Facile access to chiral alcohols with pharmaceutical relevance using a ketoreductase newly mined from Pichia guilliermondii

Article abstract of DOI:10.1002/cjoc.201201119

Chiral secondary alcohols with additional functional groups are frequently required as important and valuable synthons for pharmaceuticals, agricultural and other fine chemicals. With the advantages of environmentally benign reaction conditions, broad reaction scope, and high stereoselectivity, biocatalytic reduction of prochiral ketones offers significant potential in the synthesis of optically active alcohols. A CmCR homologous carbonyl reductase from Pichia guilliermondii NRRL Y-324 was successfully overexpressed. Substrate profile characterization revealed its broad substrate specificity, covering aryl ketones, aliphatic ketones and ketoesters. Furthermore, a variety of ketone substrates were asymmetrically reduced by the purified enzyme with an additionally NADPH regeneration system. The reduction system exhibited excellent enantioselectivity (>99% ee) in the reduction of all the aromatic ketones and ketoesters, except for 2-bromoacetophenone (93.5% ee). Semi-preparative reduction of six ketones was achieved with high enantioselectivity (>99% ee) and isolation yields (>80%) within 12 h. This study provides a useful guidance for further application of this enzyme in the asymmetric synthesis of chiral alcohol enantiomers. Copyright

Full text of DOI:10.1002/cjoc.201201119

FULL PAPER
DOI: 10.1002/cjoc.201201119
Facile Access to Chiral Alcohols with Pharmaceutical
Relevance Using a Ketoreductase Newly Mined from
Pichia guilliermondii†
Guochao Xu, Huilei Yu,* and Jianhe Xu*
Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East
China University of Science and Technology, Shanghai 200237, China
Chiral secondary alcohols with additional functional groups are frequently required as important and valuable
synthons for pharmaceuticals, agricultural and other fine chemicals. With the advantages of environmentally benign
reaction conditions, broad reaction scope, and high stereoselectivity, biocatalytic reduction of prochiral ketones of-
fers significant potential in the synthesis of optically active alcohols. A CmCR homologous carbonyl reductase from
Pichia guilliermondii NRRL Y-324 was successfully overexpressed. Substrate profile characterization revealed its
broad substrate specificity, covering aryl ketones, aliphatic ketones and ketoesters. Furthermore, a variety of ketone
substrates were asymmetrically reduced by the purified enzyme with an additionally NADPH regeneration system.
The reduction system exhibited excellent enantioselectivity (＞99% ee) in the reduction of all the aromatic ketones
and ketoesters, except for 2-bromoacetophenone (93.5% ee). Semi-preparative reduction of six ketones was
achieved with high enantioselectivity (＞99% ee) and isolation yields (＞80%) within 12 h. This study provides a
useful guidance for further application of this enzyme in the asymmetric synthesis of chiral alcohol enantiomers.
Keywords carbonyl reductase, asymmetric reduction, chiral alcohols, Pichia guilliermondii, biocatalysis
Introduction
zymes with desired properties, such as high stability,
high enantioselectivity, and wide substrate spectrum.
Various types of biocatalysts with high stereoselectivity
have been screened from nature for the purpose of pre-
paring important chiral synthons, e.g. chiral alcohols,
α-hydroxyl acid, sulfide oxides and epoxides.[9-14] All of
them showed promising potential in organic synthesis.
However, most of the above mentioned biocatalysts
were discovered through traditional screening from en-
vironmental samples and the expression level of active
biocatalysts was very low, disadvantageous for scale-up.
More recently, our interest was focused on identify-
ing biocatalysts through genome data mining and pro-
tein engineering of existing enzymes to fit non-natural
susbstrates and environmental conditions. Based on a
“genome hunting” principle,[15a] a β-ketoacyl-ACP re-
ductase (FabG) from Bacillus sp. ECU0013 was dis-
covered and heterologously overexpressed with the
ability to reduce as much as 620 g•L−1 of ethyl
2-oxo-4-phynylbutyrate (OPBE) into ethyl (S)-2-
hydroxy-4-phenylbutyrate [(S)-HPBE] with excellent
enantiomeric excess (＞99%), an important precursor
for angiotensin-converting enzyme (ACE) inhibitor. By
homology driven “genome mining”,[15b] an NADH-
dependent reductase ScCR from Streptomyces coeli-
The asymmetric reduction of ketones is one of the
most important, fundamental and practical reactions for
producing chiral alcohols,[1] which can be transformed
into various functionalities for many industrial chemi-
cals such as pharmaceuticals, agrochemical and natural
products.[2] To date, a series of routes for the asymmet-
ric transfer hydrogenation (ATH) has been developed,[3]
such as precious metal mediated ATH[4] and ketoreduc-
tase catalyzed ketone reduction.[5] The interest in bio-
catalysts has been increasing recently for several rea-
sons.[6] First of all, biocatalysts have inherently high
chemo-, regio- and enantioselectivities. Secondly, bio-
catalytic reactions may be safer because they do not
require toxic reagents or solvents. Again, they are envi-
ronmentally benign (“green”) because they are natural
catalysts. Fourth, the reaction conditions are mild,
which minimize side products by preventing isomeriza-
tion, racemization, epimerization and rearrangement
reactions.[7] However, even with all these advantages,
there are still challenges in bioprocess, such as the dis-
covery of a robust biocatalyst which could go through
large scale operation and recycling.[8]
We have a standing interest in the discovery of en-
*
E-mail: huileiyu@ecust.edu.cn; jianhexu@ecust.edu.cn; Tel.: (0086)-021-6425 0840; Fax: (0086)-021-6425 0840
Received November 13, 2012; accepted December 27, 2012; published online January 28, 2013.
Dedicated to Professor Guoqiang Lin on the occasion of his 70th birthday.
†
Chin. J. Chem. 2013, 31, 349—354
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349

Xu, Yu & Xu
FULL PAPER
color A3 (2) was identified for the asymmetric reduc-
tion of a broad range of prochiral ketones including aryl
ketones, α- and β-ketoesters, with high activity and ex-
cellent enantioselectivity (＞99% ee). Employing the
same strategy, a homologous protein of Gre2p was ex-
pressed and applied in the preparation of methyl
(R)-o-chloromandelate [(R)-CMM], the key intermedi-
ate for clopidogrel.[15c]
In the current study, a new reductase PgCR (Gen-
bank accession No. EDK41368.1), which is homolo-
gous to CmCR (carbonyl reductase from Candida mag-
noliae, Genbank accession No. BAB21578.1), was
identified from Pichia guilliermondii NRRL Y-324, and
overexpressed in Escherichia coli BL21(DE3). CmCR
was an efficient and NADPH-dependent carbonyl re-
ductase, which was used for asymmetric reduction of
ethyl 4-chloro-3-oxobutanoate (COBE).[16] Many pro-
chiral ketones, e.g. 3-oxoalkanoates, alkyl ketones and
aryl ketones could be reduced by CmCR enantioselec-
tively to anti-Prelog configurated alcohols in excellent
ee.[16] Similar or even better property was expected with
this newly expressed PgCR. The biocatalytic perform-
ance and application potential of this PgCR in the syn-
thesis of chiral secondary alcohols was investigated.
to form pET28-PgCR, which was subsequently trans-
formed into Escherichia coli BL21(DE3) cells. The cul-
tivation, induction, collection, preparation of crude en-
zyme and the purification was done as described.[20]
General method for enzyme assay
Reductase activity was assayed spectrophotometri-
cally at 30 ℃, by monitoring the decrease of the ad-
sorption of NADPH at 340 nm. The standard assay
mixture (1 mL) for reductase consisted of 100 mmol/L
phosphate buffer (pH 6.5), 2.0 mmol/L ketone substrate
and 0.1 mmol/L NADPH. One unit of activity was de-
fined as the amount of enzyme which catalyzes the oxi-
dation of 1 μmol NADPH per minute.
Effect of pH, temperature and organic solvent on
activity
The enzyme activity was estimated using the above
mentioned standard assay protocol with 2-chloroaceto-
phenone 3a as substrate. The optimum pH and tem-
perature were determined as reported.[20] The stability
was examined by incubating the purified enzymes in the
same phosphate buffer (pH 6.5) at 30, 40 or 50 ℃ for a
varied period of time and the residual activities were
assayed as described above. The influence of organic
solvent on enzyme activity was determined by incubat-
ing the enzyme solution of suitable concentration with
10% organic solvents at 30 ℃ for 12 h. The residual
activities were determined using above mentioned gen-
eral method.
Scheme 1 Asymmetric reduction of prochiral ketones employ-
ing PgCR coupled with GDH for cofactor regeneration
O
PgCR
OH
S
L
NADP+
S
L
NADPH
Enantioselectivity of the reductase
"anti-prelog"
The enantioselectivity was determined by examining
the reduction of ketone substrates using an NADPH
regeneration system consisting of the purified reductase
and externally added glucose dehydrogenase (GDH).
The reaction was carried out in a reaction mixture (0.5
mL) comprising 100 mmol/L potassium phosphate
buffer (pH 6.5), 10 mmol/L ketone substrate, 10 U of
the purified reductase to 3a, 10 U of GDH, 20 mmol/L
glucose and 0.1 mmol/L NADP＋ with 900 r/min shak-
ing at 30 ℃ for 12 h. After the reaction, each reaction
mixture was extracted for three times with ethyl acetate.
The substrate conversion and product ee were deter-
mined by GC analysis using a CP-chiralsil-DEX CB
(Varian, USA).
GDH
D-Glucose
D-Gluconic acid
Experimental
All the ketones used in this study were provided
from commercial source.
Gene expression and purification of the carbonyl
reductase from Pichia guilliermondii (PgCR)
P. guilliermondii NRRL Y-324, currently deposited
in and provided by Agricultural Research Service Cul-
ture Collection (USA), was used as the DNA donor. The
strain was cultivated as described previously.[15c] The
genomic DNA of P. guilliermondii was extracted and
purified using the TIANamp Bacteria DNA Kit (Tian-
gen, Shanghai, China). DNA fragment containing PgCR
was amplified by polymerase chain reaction using
primers based on the PgCR sequence (Genebank acces-
sion No. AB036927) (Forward: 5'-CCGGAATTCAT-
Preparative procedures
The preparative synthesis was carried out as follows:
D-Glucose (0.6 g, 3.0 mmol), D-glucose dehydrogenase
(400 U), NADP＋ (0.7 mg, 1.0 μmol) and crude extract
of PgCR (400 U) were mixed in a potassium phosphate
buffer (9.0 mL, 100 mmol/L, pH 6.5). To the mixture
was added a ketone solution (2.0 mmol in 1.0 mL of
DMSO). The mixture was stirred at room temperature
and the pH was controlled at 6.5 with addition of 1.0
mol/L NaOH solution until conversion was complete.
The mixture was centrifuged (10000×g for 5 min) to
GAAGTCTATGATCAATGA-3'
and
Reverse:
5'-CCGCTCGAGTTATGGCGCACAGTAGCCGCCAT-
3'). The amplified DNA was directly cloned into the TA
cloning site of pMD-18T and the desired gene was sub-
cloned into expression plasmid pET28a(＋) between the
EcoRI and XhoI restriction sites using standard methods
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Facile Access to Chiral Alcohols with Pharmaceutical Relevance Using a Ketoreductase
promote the phase separation; the aqueous phase was
saturated with NaCl and then extracted for three times
with ethyl acetate. The organic solution was combined,
dried over anhydrous Na2SO4 and evaporated under
vacuum.
the soluble fraction and was purified through affinity
chromatography. The specific activity of purified PgCR
to COBE was 22.9 U•mg−1 corresponding to a 1.4-fold
improvement in activity compared to the crude extract.
Protein separation by SDS-PAGE resulted in a single
band corresponding to a molecular mass of 30 kDa, in
agreement with its theoretical value. Effect of pH and
temperature on the activity of the purified PgCR re-
vealed that the optimum pH and temperature were 6.5
and 25 ℃ respectively. The half-lives of PgCR meas-
ured at 30 and 40 ℃ were 147 and 96 h respectively.
Most of prochiral ketones are hydrophobic, having
low solubility in aqueous solution.[19] Employment of a
cosolvent might improve the solubility and mass trans-
fer of substrates. Effect of cosolvents on the activity of
purified PgCR was performed using 10% of ethanol,
2-propanol, dimethyl sulfoxide (DMSO), dimethylfor-
mamide (DMF), acetone or tetrahydrofuran (THF). Af-
ter incubation for 12 h, 69% residual activity was re-
mained with DMSO. Acetone and THF was harmful for
enzyme activity and the residual activity was less than
50%. Hereafter, 10% DMSO was used as the cosolvent.
Oxidoreductases have been used for producing opti-
cally active alcohols from various prochiral ketones and
ketoesters. The substrate spectrum of purified PgCR
was investigated. The enzyme showed no activity with
NADH and full activity with NADPH. To investigate
the substrate spectrum, conversion and enantioselectiv-
ity of the purified PgCR were assayed in the reduction
of various ketoesters, aryl and aliphatic ketones.
From Table 1, it can be seen that the carbonyl reduc-
tase from P. guilliermondii efficiently catalyzed reduc-
tion of the tested aryl and aliphatic ketones with high
enantioselectivities, except for α-bromoacetophone 4a
with only 93.5% ee. Substitution at the alpha position of
carbonyl group was good for the reduction, which was
the same as various other carbonyl reductases. The re-
ductase showed the strongest activity on 2-hydroxy-
acetophenone (5a) among the substituted acetophenones,
with specific activity of 11 U•mg−1. 2,2,2-Trifluroaceto-
phenone (2a) and 2-chloroacetophenone (3a) could also
be efficiently reduced with 4.6 and 2.2 U•mg−1 respec-
tively. Substitution on the para position of phenyl ring
could promote the reduction ability of PgCR, except for
4'-chloroacetophenone (9a). In the case of multi-sub-
stitutions on phenyl ring, PgCR also showed high re-
duction activity. To our delight, the aliphatic ketones
were also good substrates for this carbonyl reductase,
and chain length exerted some effect on the enzyme
activity as shown in Table 1. The specific activity of
PgCR towards various aliphatic ketones was decreased
along with the increase of the chain length (C5＞C6＞C8
＞C9). More importantly, the aliphatic ketones were
reduced into the corresponding alcohols with high enan-
tioselectivities, which was difficult to achieve in metal-
catalyzed hydrogen transfer or hydrogenation.[22] Addi-
tionally, ketoesters could be efficiently asymmetrically
reduced with high activity and enantioselectivity. High-
The reduction products were also characterized by
1H NMR.
(S)-2-Chloro-1-phenylethan-1-ol [(S)-3b] Table
2, Entry 1, 87% yield. [α]2D5 ＋52.8° (c 1.00, CHCl3)
1
{lit.,[18a] [α]D25 ＋53.8° (c 1.00, CHCl3)}; H NMR (500
MHz, CDCl3) δ: 2.62 (s, 1H, OH), 3.66－3.69 (dd, J＝
11.2, 8.8 Hz, 1H, CH2Cl), 3.76－3.79 (dd, J＝11.2, 3.4
Hz, 1H, CH2Cl), 4.92－4.94 (dd, J＝8.8, 3.4 Hz, 1H,
CHOH), 7.33－7.44 (m, 5H, PhH).
(R)-3-Chloro-1-phenylpropan-1-ol [(R)-6b] Ta-
ble 2, Entry 2, 85% yield. [α]2D5 ＋22.9° (c 1.00, CHCl3)
1
{lit.,[18b,18c,18d] [α]D25 ＋25.3° (c 7.05, CHCl3)}; H NMR
(500 MHz, CDCl3) δ: 1.99 (s, 1H, OH), 2.11－2.16 (dd,
J＝13.6, 5.4 Hz, 1H, CH2), 2.22－2.31 (td, J＝14.3, 5.7
Hz, 1H, CH2), 3.56－3.62 (dd, J＝11.0, 5.8 Hz, 1H,
CH2), 3.74－3.79 (m, 1H, CH2), 4.96－4.99 (dd, J＝8.5,
4.7 Hz, 1H, CH2OH), 7.30－7.52 (m, 5H, Ph-5H).
(R)-1-(3,5-Bis(trifluoromethyl)phenyl)ethan-1-ol
[(R)-13b] Table 2, Entry 3, 80% yield. [α]2D5 ＋13.0° (c
1.00, CHCl3) {lit.,[18e,18f] [α]D25 ＋15.0° (c 1.40, CHCl3)};
1H NMR (500 MHz, CDCl3) δ: 1.54 (d, J＝6.5 Hz, 3H,
CH3), 2.67 (s, 1H, OH), 5.03 (q, J＝6.5 Hz, 1H, CHOH),
7.82 (d, J＝16.5 Hz, 5H, PhH).
(S)-2-Chloro-1-(3,4-dichlorophenyl)ethan-1-ol
[(S)-15b] Table 2, Entry 4, 85% yield. [α]2D5 ＋33.5° (c
1.00, CHCl3) {lit.,[18a] [α]D25 ＋34.2° (c 1.00, CHCl3)}; 1H
NMR (500 MHz, CDCl3) δ: 2.64 (s, 1H, OH), 3.62 (dd,
J＝11.3, 8.5 Hz, 1H, CH2Cl), 3.75 (dd, J＝11.3, 3.5 Hz,
1H, CH2Cl), 4.90 (dd, J＝8.4, 3.4 Hz, 1H, CHOH),
7.12－7.40 (m, 1H, PhH), 7.40－7.68 (m, 2H, PhH).
Ethyl (S)-3-hydroxy-4,4,4-trifluorobutanoate [(S)-
21b] Table 2, Entry 5, 86% yield. [α]2D5 −19.8° (c 1.00,
1
CHCl3) {lit.,[18g,18h] [α]2D5 −20.9° (c 0.70, CHCl3)}; H
NMR (500 MHz, CDCl3) δ: 1.30 (t, J＝7.1 Hz, 3H,
CH3), 2.61－2.80 (m, 2H, CH2), 3.53－3.71 (m, 1H,
OH), 4.23 (q, J＝7.1 Hz, 2H, CH2), 4.40－4.53 (m, 1H,
CHOH).
Ethyl (S)-3-hydroxy-4-chlorobutanoate [(S)-22b]
Table 2, Entry 6, 92% yield. [α]2D5 −23.0° (c 1.00, CHCl3)
1
{lit.,[18i,18j] [α]D20 −20.5° (c 1.00, CHCl3)}; H NMR (500
MHz, CDCl3) δ: 1.27 (t, J＝7.1 Hz, 3H, CH3), 2.53－
2.69 (m, 2H, CH2), 3.34 (s, 1H, OH), 3.60 (dd, J＝5.4,
1.7 Hz, 2H, CH2Cl), 4.17 (q, J＝7.1 Hz, 2H, CH2),
4.21－4.32 (m, 1H, CHOH).
Results and Discussion
PgCR was successfully expressed in E. coli cells
with genomic DNA of P. guilliermondii NRRL Y-324
as template employing standard cloning techniques. A
BLASTp search revealed that the protein showed 64%
amino acid identity to CmCR from C. magnolia.
His-tagged recombinant PgCR was mainly present in
Chin. J. Chem. 2013, 31, 349—354
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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351

Xu, Yu & Xu
FULL PAPER
est specific activity was 23 U•mg−1 using ethyl
4-chloro-3-oxobutanoate (COBE, 22a) as substrate,
which was much higher than the activity of CmCR to
COBE (6.6 U•mg−1).[16d] All the tested β-ketoesters were
completely reduced (100% conversion) into optically
pure alcohols (＞99% ee) at 10 mmol/L. In summary,
this newly mined PgCR showed the same high enanti-
oselectivity as CmCR, but relatively higher specific ac-
tivity.
To investigate the application potential of this newly
mined carbonyl reductase in the synthesis of pharma-
ceutical building blocks, several prochiral ketones were
chosen to be reduced. Firstly, a 10 mL reaction system,
containing 2.0 mmol 3a, 1.0 μmol NADP＋, 3.0 mmol
glucose and 400 U PgCR and GDH, was carried out
with the addition of NaHCO3 to maintain pH. The con-
version and ee value were dropped to 78% and 60%
respectively (data not shown). Fortunately, when a
pH-state mode was adopted with titration of 1 mol/L
NaOH automatically, a high conversion and ee value
were also obtained as Table 2 Entry 1. This indicated
that too low pH value may be deleterious to the enzyme
stability. Although the NaHCO3 was added to try to
maintain the pH of reaction system at pH 6.5, the pH
was greatly changed due to the delay in adjusting the pH
value, which had bad impact on the catalytic perform-
ance of PgCR. As shown in Table 2 chiral alcohols were
obtained in essentially optically pure form with high
isolation yields (＞80%) in no more than 12 h using
PgCR coupled with GDH for cofactor regeneration. All
these chiral alcohols are important intermediates in the
synthesis of pharmaceuticals and agrochemicals.[23]
Conclusions
In this work, a carbonyl reductase (PgCR) from P.
guilliermondii NRRL Y-324 was overexpressed and
purified to homogeneity. The enzymatic properties were
characterized, especially with respect to its specificity,
Table 1 Enantioselective reduction of aryl and aliphatic ketones with PgCR
O
OH
R2
1b − 19b
O
O
O
OH
R2
R1
PgCR
R1
PgCR
R2
R1
R1
O
R2
O
20b − 23b
20a − 23a
1a − 19a
Substrate (1a－23a)
Entry
Specific activitya
Conv.b/%
eeb/%
R1
R2
1
C6H5
C6H5
C6H5
C6H5
C6H5
C6H5
CH3
CF3
0.23
4.6
99.3
100
92.1
100
100
75.0
＞99 (R)
＞99 (S)
＞99 (S)
93.5 (S)
2
3
CH2Cl
CH2Br
CH2OH
2.2
4
0.80
11
5
＞99 (S)
＞99 (R)
＞99 (R)
＞99 (R)
＞99 (R)
＞99 (R)
＞99 (R)
＞99 (R)
＞99 (R)
＞99 (S)
＞99 (S)
＞99 (R)
＞99 (R)
＞99 (R)
＞99 (R)
＞99 (R)
＞99 (S)
＞99 (S)c
＞99 (S)c
6
CH2CH2Cl
CH3
0.69
0.53
0.19
0.11
0.61
1.2
7
Cyclohexyl
Naphthyl
99.7
71.3
90.5
99.4
8
CH3
9
4'-Cl-C6H4
4'-Br-C6H4
4'-CH3-C6H4
4'-OH-C6H4
3',4'-2CF3-C6H3
4'-Cl-C6H4
3',4'-2ClC6H3
CH3(CH2)2
CH3(CH2)3
CH3(CH2)5
CH3(CH2)6
CH2CH3
CH3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
CH3
CH3
100
CH3
0.69
0.73
4.4
87.4
24.5
CH3
CH2Cl
CH2Cl
CH3
100
0.63
1.7
100
100
CH3
0.61
0.31
0.23
0.76
0.34
23
99.5
CH3
73.1
99.2
CH3
CH3
100
CH2CH3
CF3
100
100
100
CH2CH3
CH2Cl
CH2Br
CH2CH3
15
a
The unit of specific activity was µmol•min−1•mg−1. b Conversion and ee value was measured by chiral GC analysis, the absolute con-
figuration was determined by comparing the retention time with that of standard samples.[15c,20] c Determined by GC analysis after acety-
lation of the product.[21]
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Facile Access to Chiral Alcohols with Pharmaceutical Relevance Using a Ketoreductase
Table 2 Preparation of pharmaceutical building blocks using PgCR coupled with glucose dehydrogenase
Substrate
Product
Time/hYield/% ee/%
Pharmaceutical target
Trade name & application
OH
H
N
OH
O
Sotalol, non-selective competitive
3
6
87 ＞99 (S)
3a
Cl
S
N
β-adrenergic receptor blocker
H
O
CF3
OH
Prozac, selective serotonin reuptake
inhibitor, anti-depressant
O
85 ＞99 (R)
6a
Cl
N
F
CF3
OH
Emend, Human neurokinin-1 (NK1)
antagonist
F3C
12
80 ＞99 (R)
13a
O
N
N
F3C
NH
O
HN
O
CF3
OH
Zoloft, selective serotonin reuptake
inhibitor, anti-depressant
Cl
6
6
85 ＞99 (S)
15a
21a
Cl
Cl
NHCH3
Cl
Cl
O
O
O
Befloxatone, selective monoamine
oxidase inhibitor
OH
O
N
86 ＞99 (S)
OH
F3C
OEt
F3C
O
O
OH OH
O
N
H
N
OH
Lipitor, HMG-CoA reductase inhibitor
for lowing blood cholesterol
OH
O
3
92 ＞99 (S)
22a
Cl
OEt
F
stereospecificity and ability in the preparation of opti-
cally active alcohols. Highest activity was found at pH
6.5 and 25 ℃. Substrate spectrum investigation re-
vealed that it could serve as a versatile reductase with a
broad substrate profile, including a variety of aryl and
aliphatic ketones, and keto-esters. Compared to CmCR,
much higher specific activity was found with PgCR.
Synthesis of chiral alcohols at semi-preparative scale
revealed its high catalytic efficiency and enantioselec-
tivity. This study provides systematic knowledge and
useful guidance of PgCR in biocatalytic production of
chiral alcohols. Further work will focus on the immobi-
lization and the reuse ability of this carbonyl reductase.
2011CB710800), China National Special Fund for State
Key Laboratory of Bioreactor Engineering (No.
2060204).
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Acknowledgement
This work was financially supported by the National
Natural Science Foundation of China (No 21276082),
Ministry of Science and Technology, P. R. China (No.
Chin. J. Chem. 2013, 31, 349—354
© 2013 SIOC, CAS, Shanghai, & WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.cjc.wiley-vch.de
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354
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