Genomic mining-based identification of novel stereospecific aldo-keto reductases toolbox from Candida parapsilosis for highly enantioselective reduction of carbonyl compounds

Article abstract of DOI:10.1016/j.molcatb.2014.04.003

Biocatalytic reduction of prochiral ketones offers significant potential in synthesis of optically active alcohols. However, so far the application of aldo-keto reductases (AKRs) in asymmetric reduction has been hampered due to limited availability of AKRs with high enantioselectivity and catalytic efficiency. Based on the genome sequence of Candida parapsilosis, a versatile bioresource for asymmetric reduction, eight open reading frames encoding putative AKRs were discovered and expressed, and the resulted enzymes (CPARs), comprising an AKR toolbox, were evaluated toward various carbonyl substrates. The CPARs were active to the selected substrates, especially 2-hydroxyacetophenone and ethyl 4-chloro-3-oxobutyrate. Additionally, most of them were obviously enantioselective to the substrates and gave alcohol products with optical purity up to 99%e.e. Of the enzymes, CPAR4 was outstanding with excellent enantioselectivity and broad substrate spectrum. All these positive features demonstrate that genomic mining is powerful in searching for novel and efficient biocatalysts of desired reactions for pharmaceuticals and fine chemicals synthesis.

Full text of DOI:10.1016/j.molcatb.2014.04.003

Journal of Molecular Catalysis B: Enzymatic 105 (2014) 66–73
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Genomic mining-based identification of novel stereospecific
aldo-keto reductases toolbox from Candida parapsilosis for highly
enantioselective reduction of carbonyl compounds
Rongyun Guoa, Yao Niea,∗, Xiao Qing Mua, Yan Xua,b, Rong Xiaoc
a School of Biotechnology and Key laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
b State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
c Center for Advanced Biotechnology and Medicine, Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Biocatalytic reduction of prochiral ketones offers significant potential in synthesis of optically active
alcohols. However, so far the application of aldo-keto reductases (AKRs) in asymmetric reduction has been
hampered due to limited availability of AKRs with high enantioselectivity and catalytic efficiency. Based
on the genome sequence of Candida parapsilosis, a versatile bioresource for asymmetric reduction, eight
open reading frames encoding putative AKRs were discovered and expressed, and the resulted enzymes
(CPARs), comprising an AKR toolbox, were evaluated toward various carbonyl substrates. The CPARs were
active to the selected substrates, especially 2-hydroxyacetophenone and ethyl 4-chloro-3-oxobutyrate.
Additionally, most of them were obviously enantioselective to the substrates and gave alcohol products
with optical purity up to 99%e.e. Of the enzymes, CPAR4 was outstanding with excellent enantioselectivity
and broad substrate spectrum. All these positive features demonstrate that genomic mining is powerful in
searching for novel and efficient biocatalysts of desired reactions for pharmaceuticals and fine chemicals
synthesis.
Received 6 January 2014
Received in revised form 18 March 2014
Accepted 5 April 2014
Available online 16 April 2014
Keywords:
Aldo-keto reductase
Genomic mining
Toolbox
Enantioselectivity
Asymmetric reduction
© 2014 Elsevier B.V. All rights reserved.
1. Introduction
protozoa, fungi, eubacteria, and archaebacteria [8]. Several micro-
bial enzymes have already been cloned and used for the asymmetric
Optically active alcohols are useful chiral intermediates for the
synthesis of pharmaceuticals, agricultural chemicals, and specialty
materials [1,2]. Compared with the conventional chemical pro-
cess, biocatalytic asymmetric reduction is one of the efficient ways
due to its high chemo-, enantio-, and regioselectivities [3–5]. For
the production of chiral alcohols from the corresponding prochiral
aldehydes and ketones, the aldo-keto reductases (AKRs) includ-
ing aldehyde reductase (EC 1.1.1.21) and carbonyl reductase (EC
1.1.1.184) have the inherent advantages over other enzyme sys-
tems in terms of their effectiveness, pertinence, and diversity in
catalyzing reduction [6,7].
synthesis of chiral alcohols, such as ARI from Sporobolomyces
salmonicolor [9], Conjugated polyketone reductase (CPR-C1 and
CPR-C2) from Candida parapsilosis [10], and KER from Penicillium
citrinum [11]. However, so far the resources of functional enzymes
are yet not sufficient and the application of AKRs in the reduction
of aldehydes or ketones has been hampered due to the limited
availability [12]. Therefore, discovery and identification of AKRs
with the application potential should be critical for bio-mediated
asymmetric synthesis.
With the rapid development of genomics, proteomics, and
bioinformatics, the candidates of ideal biocatalysts could be discov-
ered and characterized significantly [13,14]. One typical example
is that 18 key reductases from bakers’ yeast were overexpressed
and tested for their abilities of reducing ␣- or ␤-ketoesters after
analysis of the yeast genome [15]. Within this area, the genus
Candida spp. has been taken as an important source of oxidore-
ductases for biocatalytic redox reactions including enantioselective
keto-reductions [16]. Of them, C. parapsilosis has been described
as a highly efficient biocatalyst for kinds of asymmetric reduc-
tions [17–19], involving stereospecific alcohol dehydrogenases and
The NAD(P)H-dependent AKRs with broad physiological
roles have been found in vertebrates, invertebrates, plants,
Abbreviations: AKR, aldo-keto reductase; ORF, open reading frame; IPTG,
isopropyl-␤-d-thiogalactopyranoside; TCEP, Tris (2-carboxyethyl) phosphine; SDS-
PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
∗
Corresponding author. Tel.: +86 510 85918201; fax: +86 510 85918201.
E-mail addresses: ynie@jiangnan.edu.cn (Y. Nie), yxu@jiangnan.edu.cn (Y. Xu).
http://dx.doi.org/10.1016/j.molcatb.2014.04.003
1381-1177/© 2014 Elsevier B.V. All rights reserved.

R. Guo et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 66–73
67
Table 1
carbonyl reductases [20,21]. Besides the functional conjugated
polyketone reductase [22], however, the available AKRs are yet
limited for practical application and it would be necessary to dis-
cover and identify new enzymes systematically from the promising
functional microorganism of C. parapsilosis [23].
Oligonucleotide primers for amplification of the genes encoding CPARs from C. para-
psilosis genome, involving restriction sites of NdeI and XhoI underlined.
Gene
Primer sequence
cpar1
F: 5ꢀ-CCCGCCCGCATATGACTCCACAACCAATTGAG-3ꢀ
R: 5ꢀ-GCCCGCTCGAGCTGAAACAATGAGCCTTCACTTTG-3ꢀ
F: 5ꢀ-CCCGCCCGCATATGTCTACTACAATCAAGAAAGCC-3ꢀ
R: 5ꢀ-GCCCGCTCGAGATCAAATTCTTTACTCAAAGTGTCTC-3ꢀ
F: 5ꢀ-CCCGCCCGCATATGACCCATCAGCGCAGTCTTC-3ꢀ
R: 5ꢀ-GCCCGCTCGAGCTCATCCTTATACAATGTTGGATC-3ꢀ
F: 5ꢀ-CCCGCCCGCATATGTCAGCTCAATTGAAAGTAAAC-3ꢀ
R: 5ꢀ-GCCCGCTCGAGGTCATTGAAGTTGTTGAAGCCTG-3ꢀ
F: 5ꢀ-CCCGCCCGCATATGTCATATAGACTAATTCAATTAAAC-3ꢀ
R: 5ꢀ-GCCCGCTCGAGTGGGGCATTGGTACATTCCC-3ꢀ
F: 5ꢀ-CCCGCCCGCATATGAGCTCTCCTTTACCCTCAC-3ꢀ
R: 5ꢀ-GCCCGCTCGAGCAGGTGACCGCTTGGCCAC-3ꢀ
F: 5ꢀ-CCCGCCCGCATATGACTCAAAGTAACTTACTACC-3ꢀ
R: 5ꢀ-GCCCGCTCGAGCAAATCTTTAAATTGCTCATGGAAG-3ꢀ
F: 5ꢀ-CCCGCCCGCATATGTCAATTGAGCTCAAGTACAAT-3ꢀ
R: 5ꢀ-GCCCGCTCGAGAGAGAGGGACTGTTTGGGTTTA-3ꢀ
The disclosure of microbial genome sequence allows scientists
to search for novel enzymes with potential applications [24,25].
The genome sequence of C. parapsilosis (http://www.sanger.ac.uk/
sequencing/Candida/parapsilosis/) provides us a research avenue
to dig biocatalytic resource of new enzymes from the microor-
ganism by genomic mining [26]. In this report, by analyzing the
genome sequence of C. parapsilosis, a biocatalytic toolbox was dis-
covered, comprising eight open reading frames (ORFs) encoding
putative AKRs. After expression of these ORFs, the encoded proteins
were purified and functionalized as stereospecific AKRs catalyzing
enantioselective reduction toward various carbonyl compounds
including aryl ketones, aliphatic ketones, and ketoesters. This
study would represent a systematic investigation on C. parapsilo-
sis enzymes catalyzing stereoselective carbonyl reductions, which
would serve as a useful guideline for future development of new
AKRs and also the enzymatic processes for synthesis of optically
pure alcohols.
cpar2
cpar3
cpar4
cpar5
cpar6
cpar7
cpar8
The recombinant cells were cultivated in 4 mL LB liquid
medium containing 100 ␮g mL−1 ampicillin at 37 ◦C and 200 rpm
for 12 h. Then the culture was inoculated into a 250-ml Erlen-
meyer flask containing 50 ml fresh LB medium supplemented with
100 ␮g mL−1 ampicillin. When the culture turbidity (OD600nm
)
2. Materials and methods
increased to the level between 0.6 and 0.8, the expressions of target
recombinant proteins were initiated with the optimization of the
following conditions, where isopropyl-␤-d-thiogalactopyranoside
(IPTG) (0.1, 0.5, and 1.0 mM) or lactose (2%, 4%, and 6%) was added
as inducer and the culture was incubated under different tempera-
tures (17, 20, 25, 30, and 35 ◦C) at 200 rpm for additional 12 h. The
yield of target protein was evaluated by calculating the amount of
purified protein obtained from the corresponding culture broth.
2.1. Materials
C. parapsilosis CCTCC M203011 was obtained from the China
Center for Type Culture Collection (CCTCC, Wuhan, China).
Escherichia coli strain XL-10 Gold was used for gene cloning. E. coli
BL21 (DE3) was used for gene expression. The plasmid pET21c
was obtained from Novagen (USA) served as expression vector.
All enzymes used for DNA manipulations were obtained from
TaKaRa Biotechnology Co., Ltd (Dalian, China). The cofactors includ-
ing NAD(P)H and NAD(P)+, the substrates including aryl ketones
(acetophenone, 2-hydroxyacetophenone, o-chloroacetophenone
m-chloroacetophenone, and p-chloroacetophenone), aliphatic
ketones (2-octanone and 2-hexanone), and ketoesters (ethyl
4-trifluoro-3-oxobutyrate, methyl 3-oxobutyrate, ethyl 3-phenyl-
3-oxopropionate, and ethyl 4-chloro-3-oxobutyrate), and the
standard samples of chiral alcohol products corresponding to the
above carbonyl substrates were purchased from the Sigma–Aldrich
Chemical Co. (USA). All other used chemicals were of analytical
grade and commercially available.
2.4. Purification of recombinant enzymes
The cells were suspended in binding buffer (20 mM Tris-HCl,
pH6.5, 0.3 M NaCl, 40 mM imidazole, 1× protease inhibitors, 1 mM
Tris (2-carboxyethyl) phosphine (TCEP)) and disrupted on ice with
an ultrasonic oscillator (VCX750, Sonic). The supernatant of the cell
lysate was collected by centrifugation at 26,000 × g for 40 min at
4 ◦C and purified by an AKTAxpress system using HisTrap HP affinity
column (GE Healthcare, USA). Elution was carried out with 300 mM
imidazole in the same buffer at a flow rate of 2.0 mL min−1. Then
the purified fractions were exchanged into low salt buffer (10 mM
Tris–HCl, pH 6.5, 0.1 M NaCl, 0.02% NaN3, 5 mM d,l-dithiothreitol)
using disposable PD-10 desalting columns (GE Healthcare, USA)
[29]. The final recombinant enzymes were purified to homogene-
ity as determined by sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) with 12% polyacrylamide gels [29]. The
amount of purified protein obtained from the corresponding cul-
ture broth was used to evaluate the yield of target protein. These
final preparations of purified enzymes were used in all of the exper-
iments in this study.
2.2. Search for potential genes and sequence analysis
Discovery of potential AKRs was carried out by NCBI
server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) and BLAST against
the complete C. parapsilosis genome (http://www.sanger.ac.uk/
sequencing/Candida/parapsilosis/) [26]. Multiple sequence align-
ment was performed using Clustal
phylogenetic trees were deduced from the alignment using the
neighbor-joining method of MEGA [28].
X software [27]. The
2.5. Enzyme activity assay
2.3. Cloning and expression of genes encoding AKRs
The enzyme activity of AKR was measured by a contin-
uous spectrophotometric assay using the standard assay
mixture containing 0.1 M potassium phosphate buffer (pH
6.5), 0.5 mM NAD(P)H, 5 mM substrate, and the appro-
The genes encoding the eight AKRs were amplified from C. para-
psilosis genome using appropriate primer pairs with the restriction
sites of NdeI and XhoI (Table 1). The purified fragments were
digested with these restricted enzymes and ligated into pET21c
expression vector. Then the verified recombinant plasmids were
transformed into the E. coli BL21 (DE3) competent cells. The trans-
formants were grown in Luria–Bertani (LB) medium (tryptone
10 g L−1, yeast extract 5 g L−1, NaCl, 5 g L−1) containing 100 ␮g mL−1
ampicillin at 37 ◦C.
priate enzyme in
substrates of carbonyl compound included aryl ketones (ace-
tophenone, 2-hydroxyacetophenone, o-chloroacetophenone
a total volume of 100 ␮L. The involved
m-chloroacetophenone, and p-chloroacetophenone), aliphatic
ketones (2-octanone and 2-hexanone), and ketoesters (ethyl
4-trifluoro-3-oxobutyrate,
methyl
3-oxobutyrate,
ethyl

68
R. Guo et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 66–73
3-phenyl-3-oxopropionate, and ethyl 4-chloro-3-oxobutyrate).
One unit of enzyme activity was defined as the amount of enzyme
required to catalyze the oxidation of 1 ␮mol NAD(P)H per minute
under the given assay conditions. The decrease in the amount of
the coenzyme was measured spectrophotometrically at 340 nm
(extinction coefficient [e] = 6.22 mM−1 cm−1). Protein concen-
tration was determined using Bradford reagents (Bio-Rad) with
bovine serum albumin as a standard. All the values of enzymatic
activities were averaged from three replicates and significant
differences (p < 0.05) were measured.
code for putative AKRs (Fig. 1). From the amino acid sequence
and secondary structure prediction, the putative enzymes exhibit
a classic (␣/␤)8 structure containing the NADP-binding motif and
the catalytic tetrad DxxxxY, K, and H. Two AKR family signature
sequences (PROSITE accession numbers PS00798 and PS00062) and
an AKR family putative active site signature sequence (PROSITE
accession number PS00063) could be observed in all the sequences
of putative enzymes (Fig. 1). For AKRs, the IPKS motif, comprised
of the first four amino acid residues in the AKR family putative
active site signature sequence, has been reported to play an impor-
tant role in cofactor binding [22]. While in CPARs, some residues
were observed to be varied in the IPKS motif and also the AKR
family putative active site signature sequence described as the
pattern of [ILVM]-[PAIV]-[KR]-[ST]-{EPQG}-{RFI}-x(2)-R -{SVAF}-
x-[GSTAEQK]-[NSL]-x-{LVRI}-[LIVMFA], indicating that the CPARs
would be different in catalytic properties except for the common
nature of reducing aldehyde and ketone [31]. Therefore, the puta-
tive CPARs encoded by the newly discovered ORFs in C. parapsilosis
genome sequence would be members of the AKR family, although
perform distinct characteristics in catalyzing keto reductions due
to the diversity of functional fragment in the primary structure.
Additionally, the enantioselective oxidoreductases belonging to
different superfamilies in classification were identified from the
same functional microorganism of C. parapsilosis, of which the
previously reported carbonyl reductases, SCRs, share sequence
motif characteristic of the short-chain dehydrogenase/reductase
(SDR) superfamily with the highly conserved regions including the
cofactor binding motif Gly-x-x-x-Gly-x-Gly and the catalytic triad
of Ser-Tyr-Lys [21]. Thus the AKRs discovered in this study would
exhibit distinct characteristics from the reported SCRs in sequence
homology, substrate specificity, and enantioselectivity.
To understand the evolution relationship and classification of
CPARs, a phylogenetic tree was constructed to describe the homol-
ogous relevance of CPARs and other reported AKRs. As shown in
Fig. 2, the putative proteins of CPAR2, CPAR3, CPAR4, CPAR5, and
CPAR7 would be the members of the AKR3 subfamily, where CPAR3
and CPAR5 would be predicted to have a closer relationship. On
the other hand, CPAR1, CPAR6, and CPAR8 were supposed to phy-
logenically belong to the subfamilies of AKR8, AKR7, and AKR10,
respectively. From the map of the phylogenetic tree, additionally,
it could be observed that the subfamilies from AKR1 to AKR5 clus-
ter as one major branch with close evolution relationship, while the
rest of the subfamilies comprise another phylogenetic branch. Since
the evolutionary position may contribute to the difference of enzy-
matic characteristics, the CPARs would be diverse in physiological
property and catalytic function.
2.6. Kinetic parameters analysis
Kinetic parameters of the purified enzyme were assayed by mea-
suring initial velocity at various concentrations of substrates and
cofactors [30]. To determine the apparent Km value, the concentra-
tion of carbonyl compounds was varied from 0.5 to 4 mM with a
fixed concentration of NADPH at 0.05 mM, 0.1 mM, and 0.25 mM,
respectively. Apparent kinetic parameters were further calculated
from double reciprocal Lineweaver–Burk plots. All the data were
averaged from three replicates for each substrate and cofactor con-
centration and significant differences (p < 0.05) were measured.
2.7. Asymmetric reduction of carbonyl compounds
Asymmetric reductions of various carbonyl compounds by the
purified enzymes were carried out at 30 ◦C for 8 h with mild shak-
ing in a reaction mixture containing 0.1 M potassium phosphate
buffer (pH 6.5), 1 g L−1 substrate, 10 mM NADPH, and the purified
enzyme of appropriate amount in a total volume of 2 mL. In order
to determine the absolute configuration of chiral alcohols, the reac-
tion products were extracted with ethyl acetate or hexane and the
organic layer was used for analysis. The optical purity of the reac-
tion products were determined by chiral HPLC (HP 1100, Agilent,
USA) equipped with Chiralcel OB-H column (4.6 mm × 250 mm;
Daicel Chemical Ind. Ltd., Japan) or chiral GC (7890A, Agilent, USA)
equipped with FID detector and Chrompack Chirasil-Dex CB chiral
capillary column (25 m × 0.25 mm; Varian, USA) [21].
2.8. Nucleotide sequence accession number
The nucleotide sequences for the stereospecific AKR genes have
been deposited in the GenBank database with accession numbers
JX512911, JX512912, JX512913, JX512915, JX512916, JX512917,
JX512918, and JX512919, respectively.
3. Results and discussion
3.1. Identification of putative AKR-encoding genes
3.2. Expression and purification of recombinant CPARs
With the amino acid sequence of conjugated polyketone reduc-
tase CPR-C1 as the template [22], genomic mining based on
sequence similarity was carried out against the genome sequence
of C. parapsilosis. Then eight homologous ORFs, named here as cpar,
were revealed, which comprise around 1000 base pairs in length
and uninterruptedly encode the putative proteins of CPARs with
different theoretical molecular mass, respectively (Table 2), where
the entire gene sequences can be translated into the correspond-
ing amino acid sequences without non-coding regions and thus no
intron was found in the encoding sequences of the eight homol-
ogous genes. Of them, CPAR2, CPAR3, CPAR4, CPAR5, and CPAR7
have a higher sequence identity ranging from 25% to 42% to the con-
jugated polyketone reductase C1, while somewhat lower identity
around 15% was found between C1 and other sequences includ-
ing CPAR1, CPAR6, and CPAR8. The multiple sequence alignment
of these sequences indicated that the newly discovered ORFs all
The genes encoding CPARs were expressed in recombinant E. coli
as His-tagged fusion proteins. Because the change of the external
expression conditions has the potential to significantly increase
expression yield of genetically stable recombinant system, various
factors involved in the expression optimization of the target pro-
teins were investigated, including cultivation temperature from 17
to 37 ◦C and inducer of 0.1 mM to 1.0 mM IPTG or 2–6% lactose.
Under the optimized expression conditions involving varied tem-
perature and inducer concentration, the recombinant CPARs could
be obtained in soluble form with different expression levels and
specific activities (Table 3). Of them, CPAR2, CPAR3, CPAR4, and
CPAR7 were expressed at somewhat higher yields of target pro-
tein, while CPAR1, CPAR5, CPAR6, and CPAR8 gave relatively lower
levels of target protein yield in heterologous expression (Table 3).
Then the recombinant enzymes with His-tag at C-terminal were
purified by nickel affinity chromatography, showing distinct bands

R. Guo et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 66–73
69
Fig. 1. Amino acid sequence alignment of C1 and putative CPARs from C. parapsilosis. Gaps in the aligned sequences are indicated by dashes. The AKR family signature
sequence 1 (PROSITE accession number PS00798) and sequence 2 (PROSITE accession number PS00062) and the IPKS motif are underlined.

70
R. Guo et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 66–73
Table 2
Properties of potential genes and their coding products discovered from C. parapsilosis genome by BLAST search.
Gene
GenBank accession number
Length of open
reading frame (bp)
Residue number of
encoded protein
Theoretical molecular weight of
encoded protein (Da)
cpar1
cpar2
cpar3
cpar4
cpar5
cpar6
cpar7
cpar8
JX512911
JX512912
JX512913
JX512915
JX512916
JX512917
JX512918
JX512919
1059
987
933
888
852
996
924
1047
352
328
310
295
283
331
307
348
38,927.4
36,904.8
35,170.9
32,761.1
32,382.0
37,167.2
34,650.4
39,439.4
Table 3
Optimized conditions for expression of CPARs in recombinant E. coli.
a
b
Construct Expression conditions
Target protein yield (mg L−1
)
Specific activity (␮mol min−1 mg−1
)
IPTG concentration (mM) Lactose concentration (%) Induction temperature (◦C)
CPAR1
CPAR2
CPAR3
CPAR4
CPAR5
CPAR6
CPAR7
CPAR8
1
1
1
1
0
1
1
0
0
0
0
0
2
0
0
2
30
17
17
17
17
17
17
17
2
0.23
2.51 0.15
3.23 0.21
4.72 0.24
3.57 0.09
14.02 2.10
1.50 0.78
0.82 0.05
0.67 0.12
40 1.32
52 0.74
42 1.18
9
4
0.73
0.34
48 0.88
0.37
5
a
The target protein yield (mg L−1) was evaluated by calculating the amount of purified protein from the corresponding culture broth.
Activity assay was carried out at the pH 6.5 for each enzyme with ethyl 4-chloro-3-oxobutyrate as the substrate.
b
on SDS-PAGE, with the size in agreement with the theoretically
calculated molecular weight (Fig. 3).
(acetophenone, 2-hydroxyacetophenone, o-chloroacetophenone
m-chloroacetophenone, and p-chloroacetophenone), aliphatic
ketones (2-octanone and 2-hexanone), and ketoesters (ethyl
4-trifluoro-3-oxobutyrate, methyl 3-oxobutyrate, ethyl 3-phenyl-
3-oxopropionate, and ethyl 4-chloro-3-oxobutyrate), from which
the corresponding alcohol products are valuable building blocks
and intermediates for the synthesis of important pharmaceuticals
and functional materials, for example, semisynthetic ␤-lactam
antibiotics and cholesterol-lowering drug atorvastatin [32–34].
The CPARs all performed catalytic activity with NADPH as the
coenzyme, but no obvious activities toward NADH (data not
shown), indicating that these newly discovered enzymes all play
the role of NADPH-dependent oxidoreductases.
3.3. Substrate specificity of recombinant CPARs
Predicted as members of AKRs based on the primary
structure analysis, the capability of CPARs for catalyz-
ing reduction of carbonyl compounds was investigated
toward different kinds of substrates including aryl ketones
Then the substrate specificity of the interested enzymes
involved in the discovered AKR toolbox was assessed by
measuring the reductive activity with NADPH. For the car-
bonyl substrates with various chemical structures, it could
be observed that the enzymes generally exhibited obviously
higher activities to 2-hydroxyacetophenone of aryl ketones
and ketoesters, especially ethyl 4-chloro-3-oxobutyrate (Table 4).
Compared with other carbonyl substrates including acetophenone
derivatives and aliphatic ketones, from the viewpoint of chemi-
cal structure of the substrates, ethyl 4-chloro-3-oxobutyrate and
2-hydroxyacetophenone both possess an electron-withdrawing
substituent neighboring the carbonyl group, such as chlorine or
hydroxyl, indicating that substituent at ␣-carbon has a great effect
on the enzyme activity by forming hydrogen bond between active
site residues of the enzyme and such electron-withdrawing groups
at the ␣-position of substrates, which would be favorable for the
reaction involving electron transfer.
In addition, the specific activity and the catalytic efficiency of
the enzymes were evaluated by calculating the steady-state kinet-
ics via double reciprocal plots, using ethyl 4-chloro-3-oxobutyrate,
2-hydroxyacetophenone, acetophenone, and 2-octanone as the
exemplary substrates (Fig. 4). Except for the observed trends of the
CPARs toward various carbonyl compounds, some enzymes per-
formed distinct specificity to certain kind of the tested substrates.
Of them, CPAR2 and CPAR4 exhibited obvious activities toward
Fig. 2. Unrooted phylogenetic tree of CPARs and relevant AKRs. The
amino acid sequences of relevant AKRs were referred to AKR homepage
(http://www.med.upenn.edu/akr/).

R. Guo et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 66–73
71
Fig. 3. SDS-PAGE analysis of purified CPARs. Lanes 1 and 6, molecular mass standard; Lane 2, purified CPAR2; Lane 3, purified CPAR3; Lane 4, purified CPAR4; Lane 5, purified
CPAR7; Lane 7, purified CPAR1; Lane 8, purified CPAR5; Lane 9, purified CPAR6; Lane 10, purified CPAR8.
acetophenone, and CPAR2, CPAR3, CPAR4, and CPAR5 were observ-
ably active to 2-octanone. On the contrary, CPAR6, CPAR7, and
CPAR8 were almost inefficient in catalyzing reduction of both
acetophenone and 2-octanone. Associating the catalytic effi-
3.4. Enantioselective reduction of carbonyl compounds
The enantioselectivity of CPARs catalyzing asymmetric reduc-
tion was evaluated toward the investigated substrates including
aliphatic ketones, aryl ketones, and ketoesters (Table 4). On the one
hand, although almost all of the CPARs performed stereoselectivity
to the carbonyl compounds, they did not follow the same pattern
of asymmetric reduction, according to the stereo-configuration
outcomes of the products generated from the reduction of car-
bonyl substrates. Of them, CPAR2 and CPAR6 were supposed
to catalyze asymmetric reduction in anti-Prelog type, while the
ciency regarding the value of kcat/Km
, except for CPAR2 to
2-hydroxyacetophenone, CPAR4 generally performed relatively
higher efficiency of catalyzing reduction of carbonyl compounds
with various chemical strictures, indicating that the enzyme pos-
sesses a somewhat broad spectrum of carbonyl substrates and has
the potential for the synthesis of pharmaceuticals and fine chemi-
cals from the application point of view.
Fig. 4. Specific activities and apparent kinetic parameters of CPARs toward various carbonyl substrates including (A) ethyl 4-chloro-3-oxobutyrate, (B) 2-
hydroxyacetophenone, (C) acetophenone, and (D) 2-octanone. All reactions involved in the calculation of activities and kinetic parameters were carried out as described in
the text.

72
R. Guo et al. / Journal of Molecular Catalysis B: Enzymatic 105 (2014) 66–73
others were suggested to follow Prelog’s rule [35,36]. On the other
hand, not only in stereoselectivity, the enzymes but also exhibited
diversity in stereopreference to prepare the corresponding chi-
ral alcohol products with different optical purities, presented as
enantiomeric excess (e.e.) value. Except for CPAR3 and CPAR7 with
somewhat lower stereopreference to the tested substrates, most
of the newly discovered CPARs were able to catalyze highly enan-
tioseletive reduction of certain kinds of carbonyl compounds, even
resulting in the production of optically active alcohols with the e.e.
value over 99%, such as CPAR6 to acetophenone derivatives, CPAR2
to aliphatic ketones, and CPAR2 and CPAR8 to ketoesters.
It is worth noting that, of the enzymes involved in the newly
discovered AKRs toolbox, CPAR4 was found to be outstanding in
catalyzing asymmetric reduction due to its excellent enantioselec-
tivity to all of the examined substrates, even producing the chiral
alcohols of absolute stereo-configuration (>99% e.e.). Correlating
with the catalytic efficiency, therefore, CPAR4 was supposed to be
a promising catalyst with high stereopreference and broad sub-
strate specificity for highly efficient and enantioseletive reduction
of carbonyl compounds, and hence has the application potential for
the synthesis of pharmaceutical intermediates and fine chemicals.
4. Conclusion
In this work, a biocatalytic toolbox of novel stereospecific
AKRs was identified through genomic mining. Eight putative AKRs
encoded by the ORFs from C. parapsilosis genome were expressed,
purified, and characterized. The resulted CPARs exhibited cat-
alytic activities to the investigated carbonyl compounds, especially
2-hydroxyacetophenone and ethyl 4-chloro-3-oxobutyrate, and
most of them were stereoselective to the substrates and even
performed the capability of catalyzing highly enantioselective
reduction of ketoesters or acetophenone derivatives to the corre-
sponding alcohols with optical purity over 99%e.e. Of them, CPAR4
was impressive with remarkable enantioselectivity and broad
substrate specificity, indicating its practical application potential.
Additionally, the approach of genomic mining would be promising
to discover novel and efficient enzymes for pharmaceutical and fine
chemical industries.
Acknowledgements
Financial supports from the National Key Basic Research and
Development Program of China (973 Program) (2011CB710800),
the National Hi-Tech Research and Development Program of
China (863 Program) (2011AA02A210 and 2011AA02A209), the
National Natural Science Foundation of China (NSFC) (21376107
and 21336009), the Program of Introducing Talents of Discipline to
Universities (111 Project) (111-2-06), the High-end Foreign Experts
Recruitment Program (GDW20133200113), and the Project Funded
by the Priority Academic Program Development of Jiangsu Higher
Education Institutions are greatly appreciated.
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