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Towards designing a novel oligopeptide-based deep eutectic solvent: Applied in biocatalytic reduction Jun Li, Pu Wang, Yu-Shu He, Zhi-Ren Zhu, and Jin Huang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.8b04989 • Publication Date (Web): 20 Nov 2018 Downloaded from http://pubs.acs.org on November 22, 2018

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ACS Sustainable Chemistry & Engineering

Towards designing a novel oligopeptide-based deep eutectic solvent: Applied in biocatalytic reduction Jun Li,†‡ Pu Wang,*,† Yu-Shu He,† Zhi-Ren Zhu,† Jin Huang† †College

of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, P.R.

China ‡School

of Pharmacy, Hangzhou Medical College, Hangzhou 310053, P.R. China

The e-mail address of each author: Jun Li†‡ Email:[email protected] †18

Chaowang Road, College of Pharmaceutical Science, Zhejiang University of Technology,

Hangzhou 310032, P.R. China ‡481 Binwen

Road, School of Pharmacy, Hangzhou Medical College, Hangzhou 310053, P.R.

China

Corresponding Author Pu Wang. Email: [email protected] 18 Chaowang Road, College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, P.R. China

Yu-Shu He Email: [email protected] 18 Chaowang Road, College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, P.R. China Zhi-Ren Zhu Email: [email protected] 18 Chaowang Road, College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, P.R. China Jin Huang Email: [email protected] 18 Chaowang Road, College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, P.R. China

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ABSTRACT We report a new method for developing a deep eutectic solvent (DES) and its application as a cosolvent in biocatalytic reactions. A novel oligopeptide-based DES containing choline chloride (ChCl) and glutathione (GSH, comprised of Glu, Cys, and Gly) was designed and synthesized. Using this oligopeptide-based DES as a cosolvent, we achieved efficient asymmetric reduction of 3,5-bis(trifluoromethyl) acetophenone catalyzed by Trichoderma asperellum ZJPH0810. Under optimized conditions, the substrate loading increased 2-fold in the ChCl/GSH-containing system compared with that in aqueous buffer (100 vs. 50 mM), with a yield > 90% and enantiomeric excess value > 99%. To broaden the application of the established ChCl/GSH-containing system in biocatalysis, the asymmetric reductions of different substrates in developed reaction medium were further investigated. Compared with the aqueous system, the ChCl/GSH-containing system enhanced substrate loading (50 vs. 100 mM when catalyzed by Candida tropicalis 104), obviously improved the yield (i.e. from 70.4% to 87.6% when catalyzed by C. tropicalis 104, from 65.9% to 83.8% by Candida parapsilosis ZJPH1305), and shortened the reaction time greatly (24 vs. 30 h when catalyzed by C. tropicalis 104, or 1.0 vs. 1.5 h by recombinant Escherichia coli). These findings provide valuable insight for the design of task-specific and sustainable oligopeptide-based DESs for biocatalysis. KEYWORDS: Biocatalysis, Deep eutectic solvent, Asymmetric catalysis, Ionic liquids, Reduction, Sustainable chemistry

INTRODUCTION The past decades have witnessed a major drive to increase the efficiency of biocatalytic process in the presence of ionic liquids (ILs), which have been introduced as green alternatives to hazardous organic solvents.1-3 ILs shares many unique properties, including negligible vapor pressure, nonflammability, excellent chemical and thermal stability, and the capacity to dissolve a variety of polar and nonpolar compounds, making them of great interest in many fields.4,5 However, the difficult preparation and low biodegradability of ILs cumber their application in biocatalysis.


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Deep eutectic solvents (DESs), which have emerged as a new class of IL-related solvents, are comprising of biodegradable and readily available nontoxic ions, such as ammonium salt (e.g., choline chloride [ChCl]) and hydrogen-bond donor (HBD; e.g., amino acids, natural bases, urea and glycerol).6,7 The components of DESs (cations, anions, and HBDs at various salt/HBD molar ratios) are more diverse than conventional ILs, which comprise cations and anions only. In addition to sharing many of the virtues of conventional ILs, DESs are easily synthesized and require no further purification steps.8 In DESs, HBD interacts with anion, leading to a decrease in the melting points of the obtained mixtures. A classic example is the combination of ChCl (melting point 302 ºC) with urea (melting point 133 ºC) to form a DES with a melting point of 12 ºC, which was first proposed by Abbott et al.9 To date, a wide range of applications of DESs in extraction, electrochemical process, catalysts, metal deposition or dissolution processes have been reported, but only a few of those studies used the DES, either alone or as a co-solvent, in reaction media for biocatalytic process,

especially

in

whole-cell-catalyzed

reactions.10,11

Compared

with

enzyme-catalyzed reductions, the whole-cell-mediated bioreduction is unique in terms of in situ cofactors regeneration.12 Enantiometrically pure chiral alcohols are key intermediates for the synthesis of pharmaceuticals. (R)-[3,5-bis(trifluoromethyl)phenyl] ethanol ((R)-BTPE) is an important chiral building block in the synthesis of antiemetic drugs aprepitant (Emend®)

and

fosaprepitant

(Ivemend®),

which

are

used

to

prevent

chemotherapy-induced side effects.13 As a model reaction, (R)-BTPE was synthesized by asymmetric reduction of 3,5-bis (trifluoromethyl) acetophenone (3,5-BTAP) using whole cells of Trichoderma asperellum ZJPH0810 as biocatalyst.14 Aqueous systems are conventional media used for biocatalytic reactions, however, industrially attractive substrates generally exhibit poor water solubility and exert toxic effects on biocatalysts, rendering the application of whole-cell biocatalysis difficult in some cases.15 Therefore, the development of more efficient and benign reaction medium in green engineering requires novel and alternative solvents to improve biocatalytic efficiency. Xu et al.16 used a ChCl/U-containing system for the asymmetric reduction



of 3-chloropropiophenone to (S)-3-chloro-1-phenylpropanol catalyzed by Acetobacter sp. CCTCC M209061 whole cells and found that the substrate loading was 2.5-fold higher than that in aqueous system. Maugeri et al.17 reported the use of a ChCl/glycerol-aqueous mixtures as the reaction medium for Baker’s yeast-catalyzed asymmetric bioreduction of ethyl acetoacetate, with complete inversion of enantioselectivity from approximately 95% enantiomeric excess (ee) of the S enantiomer in pure water to 95% ee of the R enantiomer in the pure DES. Müller et al.18 demonstrated that ChCl/glycerol markedly improved the ee values in the asymmetric reduction of a broad range of aromatic ketones catalyzed by recombinant Escherichia coli whole cells. Despite a few previous reports of whole-cell catalyzed bioreduction in DESs, the search for novel and task-specific DES is still ongoing, likely because strong HBDs, such as urea or citric acid, denature proteins. Recently, some studies reported the amino acid (AA)-based DESs as biodegradable and natural alternatives solvents, and AA-based DESs showed effective for biomass pretreatment.19 To date, the application of AA-based DESs as a reaction medium for whole-cell biocatalysis has been largely unexplored, and the synthesis, task-specific properties, and applications of oligopeptide-based DESs to biocatalytic processes have rarely been reported. In the quest for environmentally friendly and efficient reaction processes, we herein provide an approach of developing DESs that exert less toxicity; they do so by combining various “green” compounds according to the specific bioreduction properties. In this study, an oligopeptide-based DES composed of ChCl and glutathione (GSH) was developed to improve bioreduction efficiency. Several parameters influencing bioreduction were investigated, and a mechanism underlying the reaction was proposed. Finally, various whole-cell-catalyzed bioreductions were conducted in the established ChCl/GSH-containing system to broaden its applications in biocatalysis.

EXPERIMENTAL SECTION


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Materials. 3,5-BTAP was supplied by Beijing Golden Olive Company (Beijing, China). (R)-BTPE and (S)-BTPE were purchased from Capot Chemical Co., Ltd. (Hangzhou, China). 5-(4-Fluorophenyl)-5-oxopentanoic acid (FPOPA, ≥ 99% purity by high-performance liquid chromatography (HPLC)) was purchased from Wuhan Huameihua Science and Technology Co., Ltd. (Wuhan, China). The racemate (4-fluorophenyl)-5-hydroxypentanoic acid (FPHPA) was synthesized in our laboratory. Briefly, methyl 5-(4-fluorobenzene)-5-oxopentanoic acid was prepared by esterification

of

FPOPA,

which

was

then

reduced

to

methyl

5-(4-fluorobenzene)-5-hydroxypentanoic acid using NaBH4; the reaction mixtures were then hydrolyzed to the final product, racemate FPHPA. Ethyl acetoacetate (EAA, 99% purity) was purchased from Shanghai Lingfeng Chemical Reagent Co., Ltd. (Shanghai,

China).

Ethyl

3-hydroxybutyrate

(EHB,

98%

purity),

ethyl

(R)-3-hydroxybutyrate ((R)-EHB, 98% purity), and trifluoro acetic anhydride were purchased from Aladdin Reagent Co., Ltd. (Shanghai, China), and (S)-EHB was obtained from Adama Reagent Co., Ltd. (Shanghai, China). Other reagents and chemicals were obtained from commercial sources and were of analytical grade. Synthesis of ChCl/GSH. The DESs ChCl/GSH were synthesized via neutralization reaction that mixed ChCl with GSH at 1:0.5, 1:1 or 1:2 molar ratio at 80 °C under rigorous agitation for 24 h. The resulting DES products were dried over P2O5 at 45 °C for at least 2 weeks. The water contents of the DESs were typically varied between 0.5% (w/v) and 1.0% (w/v). The ChCl/AA DESs, such as ChCl/γ-glutamine (Glu), ChCl/cysteine (Cys) and ChCl/glycine (Gly), were synthesized independently using a method similar to that for the ChCl/GSH. The structures of the DESs were confirmed by NMR, elemental analysis, and FT-IR (available as supplemental data). The chemical structures of ammonium salts and hydrogen bond donors are shown in Figure 1.



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Ammonium salt Cl N OH

Choline chloride (ChCl) Hydrogen bond donors O

NH2 HO

OH SH

O

OH NH2

O

Glutamine (Glu)

Cysteine (Cys) O

O

OH

O HN

O

H2N

HO

O

OH H2N

NH

HS

Glycine(Gly)

Glutathione (GSH)

Figure 1. Ammonium salts and hydrogen bond donors used to synthesize eutectic mixtures.

Measurement of solubility of ChCl/GSH. The solubility of ChCl/GSH in water is expressed as the mean weight (g) of ChCl/GSH in 5 g H2O at temperature 298.15 ± 0.02 K. ChCl/GSH and water were accurately weighed using an analytic balance (u(m) = 0.1 mg). Source of strains. Strain T. asperellum ZJPH0810 (CCTCC M 209307), Candida tropicalis 104 (CCTCC M 209034) and Candida parapsilosis ZJPH1305 (CCTCC M 2013559) were isolated from soil samples by our research group. Recombinant E. coli BL21 (DE3) overexpressing a carbonyl reductase from Leifsonia xyli was constructed by our research group.20 T. asperellum ZJPH0810 (filamentous fungi) and C. tropicalis 104 (yeast) were cultivated according to our previous reports.14,

21

The

fermentation medium for C. parapsilosis ZJPH1305 cultivation contained the


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following components (per liter): 40.77 g maltose, 24.02 g yeast extract, 2 g (NH4)2SO4, 3.58 g KH2PO4, 0.6 g NaCl, MgSO4 0.6 g·7H2O, 0.4 g CaCl2. The initial pH of medium was 6.5. After cultivation at 30 ºC with shaking at 200 rpm for 48 h, the cells were harvested by centrifugation at 4 ºC and 9,000×g for 10 min and washed twice with 200 mM KH2PO4-K2HPO4 buffer. The fermentation medium for recombinant E. coli BL21 (DE3) cultivation contained the following components (per liter): 15 g moltose, 10 g tryptone, yeast 10 g extract, 10 g NaCl, 0.01 g FeCl3, 0.15 g KH2PO4, and 0.1 g ZnSO4·7H2O. The medium was incubated at 37 °C with shaking at 180 rpm for ~2 hours until the OD600 reached ~1.0, after which the expression of carbonyl reductase was induced at 33 °C

by the addition of 0.5 mM isopropyl

β-D-1-thiogalactopyranoside (IPTG). After induction for 11 h, the incubated recombinant E. coli cells were harvested by centrifugation at 4 °C and 8000 rpm for 10 min and washed twice with saline. Substrate solubility and cell membrane permeability assay. For the determination of substrate solubility, calibration curves were firstly established using the corresponding standard dissolved in n-hexane as the reference. The solubility of 3,5-BTAP in the cosubstrate-containing or DES-containing systems were determined using a method similar to that described in our previous report.22 The cell membrane permeability assay was performed using a procedure described previously.22 Briefly, T. asperellum ZJPH0810 cells were resuspended in 10 ml distilled water along with the solvents. Reaction with distilled water was used as the control. The samples were incubated at 30 ºC with shaking at 200 rpm, and aliquots were collected at 0 and 24 h. The net increases in the OD260 and OD280 values from 0 to 24 h represent the cellular contents (primarily nucleic acids and proteins) released during that time. Asymmetric synthesis of (R)-BTPE in reaction medium containing various DES components. To form the reaction system, collected mycelia (1.2 g dry cell weight [DCW]) were resuspended in 20 ml distilled water (pH 6.0) and mixed with ethanol (6%, v/v) and glycerol (0.5%, v/v), as dual cosubstrate, followed by addition of 1.0% (w/v) GSH or AAs or 0.5% (w/v) ChCl associated with 0.5% (w/v) GSH. The reaction was initiated by the addition of 3,5-BTAP (75 mM) and incubated at 30ºC



with shaking at 200 rpm for 24 h. The samples were subjected to ethyl acetate extraction twice, and the resulting extracts were assayed by gas chromatography (GC). Synthesis of (R)-BTPE or (S)-BTPE in DES-containing system. To form the reaction system, (R)-BTPE was added to 20 ml distilled water along with 1.2 g (DCW) whole cells from T. asperellum ZJPH0810 and then mixed with different DESs (shown in Table 1). The pH value of distilled water used in this study is 6.0 ± 0.1 which was detected by a pH sensor. The reactions were initiated by the addition of 3,5-BTAP, and ethanol (6%, v/v) and glycerol (0.5%, v/v) were used simultaneously as the dual-cosubstrate. The mixtures were incubated at 30 ºC with shaking at 200 rpm, and sampled at regular intervals. The reaction system for the preparation of (S)-BTPE catalyzed by C. tropicalis 104 consisted of 1% (w/v) ChCl/GSH; 200 mM phosphate buffer (pH 8.0); 60 g l-1 isopropanol as the co-substrate, and 144 g l-1 (DCW) of resting cells incubated at 30 ºC and 200 rpm for 24 h. To evaluate the ability of our newly designed ChCl/GSH-containing system to improve the efficiency of C. tropicalis 104-catalyzed bioreduction, the performance of this system was compared with that of our optimized conventional IL containing [BMIM][PF6]. The optimal reaction conditions were as follows: 5% (v/v) [BMIM][PF6], 60 g l-1 isopropanol as the co-substrate, 200 mM phosphate buffer (pH 7.0), 144 g l-1 (DCW) resting cells, and 70 mM 3,5-BTAP incubated at 30ºC and 200 rpm for 24 h.31 General bioreduction of FPOPA to (5S)-FPHPA in DES-containing system. The biocatalytic reduction was performed in Erlenmeyer flasks on an orbital shaker (200 rpm) at 30 ºC. In each flask, 0.91 g (DCW) resting cells and 1 g glucose as co-substrate, were suspended in 10 ml 200 mM KH2PO4-K2HPO4 buffer (pH 6.0) containing 1% (w/v) ChCl/GSH. The reactions were initiated with the addition of 100 mM (21 g l-1) FPOPA and quenched by removing the cellular material via centrifugation. Samples were withdrawn at regular intervals and assayed by HPLC after extraction with ethyl acetate. General bioreduction of EAA to (R)-EHB in DES-containing system. The


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biocatalytic reduction of EAA to (R)-EHB in the DES-containing system was performed by incubating 1% (w/v) ChCl/GSH, 3.045 mL 200 mM phosphate buffer (pH 7.2), 6.4 g l-1 (DCW) recombinant E. coli cells, 1 mL 20% (v/v) 2-propanol, and 500 mM EAA at 30 °C with shaking at 180 rpm. The product and residual substrate were extracted with ethyl acetate, and assayed by GC. GC analysis. The samples were extracted twice with ethyl acetate and assayed by GC to determine the yield and product ee value. The amounts of residual 3,5-BTAP and the corresponding alcohols for the model reaction were assayed using the GC-2014 chromatograph (Shimadzu, Japan) and a flame ionization detector with a CP-Chirasil-Dex CB column (25 m × 0.25 mm × 0.25 μm, df = 0.25, CP7502, Varian, USA) under the conditions described in our previous report.23 The retention-times for 3,5-BTAP, (S)-BTPE and (R)-BTPE were 4.157, 11.915 and 12.715 min, respectively. The product EHB and the residual substrate EAA were analyzed by GC (GC-2014, Shimadzu) using a Stabilwax (Crossbond Carbowax-PEG) column (30 m×0.32 mm×0.25 μm) as reported previously.24 The retention times for EAA and EHB were 7.213 and 8.178 min, respectively. The product ee value was determined after the derivatization of EHB with trifluoroacetic anhydride and measured by GC (GC-2014, Shimadzu) using a CP-Chirasil-Dex CB column (25 m×0.25 mm×0.25 μm; Varian). The retention times for (R)-EHB and (S)-EHB were 13.11 and 13.50 min, respectively. HPLC analysis. The concentration of residual FPOPA and the corresponding FPHPA were assayed by HPLC (SPD-20A, Shimazu) using a positive-phase CHIRALPAK® AD-H column (4.6 mm×250 mm, i.d., 5 μm, Daicel). The detection wavelength was 210 nm. The mobile phase was n-hexane/ethanol (86:14, v/v), and the ﬂow rate was 0.7 ml min-1. The retention times for FPOPA, (5R)-FPHPA and (5S)-FPHPA were 18.823, 29.298 and 41.165 min, respectively.

RESULTS AND DISCUSSION Design and synthesis of an oligopeptide-based DES for the bioreduction. Because of ChCl has excellent biodegradability and low toxicity to filamentous fungi, such as



Penicillium brevicompactum, Penicillium glandicola, it is considered as the best candidate ammonium salt for DES synthesis in fungus-catalyzed reaction system.25 In this study, we evaluated the performance of ChCl/AA-based DESs in a T. asperellum ZJPH0810-catalyzed reduction, at a 1:1 or 1:2 stoichiometric ratios, using 3,5-BTAP as the model substrate. The results are summarized in Table 1 (entries 2-19). The systems with six kinds of DESs composed of the same ammonium salt (ChCl) and different AAs (Glu, Cys, and Gly) all showed higher yields compared with the system without the addition of DESs, and product ee values were above 99%. It is worth noting that GSH is a water-soluble natural tripeptide composed of Glu, Cys and Gly. Additionally, GSH plays a crucial role in cellular reactions, such as oxidoreduction. The importance of GSH in different living cells is evident from its abundance in plants, mammals, fungi and some prokaryotic organisms.26 Encouraged by our above results, we subsequently synthesized ChCl associated with GSH and found that ChCl/GSH is a hydrophilic DES according to its solubility (2.63 g) in 5 g water at 298.15 ± 0.02 K. The proposed mechanism of the formation of DESs is that the complexing agent (typically an HBD) interacts with anions, thus increasing its effective size and shielding its interaction with cations.9 To further verify the interaction between the ammonium salt (ChCl) and HBD (GSH), FT-IR characterization was conducted. As shown in Figure 2, we observed the typical bands, such as H-S stretching at ~2524.4 cm-1 for GSH and O-H stretching at ~3255.3 cm-1 for ChCl. However, the typical H-S and O-H bands were not found in ChCl/GSH. The above results might be demonstrated that a mercapto group and hydroxy group form hydrogen bonding with Cl- and GSH respectively, thus obtaining the novel DES ChCl/GSH.


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Figure 2. FT-IR characterizations of ChCl, GSH and ChCl/GSH. Table 1 Bioreduction of 3,5-BTAP to (R)-BTPE catalyzed by T. asperellum ZJPH0810 in DES-containing systems.

3,5-BTAP

(R)-BTPE

Entry

DES (molar ratio)

DES content (%, w/v)

Yield (%)

ee (%)

1

control

─

53.1 ± 0.4

>98

2

ChCl/Glu(1:1)

0.5

57.8 ± 1.3

>99

3

ChCl/Glu(1:1)

1.0

66.0 ± 0.8

>99

4

ChCl/Glu(1:1)

1.5

60.7 ± 0.9

>99

5

ChCl/Glu(1:2)

0.5

56.9 ± 0.8

>99

6

ChCl/Glu(1:2)

1.0

64.1 ± 2.6

>99

7

ChCl/Glu(1:2)

1.5

54.7 ± 1.2

>99

8

ChCl/Cys(1:1)

0.5

62.4 ± 1.9

>99

9

ChCl/Cys(1:1)

1.0

80.7 ± 1.3

>99

10

ChCl/Cys(1:1)

1.5

70.5 ± 1.1

>99



Page 12 of 26

11

ChCl/Cys(1:2)

0.5

67.6 ± 1.2

>99

12

ChCl/Cys(1:2)

1.0

77.3 ± 2.1

>99

13

ChCl/Cys(1:2)

1.5

69.6 ± 0.9

>99

14

ChCl/Gly(1:1)

0.5

56.2 ± 0.8

>99

15

ChCl/Gly(1:1)

1.0

63.1 ± 1.4

>99

16

ChCl/Gly(1:1)

1.5

60.4 ± 1.3

>99

17

ChCl/Gly(1:2)

0.5

54.2 ± 1.2

>99

18

ChCl/Gly(1:2)

1.0

61.3 ± 0.9

>99

19

ChCl/Gly(1:2)

1.5

60.6 ± 1.4

>99

20

ChCl/GSH(1:0.5)

1.0

81.7 ± 0.5

>99

21

ChCl/GSH(1:1)

1.0

90.7 ± 0.6

>99

22

ChCl/GSH(1:2)

1.0

78.3 ± 0.9

>99

Reaction conditions: 75 mM of 3,5-BTAP, 60 g l-1 T. asperellum ZJPH0810 cells (DCW) in distilled water (pH 6.0), 6% (v/v) ethanol and 0.5% (v/v) glycerol as dual-cosubstrate, 0.5~1.5% (w/v) DES, 30 ºC, 200 rpm, reaction for 24 h.

Next, the developed ChCl/GSH-containing DES system was applied to a T. asperellum ZJPH0810-catalyzed reduction to determine whether ChCl/GSH demonstrated a combined advantage of ChCl/Glu, ChCl/Cys and ChCl/Gly in the bioreduction. As expected, each DES containing ChCl/AA (AA: Glu, Cys, Gly) improve the product yield (Table 1). The highest yield (90.7%) was obtained using the 1:1 ChCl/GSH-containing system, which was 70.8% higher than the yield without the ChCl/GSH addition (53.1%). ChCl/GSH had improved bioreduction performance in comparison with ChCl/AA (AA: Glu, Cys, Gly). Additionally, bioreduction yield in 1:1 molar ratio of ChCl/GSH was higher than that in 1:0.5 or 1:2 molar ratio of ChCl/GSH (Table 1). It was likely that, in the 1:0.5 ChCl/GSH system, chlorine anions strongly engage in H-bonding formation with enzyme active site, causing inactivation of oxidoreductase in T. asperellum ZJPH0810. All DESs contain HBDs, and an increase in GSH content alleviates, to some extent, these negative interactions between chlorine anions and the enzyme active site. However, in the presence of excessive GSH, such as in the 1:2 ChCl/GSH system, the equilibrium between oxidation and reduction may shift unfavorably due to the antioxidant activity of GSH. Furthermore, the bioreduction of 3,5-BTAP to (R)-BTPE conducted in the 2% (w/v)


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GSH-containing system resulted in a decreased yield (45.2%) (see Table S1). A similar result was also obtained in the 1:2 ChCl/AA system. Effect of DESs components on the bioreduction. Bioreductions were compared in the presence of ChCl, GSH, or AAs (Glu, Cys, and Gly) or in a ChCl & GSH -containing system. As shown in Figure 3, all of the evaluated AA-containing systems exhibited improved product yields compared with the reaction in aqueous medium. ChCl & GSH-containing system gave the highest yield (78.2%) in the T. asperellum ZJPH0810-catalyzed reduction, which was nearly the same as that (77.6%) in the reaction medium containing GSH only. ChCl had a negligible influence on bioreduction compared with the aqueous medium. Thus, the ChCl/GSH-containing system was selected for further studies and systematic optimization.

Figure 3. Effect of DESs components on the bioreduction of 3,5-BTAP to (R)-BTPE catalyzed by T. asperellum ZJPH0810. Reaction conditions: 75 mM of 3,5-BTAP, 60 g l-1cells (DCW) in distilled water (pH 6.0), 1.0% (w/v) GSH or amino acids or 0.5% (w/v) ChCl mixed with 0.5% (w/v) GSH, 6% (v/v) ethanol and 0.5% (v/v) glycerol as dual-cosubstrate, 30 ºC, 200 rpm, 24 h.

Effects

of

key

parameters

on

bioreduction

performance

in

the

ChCl/GSH-containing system. Having selected the 1:1 ChCl/GSH system for further investigation, we subsequently examined the effects of initial medium pH and



ChCl/GSH content on the bioreduction of 3,5-BTAP to (R)-BTPE catalyzed by T. asperellum ZJPH0810. Figure 4A reveals the optimum pH is 6.0, achieving a yield of 64.3%. In particular, the reaction performed in distilled water (pH 6.0) had a yield of 90.7% (Figure 4B). Generally, phosphate buffer is the conventional medium used for biocatalysis, and the carbonyl reductase derived from T. asperellum ZJPH0810 is potentially different from the reductases reported previously. The effects of the ChCl/GSH content on T. asperellum ZJPH0810-catalyzed bioreduction are shown in Figure 4C. The product yield was substantially increased, from 50.7% to 90.7%, when the ChCl/GSH content was increased from 0% to 1.0% (w/v). A further increase in the ChCl/GSH content (1.5~5.0%, w/v) resulted in a decrease in yield (75.4%~42.1%). Table S2 demonstrated that a higher ChCl/GSH content (2%, w/v) led to greater cell toxicity, thus terminating the reaction. Therefore, the optimal ChCl/GSH content requires a precise balance among factors because a higher ChCl/GSH content can inactivate carbonyl reductase activity in T. asperellum ZJPH0810 cells, but meanwhile improve mass transfer of substrate/product. Thus, the DES at a mass fraction of 1% (w/v) resulted in the best yield and enantioselectivity. Furthermore, a minor proportion of ILs in reaction system overcame the IL-associated issues of low recoverability and large-scale application costs.27

(A)


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(B)

(C)



(D) Figure 4. Effects of various reaction parameters on the T. asperellum ZJPH0810-catalyzed bioreduction. Reaction conditions: 75 mM of 3,5-BTAP (A, B and C), 60 g l-1 cells (DCW), 6% (v/v) ethanol and 0.5% (v/v) glycerol as dual-cosubstrate, 30 ºC, 200 rpm, 24 h (A, B and C). (A) Effect of buffer pH. 1% (w/v) ChCl/GSH-containing buffer system in the pH range of 5.5-8.0 (NaHPO4-NaH2PO4); (B) Effect of various 1% (w/v) ChCl/GSH-containing reaction medium (pH 6.0). Symbols: 1, Na2HPO4-citric acid; 2, Na2HPO4-NaH2PO4; 3, K2HPO4-KH2PO4; 4, Na2HPO4-KH2PO4; 5, Distilled water; ■ , ee. (C) Effect of ChCl/GSH content. (D) Effect of substrate concentration in a 1% (w/v) ChCl/GSH-containing system.

In this study, some critical reaction parameters were also examined in the ChCl/GSH-distilled water system. The optimal shaking speed, reaction temperature, cell concentration, and cosubstrate concentration were 200 rpm, 30 ºC, 60 g l-1 cells (DCW), and 6% (v/v) ethanol and 0.5% (v/v) glycerol, respectively. Under the above reaction conditions, the product ee value remained intact (>99%). Based on these optimized conditions, the time courses of T. asperellum ZJPH0810-catalyzed bioreduction at different 3,5-BTAP concentration were examined (Figure 4D). The reaction efficiency was obviously improved in the DES-containing system, with a yield of 90.3% obtained using 100 mM 3,5-BTAP. Compared with the previous data reported in aqueous media,14 the addition of DES (ChCl/GSH) increased the substrate concentration from 50 mM (in the absence of DES) to 100 mM (in the presence of


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DES), and the product concentration was also increased substantially, from 46.7 to 90.3 mM. The addition of ChCl/GSH hardly affected the product ee value (> 99%). It was reported that the same reaction catalyzed by the fungus Penicillium Expansum EBK-9, only 13 mM (R)-BTPE was obtained using 17 mM 3,5-BTAP.28 The substrate loading and product concentration in our ChCl/GSH-containing system were increased by 5.9- and 6.9-fold respectively, compared with the previously reported values. Reaction

mechanism

of

T.

asperellum-catalyzed

bioreduction

in

ChCl/GSH-containing system. The reaction mechanism of 3,5-BTAP bioreduction catalyzed by microbial whole cells in the ChCl/GSH-containing system is not yet clear, thus, we propose the potential mechanism shown in Figure 5. The cationic structure of ChCl is similar to those of quaternary ammonium-based cationic surfactant, and 1.0% (w/v) ChCl/GSH moderately improved cell membrane permeability and exhibited negligible toxicity to T. asperellum cells (Table S2). As shown in Table S3, ChCl/GSH can also enhance the solubility of 3,5-BTAP in the established reaction system, consequently allowing sufficient amount of substrate to enter the cells to interact with intracellular enzyme. The carbonyl reductase responsible

for

3,5-BTAP

reduction

belongs

to

the

short-chain

dehydrogenase/reductase (SDR) family. The domain of SDR is present on the interior surface of an α-helix and contains the universally conserved sequence Ser-Tyr-Lys which is demonstrated to be the active site.20,29 Biocatalysis is initiated by proton transfer from a Tyr hydroxyl group to a carbonyl group in the substrate, this is followed by transfer of a hydrogen atom to the oxygen of a carbonyl group in 3,5-BTAP. Lys forms hydrogen bonds with the nicotinamide ribose to promote proton transfer. However, in a ChCl/GSH-containing system, GSH forms hydrogen bonds with ChCl, as well as with the carbonyl group oxygen in 3,5-BTAP, increasing its electrophilicity and thus facilitating proton acceptance from NADH and promoting coenzyme regeneration.



Figure 5. Postulated reductive reaction mechanism of SDR involving NADH, 3,5-BTAP and ChCl/GSH.

Coenzyme (NAD(P)H) regeneration is indispensible for whole-cell-catalyzed bioreduction.22,30 In our previous report, the co-substrates ethanol and glycerol were used simultaneously for effective regeneration of NADH, enabling efficient completion of bioreduction catalyzed by T. asperellum ZJPH0810.14 In the present study, ChCl/GSH promoted NADH regeneration to improve bioreduction efficiency. To determine whether ChCl/GSH can replace the dual-cosubstrate for coenzyme recycling during bioreduction, a bioreduction system was established consisting of 60 g l-1 mycelia, 50 mM 3,5-BTAP, and 1.0% (w/v) ChCl/GSH. The mixture was incubated at 30 ºC with shaking at 200 rpm for 48 h. The (R)-BTPE yield was 36.4%, which was 40.5% higher than the yield obtained in the reaction system without ChCl/GSH (composed of 60 g l-1 [DCW] mycelia and 50 mM 3,5-BTAP only). Unfortunately, replacing the dual-substrate with ChCl/GSH in the T. asperellum ZJPH0810-catalyzed bioreduction was not feasible because of the higher reaction yield (93.4 vs. 36.4%) obtained in the system containing the two co-substrates. One probable explanation for this is that the ChCl/GSH could not solubilize sufficient 3,5-BTAP to ensure a continued and efficient biocatalsis owing to its lower polarity than ethanol. As shown in Table S3, the solubility of 3,5-BTAP in the reaction system was actually higher after the addition of 6% (v/v) ethanol than after the addition of


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1.0% (w/v) ChCl/GSH (33.2 vs. 27.3 mg l-1). In our previous study, as the ethanol content increased from 6% to 8%, the yield of (R)-BTPE decreased sharply, from 73.7% to 44.0%, suggesting that productivity decreased with the addition of ethanol in the reaction medium.14 As shown in Table S2, the addition of 8% ethanol increased cell membrane permeability, yet led to greater cell toxicity, resulting in termination of the reaction. However, the addition of 1.0% (w/v) ChCl/GSH to the reaction system increased the electrophilicity of 3,5-BTAP, facilitated NADH regeneration, and improved cell membrane permeability (see Table S2) while having a negligible toxic effect on T. asperellum ZJPH0810 cells compared with the increasing addition of ethanol. Notably, compared with ethanol (40.2 vs. 34.7 mg l-1), the solubility of 3,5-BTAP slightly improved after ChCl/GSH was added to the reaction system along with the ethanol and glycerol dual-cosubstrate. Therefore, the newly designed ChCl/GSH is indispensable for improving the efficacy of T. asperellum ZJPH0810-catalyzed bioreduction in a dual-cosubstrate-containing system. Applications of ChCl/GSH-containing systems in bioreductions to synthesize value-added products. To broaden its applications, the ChCl/GSH-containing system was also evaluated in other whole cell-catalyzed bioreductions. As shown in Table 2, compared with the reduction conducted in a monophasic aqueous system31, the C. tropicalis 104-catalyzed bioreduction of 3,5-BTAP to (S)-BTPE in a conventional IL ([BMIM][PF6])/buffer biphasic system only slightly improved the substrate concentration (70 vs. 50 mM). However, the same reaction performed in the ChCl/GSH-containing system resulted in an increased 3,5-BTAP concentration of up to 100 mM and a yield of 87.6%, suggesting that ChCl/GSH was an excellent co-solvent in the bioreduction and exhibited a good compatibility between the developed DES and whole cells from C. tropicalis 104. To show the feasibility of the whole

cell-catalyzed

bioreduction

using

different

substrates

in

the

ChCl/GSH-containing system, a reaction system was established for asymmetric reduction of FPOPA to (5S)-FPHPA, a key chiral intermediate in the synthesis of cholesterol-lowering drug Ezetimibe, catalyzed by C. parapsilosis ZJPH1305. The product yield was improved by adding ChCl/GSH (1.0%, w/v) to the aqueous reaction



system (83.8 vs. 65.9% control) (Table 2). Furthermore, recombinant E. coli-catalyzed reduction of EAA to (R)-EHB was also performed in the ChCl/GSH-containing system, and reaction time was shortened compared with that in the aqueous system (Table 2). Based on these results, it is suggested that ChCl/GSH is a promising co-solvent with broad applications in whole cell-catalyzed reduction.

CONCLUSION In the present study, a novel oligopeptide-based DES ChCl/GSH was developed and applied in whole-cell-catalyzed bioreductions. Enhanced (R)-BTPE yield was achieved in our established ChCl/GSH-containing system using T. asperellum ZJPH0810 whole cells as the catalyst. Adding ChCl/GSH to the aqueous system increase the electrophilicity of 3,5-BTAP and facilitate coenzyme regeneration, thus enhancing the effectiveness of T. asperellum ZJPH0810-catalyzed bioreductions. Furthermore, different whole-cell-catalyzed reductions using various substrates were evaluated in the ChCl/GSH-containing systems. In the C. tropicalis 104-catalyzed bioreduction (from 3,5-BTAP to (S)-BTPE), ChCl/GSH was a better co-solvent than the conventional ILs [BMIM][PF6]. Furthermore, the ChCl/GSH-containing system improved the product yield and shortened the reaction times for both C. parapsilosis ZJPH1305- and recombinant E. coli-catalyzed bioreduction. Overall, we demonstrated ChCl/GSH to be a promising co-solvent for whole-cell bioreduction. The findings illustrate the potential ability of oligopeptide-based DESs to modulate biocatalytic processes. The obtained results may help in designing new task-specific and sustainable oligopeptide-based DESs for biocatalysis.


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Table 2 Comparison of different whole-cell-catalyzed reductions in ChCl/GSH-containing system. Catalyst

Substrate

Product

Substrate conc.

Product yield

Product ee

Reaction time

(mM)

(%)

(%)

(h)

Aqueous buffer

50

70.4

>99

30

[BMIM][PF6]/buffer a

70

82.5

>99

24

ChCl/GSH-buffer b

100

87.6

>99

24

Aqueous buffer

100

65.9

>99

72

ChCl/GSH-buffer

100

83.8

>99

72

Aqueous buffer

500

99.9

>99

1.5

ChCl/GSH-buffer

500

99.9

>99

1.0

Reaction system

Candida tropicalis 104

Candida parapsilosis ZJPH1305 c

Recombinant E. coli d

The data was obtained in our previous research.31 bReaction conditions: phosphate buffer (0.2 M, pH 8.0); 60 g l-1 of isopropanol; 144 g l-1 of cells (DCW); or 1% (w/v) ChCl/GSH; reaction at 30 ºC, 200 rpm. c Reaction conditions: phosphate buffer (0.2 M, pH 6.0); 91.2 g l-1 of cells (DCW); 100 g l-1 of glucose; or 1% (w/v) ChCl/GSH; reaction at 30 ºC, 200 rpm. d Reaction conditions: phosphate buffer (0.2 M, pH 7.2); 6.4 g l-1 of cells (DCW); 20% (v/v) of 2-propanol; or 1% (w/v) ChCl/GSH; reaction at 30 ºC, 180 rpm.

a



Page 22 of 26

ASSOCOATED CONTENT Supporting information Additional information obtained from this study regarding the charaterization of DESs (NMR, elemental analysis and IR spectra); effect of GSH on 3,5-BTAP to (R)-BTPE bioreduction; effect of various solvents on T. asperellum ZJPH0810 cell membrane permeability; and the solubility of 3,5-BTAP in different reaction media is available at the ACS Publications website. AUTHOR INFORMATION Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS This work was financially supported by grants from the National Natural Science Foundation of China (No. 21676250); Zhejiang Provincial Natural Science Foundation of China (LY16B060010); Educational Committee Foundation of Zhejiang Province (No. Y201738517); Project of Medical & Health Science and Technology of Zhejiang Province (No. 2017203267); Program of Science Research of Hangzhou Medical College (No. 2016B01).

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TOC

Sustainable bio-derived DESs are designed and synthesized for enhancing biocatalytic efficiency of valuable intermediates of chiral drugs.


Page 26 of 26

Towards designing a novel oligopeptide-based ... - ACS Publications

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