YtbE. Lane 1
protein markers, 2 crude extract,
3 purified enzyme. Protein
bands were visualized by silver
staining
Appl Microbiol Biotechnol (2011) 89:1111–1118 1113

4. derivatives and acetyl pyridines were accepted by the
reductase, except for propiophenone and 4′-aminoacetophe-
none. The reductase showed the strongest activity on 2,2,2-
trifluoacetophenone among the substituted acetophenones
tested. Meanwhile, both α- and β-keto esters served as
suitable substrates for the reductase. The obviously high
activity was observed on ethyl 4-chloroacetoacetate and
ethyl pyruvate. In the present work, ethyl pyruvate was
chosen as the standard substrate to assay the enzyme
activity during the enzyme characterization and to calculate
kinetic parameters of the purified reductase. The maximal
reaction rate (Vmax) and apparent Michaelis–Menten con-
stant (Km) were found to be 2.66±0.14 Umg−1
protein and
4.47±0.23 mM, respectively.
pH and temperature effects and thermostability
The effect of pH on the enzyme activity was examined in
the range of pH 4.0–10.0. As demonstrated in Fig. 2a, the
optimal activity was observed at pH 6.5 and the enzyme
showed good activity (>75%) at pH ranging from 6 to 8.
The optimum temperature of carbonyl reductase activity
was determined by measuring the enzyme activity at 20–
65°C (Fig. 2b). The maximum activity was observed at
around 50°C, being 150% higher than that under standard
assay conditions (30°C).
Thermostability of the purified carbonyl reductase was
examined at temperatures of 30°C, 40°C, and 50°C (Fig. 3).
The enzyme was quite labile at higher temperatures (40°C
and 50°C) but more stable at 30°C. The half-lives of the
enzyme measured at 30°C, 40°C, and 50°C were 268, 27,
and 10 min, respectively. This suggests that higher temper-
atures will result in a rapid loss of the reductase activity.
The relatively low protein concentration of the purified
enzyme sample (ca. 0.1 mg·ml−1
) might be responsible for
the poor thermostability.
Effect of various metal ions and additives
The effect of various compounds on the purified reductase
was assessed by incubating the enzyme with salts of
different metal ions or additives at a final concentration of
1 mM (Table 2). The enzyme displayed a slightly higher
Table 1 Substrate specificity of the reductase
Substrate (2 mM) Relative activity (%)
Aromatic ketones
Acetophenone 9.0
2-Chloroacetophenone 100
2-Bromoacetophenone 40.3
2,2,2-Trifluoroacetophenone 140
Propiophenone 0
4′-Chloroacetophenone 64.2
4′-Bromoacetophenone 32.8
4′-Aminoacetophenone 0
2-Acetylpyridine 43.3
3-Acetylpyridine 13.4
4-Acetylpyridine 34.3
Keto esters
Ethyl acetoacetate 145
Ethyl 4-chloroacetoacetate 8,776
Ethyl 4,4,4-trifluoroacetoacetate 74.6
Ethyl benzoylacetate 13.4
Ethyl 3-oxo-pentanoate 16.4
Ethyl pyruvate 7,821
Ethyl 3-methyl-2-oxo-butyrate 29.5
Methyl benzoylformate 309
Ethyl 2-oxo-4-phenylbutyrate 576
The reductase activity was estimated using the standard assay
protocol. The activity for 2-chloroacetophenone was taken as 100%
and those for others were represented as percentages of that for
2-chloroacetophenone
0
20
40
60
80
100
120
3 4 5 6 7 8 9 10 11
pH
Relativeactivity(%)
0
20
40
60
80
100
120
10 20 30 40 50 60 70
Temperature (o
C)
Relativeactivity(%)
A
B
Fig. 2 Effects of pH and temperature on activity of the purified
enzyme. a To ascertain the pH optima of the purified enzyme, enzyme
assay was performed using standard assay procedure in the following
buffers of 50 mM: (1) citrate (pH 4.0–6.0), (2) phosphate (pH 6.0–
8.5), and (3) Gly–NaOH (pH 8.5–10.0). b The optimum temperature
for the carbonyl reductase was estimated at various temperatures (20–
65°C) in phosphate buffer (50 mM, pH 7.0) using the standard assay
procedure. Relative activity was expressed as a percentage of
maximum activity under the experimental conditions
1114 Appl Microbiol Biotechnol (2011) 89:1111–1118

5. activity with the addition of Ca2+
. Metal ions that
considerably activate the reductase were not found. On
the other hand, the presence of Ag+
, Ni2+
, Pb2+
, and Zn2+
considerably diminished the activity and Fe3+
totally
inhibited the activity. Ni2+
could bind with His116
, a
catalytically important residue for the reductase activity
(Lei et al. 2009; Kilunga et al. 2005), which probably
resulted in its inhibitory effect on the activity. The presence
of EDTA, a chelating agent, slightly affected enzyme
activity, suggesting that the protein does not require metals
for its activity.
Enantioselectivity
Enantioselectivity of the enzyme was analyzed in aqueous
phosphate buffer using glucose dehydrogenase/glucose as
an external NADPH regeneration system. The results were
summarized in Table 3. It can be seen that YtbE catalyzes all
the aromatic ketones tested affording chiral alcohols with
excellent enantiomeric excess (>99%). Among them, 2,2,2-
trifluoroacetophenone and 2-acetylpyridine were efficiently
reduced by the enzyme, affording 100% conversion. The
versatility of the enzyme was further shown by using keto
esters as substrates, and it was found that YtbE functions as a
robust biocatalyst to reduce all of the five keto esters tested
with high reactivity. The enantioselectivities were very high
for the bioreduction of ethyl acetoacetate, ethyl 4,4,4-
trifluoroacetoacetate, and ethyl pyruvate (>99% ee), but only
relatively moderate for ethyl 4-chloroacetoacetate and ethyl
2-oxo-4-phenylbutyrate, giving 62% ee and 86% ee of
products, respectively. On the whole, the enantioselectivity
data indicate that the hydride of NADPH is transferred to the
Re-face of the carbonyl group, suggesting that the enantio-
preference of this enzyme generally follows Prelog’s rule,
except for the reduction of ethyl 2-oxo-4-phenylbutyrate.
Furthermore, all these aromatic ketones at 10 mM concen-
tration were also subjected to bioreduction by the whole cells
of recombinant E. coli under the same conditions, and the
optical purities of the corresponding alcohols were extremely
satisfactory (>99% ee).
Discussion
This study demonstrated the utility of screening a set of
recombinant enzymes created by using bioinformatics and
gene cloning techniques for rapid identification of useful
biocatalysts capable of transforming targeted substrates. It
would also be an efficient and easy-handling approach to
allow high-throughput screening of various substrates. In
the screening of 11 E. coli strains expressing 11 recombi-
nant oxidoreductases from Bacillus sp. ECU0013, the ytbE
gene-encoding reductase (YtbE) was found to be able to
reduce prochiral ketones. The deduced amino acid sequence
of ytbE gene is practically identical to that of putative aldo-
keto reductase from B. subtilis 168. The YtbE enzyme from
B. subtilis has been assigned as AKR5G2 of aldo-keto
reductase (AKR) superfamily on the basis of sequence
alignment. To date, although YtbE from B. subtilis has been
studied by crystallographic researchers and found to show
catalytic activity on aldehydes (Lei et al. 2009), the use of
YtbE for asymmetric reduction of prochiral carbonyl
compounds has not yet been reported so far. In this work,
the reductase was overexpressed, purified to homogeneity,
and characterized with respect to substrate specificities and
prochiral selectivities.
In addition to catalyzing the reduction of aldehyde or
aldose, the common AKR substrates, some members of
AKR superfamily were found to reduce keto esters, such as
2,5-diketo-D-gluconate reductase (YqhE) from E. coli
(Q46857; Habrych et al. 2002), carbonyl reductase from
Gluconobacter oxydans (YP_191077; Schweiger et al.
0
20
40
60
80
100
0 60 120 180 240 300
Time (min)
Residualactivity(%)
Fig. 3 Thermostability of the purified enzyme at different temperatures.
Diamond 50°C, square 40°C, triangle, 30°C. Samples were withdrawn
at different time intervals to estimate the residual activity using the
standard assay protocol. Residual activity was expressed as a percentage
of the activity measured initially without any pre-incubation
Metal ion
(1 mM)
Relative
activity (%)
Ag+
3.2
Ca2+
104
Co2+
17.1
Cu2+
7.2
Fe3+
0
Mg2+
101
Mn2+
90.4
Na+
96.3
Ni2+
4.8
Pb2+
3.2
Zn2+
2.4
EDTA 108
Table 2 Effect of metal
ions and additives on reductase
activity
The reductase was pre-incubated
with the various metal ions or
additives for 20 min and the
relative activity was determined
using the standard assay. Rela-
tive activity was expressed as a
percentage of the activity
obtained in absence of any test
compound
Appl Microbiol Biotechnol (2011) 89:1111–1118 1115

6. 2010), β-keto ester reductase from Penicillium citrinum
(CAD43583; Itoh et al. 2004), aldehyde reductase from
Sporobolomyces salmonicolor (P27800; Yamada et al.
1990; Kita et al. 1996), and 2-methylbutyraldehyde
reductase (Ypr1p) from Saccharomyces cerevisiae
(Q12458; Ford and Ellis 2002). Our results showed that
YtbE accepts both α- and β-keto esters. In fact, a BLAST
analysis revealed that the primary structure of YtbE shows
47–34% identities to those reductases. However, since few
of these aldo-keto reductases had been tested for aromatic
ketone reductions, we assessed the ability of YtbE to reduce
aromatic ketones. Fortunately, the result was very encour-
Substrate Product Conversion (%)a, b
ee (%)b
1a 1b
70.8 ± 0.9 >99 (S)
2a 2b
74.5 ± 2.1 >99 (R)
3a 3b
72.8 ± 2.0 >99 (R)
4a 4b
100 ± 0 >99 (R)
49.6 ± 1.8 >99 (S)
6b
56.0 ± 0.7 >99 (S)
7a 7b
100 ± 0 >99 (S)
8a 8b
33.2 ± 0.6 >99 (S)
9a 9b
66.8 ± 2.1 >99 (S)
10a 10b
100 ± 0 >99 (S)
11a 11b
100 ± 0 62.2 ± 0.6 (R)c
12a 12b
100 ± 0 >99 (R)
13a 13b
100 ± 0 >99 (S)
14a
O OH
Cl
O
Cl
OH
Br
O
Br
OH
CF3
O
CF3
OH
Cl
O
5a
OH
Cl 5b
O
Br 6a Br
OH
N
O
N
OH
N
O
N
OH
N
O
N
OH
O
O O
O
O OH
O
Cl
O O
O
Cl
O OH
O CF3
O O
O CF3
O OH
O
O
O
O
O
OH
O
O
O
O
O
OH
14b
100 ± 0 85.7 ± 0.4 (S)
Table 3 Stereoselective reduc-
tion of different substrates by
the purified reductase
a
Not optimized (reaction time of
12 h)
b
Conversion yields and enantio-
meric excesses were determined
by chiral GC analysis. Configura-
tions of products were determined
by comparing the retention time
with that of standard samples
c
Determined by GC analyses after
acetylation of the product
1116 Appl Microbiol Biotechnol (2011) 89:1111–1118

7. aging that YtbE serves as a versatile reductase with a broad
substrate spectrum towards aromatic ketones in addition to
keto esters. Studies on substrate specificity of the enzyme
revealed that the substituents on the phenyl ring exert some
effect on the enzyme activity. It seemed that the electron-
withdrawing substituents enhanced the reduction of the
substrate while the electron-releasing groups (like amino-)
demolished the reductase activity.
Chiral alcohols are important synthons for pharmaceut-
icals, agrochemicals, and fine chemicals, and are thus of
steadily increasing interest. All the aromatic ketones tested
could be reduced by YtbE with high optical purity of
products (>99% ee), although the enantioselectivity was only
moderate for the reduction of ethyl 4-chloroacetoacetate
(62% ee) and ethyl 2-oxo-4-phenylbutyrate (86% ee). Some
of the chiral alcohols produced have been used as valuable
intermediates for synthesis of biologically or pharmaco-
logically active compounds, for example, (R)-2b for
antidepressant fluoxetine, tomoxetine, and nisoxetine
(Ramu et al. 2002); (R)-11b for nutrient (R)-carnitine
(Kitamura et al. 1988); and (R)-12b for antidepressant
befloxatone (Rabasseda et al. 1999). (R)-4b is applicable
to the synthesis of liquid crystals (Fujisawa et al. 1993),
and optically active pyridyl alcohols [e.g., (S)-7b, (S)-8b,
and (S)-9b] are used as pharmaceutical intermediates and
chiral ligands in asymmetric synthesis (Okano et al. 2000).
YtbE generally exhibited Prelog specificity except for the
reduction of ethyl 2-oxo-4-phenylbutyrate. That might be
due to the bulkiness of the substituents, which affected the
enzyme’s enantiopreference. It was interesting to note that
the substituent at the 4-position of ethyl 3-oxo-butyrate
exerted great effect on the enzyme enantioselectivity.
Ethyl 4,4,4-trifluoroacetoacetate (12a) was found to be
reduced to ethyl (R)-4,4,4-trifluoro-3-hydroxybutanoate
[(R)-12b] with excellent enantioselectivity. Several studies
have been reported on the asymmetric reduction of 12a
using Baker’s yeast (Davoli et al. 1999), Bacillus pumilus
Phe-C3 (Zhang et al. 2006), and Saccharomyces uvarum
SW-58 (He et al. 2007). However, the unsatisfactory
enantioselectivity of these bioreactions do limit their
applications for practical production. Therefore, YtbE
was established to be attractive for the production of (R)-
12b. Further studies on asymmetric synthesis of (R)-12b
with an E. coli transformant coexpressing both the ytbE
and the glucose dehydrogenase (GDH) genes are still in
progress.
In conclusion, YtbE exhibited high stereoselectivity
towards various carbonyl substrates of interest, which is
one of the most important factors for determining the
potential value of an enzyme. As far as we know, this is the
first report of ytbE gene-encoding recombinant aldo-keto
reductase from Bacillus sp. serving as a robust biocatalyst
for stereoselective reduction of prochiral carbonyl com-
pounds. The present study will provide a useful guidance
for further application of this enzyme in the biocatalytic
production of chiral synthons. Further improvement of
enzyme functions like activity, thermostability or enantio-
selectivity might be achieved via enzyme engineering
approaches including directed evolution, immobilization,
or a combination of these techniques.
Acknowledgments This work was financially supported by the
National Natural Science Foundation of China (Nos. 20773038 and
20902023), Ministry of Science and Technology, P.R. China (Nos.
2009CB724706 and 2009ZX09501-016), China National Special
Fund for State Key Laboratory of Bioreactor Engineering (No.
2060204), and Shanghai Leading Academic Discipline Project
(No. B505).
References
Bohren KM, Bullock B, Wermuth B, Gabbay KH (1989) The aldo-
keto reductase superfamily. J Biol Chem 264:9547–9551
Davoli P, Forni A, Moretti I, Prati F, Torre G (1999) (R)-(+) and (S)-(−)
ethyl 4, 4, 4-trifluoro-3-hydroxy butanoate by enantioselective
Baker’s yeast reduction. Enzyme Microbial Tech 25:149–152
Ellis EM (2002) Microbial aldo-keto reductases. FEMS Microbiol Lett
216:123–131
Ford G, Ellis EM (2002) Characterization of Ypr1p from Saccharo-
myces cerevisiae as a 2-methylbutyraldehyde reductase. Yeast
19:1087–1096
Fujisawa T, Ichikawa K, Shimizu M (1993) Stereocontrolled synthesis
of p-substituted trifluoromethylbenzylic alcohol derivatives of
high optical purity by the baker’s yeast reduction. Tetrahedron:
Asymmetry 4:1237–1240
Goldberg K, Schroer K, Lütz S, Liese A (2007) Biocatalytic
ketone reduction—a powerful tool for the production of chiral
alcohols—part I: processes with isolated enzymes. Appl
Microbiol Biotechnol 76:237–248
Habrych M, Rodriguez S, Stewart JD (2002) Purification and
identification of an Escherichia coli β-keto ester reductase as 2,
5-diketo-D-gluconate reductase YqhE. Biotechnol Prog 18:257–
261
Hammond JR, Poston WB, Ghiviriga I, Feske DB (2007) Biocatalytic
synthesis towards both antipodes of 3-hydroxy-3-phenylpropanitrile,
a precursor to fluoxetine, atomoxetine and nisoxetine. Tetrahedron
Lett 48:1217–1219
He JY, Mao XD, Sun ZH, Zheng P, Ni Y, Xu Y (2007) Microbial
synthesis of ethyl (R)-4, 4, 4-trifluoro-3-hydroxybutanoate by
asymmetric reduction of ethyl 4, 4, 4-trifluoroacetoacetate in an
aqueous-organic solvent biphasic system. Biotechnol J 2:260–
265
Hyndman D, Bauman DR, Heredia VV, Penning TM (2003) The aldo-
keto reductase superfamily homepage. Chem Biol Interact 143–
144:621–631
Itoh N, Asako H, Banno K, Makino Y, Shinohara M, Dairi T, Wakita
R, Shimizu M (2004) Purification and characterization of
NADPH-dependent aldo-keto reductase specific for β-keto esters
from Penicillium citrinum, and production of methyl (S)-4-
bromo-3-hydroxybutyrate. Appl Microbiol Biotechnol 66:53–62
Jez JM, Penning TM (2001) The aldo-keto reductase (AKR)
superfamily: an update. Chem Biol Interact 130–132:499–525
Jez JM, Flynn TG, Penning TM (1997) A new nomenclature for the
aldo-keto reductase superfamily. Biochem Pharmacol 54:639–
647
Appl Microbiol Biotechnol (2011) 89:1111–1118 1117

8. Kaluzna IA, Matsuda T, Sewell AK, Stewart JD (2004) Systematic
investigation of Saccharomyces cerevisiae enzymes catalyzing
carbonyl reductions. J Am Chem Soc 126:12827–12832
Kaluzna IA, Feske BD, Wittayanan W, Ghiviriga I, Stewart JD (2005)
Stereoselective, biocatalytic reductions of α-chloro-β-keto esters.
J Org Chem 70:342–345
Kataoka M, Kita K, Wada M, Yasohara Y, Hasegawa J, Shimizu S
(2003) Novel bioreduction system for the production of chiral
alcohols. Appl Microbiol Biotechnol 62:437–445
Kilunga KB, Inoue T, Okano Y, Kabututu Z, Martin SK, Lazarus M,
Duszenko M, Sumii Y, Kusakari Y, Matsumura H, Kai Y,
SugiYama S, Inaka K, Inui T, Urade Y (2005) Structural and
mutational analysis of Trypanosoma brucei prostaglandin H2
reductase provides insight into the catalytic mechanism of aldo-
keto reductases. J Biol Chem 280:26371–26382
Kita K, Matsuzaki K, Hashimoto T, Yanase H, Kato N, Chung MCM,
Kataoka M, Shimizu S (1996) Cloning of the aldehyde reductase
gene from a red yeast, Sporobolomyces salmonicolor, and
characterization of the gene and its product. Appl Environ
Microbiol 62:2303–2310
Kitamura M, Ohkuma T, Takaya H, Noyori N (1988) A practical
asymmetric synthesis of carnitine. Tetrahedr Lett 29:1555–1556
Kroutil W, Mang H, Edegger K, Faber K (2004) Recent advances in
the biocatalytic reduction of ketones and oxidation of sec-
alcohols. Curr Opin Chem Biol 8:120–126
Kunst F, Ogasawara N, Moszer I, Albertini AM, Alloni G, Azevedo V,
Bertero MG, Bessieres P, Bolotin A, Borchert S, Borriss R,
Boursier L, Brans A, Braun M, Brignell SC, Bron S, Brouillet S,
Bruschi CV, Danchin A (1997) The complete genome sequence
of the Gram-positive bacterium Bacillus subtilis. Nature
390:249–256
Lei J, Zhou YF, Li LF, Su XD (2009) Structural and biochemical
analyses of YvgN and YtbE from Bacillus subtilis. Protein Sci
18:1792–1800
Matsuda T, Yamanaka R, Nakamura K (2009) Recent progress in
biocatalysis for asymmetric oxidation and reduction. Tetrahe-
dron: Asymmetry 20:513–557
Moore CJ, Pollard JD, Kosjek B, Devine NP (2007) Advances in the
enzymatic reduction of ketones. Acc Chem Res 40:1412–1419
Nakamura K, Yamanaka R, Matsuda T, Harada T (2003) Recent
developments in asymmetric reduction of ketones with biocata-
lysts. Tetrahedron: Asymmetry 14:2659–2681
Okano K, Murata K, Ikariya T (2000) Stereoselective synthesis of
optically active pyridyl alcohols via asymmetric transfer hydro-
genation of pyridyl ketones. Tetrahedron Lett 41:9277–9280
Petrash JM (2007) All in the family: aldose reductase and closely
related aldo-keto reductases. Cell Mol Life Sci 61:737–749
Rabasseda X, Sorbera LA, Castaner J (1999) Befloxatone. Antide-
pressant, MAO-A inhibitor. Drugs Fut 24:1057–1067
Ramu R, Khanna GBR, Kamal A (2002) Chemoenzymatic synthesis
of both enantiomers of fluoxetine, tomoxetine and nisoxetine:
lipase-catalyzed resolution of 3-aryl-3-hydroxy propanenitriles.
Tetrahedron: Asymmetry 13:2039–2051
Schweiger P, Gross H, Deppenmeier U (2010) Characterization of two
aldo-keto reductases from Gluconobacter oxydans 621H capable
of region- and stereoselective α-ketocarbonyl reduction. Appl
Microbiol Biotechnol 87:1415–1426
Woodley JM (2008) New opportunities for biocatalysis: making
pharmaceutical processes greener. Trends Biotechnol 26:321–327
Xie Y, Xu JH, Xu Y (2010) Isolation of a Bacillus strain producing
ketone reductase with high substrate tolerance. Bioresour
Technol 101:1054–1059
Yamada H, Shimizu S, Kataoka M, Sakai H, Miyoshi T (1990) A
novel NADPH-dependent aldehyde reductase, catalyzing asym-
metric reduction of β-keto acid esters, from Sporobolomyces
salmonicolor: purification and characterization. FEMS Microbiol
Lett 70:45–48
Yamamoto H, Matsuyama A, Kobayashi Y (2003) Synthesis of ethyl
(S)-4-chloro-3-hydroxylbutanoate using fabG-homologues. Appl
Microbiol Biotechnol 61:133–139
Zhang J, Witholt B, Li Z (2006) Efficient NADPH recycling in
enantioselective bioreduction of a ketone with permeabilized
cells of a microorganism containing a ketoreductase and a
glucose 6-phosphate. Adv Synth Catal 348:429–433
1118 Appl Microbiol Biotechnol (2011) 89:1111–1118