Cloning, expression and enzymatic characterization of an aldo-keto reductase from Candida albicans XP1463

Article abstract of DOI:10.1016/j.molcatb.2015.08.018

An aldo-keto reductase encoding gene caakr was cloned from Candida albicans XP1463 (CCTCC M 2014382), and heterologously expressed in Escherichia coli. The aldo-keto reductase CaAKR is NADH-dependent with a molecular weight of approximately 38.6 kDal including a His₆-Tag. It is active and stable at 30°C and pH 7.0. The maximal reaction rate (V_max), apparent Michaelis-Menten constant (K_m) and catalytic constant (k_cat) for t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((R)-1a) were 11.50 mmol/L min, 1.91 mmol/L and 218.50 min^-1. Besides atorvastatin's chiral synthon t-butyl 6-cyano-(3R,5R)- dihydroxy hexanoate ((R,R)-1b), it can synthesize N,N-2-dimethyl-(3S)-hydroxy-3-(2-thienyl)-1-propanine ((S)-9b) and methyl 1-[E]-2-[3-[3-[2-(7-chloro-2-quinoliny) ethenyl] phenyl]-(3S)-hydroxy propy] benzoate ((S)-10b), the chiral intermediates of duloxetine and montelukast, displaying potential applications in pharmaceutical industry. 2015 Elsevier B.V. All rights reserved.

Full text of DOI:10.1016/j.molcatb.2015.08.018

Journal of Molecular Catalysis B: Enzymatic 122 (2015) 44–50
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Cloning, expression and enzymatic characterization of an aldo-keto
reductase from Candida albicans XP1463
Ya-Jun Wanga,b, Xiao-Qing Liua,b, Xi Luoa,b, Zhi-Qiang Liua,b, Yu-Guo Zhenga,b,∗
a Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, PR China
b Engineering Research Center of Bioconversion and Biopurification of the Ministry of Education, Zhejiang University of Technology, Hangzhou, Zhejiang
310014, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 June 2015
Received in revised form 21 August 2015
Accepted 22 August 2015
Available online 28 August 2015
An aldo-keto reductase encoding gene caakr was cloned from Candida albicans XP1463 (CCTCC M
2014382), and heterologously expressed in Escherichia coli. The aldo-keto reductase CaAKR is NADH-
dependent with a molecular weight of approximately 38.6 kDal including a His6-Tag. It is active and stable
at 30 ◦C and pH 7.0. The maximal reaction rate (Vmax), apparent Michaelis–Menten constant (Km) and cat-
alytic constant (kcat) for t-butyl 6-cyano-(5R)-hydroxy-3-oxohexanoate ((R)-1a) were 11.50 mmol/L min,
1.91 mmol/L and 218.50 min−1. Besides atorvastatin’s chiral synthon t-butyl 6-cyano-(3R,5R)- dihydroxy
hexanoate ((R,R)-1b), it can synthesize N,N-2-dimethyl-(3S)-hydroxy-3-(2-thienyl)-1-propanine ((S)-
9b) and methyl 1-[E]-2-[3-[3-[2-(7-chloro-2-quinoliny) ethenyl] phenyl]-(3S)-hydroxy propy] benzoate
((S)-10b), the chiral intermediates of duloxetine and montelukast, displaying potential applications in
pharmaceutical industry.
Keywords:
Aldo-keto reductase
Candida albicans
Asymmetric bioreduction
Chiral alcohol
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
polycyclic aromatic hydrocarbons, and isoflavinoid phytoalex-
ins [6,8–11]. So far, only limited AKRs have been purified or
Enantiometrically pure alcohols are the most important chi-
ral synthons for pharmaceuticals [1,2]. Optically active t-butyl
6-cyano-(3R, 5R)-dihydroxyhexanoate ((R,R)-1b) is a key chiral
intermediate for the synthesis of atorvastatin calcium (Lipitor®),
an efficacious statin for the treatment of hypercholesterolemia [3].
Chemical strategies for (R,R)-1b have been developed, but unfortu-
nately most are hard to meet the rigorous requirements on optical
purity of chiral drugs [4]. Biocatalysis has been applied to the
synthesis of optically pure substances, which has emerged as a
powerful approach attributing to the outstanding benefits such as
mild reaction conditions, high catalytic efficiency, excellent selec-
tivity plus environmental friendliness [5].
The aldo-keto reductase (AKR) superfamily are oxidoreduc-
tases, and widely exist in mammals, amphibians, plants, yeast,
protozoa, and bacteria [6]. Members of the AKR superfamily are
NAD(P)H-dependent, monomeric (˛/ˇ)8 barrel proteins, about 320
amino acids in length, which bind NAD(P)(H) without a Rossmann-
fold motif [7]. The AKRs convert a range of substrates including
aliphatic aldehydes, monosaccharides, steroids, prostaglandins,
cloned, characterized, and applied in the asymmetric synthesis
of chiral alcohols, including Tm1743 from Thermotoga maritime
[12], YOL151W from Saccharomyces cerevisiae [13], alkyl 4-halo-
3-oxobutyrate reductase from Penicillium citrinum IFO4631 [14],
AKRs from Lodderomyces elongisporus [15,16], C. parapsilosis [17]
and Bacillus sp. ECU0013 [18]. Compared with the widely used car-
bonyl reductase and alcohol dehydrogenase, AKRs for chiral alcohol
synthesis are rare [19,20].
In this work, we reported cloning an aldo-keto reductase encod-
ing gene caakr from Canadida albicans XP1463, which possesses
NADH-dependent AKR activity toward t-butyl 6-cyano-(5R)-
hydroxy-3-oxohexanoate ((R)-1a), expressing it in Escherichia coli
BL21 (DE3), and characterizing its enzymatic features. The CaAKR
has broad substrate spectrum, displaying prospective in some high-
valued chiral alcohols synthesis.
2. Materials and methods
2.1. Strains and chemicals
C. albicans XP1463, used as the source of the AKR encoding
gene caakr, was screened and identified by our group, which has
been deposited at China Center for Type Culture Collection (Wuhan,
China) under the acquisition number CCTCC M 2014382. E. coli BL21
∗
Corresponding author at: Zhejiang University of Technology, Hangzhou, Zhe-
jiang 310014, PR China. Fax: +86 571 88320630.
E-mail address: zhengyg@zjut.edu.cn (Y.-G. Zheng).
http://dx.doi.org/10.1016/j.molcatb.2015.08.018
1381-1177/© 2015 Elsevier B.V. All rights reserved.

Y.-J. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 44–50
45
(DE3) (Novagen, USA) was used to overexpress the caakr. (R)-1a
was generously presented by Neo-Dankong Pharmaceutical Co., Ltd
(Taizhou, China). (R,R)-1b was purchased from Toronto Research
Chemicals Co. Ltd. (Toronto, Canada). Plasmids pMD18-T (TaKaRa,
Japan) and pET-28a (+) (Novagen, USA) were used as vectors for
the cloning and expression, respectively. Taq DNA polymerase and
Pfu DNA were purchased from Takara (Dalian, China). Restriction
enzymes and T4 DNA ligase were supplied by Thermo Fisher Scien-
tific Co., Ltd (Beijing, China). Primers synthesis and DNA sequencing
were conducted at Shanghai Sunny Biotechnology Co., Ltd. (Shang-
hai, China). The DNA gel extraction kit and plasmid extraction
kits were purchased from Axygen (Suzhou, China). Kanamycin and
ampicillin were purchased from Sigma-Aldrich (USA). NADH was
supplied by Sangon Biotech Co. (Shanghai, China). Unless otherwise
stated, all other chemicals and agents used were of analytical grade
and available commercially.
sis IFO 0708 (PDB ID: 4H8N and PDB ID: 3VXG) in complex with
NADPH using AutoDock vina (Accelrys Inc., CA, USA) [22].
2.5. Enzyme assays
The activity of CaAKR was assayed spectrophotometrically at
30 ◦C by continuous monitoring the absorbance decrease of NADH
at 340 nm, which was conducted in 96-well plates. The reaction
mixture with a total volume of 200 ␮L contained 200 mmol/L potas-
sium phosphate buffer (pH 7.0), 0.5 mmol/L of NADH, 12.5 mmol/L
(R)-1a and an appropriate amount of the purified CaAKR. One unit
of CaAKR activity was defined as the amount of enzyme required to
oxidize 1 ␮mol NADH per minute under the standard conditions.
2.6. Characterization of the recombinant CaAKR
2.6.1. Effect of pH
2.2. Construction of expression plasmid
The optimum pH for CaAKR activity was determined at 1 mg/mL
CaARK in three buffers within a pH range from 3.5 to 9.5 at
200 mmol/L, including acetate buffer (pH 3.5–5.5), potassium phos-
phate buffer (pH 6.0–8.0), Tris–HCl buffer (pH 8.5–9.5). The pH
stability was determined by incubating 1 mg/mL CaARK in buffers
with varying pHs between 4.0 and 8.0 at 4 ◦C for 120 h. Samples
were withdrawn for the residual activity determination, which
was carried out under standard assay conditions with the non-
incubated CaAKR as the control.
The caakr was amplified by PCR from the genomic DNA of C. albi-
cans CCTCC M 2014382 by using the primers designed as follows:
ca-F:
5ꢀ-CATATGATAGGATCCGGAGGAGGATGTATATGTATCC-3ꢀ
(the
Nde
I
site
is
underlined),
ca-R:
5ꢀ-
site
GCGGCCGCTCGCTGCAGATGTTTAACCGCGGC-3ꢀ (the Not
I
is underlined), based on caakr sequence with the GenBank
accession number of XP 711680.1.
The purified PCR product was ligated with the pMD18-T vector,
double-digested with Nde I and Not I, and then inserted into expres-
sion vector pET-28a (+). The resultant recombined plasmids pET28a
(+)-caakr was transformed into E. coli BL21 (DE3) competent cells,
and selected on Luria-Bertani (LB) agar plates supplementing with
50 ␮g/mL kanamycin. The positive clones were confirmed by DNA
sequencing.
2.6.2. Effect of temperature
The optimum temperature for CaAKR activity was investigated
at temperatures between 20 ◦C and 50 ◦C. Thermal stability was
determined by incubating the purified CaAKR at a final concentra-
tion of 1 mg/mL, temperatures between 20 ◦C and 50 ◦C for 20 h.
Samples were withdrawn for the residual activity determination,
which was carried out under standard assay conditions with the
non-heated CaAKR as the control.
2.3. CaAKR expression and purification
The positive transformants were cultivated at 37 ◦C, 150 rpm in
LB medium containing 50 ␮g/mL kanamycin until OD600 reached
0.8, and then subjected to induction by 9 g/L lactose at 28 ◦C,
150 rpm for 10 h. The resultant cells were harvested by centrifu-
gation at 4 ◦C, 9000 rpm for 10 min, and washed twice with 0.9%
NaCl (w/v) solution.
2.7. Kinetic analysis
The kinetic parameters were calculated from the initial-
velocity double-reciprocal plot [23], at 1 mg/mL CaARK, 30 ◦C,
in 200 mmol/L, pH7.0 potassium phosphate buffer. For la, v0
was detected at varying final concentrations of (R)-1a between
The cell pellet was resuspended in buffer A composed of
20 mmol/L, pH 8.0 sodium phosphate buffer, 500 mmol/L NaCl
and 20 mmol/L imidazole, and disrupted by sonication. The cell
debris was removed by centrifugation at 4 ◦C, 12,000 rpm for
20 min, and the supernatant was loaded onto a Ni-NTA column pre-
equilibrated with buffer A, and eluated with buffer B consisting of
20 mmol/L sodium phosphate buffer, pH 8.0, 500 mmol/L NaCl and
500 mmol/L imidazole. The flowrate of mobile phase was main-
tained at 1 mL/min. The active fractions were pooled and dialyzed
against 200 mmol/L, pH 7.0 potassium phosphate buffer at 4 ◦C for
12 h. The purity and molecular mass of the enzyme was analyzed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-
PAGE) with 12% separating gel. The protein concentration was
determined by the method of Bradford using BSA as the standard
[21].
0.5 mmol/L and 4.0 mmol/L, a fixed NADH concentration of
0.4 mmol/L. For NADH, the final concentration of NADH was
varied from 0.1 mmol/L to 0.4 mmol/L in the presence of
4.0 mmol/L (R)-1a. The maximal reaction rate (Vmax) and apparent
(R)−1a
Michaelis–Menten constants (Km
KmNADH) were calculated
,
from the initial-velocity double-reciprocal plot [23]. The value of
parameter kcat (the catalytic constant) was obtained by dividing
Vmax by CaAKR concentration [16].
2.8. Substrate specificity
To investigate its substrate spectrum, CaAKR-catalyzed reduc-
tion of aldehydes, ketones and ketone esters were investigated
under the standard assay conditions. The relative activity toward
(R)-1a was designated as 100%.
The bioreduction was performed in 1.5 mL Eppendorf tubes con-
taining 200 mmol/L potassium phosphate buffer (pH 7.0), 1 mmol/L
of each substrate, 0.5 mmol/L NADH, and 1 mg/mL purified CaAKR
in a total volume of 1.0 mL, shaking for 10 h at 30 ◦C. The reaction
mixture was extracted twice with an equivalent volume of 300 ␮L
ethyl acetate. The extracts were combined, dried with anhydrous
sodium sulfate. The concentrations for each product were deter-
mined by GC or HPLC analysis.
2.4. Sequence analysis and molecular modeling
The multiple sequence alignment was performed with the soft-
ware Clustal X2 and the website of Espript 3.0 (http://espript.ibcp.
fr/espript/cgi-bin/espript.cgi). Three-dimensional homology mod-
els of CaAKR were built based on the crystal structure of the
conjugated polyketone reductase C2 (CPR-C2) from C. parapsilo-

46
Y.-J. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 44–50
Fig. 1. Alignments of multiple amino acid sequence of CaAKR and other AKRs from various microbes listed at the web of www.med.upenn.edu/akr. AKR2B2: xylose reductase
from Kluyveromyces lactis (UniProtKB/Swiss-Prot accession number: P49378.1); AKR2B7: xylose reductase from Candida tropicalis (GenBank accession number: BAA19476.1);
AKR2C1: 4-dihydromethyltrisporate dehydrogenase from Mucor mucedo (GenBank accession number: CAA98021.1); AKR3B1: aldehyde reductase from Sporidiobolus salmoni-
color (GenBank accession number: AAB17362.1); AKR5G2: reductase from Bacillus subtilis (NCBI reference sequence: NP 390783.1); AKR9B3: aryl-alcohol dehydrogenase
from Saccharomyces cerevisiae (NCBI Reference Sequence: NP 010038.1); AKR11B1: aldo-keto reductase from Bacillus halodurans (UniProtKB/Swiss-Prot accession number:
Q9KE47); AKR12B1: mycarose/desosamine reductase from Saccharopolyspora erythraea (GenBank accession number: AAB84068.1); AKR13A1: aldo-keto reductase from
Schizosaccharomyces pombe (UniProtKB/Swiss-Prot accession number: Q09923.1); AKR14A1: aldehyde reductase form E. coli (GenBank accession number: AAA69168). The
conserved sites are all labeled in red. The catalytically active tetrad and active sites are marked with triangle and circle, respectively.
2.9. Analytical methods
The
conversion
of
N,N-2-dimethyl-3-oxo-3-(2-
and optical purity of
thienyl)-1-propanine
(9a)
The conversion of (R)-1a and optical purity of 1b were deter-
mined by HPLC (Shimadzu LC-10A system, Japan) equipped with
an ultraviolet detector (SPD-10A VP Plus, Shimadzu, Japan) using
an ODS HYPERSIL column (4.6 × 250 mm, Thermo Scientific, USA).
Mobile phase consisted of acetonitrile and deionized at a volumet-
ric rate of 25:75 (v/v), run at a flowrate of 1.0 mL/min. Column
temperature was maintained at 40 ◦C, detector wavelength set at
210 nm. The retention times of (R,S)-1b, (R,R)-1b and (R)-1a were
7.9 min, 8.3 min and 11.8 min, respectively.
N,N-2-dimethyl-3-hydroxy-3-(2-thienyl)-1-propanine (9b) were
measured by the same HPLC system except a Chiralcel OJ-H
column (4.6 × 250 mm, Daicel, Japan). Mobile phase was composed
of n-heptane, ethanol and diethylamine at 97:3:0.1 (v/v), and run
at a flow rate of 1.0 mL/min. Column temperature was maintained
at 40 ◦C, detector wavelength set at 235 nm. The corresponding
retention times for 9a, (S)-9b and (R)-9b were 5.9 min, 6.4 min and
7.6 min, respectively.
Methyl 2-[3-[3-[2-(7-chloro-2-quinolinyl) ethenyl] phenyl]-3-
oxopropyl] benzoate (10a) and methyl 2-[3-[3-[2-(7-chloro-2-

Y.-J. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 44–50
47
Fig. 2. SDS-PAGE analysis of CaAKR from C. albicans CCTCC M 2014382 overex-
pressed in E. coli BL21(DE3). M, protein markers; Lane 1, purified enzyme; Lane
2, the intact cell lysate of E. coli BL21 (DE3)/pET-28a (+)-caakr cells with lactose
induction; Lane 3, soluble fraction of crude extract of E. coli BL21 (DE3)/pET-28a
(+)-caakr with lactose induction.
quinolinyl) ethenyl] phenyl]-3-hydroxypropyl] benzoate (10b)
were measured by using the same HPLC system except a Chiralcel
OD-H column (4.6 × 250 mm, Daicel, Japan). Detector wavelength
was set at 287 nm, column temperatures at 40 ◦C. Mobile phase was
consisted of n-hexane and 2-propanol at volumetric ratio of 80:20
(v/v), and run at 1.0 mL/min. The retention times for 10a, (S)-10b
and (R)-10b were 12.9 min, 14.4 min and 16.4 min, respectively.
The concentration of acetophenone (2a), ethyl 4-
chloroacetoacetate (6a), ethyl 4-bromoacetoacetate (7a), ethyl
4,4,4-trifluoroacetoacetate (8a) and the corresponding prod-
ucts 1-phenylethanol (2b), ethyl 4-chloro-3-hydroxybutanoate
(6b), ethyl 4-bromo-3-hydroxybutanoate(7b) as well as ethyl
4,4,4-trifluoro-3-hydroxybutanoate (8b) were determined by GC-
14C gas chromatography (Shimadzu, Japan), equipped with FID
detector and a chiral capillary column BGB-174 (30m × 0.25 mm,
0.12 ␮m film thickness, BGB Analytic, Switzerland). Helium was
used as the carrier gas.
For 2a and 2b, the inlet and detector temperatures were both
set at 240 ◦C; the column temperature was held at 110 ◦C for 1 min,
elevated at 2 ◦C/min to 140 ◦C, and held at 140 ◦C for 15 min. The
retention times of 2a, (S)-2b and (R)-2b were 6.6 min, 7.9 min and
8.2 min respectively.
Fig. 3. Three-dimensional homology models of CaAKR (A) and PROCHECK analyses
(B). The NADH and the catalytic tetrad (Asp61, Tyr66, Lys91 and His128) are indicated
by sticks (A).
For 6a and 6b, the inlet and detector temperatures were both
set at 220 ◦C; the column temperature was initially set at 110 ◦C for
1 min, elevated at 5 ◦C/min to 140 ◦C, and held at 140 ◦C for 15 min.
The retention times of 6a, (S)-6b and (R)-6b were 7.9 min, 11.67 min
and 12.32 min, respectively.
For 7a and 7b, the inlet and detector temperatures were both set
at 220 ◦C; the column temperature was held at 110 ◦C for 25 min,
then elevated at 5 ◦C/min to 160 ◦C, and held at 160 ◦C for 2 min. The
retention times of 7a, (S)-7b and (R)-7b were 27.9 min, 34.39 min
and 34.9 min respectively.
For 8a and 8b, the inlet and detector temperatures were both
set at 220 ◦C; the column temperature was only held at 120 ◦C for
15 min. The retention times of 8a, (S)-8b and (R)-8b were 2.6 min,
6.8 min and 7.1 min, respectively.
The detection methods of p-chloroacetophenone (3a), p-
methylacetophenone (4a), ethyl acetoacetate (5a) and the
corresponding products p-chloro-1-phenylethanol (3b), p-methyl-
1-phenylethanol (4b) as well as ethyl 3-hydroxybutyrate (5b) are
as follows:
3a and 3b were determined by GC-HP Chiml 10% ␤-cyclodextrin
(30m × 0.32 mm × 0.25 ␮m, HP6890, USA). The inlet and detector
temperatures were set at 250 ◦C, the column temperature was only
held at 130 ◦C for 15 min. The retention times of 3a, (S)-3b, (R)-3b
were 6.8 min, 9.5 min and 10.3 min, respectively.
4a and 4b were determined by GC-HP 5% PEG-20 m (2 m × 3 mm,
PE, USA). The inlet and detector temperatures were set at 250 ◦C and
260 ◦C, the column temperature was only held at 180 ◦C for 10 min.
The retention times of 4a, (R)-4b were 5.32 min and 7.05 min.
5a and 5b were determined by GC (30m × 0.32 mm × 0.25 ␮m,
Agilcnt19091J-113, Japan). The inlet and detector temperatures
were set at 200 ◦C; the column temperature was held at 100 ◦C for
2 min, elevated at 10 ◦C/min to 250 ◦C, and held at 250 ◦C for 15 min.
The retention times of 5a, (S)-5b were 7.32 min and 10.15 min.

48
Y.-J. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 44–50
3. Results and discussions
100
A
3.1. Gene cloning and sequencing analysis
80
60
40
20
0
The caakr gene was amplified by PCR using C. albicans CCTCC M
2014382 genomic DNA as the template. It contained an intact open
reading frame (ORF) of 927 bp excluding termination codon, encod-
ing a subunit of 309 residues with a predicted molecular mass of
38.6 kDal. A multiple sequence alignment was performed between
CaAKR and other AKRs from various microbes listed at the website
of www.med.upenn.edu/akr. As illustrated in Fig. 1, they have low
identity, but share the conservative sites of Asp61, Tyr66, Lys91 and
His128, which constitute the catalytic tetrad of AKRs. In addition, the
residues participating in substrate binding were also conserved.
Acetate buffer
Potassium phosphate buffer
Tris-HCl buffer
3
4
5
6
7
8
9
10
pH
3.2. Expression and purification of CaAKR
After induced by lactose, the recombinant plasmid pET-28a (+)-
caakr was successfully expressed in the heterologous host E. coli
BL21 (DE3). SDS-PAGE analysis indicated that most of the recom-
binant CaAKR exists in a soluble form (Fig. 2). Thanks to a His6-Tag
presence at C-terminalof CaAKR, it was purified with Ni2+-chelating
affinity chromatography. As illustrated in Fig. 2, a notable band
was seen on the polyacrylamide gel, corresponding to approxi-
mately 38 kDal, which includes a His6-Tag. CaAKR molecular size
is in accordance with the predicted molecular mass, approximat-
ing to that of Candida magnoliae KFCC 11023 erythrose reductase
[23], larger than that of C. magnoliae AKU4643 aldehyde reductase,
35 kDal [24].
100
80
60
40
20
0
B
pH4.0
pH5.0
pH6.0
pH7.0
pH8.0
3.3. Molecular modeling
0
20
40
60
80
100
120
Three-dimensional structure of the CaAKR was built based on
the crystal structure of conjugated polyketone reductase from Can-
dida parapsilosis IFO 0708 (PDB ID: 4H8N and PDB ID: 3VXG) in
complex with NADPH [22], which has the highest sequence sim-
ilarity to the CaAKR in the PDB entries and shared 65% identity.
The molecular model was constructed using the program Modller
9.14 (Fig. 3A). The CaAKR structure was validated by using PDB-
sum entry on line of generate full PROCHECK analyses (Fig. 3B).
Asp61, Tyr66, Lys91 and His128 were composed of the highly con-
served catalytic tetrad. Together, these data strongly suggest that
it belongs to AKR superfamily. Additionally, from the Ramachan-
dran plot (Fig. 3B), we found the Asn97 located in the generously
allowed region, which has large repulsion and high conformation
energy with labile bond structure.
Time/h
Fig. 4. Optimum pH and pH stability of CaAKR: A, The optimum pH was determined
at pH3.5-9.5, 30 ◦C; B, pH stability was measured at pH4.0–8.0, 4 ◦C.
to heat. The thermal stability was determined at temperatures
between 25 ◦C and 50 ◦C (Fig. 5B). After incubation at 25 ◦C, 30 ◦C
for 10 h, approximately 55% and 80% of CaAKR activity was lost,
respectively.
3.5. Kinetic analysis
The kinetic parameters of the purified CaAKR were obtained
from the initial-velocity double-reciprocal plot [23]. Vmax, Km
for (R)-1a were calculated to be 11.50 mmol/L min and 1.91 mmol/L,
respectively, presented in Table 1. The Km
(R)−1a
3.4. Effects of pH and temperature
(R)−1a
is in the mmol/L
Studies on the effects of pH and temperature were conducted
by using (R)-1a as the substrate. Effect of pH on the purified CaAKR
activity was studied at the pHs between 3.5 and 9.5. CaAKR activ-
ity was most active at pH 7.0 (Fig. 4A), with optimum pH close to
those of the aldehyde reductases from C. magnoliae AKU4643 [24]
and C. albicans AKU4596 [25]. The pH-stability of CaAKR was exam-
ined between pH 4.0 and pH 8.0 at 4 ◦C for 120 h, shown in Fig. 4B.
Majority of CaAKR activity was lost at pH 4.0–5.0, and CaAKR is
stable under neutral pH condition.
range, close to a NADH-dependent Cattleya tenuis xylose reductase
[26] and C. magnoliae erythrose reductase [23]. Its turnover number
(R)−1a
kcat
was 218.50 min−1, approximating to those of C. magno-
liae erythrose reductase [23] but 2–4 folds lower than those of C.
tenuis xylose reductase [26].
3.6. Substrate and coenzyme specificity
Temperature plays an important role in enzyme activity regu-
lation. As presented in Fig. 5A, the highest (R)-1a reducing activity
of CaAKR was observed at 30 ◦C, and the activity decreased sharply
above the optimum temperature. In comparison, its optimum tem-
perature is much higher than that of C. albicans AKU4596 aldehyde
reductase of 15 ◦C [25], but lower than that of C. magnoliae AKU4643
aldehyde reductases of 40 ◦C [24]. Its activity declined almost lin-
early at temperatures exceeding 30 ◦C, indicating that it is liable
The majority of AKR superfamily members are strictly depen-
dent on NADPH for activity [27]. Relatively less AKRs have been
reported to utilize NADH as the coenzyme, however, they are likely
to be useful in various biotechnological applications [28]. CaAKR is
highly specific to NADH as the coenzyme, different from C. mag-
noliae AKU4643 aldehyde reductase and C. tenuis xylose reductase.
The former is highly specific for NADPH [24]; the latter is able to
utilize both NADPH and NADH, possessing dual-specificity [27].

Y.-J. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 44–50
49
Table 1
Steady-state kinetic parameters for CaAKR-catalyzed (R)-1a reduction
NADH
(R)−1a
Enzyme
Vmax (mmol/L min)
Km
(mmol/L)
Km
(mmol/L)
kcat for (R)-1a (min−1
)
CaAKR
11.50
2.69 × 10−2
1.91
218.50
NADH
(R)−1a
is the Michaelis constant (R)-1a when NADH used as
Km
is the Michaelis constant for NADH when (R)-1a was used as the substrate and its concentration fixed; Km
the coenzyme and its concentration fixed. kcat = Vmax/[E], where Vmax is the maximal reaction rate and [E] is the molar concentration of CaAKR.
Table 2
Substrate specificity and stereoselectivity of CaAKR.
Substrates
Products
Relative activity (%)a
eep /d.ep (%)b
100
>90 (R)
10
20
>90 (R)
>85 (R)
8
>85(R)
>99 (R)
27
56
45
>99 (S)
>84(S)
50
>80 (S)
160
145
>99 (S)
>99 (S)
a
The activity for 1 mmol/L (R)-1a was taken as 100%.
b
The e.e.p and d.e.p values were calculated from products concentrations measured by chiral GC or HPLC analysis. The absolute configurations of products were obtained
by comparing the retention times with those of standard samples.

50
Y.-J. Wang et al. / Journal of Molecular Catalysis B: Enzymatic 122 (2015) 44–50
4. Conclusions
100
80
60
40
20
0
A
In this study, the caark was cloned from C. albicans CCTCC
M 2014382, and overexpressed, which codes a stereoselective
aldo-keto reductase. The caark contains an open reading frame of
927 nucleotides, encoding a protein containing 309 amino acid
residues. The CaAKR has the highly conserved catalytic tetrad
(Asp61, Tyr66, Lys91 and His128). It is strictly dependent on NADH
for activity, and has a broad substrate spectrum with varying
stereoselectivity. Besides atorvastatin’s chiral synthon (R,R)-1b,
it can synthesize optically pure (S)-9b and (S)-10b, the chiral
intermediates of duloxetine and montelukast, displaying potential
applications in pharmaceutical industry.
15
20
25
30
35
40
45
50
55
Temperature /oC
Acknowledgments
This work was financially supported by the National Basic
Research Program of China (973 Program, 2011CB710800) and the
Major Program for Key Science and Technology of Zhejiang Province
of China (2014C03010).
100
80
60
40
20
0
B
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