Marked improvement in the asymmetric reduction of 2-hydroxyacetophenone with mut-AcCR in a biphasic system

Article abstract of DOI:10.1016/j.mcat.2020.110903

(S)-1-Phenyl-1,2-ethanediol (PED) is a vital chiral block in specialty chemical industries. The biotransformation of 2-hydroxyacetophenone (2-HAP) to (S)-PED was conducted successfully catalyzed with BL21(DE3)(pETDuet-gst-mut-accr-gdh) which harboring a c

Full text of DOI:10.1016/j.mcat.2020.110903

MolecularCatalysis488(2020)110903
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Molecular Catalysis
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Marked improvement in the asymmetric reduction of 2-
hydroxyacetophenone with mut-AcCR in a biphasic system
Ping Weia,b, Peng Chaoa,b, Yao-Ying Wanga, Dong-Li Lia, Qing-Jian Zoua, Min-Hua Zongb,
Wen-Yong Loub,*
a School of Biotechnology and Health Sciences, Wuyi University, Jiangmen 529020, Guangdong, China
b Lab of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
(S)-1-Phenyl-1,2-ethanediol (PED) is a vital chiral block in specialty chemical industries. The biotransformation
of 2-hydroxyacetophenone (2-HAP) to (S)-PED was conducted successfully catalyzed with BL21(DE3)(pETDuet-
gst-mut-accr-gdh) which harboring a carbonyl reductase mutant (mut-AcCR). The catalytic activity of the bio-
catalyst was 15.9 times higher than the recombinant cells harboring original AcCR. 250 mM 2-HAP was reduced,
under optimal conditions, and the yield and space-time yield were 95.2 % and 1.89 M/d in the buffer system.
Then C4MIMI·PF6 was chosen to be the second phase to improve the catalytic efficiency, and the substrate
concentration was increased to 450 mM in the C4MIMI·PF6/buffer system. After a reaction duration of 3 h, the
product yield was over 92 %, and the space-time yield increased to 2.88 M/d which was 1.52 folds higher than
that in the buffer system. Also, the product e.e. was over 99 % consistently. Overall, the preparative scale
reduction of 2-HAP (450 mM) in C4MIMI·PF6/buffer system was conducted with promising result. The developed
effective C4MIMI·PF6/buffer system of asymmetric reduction catalyzed by BL21(DE3)(pETDuet-gst-mut-accr-gdh)
will promote the preparation of the enantiomeric pureity chiral alcohol at industrial scale.
Carbonyl reductase
Asymmetric reduction
(S)-1-phenyl-1,2-ethanediol
Biocatalysis
Introduction
parapsilosis can catalyze the conversion of 2-HAP to (S)-PED with high
product e.e. (97.4 %) and a yield of 95.2 % after 6 h reaction [16].
The enantiomeric purity chiral alcohols are valuable and widely
used chiral building blocks for the chemical synthesis [1–3]. Therefore,
various synthetic methods have been developed to achieve optically
pure chiral alcohols [4–8], among which biocatalysis represents an
efficient method to produce chiral alcohols from carbonyl compounds
in an environmentally friendly and sustainable process for the chemical
and pharmaceutical industries [9–11]. The enantiomerically pure PED
is a versatile chiral intermediate. (R)-PED can be used to prepare the
antidepressant drug, fluoxetine. (S)-PED is used as a precursor for the
synthesis of biphosphines, which can be used as ligands in the cationic
rhodium complexes [12]. (S)-PED can also be used as chiral initiator for
stereoselective polymerization [13,14]. Carbonyl reductase and whole-
cell can be used to reduce 2-hydroxyacetophenone (2-HAP) to obtain
the enantiomerically pure PED. Zhou et al. developed the biocatalytic
synthesis of (R)-PED catalyzed by (R)-carbonyl reductase (RCR) from
Candida parasilosis with 6 g/L (≈44 mmol/L) 2-HAP in the buffer
system. After a reaction duration of 24 h, the product yield and the
product e.e. were both over 99 % [15]. (S)-CR II from Candida
Additionally, two carbonyl reductases with complementary stereo-
selectivity, BDHA (from Bacillus subtilis) and GoSCR (from Glucono-
bacter oxydans) were expressed in E.coli for the transformation of 2-HAP
to (S)-PED and (R)-PED with outstanding stereochemical selectivity.
The asymmetric reduction of about 400 mM 2-HAP could be conducted
by the two E.coli strains to obtain optically pure PED with 99 % yield
and product e.e. [17]. However, several limiting factor still exist in
many biocatalytic processes, for example, the large additions of coen-
zyme [NAD(P)H], substrate toxicity to the microbial cells, high loading
of the biocatalyst and the long reaction time, etc. Therefore, it is ne-
cessary to seek more oxidoreductase with excellent catalytic properties
to conduct the asymmetric reduction of 2-HAP in biocompatible system.
The reduction process catalyzed by the carbonyl reductase mostly re-
quires a mass of expensive coenzyme (NADH or NADPH), and the high
cost of coenzyme would be restricted the application of the carbonyl
reductase for the asymmetric reduction to obtain the chiral compounds.
Recently, many ways have been investigated for the regeneration of
coenzyme in the catalytic processes [18–21]. In situ coenzyme
⁎ Corresponding author.
E-mail address: wylou@scut.edu.cn (W.-Y. Lou).
https://doi.org/10.1016/j.mcat.2020.110903
Received 24 October 2019; Received in revised form 22 February 2020; Accepted 19 March 2020
Availableonline13April2020
2468-8231/©2020PublishedbyElsevierB.V.

P. Wei, et al.
MolecularCatalysis488(2020)110903
regeneration conducted by the enzyme-coupled system, which can re-
duce or avoid the addition of extra coenzyme and predigest the reaction
process, is especially preferred for biocatalysis on account of its ex-
cellent selectivity and high-efficiency [22–24].
of Chemical Physics. Ampicillin was bought from Sangon Biotech,
whereas the PCR primers were synthesized by Sangon Biotech.
Medium and cultivation
It has been reported many of the substrates and/or products have
obviously inhibitory or toxic effects on the microbial cells in the single
phase, which leads to a low productivity of the microbial cells [25–27].
Normally, a biphasic system can be used to overcome these challenges
[28–30], where the buffer phase contains the microbial cells and the
nonaqueous phase serves as a storage for the substrate or product.
In our previous study, a carbonyl reductase AcCR from Acetobacter
sp. CCTCC M209061 was characterized, and AcCR possessed the ability
to catalyze the asymmetric reduction of different prechiral compounds,
including 2-HAP with excellent stereoselectivity [31]. AcCR was mod-
ified by the site-directed mutation and the specific activity of the mu-
tant AcCR (mut-AcCR) reached 6.4 U/mg, which was 17.4-fold higher
than the wild type AcCR [32]. Therefore, it would be of great interest to
study the asymmetric reduction of 2-HAP catalyzed with the re-
combinant E.coli harboring mut-AcCR. In this study, to improve the
catalytic efficiency of 2-HAP and achieve coenzyme recycle, the E.coli
coexpressed mut-AcCR (I147 V/G152 L) gene and glucose dehy-
drogenase gene (gdh) were used to systematically investigate the
asymmetric reduction of 2-HAP to obtain (S)-PED systematically. A
biphasic system was introduced to further improve the substrate con-
centration and catalytic efficiency (Scheme 1). Moreover, the efficient
biocatalytic process in the hydrophobic ionic liquid (IL)/buffer system
for the 2-HAP reduction to (S)-PED was carried out on the preparative
scale.
Luria-Bertani (LB) medium was used to culture the recombinant
E.coli. At first, the strains were cultured at 37 °C. Once the culture
concentration met the requirement (OD600 = 1.2), the culture tem-
perature was reduced to 20 °C, Subsequently, isopropyl-β-D-thioga-
lactoside (IPTG, final concentration 0.4 mM) was added, and continued
to be cultured at 20 °C for 15−18 h. On completion of the culturing
process, the cells were collected at 4 °C by centrifugation (8000 rpm, 5
min) and using physiological saline (0.85 %) washed three times.
Further, the cells were freeze-dried, and stored at −20 °C for later use.
General procedure for the asymmetric reduction of 2-HAP in the buffer
system
The general procedure was as follows, 4 mL citrate-phosphate buffer
(pH 6.5, 200 mM) containing 100 mM 2-HAP, 200 mM glucose and 10
mg-dcw/mL BL21(DE3)(pETDuet-gst-mut-accr-gdh) formed the reaction
system. The mixture was shaken at 200 rpm and 35 °C in an air-bath
shaker, and the samples (20 μL×2) were withdrawn regularly for the
HPLC (Agilent 1260 Series) analysis. One sample was diluted by adding
the purified water before filtering with the filter membrane (0.22 μm),
followed by the HPLC analysis. Another sample was extracted with 600
μL (30 × 20 μL) ethyl acetate for the analysis of the e.e. value by HPLC.
The pH value of the buffer system was adjusted by batch-feeding the
NaHCO3 powder.
Several parameters, i.e. temperature, buffer pH and concentration of
substrate, glucose and cells, were studied with respect to the general
procedure to efficiently synthesize (S)-PED from 2-HAP.
Material and methods
Biological and chemical materials
DH5α, BL21(DE3) and plasmids pETDuet-1 were bought from
Novagen. The restricted enzymes including FastDigest BglII and
FastDigest XhoI, T4 DNA Ligase, DNA and protein marker were bought
from Thermo. pET-gst-accr-gdh and pGEX-mut-accr were constructed in
our previous study [21,32]. The kits used to construct the recombinant
plasmids were purchased from TaKaRa. 2-HAP, (R)-PED, (S)-PED and
PED were bought from Aladdin. 1-butyl-3-methylimidazolium hexa-
fluoro phosphate (C4MIMI·PF6) was purchased from Lanzhou Institute
Procedure for the asymmetric reduction of 2-HAP to (S)-PED in the biphasic
system
In a typical experiment, 2.0 mL of citrate-phosphate buffer (200
mM, pH 6.5) including dry cells (15 mg/mL, concentration based on the
whole system, similarly hereinafter) and a predetermined quantity of
glucose (glucose/2-HAP = 1.5/1) were added to a flask in duplicate,
and pre-incubated at 200 rpm and 35 °C for 10 min in bath shaker. The
Scheme 1. The asymmetric reduction of 2-HAP to (S)-PED catalyzed by recombinant BL21(DE3)(pETDuet-gst-mut-accr-gdh) cells in the C4MIMI·PF6/buffer system.
2

P. Wei, et al.
MolecularCatalysis488(2020)110903
reaction was initiated by adding the hydrophobic phase containing 200
mM 2-HAP. Aliquots (2 × 20 μL) were taken at specified time intervals
from the biphasic system. The sample from the buffer phase was diluted
by adding the purified water before filtering with the 0.22 μm filter
membrane, whereas the sample from the hydrophobic phase was di-
luted using acetonitrile or ethyl acetate for HPLC analysis. The pH value
of the buffer system was adjusted by batch-feeding the NaHCO3
powder.
Table 2
Enzymatic activity of recombinant BL21 (DE3).
Number
Strain
AcCR activity
(U/g-dw)
A
B
BL21(DE3)
(pETDuet-gst-accr-gdh)
BL21(DE3)
26.5 0.7
420.9 4.8
(pETDuet-gst-mut-accr-gdh)
Condition: 2 mL citrate phosphate buffer (100−200 mM, pH 6.5) containing 50
mM 2-HAP, 50 mM glucose, 10 mg-dcw/mL cells, 30 °C,180 rpm.
Biocatalytic reduction of 2-HAP on the preparative scale
The biocatalytic reduction of 2-HAP on preparative scale with
whole-cell BL21(DE3)(pETDuet-gst-mut-accr-gdh) was conducted by
adding 1.5 g dry-cell of BL21(DE3)(pETDuet-gst-mut-accr-gdh) (15 mg-
dcw/mL) and 675 mM glucose (675 mM, stage addition) in 50 mL
buffer phase as well as 450 mM 2-HAP to 50 mL IL phase, followed by
the execution of the reaction at 200 rpm and 35 °C. Subsequently, the
samples were analyzed by HPLC. The pH value of buffer system was
adjusted by batch-feeding the NaHCO3 powder.
respectively. It indicated that the substrate coupling for the coenzyme
regeneration with isopropanol did not provide NADH fast enough for
the reduction process. The catalytic efficiency based on the enzyme-
coupled coenzyme regeneration overmatched that of the substrate
coupling method. The initial reaction rate was 3.0 mM/min, which was
almost 2 times higher than that of the coenzyme regenerated by iso-
propanol, along with an excellent yield (90.7 %). Therefore, the coex-
pressed strain harboring mut-AcCR and GDH gene had higher catalytic
efficiency, which was chosen to be used to the follow-up study.
The catalytic activity of the recombinant E. coli harboring wild-type
AcCR and mut-AcCR was analyzed for the reduction of 2-HAP. As
shown in Table 2, the catalytic activity of strain A was only 26.5 0.7
U/g-dcw, however the stain B exhibited an activity of 420.9 4.8 U/g-
dcw, which was 15.9 times as compared to the wild-type one.
HPLC analysis
The substrate and product concentration were analyzed by HPLC
(Agilent 1260 Series) with UV detection at 245 nm and 215 nm using
C18 column (Waters, XBridge™ C18, 5 μm, 4.6 φ × 250 mm). The
mobile phase was water/acetonitrile (3/2, v/v, 0.1 % trifluoroacetic
acid in water phase) and its flow rate was 0.5 mL/min at a column
temperature of 35 °C. The retention durations for 2-HAP and PED were
7.80 and 6.24 min, respectively.
Effect of the critical variables on the reduction of 2-HAP to (S)-PED with
BL21(DE3)(pETDuet-gst-mut-accr-gdh) cells in the buffer system
The e.e. value of (S)-PED was analyzed by HPLC (Agilent 1100
Series) with UV detection at 215 nm using OB-H column (Daicel, 4.6
mm × 150 mm, 5 μm). The mobile phase was the mixture of 2-propanol
and n-hexane (1/9, v/v) and its flow rate was 0.7 mL/min at a column
temperature of 35 °C. The retention time for (R)-PED and (S)-PED was
8.51 and 10.21 min, respectively.
To investigate the asymmetric bioreduction of 2-HAP in detail and
to achieve an improvement in the initial reaction rate and yield, a
systematic study about the effect of the important variables was made.
The buffer pH plays a vital role in the biocatalytic reduction process.
The buffer pH has an effect on the activity and selectivity of the bio-
catalyst, along with coenzyme regeneration, which have an inverse on
the reduction rate [34]. Therefore, an optimum pH range needed for the
reduction process. As presented in Fig. 1a, as the buffer pH increased
from 5.0 to 6.0–6.5, both the reaction rate and yield were observed to
enhance. As the buffer pH reached 7.0, both the initial reaction rate and
yield were noted to drop. There was an insignificant change in the
product e.e. on changing the buffer pH. Obviously, the optimal buffer
pH for the reduction of 2-HAP lied in the range 6.0−6.5. At the pH
range, the initial reaction rate was 2.97 mM/min with a yield of about
90 %.
The coenzyme regeneration relies on the oxidation of glucose, cat-
alyzed by GDH, which can couple with the reduction reaction catalyzed
by the carbonyl reductase. The oxidation process produced D-Gluconic
acid, which can bring down the buffer pH of the reaction system. As
shown in Fig. 1b, the buffer pH exhibited a significant drop accom-
panied by an increase in the D-Gluconic acid concentration during the
reaction progress. Owing to the simultaneous production of D-Gluconic
acid, the buffer pH decreased to ∼ 4.5 after a reaction duration of 4 h.
As shown in Fig. 1a, the buffer pH influenced the initial reaction rate
and yield. The buffer pH was controlled by batch-feeding of the alka-
lescent carbonate (NaHCO3) to the system. The result was remarkable
as the maximum product yield reached 94.1 % after feeding NaHCO3 to
The initial reaction rate, yield and e.e. value of these reactions were
calculated as described in our previous study [27].
The gluconic acid concentration was analyzed indirectly by de-
tecting sodium gluconate, as reported previously (as the sodium glu-
conate concentration was equal to that of gluconic acid) [33]. NaHCO3
was added to the reaction solution until the pH of the mixture was
about 10. The sodium gluconate concentration was analyzed using the
XDB-C18 column (Agilent, 4.6 mm × 250 mm, 5 mm) by HPLC. The
mobile phase was the mixture of water, methanol and 1.0 % H3PO4
solution (45/ 5/ 50, v/v/v), and its flow rate was 0.6 mL/min. The
retention duration of sodium gluconate was 5.7 min.
Results and discussion
Effect of coenzyme recycling on the asymmetric reaction of 2-HAP catalyzed
by recombinant BL21(DE3)
The coenzyme recycling is an important influencing factor for the
asymmetric reduction catalyzed by the whole-cell. As shown in Table 1,
different coenzyme regenerations were investigated, and the initial
reaction rate and yield were observed to be 1.6 mM/min and 62.6 %,
Table 1
Effect of coenzyme recycling on the asymmtric reaction of 2-HAP catalyzed by recombinant BL21(DE3).
Cosubstrate
(200 mmol/L)
Strain
Initial reaction rate
Yield
(%)
e.e
(%)
Config-uration
(mmol L−1 min−1
)
Isopropanol
Glucose
BL21(DE3) (pETDuet-gst-mut-accr)
BL21(DE3) (pETDuet-gst-mut-accr-gdh)
1.6
3.0
62.6
90.7
> 99
> 99
S
S
Reaction condition: 2 mL citrate phosphate buffer (100−200 mM, pH 6.5) containing 100 mM 2-HAP, 200 mM glucose, 10 mg-dcw/mL cells, 35 °C, 200 rpm.
3

P. Wei, et al.
MolecularCatalysis488(2020)110903
Fig. 1. Effect of buffer pH on the asymmetric reduction of 2-HAP catalyzed by recombinant BL21(DE3). Reaction condition: a) 2 mL citrate phosphate buffer
(100-200 mM, pH 5.0-7.0) containing 100 mM 2-HAP, 200 mM glucose, 10 mg-dcw/mL cells, 35 °C, 200 rpm. b) 2 mL citrate phosphate buffer (100-200 mM, pH 6.5)
containing 100 mM 2-HAP, 200 mM glucose, 10 mg-dcw/mL cells, 35 °C, 200 rpm.
achieve the buffer pH of 6.5.
equivalent value was obtained as the 2-HAP concentration reached 250
mM. Further increasing the concentration of substrate led to a sig-
nificant drop in the relative activity (6.4 %–41.3 %), possibly due to the
growing inhibiting effect of 2-HAP towards the cells. Therefore, a
second phase could be introduced to relieve the inhibitory effect.
In a bio-catalyzed reaction, the cell concentration is of significant
importance for the highly efficient completion of the bioreduction
process. Therefore, the cell concentration was investigated for
achieving an efficient reaction and catalyst savings, in order to achieve
more economically competitive process. The initial reaction rate in-
creased as the cell concentration was enhanced up to 20 mg-dcw/mL
(V0 = 4.91 mmol/L/min). Further increasing the concentration led to a
minor growth in the initial reaction rate (Fig. S2). In addition, the
product yield reached to 94.7 % for the cell concentration of 15 mg-
dcw/mL with the initial reaction rate reaching to 4.22 mM/min. From
an economic standpoint, the recombinant cell concentration of 15 mg-
dcw/mL was sufficient for the bioreduction of 2-HAP to (S)-PED.
Coenzyme regeneration also plays a crucial part in the reduction
process. In our study, glucose was used as cosubstrate for the coenzyme
regeneration by the oxidation process catalyzed with GDH. As shown in
Fig. S3, as the molar ratio of glucose and 2-HAP reached 1.5, the yield
was observed to be over 94 %, and the initial reaction rate was about
4.2 mM/min. Further increase in the ratio had an insignificant effect on
the corresponding value. Hence, the optimal ratio of glucose and 2-HAP
was noted to be 1.5.
The reaction temperature can also affect the stereoselectivity, sta-
bility and activity of a biocatalyst, as well as the reaction equilibrium.
As shown in Fig. S1, the product yield was over 92 % for the reaction
temperature in the range 25−40 °C. However, the initial reaction rate
was slow for this temperature range (25−30 °C). The maximum initial
reaction rate reached 2.97 mM/min for the reaction temperature of 35
°C, which was similar to that at 40 °C (2.93 mM/min), and the product
yield was around 94 % at the two reaction temperatures. Therefore,
taking the initial reaction rate and yield into account, the temperature
range of 35−40 °C was confirmed to be optimal range for the bior-
eduction of 2-HAP.
Fig. 2a presented the effect of the substrate concentration on the
reduction of 2-HAP in the buffer system. In the range of the tested
concentrations, the e.e. value revealed was little change and was
maintained above 99 %. The reaction obviously expedited as the 2-HAP
concentration was increased from 100 to 200 mM, however, the yield
exhibited almost no change (around 94 %), with a small decrease (90.1
%) as the concentration of 2-HAP increased to 250 mM. Further in-
creasing the concentration led to a decrease in the initial rate and yield,
probably due to the increased inhibition effect and toxicity of 2-HAP
towards the cells. Therefore, the relative activities of the cells were
tested to further analyze the effect of 2-HAP concentration on the re-
combinant cells. As shown in Fig. 2b, after incubation for 5 h in dif-
ferent substrate concentration, the relative activity of the recombinant
cells varied markedly. Over 75 % relative activity was observed in case
of the substrate concentration ≤ 200 mM, whereas 60.2 % of the
Apart from the inhibition of the substrate, the product may also
have other negative effects in the aqueous system. Therefore, the effect
Fig. 2. Effect of substrate concentration on the asymmetric reduction of 2-HAP and the activity of recombinant BL21(DE3). Reaction condition: a) 2 mL
citrate phosphate buffer (100-200 mM, pH 6.5) containing 100-400 mM 2-HAP, 200 mM glucose, 10 mg-dcw/mL cells, 35 °C, 200 rpm; b) 2 mL citrate phosphate
buffer (100-200 mM, pH 6.5) containing 100-400 mM 2-HAP, 200 mM glucose, 10 mg-dcw/mL cells, 35 °C, 200 rpm, incubate for 5 h.
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P. Wei, et al.
MolecularCatalysis488(2020)110903
Table 3
Effect of different biphasic system on the asymmetric reduction of 2-HAP cat-
alyzed by recombinant BL21(DE3).
Reaction system
Initial reaction rate
Reaction time
(h)
Yield
(%)
e.e.
(%)
(mmol L−1 min−1
)
Buffer
4.2
3.7
3.2
4.1
3
3
3
3
82.2
91.1
88.8
95.1
＞99
＞99
＞99
＞99
Dibutyl Phthalate/ Buffer
Ethyl Laurate/ Buffer
C4MIMI·PF6/ Buffer
Reaction conditions: Buffer system: 2 mL citrate phosphate buffer (100−200
mM, pH 6.5) containing 300 mM 2-HAP, glucose: 2-HAP = 1.5, 15 mg-dcw/mL
cells, 35 °C, 200 rpm.
Biphasic system: 2 mL hydrophobic media containing 600 mM 2-HAP (final
concentration 300 mM), 2 mL citrate phosphate buffer (100−200 mM, pH 6.5)
containing glucose: 2-HAP = 1.5 (final concentration 450 mM), 30 mg-dcw/mL
cells (final concentration 15 mg-dcw/mL), 35 °C, 200 rpm.
Fig. 3. Process of the asymmetric reduction of 2-HAP catalyzed by re-
combinant BL21(DE3). Reaction condition: 2 mL citrate phosphate buffer
(100-200 mM, pH 6.5) containing 250-400 mM 2-HAP, glucose: 2-HAP = 1.5,
15 or 20 mg-dcw/mL cells, 35 °C, 200 rpm.
reduction in buffer system. Therefore, a biphasic system consisting of
organic solvent or ionic liquid (IL) and buffer was studied to enhance
the efficiency of the reduction process. Various studies have revealed
that the effect of different organic solvents and ILs on the bioreduction
process varies extensively [9,29,35]. In this study, two organic solvents
and one hydrophobic IL were chosen to analyze their effects on the
reduction of 2-HAP catalyzed with BL21(DE3)(pETDuet-gst-mut-accr-
gdh). The initial reaction rates had a minor decrease in the different
biphasic systems as compared to that in the buffer system (Table 3).
However, the yields after a reaction duration of 3 h were noted to be
higher than that in the buffer system for the substrate concentration of
300 mM and 15 mg-dcw/mL cells as catalyst. The maximum yield of
95.1 % was attained for the C4MIMI·PF6/buffer system. Additionally,
the product e.e. values exhibited no change in different biphasic systems
which were consistently noted to be over 99 %.
The hydrophobic phase always acts as a “storage” medium for the
substrate and/or product. The hydrophobic phase in the biphasic
system can extract the hydrophobic substrate or product, thus, reducing
the concentration of the substrate or product in the buffer phase. It
reduces the toxicity and inhibition of the substrate or product to the
microbial cells during the reaction [36,37]. Therefore, the partition
coefficients in the hydrophobic and buffer phases were tested to esti-
mate the storing ability. As shown in Table 4, the partition coefficients
of 2-HAP and PED varied in different biphasic systems, and the hy-
drophobic interaction of 2-HAP was higher than that of PED. The
product had almost no inhibition effect on the reduction process (Fig.
S4). Hence, the allocation of the substrate is vital for relieving the
substrate inhibition. As shown in Table 4, the partition coefficient of 2-
HAP was 13.4 in the C4MIMI·PF6/buffer system, which was noted to be
the best in the investigated systems and indicated strong extraction of
the IL to 2-HAP.
of product has been investigated, as presented in Fig. S4. The initial rate
and yield remained essentially unchanged at different concentrations of
exogenous-product. Thus, no significant inhibition was observed in the
range of the investigated product concentration. The observed findings
proved that the product had hardly any inhibitory effect on the asym-
metric reduction, which is beneficial for establishing an efficient re-
duction system.
Process curves for the biocatalytic asymmetric reductions of prochiral 2-HAP
The process curves were monitored at different concentration of 2-
HAP and biocatalyst. Under optimized conditions, the reduction process
at the substrate concentrations from 250 to 400 mM with 15–20 mg-
dcw/mL cells as catalyst has been presented in Fig. 3. The (S)-PED
concentration reached 237.9 mM in case of the reduction of 250 mM 2-
HAP catalyzed by 15 mg-dcw/mL BL21(DE3)(pETDuet-gst-mut-accr-
gdh) lyophilized cells for 3 h. The space-time yield and yield were 1.89
M/d and 95.2 %, respectively. As the substrate concentration increased
to 300 mM, the maximum concentration of (S)-PED was noted to be
255.9 mM after a reacted reaction duration of 4 h. The yield decreased
to 86.6 % compared with the yield when 2-HAP was 250 mM. Further
increase in the concentration of the cells revealed that the 300 mM
substrate could be reduced by 20 mg-dcw/mL cells. 284.6 mM (S)-PED
was obtained after reaction duration off 4 h, and the corresponding
yield was 94.9 %. As the concentration of 2-HAP was enhanced to 400
mM, the final yield of reduction reached 81.4 % after a reaction period
of 4 h. Therefore, 15 mg-dcw/mL and 20 mg-dcw/mL cells could reduce
250 mM and 300 mM 2-HAP with the final yield over 94 %, respec-
tively.
The catalytic activity and efficiency improved markedly as com-
pared to the recombinant strain containing the wild-type AcCR. The
E.coli BL21(DE3)(pETDuet-gst-accr-gdh) had been constructed in our
previous study [21]. The catalytic activity of this strain towards 2-HAP
was only 26.5 U/g-dw. Using this strain to catalyze the reduction of 2-
HAP with a substrate concentration of 100 mM, the maximum yield
could only reach 62.5 % after 8 h, and the space-time yield was 187.5
mM/d. On the other hand, the catalytic activity of BL21(DE3)(pETDuet-
gst-mut-accr-gdh) was noted to be 420.9 U/g-dw. As shown in Fig. 3, at a
substrate concentration was 250 mM, the maximum yield reached 95.2
% after 3 h reaction. Overall the catalytic efficiency (space-time yield)
improved 10.1 times.
The extraction of the hydrophobic solvent may lead to the in-
activation of the microbial cells [38]. For the biocatalytic reduction
taking place in the biphasic system, the catalytic performance of the
whole-cell is strongly associated with the type of the hydrophobic
phase. The relative catalytic activity of the whole-cell after incubation
at 35 °C for 5 h in different systems has been investigated in the pre-
sence or absence of the substrate. As illustrated in Fig. 4, the catalytic
Table 4
Partition coefficients of 2-HAP and PED between biphasic systems.
Media
Partition coefficients between the two phases
(hydrophobic/buffer)
2-HAP
PED
Evaluation of various biphasic systems for the asymmetric reduction of 2-
HAP catalyzed with BL21(DE3)(pETDuet-gst-mut-accr-gdh)
Dibutyl phthalate/buffer
Ethyl laurate/buffer
C4MIMI·PF6/buffer
6.33
2.73
13.4
0.65
0.23
0.26
The inhibition of substrate was unavoidable during the biocatalytic
5

P. Wei, et al.
MolecularCatalysis488(2020)110903
Fig. 5. Effect of substrate concentration on the asymmetric reduction
catalyzed by whole-cells in the C4MIMI·PF6/buffer system. Reaction con-
dition: 2 mL C4MIMI·PF6 containing 900 mM 2-HAP (final concentration 450
mM), 2 mL citrate phosphate buffer (100-200 mM, pH 6.5) containing glu-
cose:2-HAP = 1.5, 30 mg-dcw/mL cells (final concentration 15 mg-dcw/mL),
35 °C, 200 rpm.
Fig. 4. Effect on the activity of recombinant BL21(DE3) in different sys-
tems. Reaction condition: 2 mL hydrophobic media containing 600 mM 2-HAP
(final concentration 300 mM), 2 mL citrate phosphate buffer (100-200 mM, pH
6.5) containing 30 mg-dcw/mL cells (final concentration 15 mg-dcw/mL),
glucose: 2-HAP = 1.5, 35 °C, 200 rpm, incubate for 5 h. The buffer system was
as control.
With respect to C.parapsilosis/pCP-scrII, the product yield and e.e. value
reached 99.9 %, however, the optimal 2-HAP concentration was about
36 mM only [39]. The recombinant E.coli (2, 3-butanediol dehy-
drogenase from Bacillus subtilis) could catalyze the reduction of about
390 mM 2-HAP with the yield and e.e. value of (R)-PED reaching 99 %
[17]. The carbonyl reductase YaCRII from Yarrowia lipolytica and
E228S/SCRII from Candida parapsilosis were constructed in E. coli and S.
cerevisiae AN120 osw2Δ, respectively [40,41]. The two recombinant
strains could catalyze higher concentrations of 2-HAP (145−200 mM)
with excellent stereoselectivity, however, did not exhibit a high product
yield (83.7 % & 88 %, respectively). Overall, as evident in Table 5, the
asymmetric reduction of 2-HAP to (S)-PED catalyzed with BL21(DE3)
(pETDuet-gst-mut-accr-gdh) could be conducted at satisfactory substrate
concentration both in buffer and C4MIMIP%F6/buffer biphasic systems
with promising performance.
activity of BL21(DE3)(pETDuet-gst-mut-accr-gdh) cells was lower in the
tested biphasic systems as compared to the buffer system in the absence
of 2-HAP, indicating that the tested hydrophobic solvents partly da-
maged the BL21(DE3)(pETDuet-gst-mut-accr-gdh) cells. The relative
catalytic activities were observed to vary in the studied biphasic sys-
tems, of which C4MIMI·PF6 exhibited the satisfactory biocompatibility
with the microbial cells and the best relative catalytic activity (84.5 %).
Furthermore, in the presence of 2-HAP (300 mM), the relative catalytic
activity of the recombinant cells after incubation for 5 h declined in the
tested systems as compared to the system without 2-HAP, which was
attributed to the toxicity of 2-HAP to the BL21(DE3)(pETDuet-gst-mut-
accr-gdh) cells. Thus, in the presence of 2-HAP, the highest relative
catalytic activity (70 %) of the recombinant cells was observed in
C4MIMI·PF6/buffer system. This result agreed with the best initial re-
action rate, yield and partition coefficient achieved in the case of
C4MIMI·PF6-based system. Therefore, C4MIMI·PF6 was chosen to be the
second phase in the biphasic system for the reduction of 2-HAP.
Preparative scale asymmetric reduction of 2-HAP in the C4MIMI·PF6/buffer
biphasic system
Effect of 2-HAP concentration on the asymmetric reduction catalyzed with
whole-cell in the C4MIMI·PF6/buffer system
The biotransformation was conducted on a 100-mL scale to ascer-
tain the scalability of the 2-HAP reduction to (S)-PED catalyzed with
BL21(DE3)(pETDuet-gst-mut-accr-gdh). HPLC analysis was used to
monitor the reaction process and the results were shown in Fig. 6. 450
mM substrate was used to investigate the preparative scale. 407.3 mM
(S)-PED could be achieved after a reaction period of 3 h, with space-
time yield of 2.71 M/d. The yield and e.e. value were 90.5 % and > 99
%. Further prolongation of the reaction time to 4 h led to the (S)-PED
concentration of 414.6 mM with a yield of 92.1 %. Overall, the e.e.
value was satisfactory (> 99.0 %) during the whole catalytic process.
Hence, the recombinant BL21(DE3)(pETDuet-gst-mut-accr-gdh) cell-
catalyzed asymmetric reduction of 2-HAP to (S)-HAP on a preparative
scale in the C4MIMI·PF6/buffer system was confirmed to be promising
and competitive.
The concentration of 2-HAP reflects the catalytic efficiency of the
reduction process catalyzed with BL21(DE3)(pETDuet-gst-mut-accr-gdh)
in the C4MIMI·PF6/buffer system. Fig. 5 described the evidently influ-
ence of 2-HAP concentration on the reduction process in the
C4MIMI·PF6/buffer system. As the 2-HAP concentration changed from
300 to 450 mM, the reaction rate accelerated obviously, while the
yields (over 92 %) and the e.e. (> 99 %) displayed an insignificant
change. The initial reaction rate reached 6.77−6.74 mM/min for the
concentration ranging from 400 to 450 mM and the yield was over 92 %
after a reaction duration of 3 h, and the space-time yield was 2.88 M/d,
which was 1.52 times higher than that in buffer system. Further im-
proving the concentration of 2-HAP from 450 to 550 mM caused a
minor drop in the initial reaction rate, probably due to the growing
inhibition effect of 2-HAP on BL21(DE3)(pETDuet-gst-mut-accr-gdh).
Also, the yield decreased to 86.1 %-74.0 % with unchanged e.e. value.
Consequently, the most suitable 2-HAP concentration was identified to
be 450 mM in the C4MIMI·PF6/buffer system, which was 1.8 times
higher than that in the buffer system.
Conclusions
In this study, the asymmetric reduction of 2-HAP to (S)-PED cata-
lyzed with BL21(DE3)(pETDuet-gst-mut-accr-gdh) has been investigated
in buffer and biphasic systems systematically, and the results were sa-
tisfactory. A highly efficient C4MIMI·PF6/buffer system has been iden-
tified for the asymmetric reduction of 2-HAP to synthesize chiral al-
cohol (S)-PED. Using this system, 450 mM 2-HAP could be reduced in 3
h with encouraging product yield and e.e. value. The synthesis of the
Comparing the obtained results with the previously reported find-
ings for the synthesis of PED is vital for gaining further insights. The
results of the biocatalytic reduction of 2-HAP to PED in different sys-
tems catalyzed with various biocatalysts were summarized in Table 5.
6

P. Wei, et al.
MolecularCatalysis488(2020)110903
Table 5
Comparison of the PED synthesis catalyzed by different biocatalysts.
Catalyst
Catalyst dosage
Substrate concentration
Reaction system
Buffer
Product
(S)-PED
(R)-PED
(S)-PED
(R)-PED
(S)-PED
(S)-PED
Yield
e.e.
Reference
[39]
C.parapsilosis/
pCP-scrII
E. coli (BDHA-GDH)
100 g/L wet cells
5g/L
≈36 mM
54 g/L
≈390 mM
20 g/L
≈145 mM
200 mM
99.9 %
99 %
99.9 %
99 %
30 g /L
dry cells
50 g/L
wet cells
50 g/L
wet cells
20 g/L
dry cells
15 g/L
Buffer
[17]
BL21(DE3)/ pET-28a-yacrII
Dibutyl phthalate/ buffer
Ethyl acetate/ bufer
Buffer
83.7 %
88 %
99.9 %
99 %
[40]
S. cerevisiae osw2Δ(E228S-GDH)
BL21(DE3)(pETDuet-gst-mut-accr-gdh)
BL21(DE3)(pETDuet-gst-mut-accr-gdh)
[41]
300 mM
450 mM
95 %
> 99 %
> 99 %
This study
This study
[C4MIMI]•PF6/buffer
92.7 %
dry cells
[2] Y.G. Zheng, H.H. Yin, D.F. Yu, X. Chen, X.L. Tang, X.J. Zhang, Y.P. Xue, Y.J. Wang,
Z.Q. Liu, Recent advances in biotechnological applications of alcohol dehy-
drogenases, Appl. Microbiol. Biot. 101 (3) (2017) 987–1001.
[3] X. Chen, H. Zhang, J. Feng, Q. Wu, D. Zhu, Molecular basis for the high activity and
enantioselectivity of the carbonyl reductase from Sporobolomyces salmonicolor to-
ward α-haloacetophenones, ACS Catal. 8 (4) (2018) 3525–3531.
[4] R. Kadyrov, R.M. Koenigs, C. Brinkmann, D. Voigtlaender, M. Rueping, Efficient
enantioselective synthesis of optically active diols by asymmetric hydrogenation
with modular chiral metal catalysts, Angew. Chem. Int. Edit 48 (41) (2009)
7556–7559.
[5] X. Xue, P. Chen, P. Xu, Y. Wang, Highly efficient and recyclable chiral Pt nano-
particle catalyst for enantioselective hydrogenation of activated ketones, Catal.
Commun. 110 (2018) 55–58.
[6] J. Xu, S. Zhou, Y. Zhao, J. Xia, X. Liu, J. Xu, B. He, B. Wu, J. Zhang, Asymmetric
whole-cell bioreduction of sterically bulky 2-benzoylpyridine derivatives in aqu-
eous hydrophilic ionic liquid media, Chem. Eng. J. 316 (2017) 919–927.
[7] A. Li, L. Ye, X. Yang, B. Wang, C. Yang, J. Gu, H. Yu, Reconstruction of the catalytic
pocket and enzyme-substrate interactions to enhance the catalytic efficiency of a
short-chain dehydrogenase/reductase, ChemCatChem 8 (20) (2016) 3229–3233.
[8] F. Tieves, I.N. Erenburg, O. Mahmoud, V.B. Urlacher, Synthesis of chiral 2-alkanols
from n-alkanes by a P. putida Whole-Cell Biocatalyst, Biotechnol. Bioeng. 113 (9)
(2016) 1845–1852.
[9] Z.Q. Liu, S.C. Dong, H.H. Yin, Y.P. Xue, X.L. Tang, X.J. Zhang, J.Y. He, Y.G. Zheng,
Enzymatic synthesis of an ezetimibe intermediate using carbonyl reductase coupled
with glucose dehydrogenase in an aqueous-organic solvent system, Bioresource
Technol. 229 (2017) 26–32.
Fig. 6. Process of asymmetric reduction of 2-HAP catalyzed by whole-cells
in the C4MIMI·PF6/buffer system. Reaction condition: 50 mL C4MIMI·PF6
containing 900 mM 2-HAP (final concentration 450 mM), 50 mL citrate phos-
phate buffer (100-200 mM, pH 6.5) containing glucose: 2-HAP = 1.5, 30 mg-
dcw/mL cells (final concentration 15 mg-dcw/mL), 35 °C, 200 rpm.
[10] X.M. Gong, G.W. Zheng, Y.Y. Liu, J.H. Xu, Identification of a robust carbonyl re-
ductase for diastereoselectively building syn-3,5-dihydroxy hexanoate: a bulky side
chain of atorvastatin, Org. Process Res. Dev. 21 (9) (2017) 1349–1354.
[11] P. Gruber, F. Carvalho, M.P.C. Marques, B. O’Sullivan, F. Subrizi, D. Dobrijevic,
J. Ward, H.C. Hailes, P. Fernandes, R. Wohlgemuth, F. Baganz, N. Szita, Enzymatic
synthesis of chiral amino-alcohols by coupling transketolase and transaminase-
catalyzed reactions in a cascading continuous-flow microreactor system,
Biotechnol. Bioeng. 115 (3) (2018) 586–596.
enantiomerically pure (S)-PED on a 100-mL preparative scale could also
be successfully performed via the asymmetric bioreduction of 2-HAP
catalyzed with BL21(DE3)(pETDuet-gst-mut-accr-gdh) cells in
C4MIMI·PF6/buffer system, which indicating a strong prospect of the
whole-cell catalytic system for industrial application.
[12] M. Li, Y. Nie, X.Q. Mu, R. Zhang, Y. Xu, Highly selective anti-Prelog synthesis of
optically active aryl alcohols by recombinant Escherichia coli expressing stereo-
specific alcohol dehydrogenase, Prep. Biochem. Biotechnol. 46 (5) (2016) 429–433.
[13] A. Liese, M. Karutz, J. Kamphuis, C. Wandrey, U. Kragl, Enzymatic resolution of 1-
phenyl-1,2-ethanediol by enantioselective oxidation: overcoming product inhibi-
tion by continuous extraction, Biotechnol. Bioeng. 51 (5) (1996) 544–550.
[14] T.W.B. Gehr, D.M. Tenero, D.A. Boyle, Y. Qian, D.A. Sica, N.H. Shusterman, The
pharmacokinetics of carvedilol and its metabolites after single and multiple dose
oral administration in patients with hypertension and renal insufficiency, Eur. J.
Clin. Pharmacol. 55 (4) (1999) 269–277.
[15] X.T. Zhou, R.Z. Zhang, Y. Xu, H. Liang, J. Jiang, R. Xiao, Coupled (R)-carbonyl
reductase and glucose dehydrogenase catalyzes (R)-1-phenyl-1,2-ethanediol bio-
synthesis with excellent stereochemical selectivity, Process Biochem. 50 (11)
(2015) 1807–1813.
[16] B.B. Zhang, J. Cheng, W.Y. Lou, P. Wang, M.H. Zong, Efficient anti-Prelog en-
antioselective reduction of acetyltrimethylsilane to (R)-1-trimethylsilylethanol by
immobilized Candida parapsilosis CCTCC M203011 cells in ionic liquid-based bi-
phasic systems, Microb. Cell Fact. 11 (2012).
Acknowledgements
The authors gratefully acknowledge the National Natural Science
Foundation of China (21802019, 21676104 and 21878105), the
National Key Research and Development Program of China
(2018YFC1603400, 2018YFC1602100), the Science and Technology
Program of Guangzhou (201904010360), the Science and Technology
Program of Jiangmen (2019JC01018, 2019JC01019), the Science and
Technology Planning Project of Jiangmen (2017TD02), Youth Research
Fund Team Project of Wuyi University (2019td05), Research
Foundation for Advanced Talents of Wuyi University (2018AL012) and
Foundation from Department of Education of Guangdong Province
(2016KCXTD005 and 2017KSYS010) for partially funding this work.
[17] Z.M. Cui, J.D. Zhang, X.J. Fan, G.W. Zheng, H.H. Chang, W.L. Wei, Highly efficient
bioreduction of 2-hydroxyacetophenone to (S)- and (R)-1-phenyl-1,2-ethanediol by
two substrate tolerance carbonyl reductases with cofactor regeneration, J.
Biotechnol. 243 (2017) 1–9.
[18] G. Rehn, A.T. Pedersen, J.M. Woodley, Application of NAD(P)H oxidase for cofactor
regeneration in dehydrogenase catalyzed oxidations, J. Mol. Catal. B-Enzym. 134
(2016) 331–339.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.mcat.2020.110903.
[19] X. Wang, H.H.P. Yiu, Heterogeneous catalysis mediated cofactor NADH regenera-
tion for enzymatic reduction, ACS Catal. 6 (3) (2016) 1880–1886.
References
[20] J. Deng, K.L. Chen, Z.Q. Yao, J. Lin, D. Wei, Efficient synthesis of optically active
halogenated aryl alcohols at high substrate load using a recombinant carbonyl re-
ductase from Gluconobacter oxydans, J. Mol. Catal. B-Enzym. 118 (2015) 1–7.
[21] P. Wei, J.X. Gao, G.W. Zheng, H. Wu, M.H. Zong, W.Y. Lou, Engineering of a novel
[1] G.W. Zheng, Y.Y. Liu, Q. Chen, L. Huang, H.L. Yu, W.Y. Lou, C.X. Li, Y.P. Bai,
A.T. Li, J.H. Xu, Preparation of structurally diverse chiral alcohols by engineering
ketoreductase CgKR1, ACS Catal. 7 (10) (2017) 7174–7181.
7

P. Wei, et al.
MolecularCatalysis488(2020)110903
[32] P. Wei, Z.W. Guo, X.L. Wu, S. Liang, X.Y. Ou, P. Xu, M.H. Zong, J.G. Yang, W.Y. Lou,
Significantly enhancing the biocatalytic synthesis of chiral alcohols by semi-ra-
tionally engineering an anti-Prelog carbonyl reductase from Acetobacter sp. CCTCC
M209061, Mol. Catal. 479 (2019) 110613.
[33] W.P. Cui Yuhan, Peng Fei, Zong Minhus, Lou Wenyong, Efficient biocatalytic ste-
reoselective reduction of methyl acetoacetate catalyzed by whole cells of en-
gineered E. coli, RSC Adv. 8 (2018) 9970–9978.
[34] P.X. Du, P. Wei, W.Y. Lou, M.H. Zong, Biocatalytic anti-Prelog reduction of pro-
chiral ketones with whole cells of Acetobacter pasteurianus GIM1.158, Microb. Cell
Fact. 13 (1) (2014) 84.
[35] P. Wei, J. Liang, J. Cheng, M.H. Zong, W.Y. Lou, Markedly improving asymmetric
oxidation of 1-(4-methoxyphenyl) ethanol with Acetobacter sp. CCTCC M209061
cells by adding deep eutectic solvent in a two-phase system, Microb. Cell Fact. 15
(1) (2016).
[36] J.J. Yuan, Y.X. Guan, S.J. Yao, Evaluation of Biocompatible Ionic liquids for their
application in phytosterols bioconversion by Mycobacterium sp resting cells, ACS
Sustain. Chem. Eng. 5 (11) (2017) 10702–10709.
carbonyl reductase with coenzyme regeneration in E. coli for efficient biosynthesis
of enantiopure chiral alcohols, J. Biotechnol. 230 (2016) 54–62.
[22] J.-D. Zhang, Z.M. Cui, X.J. Fan, H.L. Wu, H.H. Chang, Cloning and characterization
of two distinct water-forming NADH oxidases from Lactobacillus pentosus for the
regeneration of NAD, Bioproc. Biosyst. Eng. 39 (4) (2016) 603–611.
[23] C. Zhang, J. Pan, C.X. Li, Y.P. Bai, J.H. Xu, Asymmetric bioreduction of keto groups
of 4-and 5-oxodecanoic acids/esters with a new carbonyl reductase, Catal.
Commun. 102 (2017) 35–39.
[24] M. Zhao, L. Gao, L. Zhang, Y. Bai, L. Chen, M. Yu, F. Cheng, J. Sun, Z. Wang,
X. Ying, Asymmetric reduction of ketopantolactone using a strictly (R)-stereo-
selective carbonyl reductase through efficient NADPH regeneration and the sub-
strate constant-feeding strategy, Biotechnol. Lett. 39 (11) (2017) 1741–1746.
[25] P. Wei, P. Xu, X.T. Wang, W.Y. Lou, M.H. Zong, Asymmetric reduction of ethyl
acetoacetate catalyzed by immobilized Acetobacter sp CCTCC M209061 cells in
hydrophilic ionic liquid hybrid system, Biotechnol. Bioprocess Eng. 20 (2) (2015)
324–332.
[26] Y. Xu, X.T. Wang, W.Y. Lou, M.H. Zong, Biocatalytic asymmetric reduction of ethyl
acetoacetate with immobilized Acetobacter sp. CCTCC M209061 cells in a micro-
water-containing organic solvent, Modern Food Sci. Technol. 30 (7) (2014) 91 31-
37 and.
[37] S.H. Krishna, Developments and trends in enzyme catalysis in nonconventional
media, Biotechnol. Adv. 20 (3-4) (2002) 239–267.
[38] H. Nakaya, O. Miyawaki, K. Nakamura, Determination of logP for pressurized
carbon dioxide and its characterization as a medium for enzyme reaction, Enzyme
Microb. Technol. 28 (2–3) (2001) 176–182.
[39] Y. Li, R. Zhang, Y. Xu, R. Xiao, L. Wang, X. Zhou, H. Liang, J. Jiang, Efficient
bioreduction of 2-hydroxyacetophenone to (S)-1-phenyl-1, 2-ethanediol through
homologous expression of (S)-carbonyl reductase II in Candida parapsilosis CCTCC
M203011, Process Biochem. 51 (9) (2016) 1175–1182.
[40] H.L. Zhang, C. Zhang, C.H. Pei, M.N. Han, W. Li, Enantioselective synthesis of en-
antiopure chiral alcohols using carbonyl reductases screened from Yarrowia lipoly-
tica, J. Appl. Microbiol. 126 (1) (2019) 127–137.
[41] J. Rao, R. Zhang, H. Liang, X.D. Gao, H. Nakanishi, Y. Xu, Efficient chiral synthesis
by Saccharomyces cerevisiae spore encapsulation of Candida parapsilosis Glu228Ser/
(S)-carbonyl reductase II and Bacillus sp. YX-1 glucose dehydrogenase in organic
solvents, Microb. Cell Fact. 18 (1) (2019) 87.
[27] X.T. Wang, D.M. Yue, M.H. Zong, W.Y. Lou, Use of ionic liquid to significantly
improve asymmetric reduction of ethyl acetoacetate catalyzed by Acetobacter sp.
CCTCC M209061 cells, Ind. Eng. Chem. Res. 52 (35) (2013) 12550–12558.
[28] X.T. Wang, X.H. Chen, Y. Xu, W.Y. Lou, H. Wu, M.H. Zong, Biocatalytic anti-Prelog
stereoselective reduction of ethyl acetoacetate catalyzed by whole cells of
Acetobacter sp CCTCC M209061, J. Biotechnol. 163 (3) (2013) 292–300.
[29] P. Xu, G.W. Zheng, P.X. Du, M.H. Zong, W.Y. Lou, Whole-cell biocatalytic processes
with ionic liquids, ACS Sustain. Chem. Eng. 4 (2) (2016) 371–386.
[30] J.Y. He, Z.H. Sun, W.Q. Ruan, Y. Xu, Biocatalytic synthesis of ethyl (S)-4-chloro-3-
hydroxy-butanoate in an aqueous-organic solvent biphasic system using
Aureobasidium pullulans CGMCC 1244, Process Biochem. 41 (1) (2006) 244–249.
[31] P. Wei, Y.H. Cui, M.H. Zong, P. Xu, J. Zhou, W.Y. Lou, Enzymatic characterization
of a recombinant carbonyl reductase from Acetobacter sp. CCTCC M209061,
Bioresources Bioproc. 4 (1) (2017) 39–50.
8