The Asymmetric Reduction of Acetophenone and Its Derivatives to (S)-Aromatic Secondary Alcohols by Rhodotorula mucilaginosa CCTCC M2014255 Resting Cells

Article abstract of DOI:10.1007/s10562-016-1730-9

Abstract: Enantiomerically enriched aromatic secondary alcohols and its derivatives are important chiral intermediates utilized widely in pharmacy, chemical, hormone, spices and chiral auxiliary materials. Thus, the key factors affecting the asymmetric reduction were successfully analyzed using R. mucilaginosa CCTCC M2014255 resting cells. The optimal temperature, buffer pH, co-substrate and its concentration, resting cells concentration were systematically investigated. Under the optimized conditions, acetophenone and its derivatives were effectively asymmetrically reduced by R.mucilaginosa CCTCC M2014255 while the conversion reached >99.0?% and e.e. of product were >99.0?%, respectively. These results provide a theoretical and technical support for realizing industrialized production of enantiomerically enriched aromatic secondary alcohols by microbial catalytic asymmetric synthesis. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s10562-016-1730-9

Catal Lett (2016) 146:1079–1086
DOI 10.1007/s10562-016-1730-9
The Asymmetric Reduction of Acetophenone and Its Derivatives
to (S)-Aromatic Secondary Alcohols by Rhodotorula mucilaginosa
CCTCC M2014255 Resting Cells
1
2
1
1
1
•
Dan Wang
1
Lang He
•
Zhirong Yang
•
Jinhua Zhang
•
Yunlei Han
•
Junli Hao
Received: 20 February 2016 / Accepted: 13 March 2016 / Published online: 24 March 2016
Ó Springer Science+Business Media New York 2016
Abstract Enantiomerically enriched aromatic secondary
alcohols and its derivatives are important chiral interme-
diates utilized widely in pharmacy, chemical, hormone,
spices and chiral auxiliary materials. Thus, the key factors
affecting the asymmetric reduction were successfully ana-
lyzed using R. mucilaginosa CCTCC M2014255 resting
cells. The optimal temperature, buffer pH, co-substrate and
its concentration, resting cells concentration were system-
atically investigated. Under the optimized conditions,
acetophenone and its derivatives were effectively asym-
metrically reduced by R.mucilaginosa CCTCC M2014255
while the conversion reached [99.0 % and e.e. of product
were [99.0 %, respectively. These results provide a theo-
retical and technical support for realizing industrialized
production of enantiomerically enriched aromatic sec-
ondary alcohols by microbial catalytic asymmetric
synthesis.
Graphical Abstract
Keywords Rhodotorula mucilaginosa CCTCC
M2014255 Á Asymmetric reduction Á Acetophenone Á
Aromatic secondary alcohols
1
Background
As the versatile chiral drug intermediates, single enan-
tiomers of phenylethanol and its derivatives are utilized as
the starting material to synthesize various chiral drugs such
as (S)-clorprenaline, (S)-fluoxetine, (R)-atomoxetine and
(
R)-salbutamol [1, 2]. In order to gain phenylethanol and its
derivatives with enantiomerically pure state, the efficient
method is the catalytic asymmetric synthesis [3–6].
Recently, the asymmetric reduction of carbonyl com-
pounds by biocatalysts has attracted extensive attention due
to the performance under milder reaction conditions,
shorter reaction time, environmental benignancy and the
excellent stereoselectivity of the reaction [6, 7].
&
&
Junli Hao
haojunli00@163.com
Lang He
helang79@sohu.com
1
2
Chengdu Medical College, Sichuan 610500, China
Sichuan University, Sichuan 610065, China
123

1
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D. Wang et al.
The biocatalysts was divided into isolated enzyme
Table 1 Comparison of the biocatalytic asymmetric reduction of
acetophenone with various strains
catalysis and incubated whole-cell catalysis [8–10]. In
contrast, incubated whole-cell catalysis can save not only
the processes of separation and purification of enzymes, but
also the co-factor regeneration system such as nicotinamide
adenine dinucleotide phosphate (NADPH) or (NADH).
When prochiral ketoneswere asymmetrically reduced by
carbonyl reductase, coenzyme NAD(P)H as a hydrogen
donor was needed in the reduction reaction. However,
glucose and other cheap energy substances can be utilized
to regenerate the coenzyme NAD(P)H required by car-
bonyl-reductase in the whole-cell based catalysis [11–13].
Microbial resting cells are starved cells suspended in
physiological saline or buffer for a period to consume
intracellular nutrients. Although resting cells are in hiber-
nation without reproduction, they still contain a variety of
enzymes and the abilities to degrade or synthesis of pro-
teins and lipids and secret proteins outside to support some
vegetative tissues [14, 15]. Therefore, there are growth and
catalytic properties for microbial resting cells under the
reaction conditions without nitrogen source. So far, many
literatures on catalytic asymmetric reduction reaction by
resting cells have been reported [16, 17]. Narancic et al.
found that highly efficient Michael-type addition of
acetaldehyde to b-nitrostyrenes by whole resting cells of
Escherichia coli expressing 4-oxalocrotonate tautomerase
Strain
Conversion%
e.e.%
Config
ma6-3
cmq6-8
WD-B5
cs5-1
38.4
49.4
43.0
26.9
20.7
28.7
32.1
34.3
95.9
94.8
96.8
94.7
90.5
94.6
93.2
94.3
S
R
S
S
S
S
S
S
1
3
6
m6-8
m6-2
Reaction conditions: 10 mL phosphate buffer (0.1 mol/L, pH 6.6),
2 g resting cells of different generations, 2 % (W/V) glucose,
6
0 mmol/L acetophenone, 30 °C, 150 r/min, 24 h
can be shown in Table 1, the strain WD-B5 with high
biotransformation activities and inheritable qualities was
obtained by rescreening. Based on the analysis of physio-
logical and biochemical characteristics and 26S rDNA D1/
D2 domain sequence with its general morphology, WD-B5
deposited at China Center for Type Culture Collection
(CCTCC) with number CCTCC M2014255 was identified
as Rhodotorula mucilaginosa.
[
18]. Moreover, our previous work also showed that the
2.2 Catalytic Activity and Stability of
R. mucilaginosa CCTCC M2014255 Cells
efficient asymmetric reduction of acetone acid ethyl ester
to (S)-lactic acid ethyl ester by yeast resting cells [19].
Nevertheless, there are only few microorganisms reported
to catalyze acetophenone to aromatic secondary alcohols
with (S)-absolute configuration used in the industry, due to
the low catalytic activity and stereoselectivity. Taken
together, finding a microbial catalyst with high catallytic
activities and stabilities is an effective way to promote
asymmetric reduction for industrial application. In the
present study, the effects of important variables on the
bioreduction of acetophenone and its derivatives with R.
mucilaginosa CCTCC M2014255 resting cells obtained by
rescreening were systematically investigated.
To assess the stability of catalytic activity of strain cells, R.
mucilaginosa CCTCC M2014255 was sub-cultured for 20
generations. The results showed that the asymmetric
reduction capability of the first, fifth, tenth, fifteenth and
twentieth generation strains were quite stable while the
conversion always reached [40.8 %, and the e.e. of pro-
duct was stable at [96.3 %.
2.3 Effects of Several Key Variables
on the Biocatalytic Asymmetric Reduction
of Acetophenone to (S)-Aromatic Secondary
Alcohols with R. mucilaginosa CCTCC
M2014255 Cells
2
Results and Discussion
To gain a deeper insight into the reduction and improve the
initial reaction rate, the effects of several important vari-
ables were systematically investigated such as reaction
time, temperature, buffer pH, co-substrate concentration,
cell concentration and substrate concentration. As shown in
Fig. 1, the conversion raised accompanied with the pro-
gress of the reaction and reached the maximum at 36 h.
However, the conversion did not increase after 36 h, which
was due to the inhibition of the cell growth and product
accumulation [20]. Moreover, the results showed that e.e.
2
.1 Comparison of the Biocatalytic Asymmetric
Reduction of Acetophenone with
R. mucilaginosa CCTCC M2014255 and Other
Potential Microorganisms
Eight strains microorganisms with the abilities of asym-
metric reduction of acetophenone to (S)-1-phenylethanol
were isolated and purified from sewage and the neighbor-
ing soil samples from a chemical factory in Chengdu. As
1
23

The Asymmetric Reduction of Acetophenone and Its Derivatives to (S)-Aromatic Secondary…
1081
decreased while the temperature kept rising from 30 to
8 °C, which could be attributed to the partial denaturation
3
and deactivation of the enzymes at a higher temperature.
On the other hand, the e.e. values of products showed no
significant variation in the range of 20–38 °C. Hence, the
optimum temperature of asymmetric reduction was con-
sidered as 30 °C, which was similar to the optimum tem-
perature 25 °C of a novel carbonyl reductase from
Acetobacter sp. CCTCC M209061 [22].
The initial pH of the reaction not only affects the con-
figuration and stability of the enzymes, but also affects the
substrate dissociation which in turn affects the reduction
rate markedly. Therefore, a suitable pH will promote the
combination of enzymes and substrate, improve the enzy-
matic activity and reaction rate [23]. To investigate the
influence of pH on catalytic activity of R. mucilaginosa
CCTCC M2014255, the conversion and e.e. of the product
were both detected while the phosphate buffer solution was
pH 5, 6, 6.2, 6.6, 6.8, 7.0 and 8.0 respectively. As shown in
Fig. 3, the catalytic activity of the cells reached maximum
with pH 7.0. The conversion dropped significantly with pH
from 7.0 to 8.0 while the e.e. of product was still over
Fig. 1 Time course of asymmetric reduction of acetophenone to (S)-
aromatic secondary alcohols by R. mucilaginosa CCTCC M2014255.
Reaction conditions: 10 mL phosphate buffer (0.1 mol/L, pH 6.6),
2
3
g resting cells, 2 % (W/V) glucose, 60 mmol/L acetophenone,
0 °C, 150 r/min, gradient time (6–60 h)
of product showed small changes after 36 h, thus, the best
conversion time was 36 h.
99.0 %. However, the conversion and e.e. of the product
As we all know, the effects of temperature on the
enzymatic reaction had two sides. The activity, the selec-
tivity and the stability of a biocatalyst and the equilibrium
of reactions can be affected by temperature [21]. To
explore the effect of temperature on the reaction, the bio-
reduction was conducted at different temperatures. As
shown in Fig. 2, the conversion increased markedly in the
range of 20–30 °C. The substrate conversion reached the
highest at 30 °C. However, the substrate conversion ratio
were the minimum while the pH was 9.0, indicating that
oxidoreductases from R. mucilaginosa CCTCC M2014255
were more tolerant in acidic environment. Obviously, the
optimal initial pH of the reaction system was 7.0 corre-
sponding a broad pH optimum of a novel ketoreductase
from the Cyanobacterium Synechococcus sp. Strain
PCC7942 between pH 7.0 and pH 9.0 [24].
It is well known that the asymmetric reduction of ace-
tophenone to (S)-1-phenylethanol is catalyzed by the
Fig. 2 Effect of temperature on the asymmetric reduction by R.
mucilaginosa CCTCC M2014255. Reaction conditions: 10 mL phos-
phate buffer (0.1 mol/L, pH 6.6), 2 g resting cells, 2 % (W/V)
glucose, 60 mmol/L acetophenone, 36 h, 150 r/min, gradient tem-
perature (24–38 °C)
Fig. 3 Effect of pH on the asymmetric reduction of acetophenon to
(S)-aromatic secondary alcohols by R. mucilaginosa CCTCC
M2014255. Reaction conditions: 10 mL phosphate buffer (0.1 mol/
L, pH gradient), 2 g resting cells, 2 % (W/V) glucose, 60 mmol/L
acetophenone, 36 h, 150 r/min, 30 °C
123

1
082
D. Wang et al.
intracellular oxidoreductase and coenzyme NAD(P)H as an
electron donor. The reductions could proceed efficiently
without adding expensive coenzyme NAD(P)H only if a
co-substrate (glucose, glycerol or other energy substances)
is present, which can be used to realize the regeneration of
NADPH by the whole-cell [25]. Therefore, according to
previous studies [19], glucose was selected as the auxiliary
substrate to regenerate NADPH in this study. As shown in
Fig. 4, a small amount of substrates were catalyzed without
adding glucose in the reaction system (control), indicating
that a certain amount of energy substances and coenzymes
had already been accumulated in microbial cells prior to
the reduction. Furthermore, the substrate conversion was
markedly influenced as glucose concentration increasing
from 0 to 2 %. When the glucose concentration was up to
Fig. 5 Effect of resting cell concentrations on the asymmetric
reduction of acetophenone to (S)-aromatic secondary alcohols by R.
mucilaginosa CCTCC M2014255. Reaction conditions: 10 mL phos-
phate buffer (0.1 mol/L, pH 7.0), 2 % (W/V) glucose, 60 mmol/L
acetophenone, 36 h, 150 r/min, 30 °C, gradient concentrations of
resting cells
2
%, the substrate conversion reached the maximum.
However, the substrate conversion began to decline as the
glucose concentration further increased. The possible rea-
sons for this phenomenon might be due to the changes of
osmotic pressure in the cells. Considering the e.e. of pro-
duct kept constantly above 98.5 % with the tested range of
glucose concentration, the optimal glucose concentration
was settled to be 2 %.
uniformly dispersed and not be sufficiently contacted with
the substrate in reaction system. In this study, lots of cells
deposited to the wall of flask bottles were observed while
the cell concentration was too high. Moreover, The e.e. of
product remained constant (about 99.0 %) in the range of
cell loading.
In this study, the amount of incubated whole cells (cell
loading) has also been taken into account in order to save
biocatalyst as much as possible. When the amount of
substrate was fixed, the conversion reaction had an obvious
enhancement as the yeast cell loading increased. As shown
in Fig. 5, the conversion reached the maximum when the
resting cell loading increased to 0.25 g/mL. However, the
substrate conversion lowered as the cell loading increased.
This observation suggests that excessive cells could not be
Under the improved reaction conditions, the best tran-
sition temperature was 30 °C; the pH was 7.0; the auxiliary
substrate glucose concentration was 2 %; the amount of
resting cells was 0.25 g/mL and the reaction time was 36 h.
Fig. 6 Effect of substrate concentrations on the asymmetric reduc-
tion of acetophenon to (S)-aromatic secondary alcohols by R.
mucilaginosa CCTCC M2014255. Reaction conditions: 10 mL phos-
phate buffer (pH 7.0), 2 % (W/V) glucose, 36 h, 150 r/min, 30 °C,
0.25 g/mL(wet weight) resting cells, gradient concentions of
acetophenone
Fig. 4 Effect of glucose concentration on the asymmetric reduction
of acetophenon to (S)-aromatic secondary alcohols by R. mucilagi-
nosa CCTCC M2014255. Reaction conditions: 10 mL phosphate
buffer (0.1 mol/L, pH 7.0), 2 g resting cells, 60 mmol/L acetophe-
none, 36 h, 150 r/min, 30 °C, different concentrations of glucose
1
23

The Asymmetric Reduction of Acetophenone and Its Derivatives to (S)-Aromatic Secondary…
1083
The suitable concentration of substrate was also examined
under these conditions. As shown in Fig. 6, the conversion
increased to the maximum value 96.8 % with the range of
substrate concentration from 40 to 50 mmol/L, while the
e.e. of product was constantly over 99.0 %. However, the
substrate conversion decreased while the substrate con-
centration exceeded 50 mmol/L and the potential reason
may be some substrate inhibitory effect. Taken together,
the most suitable substrate concentration was 50 mmol/L,
which was similar to the optimal substrate concentration
improved conversion conditions, the R.mucilaginosa
CCTCC M2014255 could asymmetrically reduce ace-
tophenone and its derivatives with better substrate trans-
formation abilities.
In future work, to further increase the production and
reduce the toxicity of high concentration of substrate on
microbial cells, on one hand, we will try other methods
such as cell immobilization, ionic liquid reaction system
[26]. On the other hand, to obtain a new biocatalysts by
genetic mutation or recombination through analyzing pro-
tein structure of carbonyl reductase from R.mucilaginosa
CCTCC M 2014255 [27–29].
4
0 mmol/L in the reduction of prochiral ketones with
whole-cell of Acetobacter pasteurianus GIM1.158 [11].
To further improve the conversion and reduce the tox-
icity of substrate to the strain cells, addition of substrates
were divided into twice. For example, in the initial reac-
tion, 50 % substrates (acetophenone) were added, and the
other half were added after 12 h. In these conditions, both
of the maximum conversion and the e.e. of product were
more than 99.0 %.
4 Materials and Methods
4.1 Biological and Chemical Materials
R. mucilaginosa CCTCC M2014255 was isolated and
purified from sewage and the neighboring soil samples
from a chemical factory in Chengdu, Sichuan, China. (R)-
1-phenylethanol and (S)-1-phenylethanol (99 % purity)
were purchased from Sigma-Aldrich. Acetophenone
(ACP), other prochiral ketones and the corresponding
alcohols were obtained from Chengdu Asta Tech Trading
Co. Ltd (Chengdu, China). All other chemical and bio-
chemical reagents (analytical grade) were from Chengdu
Kelong Chemical Reagent Company.
2
.4 Stereoselective Reduction of Various Prochiral
Carbonyl Compounds
To rationally evaluate the potential of R. mucilaginosa
CCTCC M2014255 cells for the biocatalytic asymmetric
reduction of carbonyl compounds, under the above men-
tioned conditions for acetophenone, eight derivatives of ace-
tophenone, such as o-chloroacetophenone, o-methoxyace-
tophenone, o-methylacetophenone, p-fluoroacetophenone,
propiophenone, 2-acetylthiophene and 2-acetylpyridine were
tested for reduction. The results were shown in Table 2, the
conversions of acetophenone, o-chloroacetophenone and
o-methylacetophenonewerehigher([90.0 %)respectively to
give the product with (S)-configuration. The results showed
the oxidoreductases of R. mucilaginosa CCTCC M2014255
had similar enantioselectivities of substrate, facially with
relatedstructure.However,theconversionofotherderivatives
was lower than that of acetophenone itself indicating the
elaborated conversion conditions of acetophenone derivatives
need to be further explored which also proved that enzymes
had the high selectivity and specificity characteristics.
4.2 Strain Cell Cultivation
R. mucilaginosa CCTCC M2014255 strain cells were
activated firstly. A full ring of strain cells from the bottom
of the activated slant medium were inoculated in a 20 mL
seed medium for 24 h with the condition of 30 °C, 150 r/
min. Then, seed culture liquid was inoculated in a 50 mL
fermentation medium with 5 % final concentration. Fer-
mentation conditions: 30 °C, 150 r/min and 48 h. The
fermentation medium with pH 7.0 contains 10 g/L peptone,
5 g/L yeast extract, 2 g/L KH PO , 5 g/L (NH ) SO and
2
4
4 2
4
20 g/L Glucose. Seed medium was yeast extract peptone
dextrose (YPD) medium and slant medium was potato
dextrose agar (PDA) medium.
3
Conclusions
4.3 Resting Cell Collection and Biocatalytic
Asymmetric Reductions of Prochiral Ketones
The conditions of asymmetric reduction of acetophenone to
S)-1-phenethylalcohol by R. mucilaginosa CCTCC
(
M2014255 were systematically studied. The improved
conditions are 30 °C, pH 7.0, 2 % glucose, resting cell
concentration 0.25 g/mL (wet weight), the final substrate
concentration 50 mmol/L adding in batch: 0 h; 50 %, 12 h;
2 g strain cells were collected by centrifuging at 4 °C,
6000 r/min for 10 min. Then, the collected strain cells were
repeatedly purged for three times by 0.1 mol/L phosphate
buffer (pH 6.6) and suspended with 10 mL 0.1 mol/L
phosphate buffer (pH 6.6) with 60 mmol/L final substrate
concentration and 2 % (w/v) glucose. The conditions of
5
0 %, 2 % ethanol for dissolution of substrate. The con-
version could reach [99.0 % with e.e. [99.0 %. Under
123

1
084
D. Wang et al.
Table 2 Asymmetric reduction
of acetophenone and its
derivatives by R. mucilaginosa
CCTCC M2014255
Prochiral ketones
Acetophenone
Structure
Rentention time (min) conversion% e.e.%
Config
9.94
[99.0
[99.0
S
o-Chloroacetophenone
o-Methylacetophenone
11.4
94.4
97.9
S
S
12.35
12.47
9.92
90.4
71.6
74.2
83.2
[99.0
[99.0
97.4
o-Methoxyacetophenone
p-Fluorineacetophenone
Propiophenone
S
S
S
12.78
[99.0
2
2
-Acetylthiophene
-Acetylpyridine
12.05
7.58
51.1
0.5
93.3
60.1
S
S
Reaction conditions: 10 mL phosphate buffer (pH 7.0), 150 r/min, 30 °C, 2 % (W/V) glucose, 0.25 g/
mL(wet weight) resting cells, 50 mmol/L substrates (adding in two batches: 0 h 50 %, 12 h 50 %), 2 %
ethanol (substrate solvent). The detection conditions of substrates had slightly different, the column tem-
perature was 115 °C: acetophenone, p-fluoroacetophenone; the column temperature was held at 110 °C: o-
chloroacetophenone, o-methylacetophenone, o-methoxyacetophenone, propiophenone, 2-acetylthiophene;
The column temperature was held at 120 °C: 2-acetylpyridine
biocatalytic asymmetric reductions were 30 °C, pH 6.6,
50 r/min, 24 h. At the end of reactions, the product and
4.4 GC Analysis
1
the residual substrate were extracted by ethyl acetate (1:1,
Products were analyzed by GC instrument with an FID
detector and b-DEX120 capillary column (30 m 9 0.32
mm 9 0.25 lm). Detection conditions: carrier gas was
nitrogen ([99.9 %), injector temperature was 220 °C, the
column temperature was 110–120 °C, FID detector tem-
perature was 200 °C; split ratio was 1:100; the injection
volume was 100 lL. The retention time of products were as
follows.
v/v) and dried by MgSO . The substrate conversion and
4
product e.e. value were determined by GC analysis. Sub-
strate conversion (C) and enantiomeric excess (e.e.) was
calculated as follows:
C% ¼ C =C Â 100
ð1Þ
ð2Þ
P
0
e:e:% ¼ ðC À C Þ=ðC þ C Þ Â 100
S
R
S
R
The column temperature was held at 115 °C: (R)-1-
phenylethanol (17.65 min), (S)-1-phenylethanol (18.77
min); (R)-p-fluorophenethylicalcohol (21.20 min), (S)-p-
fluorophenethylicalcohol (23.81 min). The column tem-
perature was held at 110 °C: (R)-o-chlorophenethylic
C was the initial concentration of the substrate (mmol/
0
L), C was products concentration when the reaction was
P
terminated (mmol/L), C and C represented the concen-
S
R
tration of (R)-1-phenylethanol and (S)-1-phenyl ethanol
mol/L) respectively.
(
1
23

The Asymmetric Reduction of Acetophenone and Its Derivatives to (S)-Aromatic Secondary…
1085
alcohol (21.03 min), (S)-o-chlorophenethylicalcohol (23.07
min); (R)-o-methylphenethylicalcohol (20.00 min), (S)-
methylphenethylicalcohol (22.30 min); (R)-o-methoxy-
phenethylicalcohol (18.03 min), (S)-o-methoxyphenethyli-
calcohol (19.27 min); (R)-phenylpropanol (24.13 min),
Complance with Ethical Standards
Conflict of interest The authors declare that they have no conflict
of interests.
(
S)-phenylpropanol (26.53 min); (R)-1-thiophen-2-yl-etha-
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.5 The Stability of Catalytic Activities of
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.6 Several Key Variables of the Biocatalytic
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Phenylethanol with R. mucilaginosa CCTCC
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6
7
To further improve the conversion and e.e. of product, the
key variables of the biocatalytic asymmetric reduction of
acetophenone with R. mucilaginosa CCTCC M2014255
resting cells were studied, such as reaction temperature,
buffer pH, different co-substrates and their concentration,
cell concentration, substrate concentration and the way of
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Acknowledgments This work was supported by grants from sci-
entific research project of Sichuan Provincial Health Department (No.
1
30295), open project of key laboratory of environmental and applied
microbiology of Chinese academy of sciences (No. KLCAS-2014-
4), national innovation experiment program for university students
Nos. 201513705006 and 201413705002), science and technology
support program of Sichuan Science and Technology Department
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