Using deep eutectic solvents to improve the resolution of racemic 1-(4-methoxyphenyl)ethanol through Acetobacter sp. CCTCC M209061 cell-mediated asymmetric oxidation

Article abstract of DOI:10.1039/c4ra12905a

As novel low-viscosity and environmentally-friendly reaction media, deep eutectic solvents (DESs) have gained much attraction in biocatalysis. In this study, various DESs were prepared and their effects on the asymmetric oxidation of racemic 1-(4-methoxyphenyl)ethanol (MOPE) catalyzed by Acetobacter sp. CCTCC M209061 cells to give enantiopure (S)-MOPE were examined. The best results were obtained with the DES [ChCl][Gly] and its concentration exerted a significant influence on the reaction, with the optimal content being 10% (v/v). In the [ChCl][Gly]-containing system, the substrate concentration was substantially increased (55 mmol L^-1vs. 30 mmol L^-1) as compared with the [ChCl][Gly]-free aqueous system, while the residual substrate e.e. was kept as high as 99.9%. The good biocompatibility of [ChCl][Gly] with the cells and the improved cell membrane permeability in the [ChCl][Gly]-containing system could partly account for the clearly enhanced reaction efficiency.

Full text of DOI:10.1039/c4ra12905a

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Using deep eutectic solvents to improve the
resolution of racemic 1-(4-methoxyphenyl)ethanol
through Acetobacter sp. CCTCC M209061 cell-
mediated asymmetric oxidation†
Cite this: RSC Adv., 2015, 5, 6357
a
a
ab
and Min-Hua Zongb
*
Pei Xu,‡ Jing Cheng,‡ Wen-Yong Lou
As novel low-viscosity and environmentally-friendly reaction media, deep eutectic solvents (DESs) have
gained much attraction in biocatalysis. In this study, various DESs were prepared and their effects on the
asymmetric oxidation of racemic 1-(4-methoxyphenyl)ethanol (MOPE) catalyzed by Acetobacter sp.
CCTCC M209061 cells to give enantiopure (S)-MOPE were examined. The best results were obtained
with the DES [ChCl][Gly] and its concentration exerted a significant influence on the reaction, with the
optimal content being 10% (v/v). In the [ChCl][Gly]-containing system, the substrate concentration was
substantially increased (55 mmol Lꢀ1 vs. 30 mmol Lꢀ1) as compared with the [ChCl][Gly]-free aqueous
system, while the residual substrate e.e. was kept as high as 99.9%. The good biocompatibility of [ChCl]
[Gly] with the cells and the improved cell membrane permeability in the [ChCl][Gly]-containing system
could partly account for the clearly enhanced reaction efficiency.
Received 22nd October 2014
Accepted 16th December 2014
DOI: 10.1039/c4ra12905a
www.rsc.org/advances
relatively low product e.e. (88%) and conversion of 18% were
recorded. In addition, lipase was found to give high yield (99%)
Introduction
and product e.e. (99.9%) in the dynamic kinetic resolution of
racemic MOPE, but with a poor substrate concentration
(20 mmol Lꢀ1) and a long reaction time (10 h). In our previous
study, the asymmetric oxidation of MOPE catalyzed by immo-
bilized Acetobacter sp. CCTCC M209061 cells was conducted
successfully in an aqueous monophasic system with the e.e. of
residual substrate (S)-MOPE being more than 98.0%. However,
the substrate concentration was only 30 mmol Lꢀ1, possibly due
to the pronounced inhibitory and toxic effects of the substrate
and the product at higher substrate loading.
On the other hand, deep eutectic solvents (DESs) have
emerged as a good alternative to conventional ones with
tailored properties, depressed toxicological traits, adequate
biodegradability and acceptable cost, and a large number of
promising application have already appeared in current litera-
ture.9–13 Some DESs have been used as benign solvents for bio-
catalytic processes.14–17 To our knowledge, only one work on
whole-cell biocatalysis in DESs has been published so far,
wherein enantioselective reduction of ketone was observed with
baker's yeast as biocatalyst.18
Chiral alcohols are important building blocks for the synthesis
of chiral pharmaceuticals, agrochemicals, avors, fragrances
and functional materials.1,2 For instance, enantiopure (S)-1-(4-
methoxyphenyl)ethanol {(S)-MOPE} is a key chiral building
block for the synthesis of cycloalkyl [b] indoles used to treat
general allergic responses.3,4 (S)-MOPE can be prepared through
asymmetric resolution of racemic MOPE either chemically or
biologically. For economic, environmental and social reasons,
biological approaches have become a subject of great interest.
Recently, whole-cells based biocatalyst has attracted much
attention due to the unique advantages such as outstanding
enantioselectivity, environmental friendliness and regeneration
of cofactors in situ.5,6 In addition, immobilized cells are recy-
clable and reusable, thus their use greatly simplies the process
and lowers the cost of production.
To date, there have been a few published accounts on the
resolution of racemic MOPE via biocatalytic routes.7,8 For
example, feruloyl esterase was used for the asymmetric trans-
esterication of racemic MOPE with vinyl acetate and the
Herein, we for the rst time report the use of various DESs as
co-solvents to improve the performance of immobilized Aceto-
bacter sp. CCTCC M209061 cells for asymmetric oxidation of
MOPE, and the effects of these DESs on the asymmetric oxida-
tion reactions with MOPE as an initial model substrate (Scheme 1).
In the scheme, MOPE is oxidized to 40-methoxyacetophenone
(MOAP) to give enantiopure (S)-MOPE, while NAD(P)+ is
aLaboratory of Applied Biocatalysis, College of Light Industry and Food Sciences, South
China University of Technology, Guangzhou 510640, China. E-mail: wylou@scut.edu.cn;
Tel: +86-20-22236669
bState Key Laboratory of Pulp and Paper Engineering, South China University of
Technology, Guangzhou 510640, Guangdong, China
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c4ra12905a
‡ These authors contributed equally to this work.
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cells were capable of catalyzing the asymmetric oxidation of
racemic MOPE in the presence of DESs with the hydrogen bond
donors being glycerol, urea, ethylene glycol or acetamide. In
general, the initial reaction rate (90.2 mmol minꢀ1), the
conversion (49.4%) and the residual substrate e.e. (98.7%)
obtained in [ChCl][Gly]-containing system were higher than
those with the control under the same conditions. To better
understand the effects of [ChCl][Gly] on the reaction, the
oxidation reaction following the addition of [ChCl][Gly], choline
chloride or glycerol was investigated. As depicted in Table 2, the
initial reaction rate and the conversion in [ChCl][Gly]-
containing system were higher than those when choline chlo-
ride or glycerol was added individually. The reason for this
might be that the hydrogen bonding interactions between
glycerol molecules and the chloride ion would increase the
nucleophilicity of the substrate, MOPE, thus leading to a faster
oxidation reaction rate.19
Scheme 1 The asymmetric oxidation of racemic MOPE catalyzed by
immobilized Acetobacter sp. CCTCC M209061 cells.
converted to NAD(P)H and the co-substrate, acetone, is simul-
taneously reduced, driving the asymmetric oxidation by regen-
erating NAD(P)+ from NAD(P)H.
Results and discussion
As a result, [ChCl][Gly] was chosen as the co-solvent in the
DESs-containing system for subsequent experiments.
The comparison of the asymmetric oxidation of MOPE using
immobilized and free Acetobacter sp. CCTCC M209061 cells
showed that although immobilized cells afforded nearly the
same maximum conversion (49.4% vs. 49.7%) and residual
substrate e.e. (99.0% vs. 99.4%) as the free cells, the stability of
immobilized cells was much better (Fig. S1–S4 in ESI†).
Consequently, immobilized cells were used in this work.
Effects of several key variables on the asymmetric oxidation of
MOPE catalyzed by immobilized Acetobacter sp. CCTCC
M209061 cells
To see the potential of [ChCl][Gly]-containing system for the
reaction, several key variables were examined. As evident in
Table 3, the initial reaction rate increased with increasing
[ChCl][Gly] content up to 10% (v/v), and a further rise in [ChCl]
[Gly] content led to a clear fall in the initial reaction rate.
Additionally, adding [ChCl][Gly] allowed a residual substrate
Effects of various DESs on the asymmetric oxidation of
racemic MOPE catalyzed by immobilized Acetobacter sp.
CCTCC M209061 cells
According to the studies on biocatalytic reactions in various e.e. of 99.9% due to higher maximum conversions.
DESs-containing systems, the catalytic performance exhibited To get deeper insight into the inuence of [ChCl][Gly]
by a biocatalyst was closely related to the hydrogen bond donor content, we investigated its effects on buffer pH. Unexpectedly,
types of the DESs, and the effects of different DESs on bio- with [ChCl][Gly] concentration increasing from 0 to 20%, the
catalytic reactions have been found to vary widely.9 Therefore, it corresponding buffer pH varied little (from 6.47 to 6.52). Obvi-
is of great signicance to explore the effects of DESs with ously, the effects of [ChCl][Gly] concentration on the reaction
different hydrogen bond donors on the asymmetric oxidation of could not attributed to the changes in buffer pH.
MOPE catalyzed by immobilized Acetobacter sp. CCTCC
A great effect of buffer pH on the reaction in the [ChCl][Gly]-
M209061 cells in various DES-containing systems (Table 1). It containing system was observed (Fig. S5†). When buffer pH
was noted that immobilized Acetobacter sp. CCTCC M209061 increased from 4.0 to 6.5, the reaction sped up (83 mmol minꢀ1
Table 1 Effect of various DESs on the asymmetric oxidation of racemic MOPE catalyzed by immobilized Acetobacter sp. CCTCC M209061
cellsa,b
Initial reaction rate
Media
(mmol minꢀ1
)
Reaction time (h)
Conversionc (%)
e.e.d (%)
Aqueous buffer
80.0
29.6
90.2
63.8
48.2
77.7
34.3
23.5
11.0
22.5
9.0
12.0
13.0
10.5
22.0
23.0
47.5
5.0
91.4
53.6
98.7
76.2
67.1
92.3
65.1
34.1
[ChCl][MA]/buffer
[ChCl][Gly]/buffer
[ChCl][EG]/buffer
[ChCl][Acet]/buffer
[ChCl][U]/buffer
[ChCl][IM]/buffer
[Bu4NBr][IM]/buffer
49.4
42.9
39.8
47.7
6.1
3.2
a
ChCl: choline chloride, MA: malonic acid, Gly: glycerol, EG: ethylene glycol, Acet: acetamide, U: urea, IM: imidazole, Bu4NBr:
tetrabutylammonium bromide. Reaction conditions: 5.0 mL TEA–HCl buffer (100 mmol Lꢀ1, pH 6.5) consisting of 20% (v/v) various DESs,
b
40 mmol Lꢀ1 MOPE, 0.3 g immobilized cells, 50 mmol Lꢀ1 acetone, 30 ꢁC, 200 rpm. c The maximum conversion. d The residual substrate e.e.
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Table 2 Effect of [ChCl][Gly] and its components on the asymmetric oxidation of racemic MOPE with immobilized Acetobacter sp. CCTCC
M209061 cellsa
Initial reaction rate
[ChCl][Gly] or its componentsb
(mmol minꢀ1
)
Reaction time (h)
Conversionc (%)
e.e.d (%)
Control
ChCl
[ChCl][Gly]
Gly
101.6
99.1
125.3
100.8
9.0
9.0
7.0
8.0
49.3
49.3
52.0
48.6
98.2
98.2
99.9
95.5
a
Reaction conditions: 5.0 mL TEA–HCl buffer (100 mmol Lꢀ1, pH 6.5) containing 10% (v/v) [ChCl][Gly], 55 mmol Lꢀ1 MOPE, 0.3 g immobilized
cells, 50 mmol Lꢀ1 acetone, 30 ꢁC, 200 rpm. ChCl or Gly was added according to the mass fraction in [ChCl][Gly]. The maximum conversion.
b
c
d
The residual substrate e.e.
vs. 107.9 mmol minꢀ1), and the maximum conversion increased was increased from 160 rpm to 220 rpm, suggesting that mass
(39.0% vs. 50.5%) with the residual substrate e.e. rising from transfer was the rate-limiting step (Fig. S8†). However, a further
63.9% to 99.9%. Further increases in buffer pH, however, led to increase in the shaking rate had little effect on the initial
a remarkable drop in initial reaction rate, the maximum reaction rate, maximum conversion, and residual substrate e.e.,
conversion and the residual substrate e.e. Thus, pH 6.5 was indicating that 220 rpm was the optimal shaking rate.
regarded as the optimal buffer pH.
As shown in Table 4, the initial reaction rate increased with
Increasing the reaction temperature from 20 ꢁC to 30 ꢁC increasing substrate concentration up to 55 mmol Lꢀ1, while
caused increased initial reaction rate (79.8 mmol minꢀ1 vs. 109.1 the maximum conversion and the residual substrate e.e.
mmol minꢀ1), maximum conversion (44.4% vs. 52.8%) and showed no clear variation. However, further increases in
residual substrate e.e. (97.6% vs. 99.9%) (Fig. S6†). However, the substrate concentration from 55 mmol Lꢀ1 to 65 mmol Lꢀ1 led
initial reaction rate (109.1 mmol minꢀ1 vs. 88.5 mmol minꢀ1), the to a clear drop in the initial reaction rate, the maximum
maximum conversion (52.8% vs. 46.5%) and the residual conversion and residual substrate e.e., possibly due to inhibi-
substrate e.e. (99.9% vs. 85.2%) decreased with increase in tory and toxic effects of the substrate and the product. It is
reaction temperature from 30 ꢁC to 45 ꢁC. So the most suitable worth noting that the optimal substrate concentration in the
ꢁ
reaction temperature was 30 C.
[ChCl][Gly]-containing system was much higher than that in the
Raising the concentration of co-substrate acetone from aqueous monophasic system (55 mmol Lꢀ1 vs. 30 mmol Lꢀ1
)
40.0 mmol Lꢀ1 to 50.0 mmol Lꢀ1 gave rise to increased initial and this will doubtlessly improve the reaction efficiency
reaction rate (109.1 mmol minꢀ1 vs. 126.5 mmol minꢀ1), substantially.
maximum conversion (47.5% vs. 52.0%) (Fig. S7†) and residual
In order to understand why [ChCl][Gly]-containing system
substrate e.e. (97.0% vs. 99.9%). However, further rise in could allow a higher substrate loading, we examined the
acetone concentration from 50.0 mmol Lꢀ1 to 65.0 mmol Lꢀ1 biocompatibility of various DESs with Acetobacter sp. CCTCC
led to declined initial reaction rate (126.5 mmol minꢀ1 vs. M209061 cells and the effects of DESs on the cells' membrane
101.2 mmol minꢀ1), maximum conversion (52.0% vs. 48.9%) integrity.
and residual substrate e.e. (99.9% vs. 95.7%). Therefore,
the optimal acetone concentration was considered to be
50.0 mmol Lꢀ1
Biocompatibility of various DESs with Acetobacter sp. CCTCC
M209061 cells
.
The initial reaction rate increased rapidly from
Despite being regarded as green solvents, DESs have been found
to be toxic to microorganisms.14 Thereby, it is necessary to
100.5 mmol minꢀ1 to 126.0 mmol minꢀ1 when the shaking rate
Table 3 Effect of [ChCl][Gly] content on the asymmetric oxidation of racemic MOPE with immobilized Acetobacter sp. CCTCC M209061 cellsa
Initial reaction rate
[ChCl][Gly] content
(mmol minꢀ1
)
Reaction time (h)
Conversionb (%)
e.e.c (%)
0
5%
8%
10%
12%
15%
20%
89.0
94.1
97.1
102.3
98.0
94.2
90.3
10.0
9.0
8.0
7.0
8.0
8.5
9.0
48.0
50.0
50.5
51.7
50.3
49.8
49.3
93.5
99.9
99.9
99.9
99.9
99.2
98.3
a
Reaction conditions: 5.0 mL TEA–HCl buffer (100 mmol Lꢀ1, pH 6.5) containing various concentrations of [ChCl][Gly], 40 mmol Lꢀ1 MOPE, 0.3 g
immobilized cells, 50 mmol Lꢀ1 acetone, 30 ꢁC, 200 rpm, incubation time 7 h. The maximum conversion. c The residual substrate e.e.
b
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Table 4 Effect of substrate concentration on the asymmetric oxidation of racemic MOPE with immobilized Acetobacter sp. CCTCC M209061
cellsa
Initial reaction rate
Substrate concentration (mmol Lꢀ1
)
(mmol minꢀ1
)
Reaction time (h)
Conversionb (%)
e.e.c (%)
40.0
45.0
50.0
55.0
60.0
65.0
109.1
118.2
126.5
126.7
107.0
93.1
6.0
6.5
7.0
7.5
9.0
52.8
52.2
51.6
51.5
49.2
48.3
99.9
99.9
99.9
99.9
97.8
94.5
10.0
a
Reaction conditions: 5.0 mL TEA–HCl buffer (100 mmol Lꢀ1, pH 6.5) containing 10% (v/v) [ChCl][Gly], various concentrations of MOPE, 0.3 g
immobilized cells, 50 mmol Lꢀ1 acetone, 30 ꢁC, 200 rpm. b The maximum conversion. The residual substrate e.e.
c
assess the biocompatibility of the DESs used with Acetobacter imidazole, which could explain the catalytic efficiency shown
sp. CCTCC M209061 with the sugar metabolic activity retention in Table 1.
(MAR) of the microbial cells as a criterion.20 As shown in Fig. 1,
It has been speculated that the good biocompatibility of the
the MAR value of the cells in the presence of substrate was DESs with glycerol, urea or ethylene glycol with microbial cells
lower than that of cells in the absence of substrate, suggesting stem from the OH-bridge protic microenvironment formed by
that MOPE was toxic to the cells. In addition, the cells in the their hydroxyl groups.21 More attention, however, should be
aqueous monophasic system showed higher MAR value than paid to the underlying mechanisms and relevant studies are
those in all tested DESs-containing system without substrate, now underway in our laboratory. The highest MAR value ach-
implying that the examined DESs were toxic to the cells to some ieved with the cells in [ChCl][Gly]-based co-solvent system,
extent.
agreed with the observation in Table 1. It is interesting to note
The MAR value varied greatly in different DESs-containing that the MAR value of the cells with substrate was only 3% lower
systems, of which [ChCl][Gly] exhibited the best biocompati- than that without substrate in the [ChCl][Gly]-containing
bility with the cells and the cells displayed the highest MAR system, while the corresponding value was around 25% in
value of 94.5%. In the case of ChCl-based DESs, the variation of aqueous buffer, demonstrating that the addition of [ChCl][Gly]
MAR value in the presence of substrate with different hydrogen to the reaction system substantially lowered the toxic effect of
bond donors in the DESs (Fig. 1) could well account for the the substrate and the product to the cells, which contributes to
signicant inuence of these hydrogen bond donors in DESs the higher substrate loading.
on the reaction (Table 1). In addition, the MAR value of the
cells with [Bu4NBr][IM] was inferior to that of [ChCl][IM] irre-
spective of the presence of substrates. For all the tested DESs,
Effect of various DESs on cell membrane integrity
the MAR values of the cells when the hydrogen bond donors
were glycerol, urea, and ethylene glycol were higher than those
when the hydrogen bond donors were carboxylic acid and
The enhanced catalytic efficiency in [ChCl][Gly]-containing
system might result partly from the improved cell membrane
permeability, which could accelerate the mass transfer, thus
leading to a higher initial rate, reduced toxic and inhibitory
effects of the product and limited reverse reaction. On the other
hand, cell death may be along with the damage to cell
membrane and thus lower the availability of reaction equiva-
lents for the reaction.15,22 Hence, it is of great interest to inves-
tigate the inuence of various DESs on the cell membrane
integrity.
Flow cytometry (FCM) with propidium iodide (PI) as cell
uorescein dye23 was adopted as a simple and accurate method
of measuring the membrane integrity of Acetobacter sp. CCTCC
M209061 cells incubated with various DESs (10%, v/v)-
containing systems for 24 h.
As can be seen in Fig. 2, the cell membrane integrity
decreased signicantly, indicating that the examined DESs
damaged and permeated the cell membrane. The different types
of DESs exerted distinctly different effects on the cell membrane
integrity and thus on the cell membrane permeability. The cell
membrane integrity as well as the cell membrane permeability
was clearly dependent on the different hydrogen bond donors.
Fig. 1 Metabolic activity retention of immobilized Acetobacter sp.
CCTCC M209061 cells after 24 h exposure to co-solvent systems
consisting of 10% various DESs and TEA–HCl buffer (100 mmol Lꢀ1
,
pH 6.5), with and without substrate (40 mmol Lꢀ1 MOPE).
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Fig. 2 Membrane integrity of Acetobacter sp. CCTCC M209061 cells measured by FCM with PI as cell fluorescein dye after being incubated in
various DES (10%, v/v)-containing co-solvent systems for 24 h.
With DESs in which the hydrogen bond donors were carboxylic
acid and imidazole, the cell membrane was relatively less inte-
grated, indicating that these DESs seriously damaged the cell
membrane, leading to cell death. As a result, in the presence of
these DESs, the microbial cells displayed a very low activity
(Table 1) and MAR (Fig. 1). For DESs in which the hydrogen
bond donors was glycerol, urea, or ethylene glycol, the cell
membrane integrity decreased slightly. Of all the DESs
assessed, the highest cell membrane integrity was recorded
with [ChCl][Gly], which correlated with the best performance of
the cells in the [ChCl][Gly]-containing system. Obviously, only a
moderate increase in cell membrane permeability could
enhance the reaction efficiency.
Preparative scale biotransformation in the [ChCl][Gly]-
containing system
To further show the feasibility of applying [ChCl][Gly]-
containing system to practical use, the reaction was con-
ducted on a 500 mL preparative scale under the optimal reac-
tion conditions {55 mmol Lꢀ1 MOPE, [ChCl][Gly]-containing
system (10% [ChCl][Gly], 100 mmol Lꢀ1, pH 6.5), 50 mmol Lꢀ1
acetone, 0.3 g mLꢀ1 immobilized cells, 30 ꢁC, and 220 rpm}. The
reaction process was monitored by GC analysis, and
the product was extracted from the reaction mixture with iso-
propyl ether upon the exhaustion of (R)-MOPE. The residual
substrate e.e. was above 99.9% aer reacting for 7 h when the
conversion was 51.5%. Therefore, the whole-cell biocatalytic
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resolution of MOPE in the [ChCl][Gly]-containing system is ask capped with a septum. Predetermined free or immobilized
though promising.
Acetobacter sp. CCTCC M209061 cells were added as well as
50 mmol Lꢀ1 acetone. The reaction mixture was pre-incubated
at an appropriate temperature and shaking rare for 15 min.
The reaction was then initiated by adding MOPE (precalculated
concentration) to the reaction system. Aliquots (50 mL) were
withdrawn at specied time intervals, and then the product and
the residual substrate were extracted with isopropyl ether
(50 mL) containing 5.04 mmol Lꢀ1 n-tetradecane (as an internal
standard) prior to GC analysis. Details of the used DESs, content
of DESs and substrate are specied in each case.
Experimental
Biological and chemical materials
Acetobacter sp. CCTCC M209061 was isolated from Chinese ker
grains by our research group and conserved in our laboratory.24
MOPE (98% purity) was purchased from Alfa Aesar (USA).
40-Methoxyacetophenone (99%) and n-tetradecane (>99%) were
purchased from TCI (Japan). All other chemicals were from
commercial sources and were of analytical grade.
Stability of biocatalysts
Cultivation and immobilization of Acetobacter sp. CCTCC
Thermal stability. Wet-free cells (2.5 g) or immobilized beads
(16.7 g, with a load of approximately 2.5 g cwm of Acetobacter sp.
CCTCC M209061 cells) in 60 mL TEA–HCl buffer
(100 mmol Lꢀ1, pH 6.5) were incubated at 30, 40, and 50 ꢁC.
Samples (0.3 g wet-free cells or 2.0 g immobilized beads) were
withdrawn at specied time intervals and cooled to 30 C. The
residual activity of biocatalysts was determined using the
activity test described above.
M209061 cells
Acetobacter sp. CCTCC M209061 was cultivated according to our
previous described methods.25 The medium was sterilized by
autoclaving at 121 ꢁC for 20 min. Aer 30 h incubation, the cells
were in the late exponential growth phase and were harvested by
centrifugation (6010ꢂg, 10 min, 4 ꢁC) to give a biomass of 6.11
ꢃ 0.17 g Lꢀ1 (wet weight). The wet cells were immobilized as
described below before use in the asymmetric oxidation. A
homogenous cell/chitosan suspension was prepared at 25 ꢁC by
adding 6 g of fresh cell suspension (3 g wet cells in 3 mL water)
into 47 mL of a homogeneous aqueous chitosan solution (3%,
w/v), which was prepared by dissolving chitosan in acetate
buffer (pH 4.2), heating and ultrasonic processing (20 KHz,
30 min).
ꢁ
Storage stability. Wet-free cells (2.5 g) or immobilized beads
(16.7 g, with a load of approximately 2.5 g cwm of Acetobacter sp.
CCTCC M209061 cells) were incubated in TEA–HCl buffer
(100 mmol Lꢀ1, pH 6.5) and stored at 4 ꢁC. Samples (0.3 g wet-
free cells or 2.0 g immobilized beads) were withdrawn at spec-
ied time intervals and the residual activity was determined.
Operational stability. In order to text the operational stability
of the cells, the re-use of immobilized and free Acetobacter sp.
CCTCC M209061 cells was investigated in the aqueous mono-
phasic system. Initially, an aliquot of the immobilized cells
(0.3 g mLꢀ1) or free cells (0.045 g mLꢀ1) was added to the
TEA–HCl buffer system (100 mmol Lꢀ1, pH 6.5) containing
50 mmol Lꢀ1 acetone and 30 mmol Lꢀ1 MOPE. The reactions
The suspension was added dropwise from a syringe to a
cross-linking solution, which was mixed by 4% (w/v) glyoxal
solution with an equal volume of 3% (w/v) tetrasodium pyro-
phosphate solution (pH 8.0). The gel beads were hardened for
30 min at room temperature. The beads were then transferred
to 0.05% glutaraldehyde for reinforcement treatment.26,27 The
coated beads were collected by ltration and washed with
sterilized water to remove the residual solution. The beads had
a load of 15% (w/w) of Acetobacter sp. CCTCC M209061 cells
(based on cell wet mass). The resulting beads were stored in
triethanolamine (TEA)–HCl buffer (100 mmol Lꢀ1, pH 6.5) at
ꢁ
were then carried out at 30 C and 200 rpm and were repeated
over 10 batches (12 h per batch) without changing the bio-
catalysts. Between batches, immobilized cells were ltered from
the reaction mixture, washed three times with distilled water,
and added to a fresh batch of reaction medium. Free cells were
recycled by centrifugation aer each batch (6010ꢂg, 10 min,
ꢁ
4 C for later use.
4
ꢁC). Other conditions were the same as those used with
Preparation of DESs
immobilized cells. The activity of the cells, the conversion and
the residual substrate e.e. were assayed in each batch. The
relative activity of the cells employed for the rst batch was
dened as 100%.
The preparation of DESs involved the reaction of choline chlo-
ride (1 mol) with different hydrogen bond donors (2 mol) at
100 ꢁC in a ask with stirring for 2 h until a clear solution was
obtained which was used for the reactions without purication.
This method gave DESs with 100% atom economy as it
completely formed a eutectic mixture with no by-product
formation. Another DES was similarly prepared by combining
tetrabutyl ammonium bromide (3 mol) with imidazole (7 mol)
under the same conditions.18
Preparative scale biocatalytic resolution of MOPE in the
[ChCl][Gly]-based system
The preparative scale biocatalytic resolution of MOPE was per-
formed by adding 150.0 g immobilized Acetobacter sp. CCTCC
M209061 cells and 55 mmol Lꢀ1 MOPE to 500 mL of the mixing
system consisting of 50 mL [ChCl][Gly] and 450 mL TEA–HCl
buffer (100 mmol Lꢀ1, pH 6.5) containing 50 mmol Lꢀ1 acetone
at 220 rpm and 30 ꢁC. The reaction was terminated when no (R)-
General procedure for asymmetric oxidation of MOPE
(oxidation activity assay)
In a typical experiment, a system (5.0 mL) consisted of DESs and MOPE was detected by GC analysis. The immobilized cells were
TEA–HCl buffer (100 mmol Lꢀ1), added to a 10 mL Erlenmeyer removed by ltration, and the reaction mixture was extracted
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with acetic ether. The residual substrate e.e. and the conversion
were determined by GC analysis.
The average error for this determination was less than 1.0%.
All reported data averages of the experiments were performed at
least twice.
Cell metabolic activity retention measurement
Conclusions
The metabolic activity retention (%, MAR) of immobilized Ace-
tobacter sp. CCTCC M209061 cells was dened as the ratio of the
amount of glucose consumed by the immobilized cells pre-
treated in various media to that by the immobilized cells pre-
treated in aqueous buffer (as the control).20 The MAR value of
immobilized Acetobacter sp. CCTCC M209061 cells was assayed
aer 24 h exposure to various systems consisting of various
DESs (10%, v/v)/TEA–HCl buffer (100 mmol Lꢀ1, pH 6.5) or in a
TEA–HCl buffer (100 mmol Lꢀ1, pH 6.5) monophasic system in
the presence and absence of substrate (40 mmol Lꢀ1 MOPE,
based on the volume of the entire system), respectively, in a
rotary incubator set at 30 ꢁC and 200 rpm. Aer separation from
the reaction medium and washed three times with fresh water,
The DES [ChCl][Gly] could greatly enhance the efficiency of
resolution of racemic MOPE using immobilized Acetobacter sp.
CCTCC M209061 cells and improve the stability of the bio-
catalyst owing to the excellent solvent property of [ChCl][Gly] for
MOPE and its benign biocompatibility with Acetobacter sp.
CCTCC M209061 cells. Furthermore, the Acetobacter sp. CCTCC
M209061 cells-catalyzed process with co-solvent [ChCl][Gly] is
promising for industrial production of enantiopure (S)-MOPE.
Further developments of the applications of DES-containing
system in biocatalysis process are awaited with great interest.
the beads of immobilized cells were transferred to glucose Acknowledgements
solution (10 mL, 1.0 g Lꢀ1), and then incubated at 30 ꢁC and 200
rpm for 4 h. The glucose concentration in the medium was then
determined by HPLC.
We thank the National Science Found for Excellent Young
Scholars (21222606), the State Key Program of National Natural
Science Foundation of China (21336002), the NSFC (21376096),
the Key Program of Guangdong Natural Science Foundation
(S2013020013049) and the Fundamental Research Funds for
SCUT (2013ZG0003) for partially funding this work.
Cell membrane integrity assay
In a typical experiment, 5 mL of a DES (10%, v/v)-containing
system or aqueous TEA–HCl buffer (100 mmol Lꢀ1, pH 6.5)
containing 0.05 g mLꢀ1 Acetobacter sp. CCTCC M209061 cells
were incubated for 24 h in a 10 mL Erlenmeyer ask capped
Notes and references
ꢁ
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6364 | RSC Adv., 2015, 5, 6357–6364
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