De novo construction of multi-enzyme system for one-pot deracemization of (R,S)-1-phenyl-1,2-ethanediol by stereoinversion of (S)-enantiomer to the corresponding counterpart

Article abstract of DOI:10.1016/j.molcatb.2016.04.003

Deracemization via oxidoreductive stereoinversion is one of the most attractive methods for the preparation of enantiomerically pure compounds. However, available enzymatic system is yet limited for efficiently catalyzing deracemization to produce optically pure alcohol in certain configuration. Through evaluation of available stereoselective oxidoreductases on activity, selectivity, and cofactor dependency, the suitable candidates were obtained to construct the enzymatic deracemization system involving cofactor self-recycling. For deracemizing (R,S)-1-phenyl-1,2-ethanediol (PED) to (R)-PED, a facile one-pot system was established by combination of two stereoselective oxidoreductases, the stereospecific carbonyl reductase 1 (SCR1) and the ketoreductase (KRD). To rebalance the activities and catalytic functions of different enzymes involved in the multi-enzyme system, the reaction conditions of SCR1-catalyzed oxidation and KRD-mediated reduction were optimized, respectively. Consequently, the deracemization system involving cofactor self-recycling was built to produce (R)-PED with the optical purity of 95.50%e.e. and the yield of 91.62% from the corresponding racemate (1 g L^-1), under the optimal reaction conditions including activity ratio of SCR1/KRD 1:4 (SCR1 10 U mL^-1 and KRD 40 U mL^-1), molar ratio of NADP⁺/NADPH 3:1, 30°C, and pH 7.0. Therefore, the developed strategy would be useful to construct the multi-enzyme deracemization system based on systematic evaluation of enzyme features.

Full text of DOI:10.1016/j.molcatb.2016.04.003

Journal of Molecular Catalysis B: Enzymatic 129 (2016) 21–28
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
De novo construction of multi-enzyme system for one-pot
deracemization of (R,S)-1-phenyl-1,2-ethanediol by stereoinversion
of (S)-enantiomer to the corresponding counterpart
a
a,∗
a
a,b,∗
Bin Li , Yao Nie , Xiao Qing Mu , Yan Xu
a
School of Biotechnology and Key laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi 214122, China
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Deracemization via oxidoreductive stereoinversion is one of the most attractive methods for the prepa-
ration of enantiomerically pure compounds. However, available enzymatic system is yet limited for
efficiently catalyzing deracemization to produce optically pure alcohol in certain configuration. Through
evaluation of available stereoselective oxidoreductases on activity, selectivity, and cofactor dependency,
the suitable candidates were obtained to construct the enzymatic deracemization system involving
cofactor self-recycling. For deracemizing (R,S)-1-phenyl-1,2-ethanediol (PED) to (R)-PED, a facile one-pot
system was established by combination of two stereoselective oxidoreductases, the stereospecific car-
bonyl reductase 1 (SCR1) and the ketoreductase (KRD). To rebalance the activities and catalytic functions
of different enzymes involved in the multi-enzyme system, the reaction conditions of SCR1-catalyzed
oxidation and KRD-mediated reduction were optimized, respectively. Consequently, the deracemiza-
tion system involving cofactor self-recycling was built to produce (R)-PED with the optical purity of
Received 26 January 2016
Received in revised form 1 April 2016
Accepted 6 April 2016
Available online 7 April 2016
Keyword:
Deracemization
Oxidoreductase
Stereoinversion
Cofactor recycling
Enzyme-coupled system
−1
9
5.50%e.e. and the yield of 91.62% from the corresponding racemate (1 g L ), under the optimal reaction
−1
−1
conditions including activity ratio of SCR1/KRD 1:4 (SCR1 10 U mL and KRD 40 U mL ), molar ratio of
+
◦
NADP /NADPH 3:1, 30 C, and pH 7.0. Therefore, the developed strategy would be useful to construct the
multi-enzyme deracemization system based on systematic evaluation of enzyme features.
©
2016 Elsevier B.V. All rights reserved.
1
. Introduction
antibiotics, which can be used for the treatment of psychiatric dis-
orders and metabolic problems [8–10].
Microbial oxidoreductive systems involving stereoselective oxi-
A series of bio-methods for preparation of (R)-PED have been
explored, including stereospecific dihydroxylation of styrene cat-
alyzed by naphthalene dioxygenase, lipase-mediated resolution of
racemic PED, biocatalytic reduction of phenylglyoxal, asymmet-
ric reduction of 2-hydroxy-1-phenylethanone by microbial cells
or isolated ketoreductases, and enantioconvergent hydrolysis of
racemic styrene oxide using recombinant whole cells [10–15].
However, the involvement of the carcinogenic styrene oxide inher-
ently occurs in some strategies and the maximal yield can never
exceed 50% for the approach of kinetic resolution. Regarding
the methods involving bio-oxidoreduction, the reported exam-
ples mainly employed asymmetric reduction of the corresponding
prochiral ketone to produce (R)-PED. However, reductase-involved
doreductases have been increasingly used in catalyzing reactions
of commercial interest and applied in the preparation of optically
active alcohols for pharmaceuticals, agrochemicals, pheromones,
and fine chemical industries [1–7]. Of the optically active alco-
hols as important chiral building blocks, enantiomerically pure
(
R)-1-phenyl-1,2-ethanediol(PED)generally serves as a synthon for
the preparation of (R)-norfluoxetine, (R)-fluoxetine, and ␤-lactam
Abbreviations: PED, 1-phenyl-1,2-ethanediol; SCR1, stereospecific carbonyl
reductase 1; KRD, ketoreductase; 2-HAP, 2-hydroxyacetophenone; HPLC, high
performance liquid chromatography; IPTG, isopropyl-␤-d-thiogalactopyranoside;
TCEP, Tris (2-carboxyethyl) phosphine; SDS-PAGE, sodium dodecyl sulfate-
polyacrylamide gel electrophoresis.
∗ Corresponding author at: School of Biotechnology and Key laboratory of Indus-
−1
wet cells of relatively high concentration (100–300 g L ) were gen-
−
1
erally used for the substrate of limited concentration (1–5 g L ),
where a relatively long reaction time (36–48 h) was usually
required for the whole-cell reaction system, and on the other hand,
in general large amount of expensive coenzyme was stoichiometri-
trial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road,
Wuxi 214122, China.
E-mail addresses: ynie@jiangnan.edu.cn (Y. Nie), yxu@jiangnan.edu.cn (Y. Xu).
http://dx.doi.org/10.1016/j.molcatb.2016.04.003
1
381-1177/© 2016 Elsevier B.V. All rights reserved.

2
2
B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 129 (2016) 21–28
Table 1
Enzymes used in the work and their sources.
Enzyme
GenBank ID
Source
Reference
ADHR
C1
C2
CR2
CR4
KRD
OYE
RCR
SCR
AY267012
AB084515
AB084516
AB183149
E59061
Lactobacillus kefiri DSM 20587
Candida parapsilosis IFO 0708
C. parapsilosis IFO 0708
Kluyveromyces marxianus AKU 4588
K. aestuarii DC 6752
[41]
[42]
[42]
[36]
[39]
[35]
[37]
[33]
[34]
[34]
[34]
[32]
[28]
[28]
[28]
[28]
[28]
[28]
[28]
[28]
AF178079
AB126227
DQ295067
DQ675534
FJ939565
FJ939564
AB036927
JX512911
JX512912
JX512913
JX512915
JX512916
JX512917
JX512918
JX512919
Zygosaccharomyces rouxii ATCC14462
K. marxianus AKU 4588
C. parapsilosis CCTCC M203011
C. parapsilosis CCTCC M203011
C. parapsilosis CCTCC M203011
C. parapsilosis CCTCC M203011
C. magnoliae AKU 464
C. parapsilosis CCTCC M203011
C. parapsilosis CCTCC M203011
C. parapsilosis CCTCC M203011
C. parapsilosis CCTCC M203011
C. parapsilosis CCTCC M203011
C. parapsilosis CCTCC M203011
C. parapsilosis CCTCC M203011
C. parapsilosis CCTCC M203011
SCR1
SCR3
S1
Fig. 1. Deracemization of racemic PED to (R)-enantiomer through selective oxi-
CPAR1
CPAR2
CPAR3
CPAR4
CPAR5
CPAR6
CPAR7
CPAR8
dation and asymmetric reduction involving cofactor self-recycling. By enzyme 1,
(
S)-PED was oxidized to 2-HAP and NADP+ was reduced to NADPH simultaneously.
Subsequently, by enzyme 2, 2-HAP was reduced to (R)-PED and NADPH was oxidized
+
to NADP meanwhile.
cally in demand for the biocatalytic system using isolated enzyme
[
10–15]. Thus, the biocatalytic methods are yet limited for stereos-
electively asymmetric synthesis of (R)-PED enantiomer.
investigated the catalytic properties of 20 stereoselective oxidore-
ductases [28–42]. By the systematic evaluation of these enzymes
and optimization of the catalytic conditions, NADPH-dependent
stereospecific carbonyl reductase 1 (SCR1) and ketoreductase
As a hot topic in microbial catalysts, deracemization by micro-
bial stereoinversion in one-pot is one of the most attractive
methods for the preparation of enantiomerically pure compounds
in 100% theoretical yield from racemic mixtures [16–19]. Tandem
oxidation and reduction are carried out sequentially in one pot, and
the key oxidoreductases involved in the oxidation-reduction proce-
dure are of great importance. During the reaction, one enantiomer
is usually oxidized to the carbonyl intermediate, while the other
enantiomer remains unchanged, then the intermediate is reduced
to the opposite enantiomer subsequently. Thus, this approach is
more efficient because it allows complete transformation of race-
mate into single stereoisomeric product [20].
(KRD) were selected to be responsible for the tandem oxidation and
reaction in deracemization, respectively. Then the multi-enzyme
system containing SCR1 and KRD was achieved for deracemization
of racemic PED to give optically active product of (R)-PED.
2. Materials and methods
2.1. Materials
However, for bioredox-based deracemization, most of the
examples were merely applied to the whole-cell system, involving
the inherent disadvantage on substance transfer. Furthermore, the
reaction direction and product configuration of deracemization by
whole-cell system is usually fixed due to the existence of intracel-
lular functional enzymes involved in deracemization. It is generally
not feasible to produce desired chiral alcohol of certain configura-
tion by using available cells, especially wild-type cells from nature.
Thus it is obviously not easy to control the reaction process of
deracemization by in vitro manipulating the in vivo enzymes. Other-
wise, the catalytic system using crude enzymes generally involves
other enzymes acting to the substrate and the coenzyme, which
may lead to the interference and competition effects toward the
target reaction. Therefore, artificial construction of multi-enzyme
system in purified form would be reasonable and practicable to
solve these problems, although the reported one-pot deracemiza-
tion systems with coupled oxidoreductases are yet limited so far.
On the other hand, the practical applications of the key oxi-
doreductases can be quite challenging since they require expensive
cofactors, such as NAD(H) or NADP(H) [21,22]. Cofactor regenera-
tion would be essential to overcome such oxidoreductive reaction
limitations. However, cofactor recycling can usually be achieved by
adding coupled enzyme acting to co-substrate for cofactor regener-
ation, such as formate dehydrogenase, glucose dehydrogenase, and
alcohol dehydrogenases [23–27]. In the one-pot deracemization,
there are both oxidation and reduction catalyzed by oxidore-
The cofactors including NAD(P)H and NAD(P)+, 2-
hydroxyacetophenone (2-HAP), (R)-PED and (S)-PED were
purchased from Sigma-Aldrich (St. Louis, USA). Hexane and
isopropanol used for high performance liquid chromatography
HPLC) were of chromatographic grade from Sigma-Aldrich (St.
Louis, USA). All other used chemicals were purchased from local
suppliers and were of analytical grade.
(
2.2. Microorganisms and culture conditions
All recombinant strains expressing the enzymes used in this
study were constructed previously in our laboratory (Table 1). The
recombinant strains were cultivated overnight in 4 mL LB liquid
−
1
◦
medium supplemented with 100 ␮g mL ampicillin at 37 C and
200 rpm. Then the culture was inoculated into a 250-ml Erlenmeyer
−
1
flask containing 50 mL fresh LB medium with 100 ␮g mL ampi-
cillin. When the OD600 nm increased to the level between 0.6 and
0.8, the expressions of target recombinant proteins were subjected
to the optimization of the following conditions, where 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 temperatures (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.
+
+
ductases. If the cofactor couple NAD /NADH or NADP /NADPH is
required alone and the two reactions have matchable reaction
rate, it would be possible to build a cofactor self-recycling system
between the two oxidoreductases (Fig. 1).
In this work, in order to obtain the suitable candidates for con-
struction of the cofactor self-recycling deracemization system, we
2.3. Purification of recombinant enzymes
The recombinant cells were cultivated under the optimum
expression conditions and then harvested by centrifugation and
washed twice with physiological saline. The bacterial pellet was
resuspended in the binding buffer (20 mM Tris-HCl, pH 7.0, 0.3 M

B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 129 (2016) 21–28
23
Table 2
Optimized conditions for expression of recombinant enzymes.
Construct
Expression conditions
Target protein yield
−
1 a
(mg L
)
◦
IPTG concentration (mM)
Lactose concentration (%)
Induction temperature ( C)
ADHR
C1
C2
CR2
CR4
KRD
OYE
RCR
SCR
1
1
1
1
0.5
0.5
1
1
0.5
1
1
1
1
1
1
1
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
0
0
2
17
17
17
17
17
17
17
17
30
17
17
17
30
17
17
17
17
17
17
17
2 ± 0.53
7 ± 0.70
12 ± 0.82
16 ± 0.40
14 ± 0.99
13 ± 0.14
23 ± 0.60
15 ± 0.78
22 ± 0.50
24 ± 0.55
57 ± 0.47
50 ± 0.53
2 ± 0.23
SCR1
SCR3
S1
CPAR1
CPAR2
CPAR3
CPAR4
CPAR5
CPAR6
CPAR7
CPAR8
40 ± 1.32
52 ± 0.74
42 ± 1.18
9 ± 0.73
4 ± 0.34
1
0
48 ± 0.88
5 ± 0.37
a
−1
The target protein yield (mg L ) was evaluated by calculating the amount of purified protein from the corresponding culture broth.
Fig. 2. SDS-PAGE analysis of purified recombinant enzymes. Lane M, molecular weight standard; lanes 1–20, purified recombinant enzyme ADHR, C1, C2, CR2, CR4, KRD,
OYE, RCR, S1, SCR, SCR1, SCR3, CPAR1, CPAR2, CPAR3, CPAR4, CPAR5, CPAR6, CPAR7, and CPAR8.
NaCl, 40 mM imidazole, 1 × protease inhibitors, 1 mM Tris (2-
carboxyethyl) phosphine (TCEP)) and disrupted on ice with an
ultrasonic oscillator (VCX750, Sonic). After centrifugation, the
supernatant was applied to a HisTrap HP affinity column (GE
Healthcare, USA) previously equilibrated with the binding buffer.
The C-terminal His-tag enzymes were eluted with the elution
buffer (20 mM Tris-HCl, pH 7.0, 0.3 M NaCl, 300 mM imidazole,
100 ␮L. One unit of enzyme activity was expressed as the amount
of enzyme required to catalyze oxidation of 1 ␮mol NAD(P)H or
reduction of 1 ␮mol NAD(P)+ per minute under the given assay
conditions. The decrease or increase in the amount of the coen-
zyme was measured spectrophotometrically at 340 nm (extinction
−
1
−1
coefficient [e] = 6.22 mM cm ). The protein concentration was
determined using Bradford reagents as a standard.
1
× protease inhibitors, 1 mM TCEP) after washing the column with
the binding buffer. Then the purified fractions were exchanged into
the low salt buffer (10 mM Tris-HCl, pH 7.0, 0.1 M NaCl, 0.02% NaN3,
2.5. Kinetic parameters
5
mM d,l-dithiothreitol) using disposable PD-10 desalting columns
Kinetic parameters of the purified enzyme were assayed by mea-
suring initial velocity at various concentrations of substrates [44].
To determine the apparent Km value, various concentrations of sub-
strate 2-HAP (0.05–0.4 mM) or (S)-PED (0.05–1 mM) were used. The
kinetic parameterswere further determinedfrom Lineweaver-Burk
plots.
(
GE Healthcare, USA) and used as the purified enzyme [43]. The
homogeneity of purified enzymes was judged by sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with 12%
polyacrylamide gels [43]. The amount of purified protein obtained
from the corresponding culture broth was used to evaluate the yield
of target protein. These final preparations of purified enzymes were
used in all of the experiments in this study.
2.6. Biotransformation and analytical methods
2.4. Enzyme assay
The biotransformation reductions of 2-HAP by the purified
enzymes were carried out at 30 C for 8 h with mild shaking in
◦
The oxidation or reduction activities of the recombinant
a 2-mL reaction mixture containing 0.1 M potassium phosphate
−
1
enzymes were measured by a continuous spectrophotometric
assay using the standard assay mixture containing 0.1 M potassium
buffer (pH 7.0), 1 g L substrate, 10 mM NAD(P)H, and the puri-
fied enzyme of appropriate. The reaction products were extracted
out using ethyl acetate and the organic layer was used for analy-
sis. The optical purity of (R)- or (S)-PED and the yield of (R)-PED
+
phosphate buffer (pH 7.0), 0.5 mM NAD(P)H or NAD(P) , 5 mM 2-
HAP or (S)-PED, and the appropriate enzyme in a total volume of

2
4
B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 129 (2016) 21–28
or 2-HAP were determined by HPLC (Agilent 1100) using a Chiral
OB-H column (4.6 mm × 250 mm; Daicel Chemical Inc., Ltd., Japan)
with hexane and 2-propanol (9:1) as the mobile phase, where the
concentration of PED enantiomer or 2-HAP was calculated from the
peak area with the standard curve [45].
2.7. Optimization of catalytic conditions for selected candidates
To determine the optimal conditions of asymmetric reduc-
tion by CR2 or KRD, the reaction parameters, including pH values
covering pH 6.0–8.0 (0.1 M potassium phosphate buffer) and tem-
◦
◦
peratures ranging from 25 C to 50 C, were investigated for their
effects on (R)-PED production from enzymatic asymmetric reduc-
tion of HAP. Additionally, the effect of metal ions, including CaCl2,
CuSO , FeCl , FeSO , MgSO , MnCl , NiSO , and ZnSO at the con-
4
3
4
4
2
4
4
centration of 1 mM, on KRD-catalyzed asymmetric reduction of
HAP was further investigated.
To determine the optimal conditions of (S)-PED oxidation by
SCR1, the reaction parameters, including pH values covering pH
6
.0–8.0 (0.1 M potassium phosphate buffer) and temperatures
◦
◦
ranging from 25 C to 50 C, were investigated for their effects on
HAP production from SCR1-catalyzed oxidation of (S)-PED.
2.8. Deracemization of (R,S)-PED by combination of SCR1 and
KRD
The biocatalytic deracemization of racemic (R,S)-PED was per-
formed by the following methods: a reaction mixture (2.0 mL)
−
1
containing 1 g L (R,S)-PED, 1 mM ZnSO , and the purified enzymes
4
◦
of SCR1 and KRD. The reaction was performed at 30 C and 200 rpm
for 8 h. The analytical methods were carried out as described above.
The reaction parameters, including activity ratio of SCR1 (20–100 U)
to KRD (20–100 U), molar ratio of NADP+ (0.05-0.2 mM) to NADPH
−
1
(
0.05–0.2 mM), and substrate concentration (1–5 g L ), were eval-
uated for the deracemization of (R,S)-PED to (R)-enantiomer.
3
. Results and discussion
3.1. Expression and purification of recombinant enzymes
To significantly increase expression yield of recombinant
enzymes, various factors involved in the expression of the target
proteins were optimized, including cultivation temperature from
◦
1
7 to 37 C and inducer of 0.1–1.0 mM IPTG or 2–6% lactose. Under
the optimized expression conditions involving varied temperatures
and inducer concentrations, the recombinant enzymes could be
obtained in soluble form with different expression levels and spe-
cific activities (Table 2). Of them, SCR3, S1, CPAR2, CPAR3, CPAR4,
and CPAR7 were expressed at somewhat higher yields, while ADHR,
C1, CPAR1, CPAR5, CPAR6, and CPAR8 gave relative lower levels of
target protein yield in heterologous expression (Table 2).
To avoid the interference from other oxidoreductases in recom-
binant cells, the purified enzymes were applied to construct the
one-pot deracemization system, instead of using crude enzymes.
Then the recombinant enzymes with His-tag at C-terminal were
purified by nickel affinity chromatography, showing distinct bands
on SDS-PAGE, with the sizes in agreement with the theoretically
calculated molecular weights (Fig. 2).
Fig. 3. Optimization of catalytic conditions of 2-HAP reduction by KRD or CR2 and
PED oxidation by SCR1. The enzymatic reactions were conducted under the different
reaction parameters: (A) 0.1 M potassium phosphate buffer at various pH values (pH
3
.2. Selection of suitable enzymes for HAP reduction or (S)-PED
◦
6.0–8.0), (B) temperatures (25–50 C), and (C) 1 mM metal ion (CaCl2, CuSO4, FeCl3,
oxidation
FeSO4, MgSO4, MnCl2, NiSO4, or ZnSO4). After reaction for 8 h, the reaction mixtures
were centrifuged and the concentrations of PED and 2-HAP in the supernatant were
analyzed by HPLC. Results were the average of three parallel replicates.
The reduction activities of the recombinant enzymes were
investigated toward 2-HAP. The available enzymes performed
catalytic activities with NADPH as the coenzyme, but no obvi-
ous activities with NADH, except for the NADH-dependent RCR

B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 129 (2016) 21–28
25
Table 3
Activities of the recombinant enzymes catalyzing 2-HAP reduction and (S)-PED oxidation, respectively.
Specific activity (␮mol min 1 mg )/Product configuration and optical purity (%e.e.)a
−
−1
Oxidation of (S)-PEDb
Enzyme
Reduction of 2-HAP
ADHR
C1
C2
CR2
CR4
KRD
OYE
RCR
SCR
–
0.08 ± 0.01
0.61 ± 0.08/-
–
–
0.09 ± 0.01
2.21 ± 0.31/64.68 ± 0.51(R)
1.34 ± 0.01/62.37 ± 0.24(S)
3.77 ± 0.26/94.08 ± 1.95(R)
0.49 ± 0.03/-
–
–
–
–
1.15 ± 0.09/ > 99.90(R)
0.30 ± 0.03/98.29 ± 0.41(S)
0.55 ± 0.06/99.10 ± 0.81(S)
2.23 ± 0.41/98.46 ± 0.23(S)
1.20 ± 0.13/99.21 ± 0.31(S)
0.57 ± 0.10/ > 99.90(R)
0.63 ± 0.03/ > 99.90(S)
0.98 ± 0.04/16.27 ± 0.09(R)
1.08 ± 0.12/ > 99.90(R)
3.19 ± 0.25/46.30 ± 0.08(R)
0.73 ± 0.11/-
–
0.02 ± 0.01
SCR1
SCR3
S1
0.97 ± 0.09
0.07 ± 0.01
0.40 ± 0.06
CPAR1
CPAR2
CPAR3
CPAR4
CPAR5
CPAR6
CPAR7
CPAR8
–
–
0.27 ± 0.02
–
–
–
–
–
1.29 ± 0.06/22.25 ± 0.11(R)
1.07 ± 0.13/ > 99.90(R)
a
Measurements of product configuration and optical purity were only carried out for PED enantiomers produced from 2-HAP reduction, but not applied to the product of
(
S)-PED oxidation.
b
The activities of the recombinant enzymes toward (S)-PED were only shown as specific activity.
[
28,31,34]. Hence, most of the recombinant enzymes had the
optical purity (94.08%e.e.) and yield (80.04%) of the product were
◦
possibility to built a cofactor self-recycling system using the cofac-
tor couple NADPH/NADP+ between the two oxidoreductases. As
shown in Table 3, the recombinant enzymes used in this work had
different capability of catalyzing reduction of 2-HAP, where the
enzymes CR2, KRD, CPAR4, CPAR5, CPAR7, and CPAR8 catalyzed
the reduction of 2-HAP to produce (R)-PED with high optical purity.
Consequently, based on the results of activity and selectivity, KRD
and CR2 were selected as the candidates responsible for the asym-
metric reduction of 2-HAP in deracemization.
The oxidation activities of the recombinant enzymes were
investigated toward (S)-PED. Of the tested enzymes, ADHR, C2, SCR,
SCR1, SCR3, S1, and CPAR3 exhibited the activity to catalyze the
oxidation of (S)-PED and produce 2-HAP (Table 3). Of the screened
enzymes, SCR1 performed the highest activity for catalyzing oxi-
dation of (S)-PED to produce the corresponding 2-HAP, while still
possessed weak ability of reversibly catalyzing 2-HAP reduction
with NADPH at pH 7.0. The enzyme exhibited high stereoselectivity
toward (S)-PED (99.10%e.e.) but no activity toward (R)-PED. There-
fore, considering the main reaction direction, SCR1 was selected as
the suitable enzyme to catalyze oxidation of (S)-PED in deracem-
ization.
both achieved at 30 C for KRD catalyzing asymmetric reduction of
◦
2-HAP. However, CR2 was more stereospecific at 30 C, giving the
optical purity of 68.64%e.e. for the product, but was more active at
◦
35 C, showing the product yield of 33.30%. Therefore, concerning
the catalytic efficiency, KRD was determined as the suitable enzyme
catalyzing the asymmetric reduction of 2-HAP to (R)-PED, i.e. the
second step in the whole reaction process of deracemization.
Based on the above results on condition optimization, the effect
of metal ion on the KRD-catalyzed bioreduction was investigated
by using CaCl , CuSO , FeCl , FeSO , MgSO , MnCl , NiSO , and
2
4
3
4
4
2
4
ZnSO (Fig. 3C). By adding ZnSO , the yield of product was obviously
4
4
improved to 92.13% with the optical purity of 95.61%e.e., indicating
that Zn2+ would be useful to activate KRD and enhance its activity
in catalyzing reaction. Thus, for KRD responsible for the reduction
of 2-HAP, the reaction would be carried out in 0.1 M potassium
◦
phosphate buffer (pH 7.0) comprising 1 mM ZnSO4 at 30 C.
For the oxidation of (S)-PED to 2-HAP, the first step in the
deracemization process, the catalytic conditions of SCR1 were opti-
mized as described above with minor modifications. According to
the reaction route of SCR1 catalyzing oxidation, using the cofactor
+
of NADP , (S)-PED was the substrate to produce prochiral 2-HAP
without optical activity. As shown in Fig. 3A and B, corresponding
to the optimized conditions of KRD, SCR1 gave higher yield of 2-
HAP at pH 7.0 and 30 C, which would make it feasible to construct
3.3. Optimization of catalytic conditions for candidate enzymes
◦
the enzyme-coupled system catalyzing deracemization involving
tandem oxidation and reduction.
For the reduction of 2-HAP involved in deracemization, the reac-
tion conditions were optimized for the selected enzymes, KRD and
CR2, respectively. The effect of pH value on the bioreduction was
◦
investigated with the purified enzyme at 30 C for 8 h, in 2-mL reac-
−
1
tion mixture comprising 1 g L 2-HAP, 10 mM NADPH, and 0.1 M
potassium phosphate buffer with the pH values ranging from 6.0
to 8.0. As shown in Fig. 3A, pH value of the reaction mixture had a
significant effect on the enzyme-mediated reductions of 2-HAP. For
KRD, the optimum pH value was determined as pH 7.0, at which the
highest e.e. value and yield of the product were achieved as 94.08%
and 80.04%, respectively, while CR2 did not exhibited enhanced
catalytic efficiency in the tested pH range. The effect of reaction
temperature on the bioreduction was investigated at various tem-
3.4. Deracemization of (R,S)-PED by combination of SCR1 and
KRD
Based on the results of enzyme screening and catalytic condi-
tions optimization, the positive features of SCR1 and KRD provided
the solid basis for combining these two enzymes to construct a
one-pot system for the deracemize of (R,S)-PED. Using KRD and
SCR1 as the catalysts, the deracemization reaction was carried out
under the optimized conditions, involving 0.1 M potassium phos-
◦
◦
◦
peratures covering 25 C to 50 C, with the optimized pH 7.0 for KRD
and pH 7.5 for CR2, respectively. As shown in Fig. 3B, the highest
phate buffer (pH 7.0) with the addition of 1 mM ZnSO at 30 C. Then
4
the composition of the multi-enzyme system was further investi-

2
6
B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 129 (2016) 21–28
Fig. 4. Optimization of reaction conditions of deracemization of PED racemate by coupled enzymes of SCR1 and KRD. The enzymatic reactions were conducted under the
+
different reaction parameters including (A) the activity ratio of SCR1/KRD, (B) the molar ratio of NADP /NADPH, (C) the concentration of (R,S)-PED, and (D) the reaction time.
After reaction for 8 h, the reaction mixtures were centrifuged and the concentrations of PED in the supernatant were analyzed by HPLC. Results were the average of three
parallel replicates.
Table 4
gated concerning the ratio of the two enzymes as well as the type
Specific activity and kinetic parameters of the enzymes involved in the deracemiza-
tion process .
and concentration of the cofactor.
a
For the two coupled enzymes sequentially catalyzing tandem
Enzymeb
Specific
activity
(
Km
(mM)
kcat
kcat/Km
( × 10
reactions of the one-pot deracemization, it would be necessary to
keep the key enzymes working in a compatible and synergistic way.
In this study, the reaction process of deracemization was firstly
conducted by controlling the activity ratio between the oxidation-
catalyzing SCR1 (20–100 U) and the reduction-catalyzing KRD
(s−1)
3
−1 −1
s M )
U mg−1)
KRD2-HAP
SCR1(S)-PED
5.22 ± 0.32 0.27 ± 0.02 0.92 ± 0.03 3.35
1.05 ± 0.13 0.50 ± 0.03 1.06 ± 0.04 2.11
a
Enzymatic activities involved in the calculation of kinetic constants were
(
20–100 U). As show in Fig. 4A, when the SCR1/KRD ratio was higher
assayed in 0.1 M potassium phosphate buffer (pH 7.0) comprising 1 mM ZnSO4 at
than 1:2, the yield and e.e. value of (R)-PED were both below 60%.
However, when the SCR1/KRD ratio was 1:4, the yield and optical
purity of the product reached 86.42% and 85.31%e.e., respectively.
Since SCR1 had weak ability to catalyze 2-HAP reduction with
NADPH at pH 7.0, i.e. the reverse reaction of the first oxidation
step in deracemization, strengthening KRD catalyzing reduction
would be more beneficial to drive the whole deracemization to
produce the final product. Table 4 showed the comparison of spe-
cific activity and kinetic parameters between SCR1 and KRD. Under
the assay conditions, the specific activity of KRD was almost five
times as much as that of SCR1, although the kcat values of these two
enzymes were in a similar level. Therefore, when the activity ratio
of SCR1/KRD was 1:4 or lower, the oxidation of PED and reduction
of 2-HAP by the coupled system containing SCR1 and KRD would
keep the reaction balance toward the production of (R)-PED.
Due to the requirement and restriction of necessary cofac-
tor, addition and regeneration of corresponding cofactors would
obviously improve the efficiency of oxidoreductase-catalyzed
cofactor-dependent reactions [39,46–48]. To improve the deracem-
ization efficiency, the coupled enzymes catalyzing oxidoreduction
was further optimized by adding the necessary cofactors of NADP+
◦
3
0
C.
b
KRD2-HAP means the reduction-catalyzing KRD with 2-HAP as substrate and
SCR1(S)-PED means the oxidation-catalyzing SCR1 with (S)-PED as substrate. For activ-
+
ity assay to calculate kinetic parameters, the cofactor of NADPH or NADP was added
in the reaction mixture at saturated concentration toward the enzyme.
and NADPH (0.05–0.2 mM) at various molar ratios (Fig. 4B). Since
both KRD and SCR1 were strictly cofactor-dependent enzymes,
using purified enzymes for catalyzing the corresponding reac-
tions, the reaction was not carried out without the addition of
+
the required cofactors. When both NADP and NADPH were added
in the reaction mixture, the production of (R)-PED was obviously
+
varied due to the difference of the NADP /NADPH ratio. When
+
the NADP /NADPH ratio was higher than 1:1, the yield and e.e.
value of the product could be both achieved over 80%. At the
+
NADP /NADPH ratio of 3:1, (R)-PED was produced from enzyme-
coupled one-pot deracemization at the yield of 91.62% and the
+
optical purity of 95.50%e.e. However, by only adding NADP (the
+
NADP /NADPH ratio at 1:0) to initiate the recycling of the nec-
essary cofactor, the production of (R)-PED was much lower than
that of adding both NADP+ and NADPH. In the reaction process,

B. Li et al. / Journal of Molecular Catalysis B: Enzymatic 129 (2016) 21–28
27
SCR1 was responsible for catalyzing the first step of (S)-PED oxida-
tion with NADP+ as the necessary cofactor, while KRD catalyzed
the second step of 2-HAP reduction to produce (R)-PED using
NADPH as the cofactor. Since SCR1 with NADPH was also capa-
as solvent engineering or in situ extraction strategy, it would be
possible to increase the optical purity of (R)-PED to a very high
level.
ble of catalyzing the reverse reaction of the first oxidation step, i.e.
4. Conclusion
+
2
-HAP reduction to (S)-PED, more NADP would be necessary to
initiate the first step of (S)-PED oxidation in the whole deracem-
ization. Additionally, KRD exhibited higher specific activity and
catalytic efficiency than SCR1 (Table 4), and moderate increase of
Through the systematic evaluation of 20 unique stereoselective
oxidoreductases concerning the features including activity, selec-
tivity, and cofactor dependency, SCR1 and KRD were selected to
construct the multi-enzyme system for one-pot deracemization
involving cofactor self-recycling. For this enzyme-coupled sys-
tem, reaction temperature, pH value, proportion of SCR1/KRD, and
+
NADP could strengthen SCR1-catalyzed (S)-PED oxidation. There-
fore, a cofactor self-recycling system was formed by the coupled
+
enzymes, and suitable ratio of NADP /NADPH would keep the bal-
ance between NADP(H)-dependent SCR1 and NADPH-dependent
KRD in catalyzing tandem reactions and be helpful to drive the
reaction for (R)-PED production.
+
NADP /NADPH ratio were found to significantly affect the yield and
optical purity of the desired product. Under the optimized con-
ditions, optically active (R)-PED was produced with the yield of
In addition, the effect of the concentration of (R,S)-PED on
the deracemization was further investigated (Fig. 4C). The opti-
cal purity of the product could be maintained over 90%e.e. when
the substrate concentration was increased to 4 g L , while the
yield decreased below 80% when the substrate concentration was
9
1.62% and the optical purity of 95.50%e.e. with the activity ratio
+
of SCR1/KRD 1:4 and the molar ratio of NADP /NADPH 3:1 at pH
◦
7
.0 and 30 C for 8 h. Thus, de novo construction of multi-enzyme
−
1
system was achieved for one-pot deracemization of racemic PED to
R)-PED. The strategy developed in this work would be promising
(
−1
−1
more than 3 g L . For the substrate concentration of 3 g L , (R)-
PED was produced with the yield of 88.29% and the optical purity
of 92.60%e.e. by using a fixed concentration of coupled enzymes.
Under the optimized conditions, the coupled enzymes were both
active of catalyzing the deracemization of racemic PED to produce
to establish an efficient multi-enzyme system for certain biocat-
alytic reaction route via systematic evaluation and combination of
functional enzymes as well as optimization of reaction conditions.
Acknowledgements
(
R)-PED and the stabilities of the enzymes were less affected dur-
ing the reaction process. Therefore, as the general phenomenon
of bio-mediated reactions [47], the effect of substrate concentra-
tion on the reaction efficiency, especially the yield of (R)-PED, was
mainly due to the inhibition of the substrate at higher concentration
toward the enzymes. Since the deracemization reaction was con-
ducted with certain concentrations of the enzymes, it was expected
to raise the substrate concentration by increasing the amounts of
the purified enzymes in the reaction system [49].
Financial supports from the National Natural Science Founda-
tion of China (NSFC) (21376107, 21336009), the Natural Science
Foundation of Jiangsu Province (BK20151124), the 111 Project
(
(
111-2-06), the High-end Foreign Experts Recruitment Program
GDT20153200044), the Program for Advanced Talents within
Six Industries of Jiangsu Province (2015-NY-007), the National
Program for Support of Top-notch Young Professionals, the Funda-
mental Research Funds for the Central Universities (JUSRP51504),
the Project Funded by the Priority Academic Program Development
of Jiangsu Higher Education Institutions, and the Jiangsu province
Based on the above optimized conditions concerning the com-
positions of functional enzymes and cofactors, the deracemization
process was investigated by measuring the change of amounts of
the substrate and the product with the reaction time. As shown
in Fig. 4D, the concentration of (S)-PED in the racemic substrate
was reduced gradually and on the contrary, that of (R)-PED was
in an increasing trend, where (S)-PED was converted into (R)-PED
by the coupled enzymes, SCR1 and KRD, catalyzing stereoinversion
involving selective oxidation and asymmetric reduction. When the
reaction was carried out for 8 h, (R)-PED with the yield of 91.62% and
the optical purity of 95.50%e.e. was achieved under the conditions
including the activity ratio of SCR1/KRD 1:4 and the molar ratio of
“Collaborative Innovation Center for Advanced Industrial Fermen-
tation” industry development program are greatly appreciated.
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