Using deep eutectic solvents to improve the biocatalytic reduction of 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol by Kurthia gibsonii SC0312

Article abstract of DOI:10.1016/j.mcat.2020.110773

The effects of five deep eutectic solvents (DESs) on the production of (R)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone catalyzed by Kurthia gibsonii SC0312 were investigated in this study. Of these, choline chloride/1,4-butanediol (ChCl/Bd) showed excellent biocompatibility and suitably increased the cell membrane permeability while having a little impact on the structure of DNA. Indeed, ChCl/Bd at the concentration of 2 % increased the catalytic rate of the cells by 22 %. The other DESs did not stimulate the catalytic capacity of the cells, despite some increases in the cell membrane permeability. Additionally, the conformation of DNA was visibly changed when adding the other examined DESs except for choline chloride/triethylene glycol. The DESs modified the fatty acid composition of cellular membrane, decreased the relative amount of iso-C14:0 and increased the relative amount of normal C15:0. Meanwhile, the DESs were able to improve the relative ratio of normal fatty acids to branched fatty acids. Finally, a highly efficient reduction of 80 mM 2-hydroxyacetophenone by K. gibsonii SC0312 in the ChCl/Bd-containing system was established, affording (R)-1-phenyl-1,2-ethanediol in 80 % yield and optical purity >99 % at 30 mg/mL wet cells. This work offers a promising approach for the preparation of (R)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone using K. gibsonii SC0312.

Full text of DOI:10.1016/j.mcat.2020.110773

Molecular Catalysis 484 (2020) 110773
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.elsevier.com/locate/mcat
Using deep eutectic solvents to improve the biocatalytic reduction of 2-
hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol by Kurthia gibsonii
SC0312
Fei Peng, Qing-Sheng Chen, Fang-Zhou Li, Xiao-Yang Ou, Min-Hua Zong, Wen-Yong Lou*
Laboratory of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Kurthia gibsonii SC0312
Whole cell catalysis
The effects of five deep eutectic solvents (DESs) on the production of (R)-1-phenyl-1,2-ethanediol from 2-hy-
droxyacetophenone catalyzed by Kurthia gibsonii SC0312 were investigated in this study. Of these, choline
chloride/1,4-butanediol (ChCl/Bd) showed excellent biocompatibility and suitably increased the cell membrane
permeability while having a little impact on the structure of DNA. Indeed, ChCl/Bd at the concentration of 2 %
increased the catalytic rate of the cells by 22 %. The other DESs did not stimulate the catalytic capacity of the
cells, despite some increases in the cell membrane permeability. Additionally, the conformation of DNA was
visibly changed when adding the other examined DESs except for choline chloride/triethylene glycol. The DESs
modified the fatty acid composition of cellular membrane, decreased the relative amount of iso-C14:0 and in-
creased the relative amount of normal C15:0. Meanwhile, the DESs were able to improve the relative ratio of
normal fatty acids to branched fatty acids. Finally, a highly efficient reduction of 80 mM 2-hydroxyacetophenone
by K. gibsonii SC0312 in the ChCl/Bd-containing system was established, affording (R)-1-phenyl-1,2-ethanediol
in 80 % yield and optical purity > 99 % at 30 mg/mL wet cells. This work offers a promising approach for the
preparation of (R)-1-phenyl-1,2-ethanediol from 2-hydroxyacetophenone using K. gibsonii SC0312.
1
2
-Phenyl-1,2-ethanediol
-Hydroxyacetophenone
Deep eutectic solvents
1. Introduction
solvents, organic solvents and ionic liquids [9–12]. Since the beginning
of the 21th century, DESs [12,13] have been applied as novel green
(
R)-1-phenyl-1,2-ethanediol (PED) serves as a pivotal synthon for
obtaining enantiopure medicines such as β-adrenergic blocking agents
1], which are suitable for treating cardiovascular disease and sympa-
solvents in the bioconversion either as the main reaction medium
[14,15] or as a co-solvent [16–18]. Some studies have introduced DESs
as co-solvents to promote reaction efficiency [19], as exemplified by the
use of choline chloride/urea (ChCl/U) and choline chloride/glycerol
(ChCl/Gly) [17] to increase the initial reaction rate or the biocatalytic
stability [20], or even to change the stereoselectivity of biocatalyst
[21]. A moderate amount of DESs can stimulate biocatalytic reaction
efficiency chiefly through increasing the solubility of a substrate [22],
or changing enzymatic structure [23]. In comparison to enzyme bio-
catalysts, the cell membrane of whole cell biocatalysts protects in-
tracellular enzyme from potential harm and hinders the contact be-
tween intracellular enzyme and extracellular substance. The impact of
none-aqueous solvents (organic solvents, ionic liquids, and DESs, etc.)
on whole cell biocatalysts is therefore more complex than on the en-
zyme biocatalysts. Prior researches have reported the effect of ionic
liquids or DESs on the cellular membrane and confirmed that ionic li-
quids could pass through cellular membrane and interact with inter-
cellular enzymes [24–26]. Some studies also found that DESs as a co-
[
thetic nervous system disorder [2–4]. Up to date, some researchers have
directed considerable attention towards biocatalytic approach for pre-
paring enantiomeric PED owing to mild reaction conditions, high se-
lectivity and environmental friendliness [5,6]. Enantiomerically pure
(
R)-PED is typically fabricated by asymmetric hydrolysis of epoxides,
asymmetric resolution of racemic PED, or asymmetric reduction of
prochiral ketones. Compared with enzymatic biocatalysis, whole cell
biocatalytic reactions have characterized as extraordinarily superior
features, including easy accessibility, strong resistance and cofactor
regeneration [7,8]. Therefore, utilization of whole cell biocatalytic re-
action for chiral PED production becomes potential and meaningful.
Deep eutectic solvents (DESs) generally encompass two or three
structural components, which are usually sustainable, biodegradable or
inexpensive, and form a eutectic mixture characterized by low toxicity,
negligible volatility and biodegradation compared with conversional
⁎
Corresponding author.
E-mail address: wylou@scut.edu.cn (W.-Y. Lou).
https://doi.org/10.1016/j.mcat.2020.110773
Received 2 October 2019; Received in revised form 11 January 2020; Accepted 11 January 2020
2468-8231/ © 2020 Elsevier B.V. All rights reserved.

F. Peng, et al.
Molecular Catalysis 484 (2020) 110773
Scheme 1. The reduction of HAP to (R)-PED by K. gibsonii SC0312 in the system containing deep eutectic solvents.
solvent would benefit the expansion of cellular membrane to enhance
catalytic efficiency [27–29]. To our knowledge, however, little in-
formation is available regarding the change of fatty acid components in
microbial cell membrane within DESs-containing systems. DNA is an
important carrier of genetic information and the maintenance of its
structure is of great significance to life activities. Calf thymus DNA
previous work [35] and are all water-soluble. In a typical preparation,
choline chloride and triethylene glycol were first mixed and stirred by a
magnetic agitator at 80 °C for approximately 2 h until a stable homo-
geneous liquid formed. ChCl/Teg was subsequently dried in a vacuum
oven at 70 °C for 48 h.
K. gibsonii SC0312 was stored in our laboratory and cultured in
Luria-Bertani (LB) broth. The strain was first cultured at 37 °C and
180 rpm in LB broth overnight, and then 0.1 % of seed liquid was in-
jected to a fresh LB broth to reproduce for 12–16 h. The cells were
collected by centrifugation (6000×g, 5 min) and washed twice with
phosphate buffer (PB, 100 mM, pH 6.5) prior to their deployment for
catalytic reaction.
(
ctDNA) has often employed in studying the interactions with mole-
cules, such as farrerol [30] and isoxazolcurcumin [31]. There were
reports indicating the structure of DNA would be changed by DESs
[
32,33]. However, few studies have addressed the relationship between
the DNA structure and the catalytic properties of whole cells. Moreover,
the DESs for different biocatalytic reactions may be diverse and little
information concerning the application of DESs in the reduction of 2-
hydroxyacetophenone (HAP) to (R)-PED has hitherto been published.
We previously isolated a strain of Kurthia gibsonii SC0312 (K. gibsonii
SC0312) from soil, which exhibited high enantioselectivity in the
synthesis of chiral PED [34]. In this study, our aims were to investigate
the impact of DESs on whole-cell catalytic properties on the reduction
of HAP to (R)-PED by K. gibsonii SC0312, and establish an approach for
preparing chiral PED based on the system containing a DES (Scheme 1).
Firstly, the effects of DESs on the catalytic activity of the cells, meta-
bolic activity of the cells, cell membrane permeability, fatty acid
compositions of cellular membrane and DNA were investigated. Sub-
sequently, we selected a DES as the co-solvent and tested the variation
in catalytic properties with various reaction factors for asymmetric
reduction of HAP by K. gibsonii SC0312.
2.2. General procedure for biocatalytic reduction of HAP
In a typical experiment, PB (100 mM, pH 7.5, 4 mL) containing 20
mM HAP, 30 mM glycerine, 30 mg/mL wet cells and a DES (4 %, v/v)
was incubated at 35 °C and 180 rpm. Aliquots were withdrawn at reg-
ular intervals to monitor the initial reaction rate, enantiomeric excess
(
ee) and yield of (R)-PED. The initial reaction rate was based on the
generated amount of PED after catalytic reaction for 30 min. The pro-
duct yield was defined as the ratio of the generated amount of (R)-PED
to the theoretical amount. The ee of (R)-PED was calculated based on
the following equation.
CR − CS
ee =
× 100%
CR + CS
where C
S
and C
R
were the concentrations of (S)-PED and (R)-PED, re-
2. Materials and methods
spectively.
2.1. Materials and strain
2.3. Cell metabolic activity in the system containing DESs
HAP (purity 97 %) was purchased from Acros Organics Technology
Co., Ltd., China. (S/R)-PED (purity 97 %) was purchased from
Metabolic activity of the cells was assessed by determining cell
Guangzhou Qiyun Bioscience Co., Ltd., China. Choline chloride (purity
growth in LB broth containing the DESs. In brief, the cells were firstly
incubated at 37 °C and 180 rpm in LB broth for 10–12 h, and then a seed
sample with an inoculation amount of 0.1 % (v/v) was added to LB
broth containing a DES, and the mixture was cultured at 37 °C and
180 rpm for 6 h. The biomass was determined at 600 nm by UV–vis
spectrophotometry. The results were expressed in terms of relative
biomass, with the biomass in the DESs-free LB broth being defined as
100 %.
98 %) and 1,4-butanediol (purity 99 %) were purchased from
Sinopharm Chemical Reagent Co., Ltd., China and Tianjin Kermel
Chemical Reagent Co., Ltd., China, respectively. ctDNA was obtained
from Shanghai Yuanye Biotechnology Co., Ltd. (Shanghai, China).
Other reagents were from commercial sources and were of analytical
grade.
The DESs used in this study (Table 1) were prepared according to a
Table 1
DESs and their characteristics.
Entry
DESs
Abbreviation of DESs
Mole ratio (HA:HD)a
Viscosity (Pa·s)b
1
2
3
4
5
choline chloride/urea
choline chloride/ethanediol
choline chloride/glycerol
choline chloride/triethylene glycol
choline chloride/1,4-butanediol
ChCl/U
1:2
1:2
1:2
1:4
1:4
0.214
0.025
0.177
0.044
0.047
ChCl/Et
ChCl/Gly
ChCl/Teg
ChCl/Bd
a
HA: hydrogen-bond acceptor; HD: hydrogen-bond donor.
These values were referenced by one of our publications [32].
b
2

F. Peng, et al.
Molecular Catalysis 484 (2020) 110773
Fig. 1. The changes in the catalytic rate of K. gibsonii SC0312cell in the different reaction systems. Reaction conditions: 20 mM HAP, 30 mg/mL wet cell dosage, 35 °C
and 180 rpm, 100 mM phosphate buffer, 2 % of DESs (v/v), reaction time 5 h.
2.4. Cell membrane permeability assay
Standards and Technology mass spectral library (2001).
The porous membrane of cell facilitates the leakage of intracellular
2.6. Circular dichroism (CD) assay
nucleic acids and proteins. To assess cell membrane permeability, K.
gibsonii SC0312 cells (OD600 = 0.2–0.3) were incubated in the saline
solution containing a DES (2 %, v/v) at 35 °C and 180 rpm. The system
without DESs was set as the control group. Samples were withdrawn at
ctDNA is often used to access the interaction with molecules. This
study used ctDNA as a temple and accessed the effect of DESs on the
structure of DNA by CD spectra assay. CD spectra of ctDNA in the ab-
sence and presence of the DESs were recorded using a Chirascan
spectrometer (Applied Photophysics, UK). The concentrations of the
DESs were set at 0 %, 2 %, 8 %, and 16 %, respectively, with a ctDNA
concentration of 4 mg/mL. The observed CD spectra were corrected by
the buffer signal (PB, 30 mM, pH 7.4).
0
h and 24 h to analyze the release of intracellular components by de-
termining the cell-free supernatants at 260 nm and 280 nm. The final
values are expressed in terms of the increase in absorbance from 0 h to
24 h.
The membrane integrity of the cells was also assayed by flow cy-
tometry (FCM). Briefly, K. gibsonii SC0312 cells at 30 mg/mL were in-
cubated for 12 h in the presence of the respective DES, and then diluted
to 10 CFU/mL with saline solution. After incubating with 2.5 μg/mL
2.7. Analytical methods
7
propidium iodide (PI) at 4 °C in the dark for 15 min, membrane in-
tegrity of the strain suspension was measured using a Gallios flow
cytometer (Beckman Coulter Inc., USA). The fluorescence emission was
excited at 488 nm and recorded at 600–620 nm.
The yield and optical purity of (R)-PED produced from HAP cata-
lyzed by K. gibsonii SC0312 were determined by means of reversed-
phase high-performance liquid chromatography (HPLC) and normal-
phase HPLC, respectively. A Waters HPLC set-up equipped with a UV
detector and an XBridge C18 column (4.6 mm × 250 mm, 5 μm, Waters,
USA) was applied to monitor changes in the concentrations of PED at
2.5. Assay of cell membrane fatty-acid composition
215 nm. The mobile phase consisted of water and acetonitrile (60:40, v/
The effect of DESs on the cell membrane fatty acid composition was
v) and was applied at a flow rate of 0.5 mL/min. The retention time of
PED was at 6.5 min. For analysis of the product ee value, the sample was
extracted and suitably diluted with ethyl acetate. The product ee was
assayed by means of an Agilent HPLC 1100 equipped with a UV de-
tector set at 215 nm using an OB-H analytical column
(4.6 mm × 150 mm, 5 μm, Agilent, USA). The mobile phase comprised
n-hexane/isopropanol (9:1, v/v) and was applied at a flow rate of
0.7 mL/min. The retention times of (R)-PED and (S)-PED were at
7.1 min and 8.7 min, respectively. The results were expressed as
mean ± standard deviation and all experiments were performed at
least in duplicate.
studied in PB (100 mM, pH 7.5) containing respective DESs (8 %, v/v)
and 25 mg/mL wet cells. A DESs-free system was prepared as the con-
trol. Each system was incubated at 35 °C and 180 rpm for 12 h, and then
the cells were collected to extract and analyze the fatty acid composi-
tion. Cell membrane fatty acids were extracted as described in a pre-
vious report [36] with some modifications. The collected cells were first
mixed with 1.0 mL of saponification solution in a tube immersed in a
boiling water bath for 30 min. The stock saponification solution con-
sisted of distilled water (15.0 mL), methanol (15.0 mL), and NaOH
(
4.5 g). The mixture was then cooled to room temperature with cold
water, whereupon 2.0 mL methylation solution was injected and the
mixture was incubated at 80 °C for 10 min. The methylation solution
consisted of methanol (27.5 mL) and 6.0 M aqueous HCl solvent
3. Results and discussion
(
1
32.5 mL). Fatty acids in the above mixture were then extracted with
.25 mL of extraction solution, which consisted of hexane and methyl
3.1. Effects of various DESs on the reduction of HAP
tert-butyl ether (1:1, v/v). Finally, the extracted fatty acids were washed
with 0.3 M aqueous NaOH solution (3 mL) and the organic phase was
used to analyze the fatty acid composition.
The fatty acid methyl esters were determined by means of an
Agilent 7890B gas chromatograph coupled to an Agilent 7000C GC/MS
triple-quadrupole set-up operating in electron-ionization mode
Fig. 1 describes the effects of five DESs on the catalytic performance
of K. gibsonii SC0312 for production of (R)-PED by asymmetric reduc-
tion of HAP. As depicted in Fig. 1a, 92.5–98.3 % of (R)-PED yields and
more than 99 % of the product optical purities were obtained in various
reaction systems. Amidst the reaction systems, ChCl/Bd-containing
system showed the highest (R)-PED yield. As to the initial reaction rate,
ChCl/Bd was capable of improving catalytic rate by 22 % compared
with the control. However, the other four DESs (ChCl/U, ChCl/Et,
ChCl/Teg, ChCl/Gly) had no such ability to enhance the catalytic rate.
According to our previous study [37], the pH values of phosphate buffer
(
(
Agilent, USA) and equipped with HP-5MS Ultra Inert capillary column
30 m × 0.25 mm ×0.25 μm; Agilent, USA). Ultra-pure helium was
used as carrier gas at a constant flow rate of 1.0 mL/min. Finally, the
fatty acids were identified by reference to the National Institute of
3

F. Peng, et al.
Molecular Catalysis 484 (2020) 110773
changed slightly when each examined DES above was added to the
system at the concentration of 10 % (v/v). Thus, the catalytic activity of
K. gibsonii SC0312 cells should not be obviously impacted due to trivial
change of the pH in reaction system. Furthermore, the assay on the
results of Table 1 and Fig. 1a showed the initial reaction rates had no
clear correlations with the viscosities of the DESs. In terms of the ste-
reoselectivity, Gotor-Fernández and Paul [38] reviewed many informed
investigations that DESs enhanced the selectivity of various microbial
cells and alcohol dehydrogenases for asymmetric reduction of prochiral
ketones, and even led to inversion of stereoselectivity. Whereas some
literatures described that the sterepreferences of whole cells [29] and
alcohol dehydrogenases/carbonyl reductases [21] for asymmetric re-
duction of prochiral ketones were not visibly influenced by the DESs.
The results shown in Table S1 indicate that the sterepreference of K.
gibsonii SC0312 cells for the preparation of (R)-PED is not changed by
the examined DESs during the biocatalytic processes. To better under-
stand the delightful effects of ChCl/Bd, we compared the impacts of
each component of ChCl/Bd on the initial rate of the reaction. The
relative reaction rates of Bd-containing system and ChCl-containing
system were 95.4 % and 59.5 % of that of the system containing ChCl/
Bd (Fig. 1b), respectively, suggesting the stimulative impact on catalytic
activity stemmed from ChCl/Bd. The results could provide a guidance
for the application of ChCl/Bd in biocatalytic reactions.
containing good biocompatible none-aqueous media. Our results from
Figs. 1 and 2 are in parallel with their studies. These results suggest that
the biocompatibility of the solvents is important for biocatalytic reac-
tion of biocatalysts.
3.3. The effects of DESs on the structure of DNA
The carrier of genetic information DNA is inextricably linked with
the reproduction and function of cells. We here used ctDNA as a tem-
plate and analyzed the impact of DESs thereon by CD spectroscopy. The
impacts of the DESs on the structure of ctDNA were shown in Fig. 3. A
positive band and a negative peak emerged at approximately 278 nm
and 248 nm in the CD spectra of ctDNA, respectively, due to π-π base-
stacking and helicity [33]. All examined DESs had no interference
patterns at 220−320 nm, and B-conformation of ctDNA was not des-
tructed by the DESs when the concentrations of the additives were from
0 % to 20 %. Some changes in the base-stacking and helicity of the DNA
were observed after injection of the DESs. A high concentration of
ChCl/U, such as 16 %, compressed the extended double-helix to a more
compact form [40], presumably due to the presence of denaturant urea
[32]. Adding ChCl/Et or ChCl/Gly rendered the positive band slightly
red-shifted and less intense. The results suggest that structural changes
of ctDNA ensue following the addition of ChCl/U, ChCl/Et, or ChCl/
Gly. However, a slight impact on the ctDAN structure was observed
when adding ChCl/Teg or ChCl/Bd at the examined concentrations. The
change in the DNA structure may disturb gene expression as well as
protein synthesis, showing difference in metabolic and catalytic activ-
ities. However, the correlation between structural change of DNA and
catalytic properties remains unknown and requires in-depth study.
Additionally, the change in the performances of some enzymes parti-
cipating in duplication, transcription and translation of genes requires
investigations.
3.2. Biocompatibility of DESs with K. gisbonii SC0312
The growth of the cells in LB broth containing DESs was used to
characterize the change of cell metabolism. Fig. 2 demonstrates the
influences of DESs on the growth of K. gibsonii SC0312 cells. When DESs
concentration was at 2 %, the examined DESs except for ChCl/Bd
contributed little to shift the biomass compared with the control. The
biomass increased slightly following the addition of ChCl/Et, and de-
creased slightly after adding ChCl/U, ChCl/Teg and ChCl/Gly, respec-
tively. However, a visibly higher biomass was achieved in the system
containing 2 % ChCl/Bd in comparison to the control. The results
manifest that ChCl/Bd and ChCl/Et have favorable biocompatibility to
K. gibsonii SC0312. In terms of ChCl/Bd, the increase in the con-
centration would decrease the cell biomass, such as affording ap-
proximately 10 % biomass of the control at 20 % ChCl/Bd. The result
indicates that a moderate amount of ChCl/Bd can accelerate the cells
growth. Many reports have introduced none-aqueous media to enhance
the catalytic efficiency of whole cells biocatalysts including organic
solvents, ionic liquids and DESs [25,26,39]. In general, the enhance-
ment in catalytic activity of whole cells was observed in the systems
3.4. Effects of DESs on the membrane permeability and fatty acid
composition of the cells
The change in catalytic rate of the cells indicates that some inter-
actions occur between K. gibsonii SC0312 and the DESs. The cell
membrane is the first target of solvents [28], and the intracellular
components (proteins and nucleic acids) release easily when it is da-
maged. Fig. 4a showed that higher absorbance values at 260 nm and
280 nm were observed in the presence of the DESs when compared with
the control. The result suggests the DESs lead to more damaged K.
gibsonii SC0312 cells within 12 h. In addition, the capacity of the DESs
to expand the cell membrane was strengthened in the order of ChCl/
Gly < ChCl/Teg < ChCl/Bd < ChCl/Et < ChCl/U. In terms of
ChCl/Bd, the leakage of the intracellular material displayed an im-
provement with increased concentration, up to a maximum at 8 %, and
then decreased.
The damaged and dead cells can be distinguished by flow cytometer
using PI as fluorescein dye [41,42]. As shown in Fig. 4b, compared with
the control, ChCl/Teg, ChCl/U and ChCl/Bd led to slight increase in the
proportion of the damaged cells when the concentration of DESs was at
2
%. However, visible increases in the ratio of damaged cells were
observed at the presence of the other two DESs, especially ChCl/Gly.
The low ratio of damaged cells in the ChCl/Bd-containing system stood
line with the bioconversion data, showing an enhancement in catalytic
rate (Fig. 1). To better understand the effects of ChCl/Bd, the propor-
tion of the damaged cells were measured with the increase of the DES.
When the concentrations of ChCl/Bd varied from 2 % to 8 %, the ratio
of damaged cells changed slightly. Thereafter, the proportion rapidly
increased with the increment of ChCl/Bd level, up to the maximum at
16 %, then followed by a slight decrease. The results imply the mem-
brane integrity is impacted by the level of ChCl/Bd, which is likely
owing to the changed osmotic pressure in buffer.
Fig. 2. The increase of cell biomass in the various DESs-containing system.
Culture conditions: 37 °C, 6 h, 180 rpm, DESs concentration (ChCl/U, ChCl/Et,
ChCl/Gly, ChCl/Teg) 2 %, and ChCl/Bd concentration: 2 %, 4 %, 8 %, 12 %, 16
%
, 20 %.
Fig. 4 shows that the additives can enhance the membrane
4

F. Peng, et al.
Molecular Catalysis 484 (2020) 110773
Fig. 3. CD spectra of ctDNA in the presence of DESs.
permeability of K. gibsonii SC0312, which is favorable not only in the
contact between intracellular enzymes and the substrate but also in the
easing of product inhibition so as to enhance catalytic efficiency. Ni
et al. [43] has certified the catalytic activity of cells would be aug-
mented by outer membrane mutation due to the enhancement of
membrane permeability. A comparison of data between UV–vis spec-
trophotometry and flow cytometry shows some differences in the
changing trend. For instance, ChCl/Gly led to a small leakage of in-
tracellular materials, but resulted in a tremendous increase in the im-
paired degree of the cell membranes. We considered the higher visc-
osity of ChCl/Gly [35] to be an important factor for this marked
discrepancy, which can restrict mass transfer. In the case of ChCl/Bd,
the opposite change between UV–vis spectrophotometry and flow cy-
tometry was seen with the increase of the additive, which again
Fig. 4. The impacts of various DESs on the membrane integrity of K. gibsonii SC0312 by ultraviolet spectroscopy (a) and Flow cytometer (b). Incubation conditions:
DESs concentration (ChCl/U, ChCl/Et, ChCl/Gly, ChCl/Teg) 2 %, and ChCl/Bd concentration: 2 %, 4 %, 8 %, 12 %, 16 %, 20 %.
5

F. Peng, et al.
Molecular Catalysis 484 (2020) 110773
Fig. 5. The effects of DESs on the cell membrane fatty acid compositions (a)and the ratio of UBFA to BFA (b).
suggests that the viscosity increase may account for the discrepancy.
Membrane lipids are capable of changing in response to perturba-
tions induced by external factors [44,45]. Relative levels of individual
major fatty acids and the ratios of branched to unbranched fatty acids
obtained at 30−40 mM HAP. Further increasing HAP concentration
would lead to a certain amount of decline in the yield, for instance
affording 80 % the product yield at 80 mM. The toxicity of HAP to the
strain might give rise to the decrease of the product yield. [48]. Ad-
ditionally, we studied the reduction of HAP for the fabrication of (R)-
PED by K. gibsonii SC0312 in DESs-free reaction system (Fig. S1). A
comparison of (R)-PED yield in reaction systems with ChCl/Bd and
without the DES showed that the obviously higher product yields were
achieved in the ChCl/Bd-containing system when HAP concentrations
were at 70 mM and 80 mM, respectively. The results indicate ChCl/Bd
can improve catalytic efficiency of K. gibsonii SC0312 for preparation of
(R)-PED.
(
7
BFA/UBFA) are shown in Fig. 5. The fatty acid iso-C14:0 accounted for
8.3 % of the total fatty acids of K. gisbonii C0312 suspended in the
buffer. When the cells were exposed to the buffer systems containing
the DESs, the relative level of iso-C14:0 would approach to 0 % as well
as C15:0 increased strikingly, even reached to 78.8–83.8 % in the
presence of the DESs except for ChCl/Gly (Fig. 5a). A significant im-
provement in the relative level of anteiso-C14:0 was also observed after
injection of ChCl/Gly, reaching to 45.2 %. Furthermore, the additional
DESs induced the content of branched-chain fatty acids to decrease,
affording the ratio of branched-chain fatty acids to unbranched fatty
acids from 5.7 % to 0.1–1.1 % (Fig. 5b). K. gibsonii SC0312 possesses a
high level of branched-chain saturated fatty acids since it is a Gram-
positive bacterium [46]. An increase in fatty acid chain length will
change the membrane permeability [46] and improve the survival rate
The increase in DESs concentration could be attributed to the im-
provement of hydrogen bond network, with a higher viscosity of buffer
system. The increase in the viscosity of buffer system is able to increase
the mass-transfer limitations, leading to a decline in catalytic activity.
Furthermore, the additional amount of ChCl/Bd also impacted the os-
motic pressure and polarity of reaction system, which were deemed as
important factors to the catalytic properties of biocatalysts [49]. Ad-
ditionally, the results from the cell biomass and the ratio of the da-
maged cells showed excessive ChCl/Bd could not benefit the whole cell
catalytical reaction. The results in the reaction temperatures showed
the desirable initial rates and yields of (R)-PED could be achieved at the
temperature of 30−45 °C, suggesting that the cell biocatalyst may be
employed in the reduction of HAP at room or higher temperature.
Buffer pH is an important factor to the catalytic performances of the
cells. The addition of DESs may make the cells more sensitive to en-
vironmental pH, due to the expansion of cell membrane. Additionally,
the results indicate a neutral or alkalescent condition could be accep-
table when considering appropriate range of buffer pH for the reduction
of HAP by the functional enzymes from K. gibsonii SC0312. In com-
parison to previous works, using K. gibsonii SC0312 to prepare (R)-PED
in ChCl/Bd-containing system presented some superiorities, such as
high HAP concentration. Saccharomyces cerevisiae JUC15 produced (R)-
PED by asymmetrically reducing of HAP at 8 g/L (58 mM) under op-
timum conditions, affording 92.4 % yield and > 99 % ee [50]. A co-
factor self-sufficiently recombinant Escherichia coli BL21(DE3) by ex-
pression of glucose dehydrogenase and carbonyl reductase from
Candida parapsilosis CCTCC M203011 was capable of asymmetrically
reducing HAP to (R)-PED in 95.5 % yield at 6 g/L of substrate con-
centration (44 mM) [51]. In summary, an efficient reaction system
containing ChCl/Bd for the production of (R)-PED from HAP catalyzed
by K. gibsonii SC0312 has been constructed, giving the product in 80 %
yield with > 99 % ee at a HAP concentration of 80 mM.
[
47] in response to environmental stresses. In addition, the main-
tenance of an abundance of straight-chain fatty acids with respect to
branched fatty acids in a cell membrane is viewed as an adaptive re-
sponse to sublethal stresses, and straight-chain fatty acids in a cell
membrane are beneficial for its stability [44,46]. The above results
indicate that the cells will change fatty acid compositions in response to
the addition of DESs.
3.5. Effects of main reaction conditions on the bioreduction of HAP in the
ChCl/Bd-containing system
The impacts of key factors on the reduction of HAP by K. gibsonii
SC0312 in the buffer system containing ChCl/Bd were studied (Fig. 6).
Fig. 6a shows the influences of ChC/Bd concentration on the prepara-
tion of (R)-PED by K. gibsonii SC0312 cells. Highly optical purities of the
product (> 99 %) were achieved as well as slight changes in the pro-
duct yields were observed when the concentrations of ChCl/Bd varied
from 2 % to 20 %. The initial reaction rates were slightly different when
ChCl/Bd concentrations were no more than 16 %. As the concentration
was at 20 %, a decline trend in the initial reaction rate was observed.
The effects of reaction temperatures on the asymmetric reduction of
HAP were shown in Fig. 6b. The reaction temperature had a substantial
effect on the initial rate, but a slight impact on the yield of the product,
except that a lower yield was obtained at 20 °C. In addition, excellent
optical purities of the product were observed at the different reaction
temperatures. Fig. 6c depicts the impacts of buffer pH on the production
of (R)-PED. The optimal buffer pH for the catalytic rate was at 7.5 as
well as the neutral or weakly alkaline environment were beneficial to
achieve high reaction rates. Moreover, negligible variations in the
yields and ee values of the product were observed within the pH range
tested. Fig. 6d shows the effects of HAP concentrations on the reduction
of HAP by K. gibsnoii SC0312 cells. Relatively high product yields were
4. Conclusion
We have studied the effects of five DESs on the K. gibsonii SC0312
cells for the preparation of (R)-PED by asymmetric reduction of HAP. Of
the additives, ChCl/Bd showed a pleasurable biocompatibility for the
6

F. Peng, et al.
Molecular Catalysis 484 (2020) 110773
Fig. 6. The effects of several key factors on the catalytic properties of K. gibsonii SC0312. Reaction conditions: 4 % ChCl/Bd (b–d), 30 mg/mL wet cells, 180 rpm,
0 mM HAP (a–c), reaction time 5 h (a–c), temperature 35 °C (a, c, d).
2
cells, stimulating the cells growth. On basis of the results from UV–vis
and FCM, all examined co-solvents were capable of enhancing the
membrane permeability of K. gibsonii SC0312 cells. Further assay on the
membrane fatty acids of the cells confirmed that the strain could
change membrane components in response to the addition of the DESs.
Finally, a highly efficient reduction of 80 mM HAP by K. gibsonii SC0312
in the ChCl/Bd-containing system was established, affording (R)-PED in
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.2020.110773.
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