Highly enantioselective resolution of racemic 1-phenyl-1,2-ethanediol to (S)-1-phenyl-1,2-ethanediol by Kurthia gibsonii SC0312 in a biphasic system

Journal Pre-proof Highly enantioselective resolution of racemic 1-phenyl-1,2-ethanediol to (S)-1-phenyl-1,2-ethanediol by Kurthia gibsonii SC0312 in a biphasic system Fei Peng (Investigation) (Data curation) (Writing - original draft) (Writing - review and editing), Ying Zhao (Investigation) (Data curation), Fang-Zhou Li (Writing - review and editing), Xiao-Yang Ou (Writing - review and editing), Ying-Jie Zeng (Writing - review and editing), Min-Hua Zong (Methodology) (Writing - review and editing), Wen-Yong Lou (Supervision) (Funding acquisition)

PII:

S0168-1656(19)30922-8

DOI:

https://doi.org/10.1016/j.jbiotec.2019.11.012

Reference:

BIOTEC 8551

To appear in:

Journal of Biotechnology

Received Date:

15 October 2019

Revised Date:

19 November 2019

Accepted Date:

20 November 2019

Please cite this article as: Peng F, Zhao Y, Li F-Zhou, Ou X-Yang, Zeng Y-Jie, Zong M-Hua, Lou W-Yong, Highly enantioselective resolution of racemic 1-phenyl-1,2-ethanediol to (S)-1-phenyl-1,2-ethanediol by Kurthia gibsonii SC0312 in a biphasic system, Journal of Biotechnology (2019), doi: https://doi.org/10.1016/j.jbiotec.2019.11.012

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Highly enantioselective resolution of racemic 1-phenyl-1,2ethanediol to (S)-1-phenyl-1,2-ethanediol by Kurthia gibsonii SC0312 in a biphasic system

Fei Peng, Ying Zhao, Fang-Zhou Li, Xiao-Yang Ou, Ying-Jie Zeng, Min-Hua Zong, Wen-Yong Lou*

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Laboratory of Applied Biocatalysis, School of Food Science and Engineering, South China University of Technology, Guangzhou 510640, Guangdong, China. * Corresponding author: Prof. Wen-Yong Lou

School of Food Science and Engineering, South China University of Technology,

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Wushan Street 381, Tianhe District, Guangzhou, China.

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Tel/Fax: +86-20-22236669; E-mail: [email protected]

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Highlights

Organic solvents can change metabolic activity and membrane integrity of cells

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Oxidation product can be efficiently extracted by dibutyl phthalate

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A biphasic system for the fabrication of (S)-PED by biocatalysis was constructed

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The reaction efficiency was higher in the biphasic system than aqueous system

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Abstract The asymmetric resolution of racemic 1-phenyl-1,2-ethanediol (PED) to (S)-PED by Kurthia gibsonii SC0312 (K. gibsonii SC0312) was conducted in a biphasic system comprised of an organic solvent and aqueous phosphate buffer. The impacts of organic solvents on the whole cell catalytic activity, metabolic activity, membrane integrity, and material distribution were first evaluated. The results showed that all organic solvents, except for dibutyl phthalate, showed a detrimental effect on the metabolic activity of the cells, especially for those with low log P values. All organic solvents were

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capable of changing the membrane permeability and membrane integrity of the cells. Moreover, some organic solvents showed a good extraction of the oxidation product. Finally, a high yield of 47.7% of (S)-PED was obtained by the asymmetric resolution of racemic PED using K. gibsonii SC0312 in a biphasic system under the optimal

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conditions: racemic PED 120 mM, temperature 35 °С, reaction time 6 h, 180 rpm, and a volume ratio of dibutyl phthalate to aqueous phosphate buffer of 1:1. The optical

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purity of (S)-PED increased from 51.3% to >99%. This work described an efficient approach to improve reaction efficiency, and constructed a highly effective biphasic

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reaction system for the fabrication of (S)-PED via K. gibsonii SC0312.

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Keywords: Kurthia gibsonii SC0312; (S)-1-phenyl-1,2-ethanediol; Asymmetric

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resolution; Dibutyl phthalate; Biphasic system

1 Introduction (S)-1-phenyl-1,2-ethanediol (PED) is a crucial chiral intermediate, which can be useful for the preparation of asymmetrically hydrogenated olefinic compounds (Brown and Murrer, 1982; Kathryn and Wong, 2001; Schmid et al. 2001). Kinetic resolution of racemates plays an important role in the production of chiral alcohols (He et al. 2013; Hwang et al. 2008; Turner, 2010). The fabrication of chiral PED by biocatalytic approaches mainly includes the asymmetric hydrolysis of styrene oxide by epoxide hydrolases (Peng et al. 2018), the selective hydrolysis of the dicarboxyester of PED

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(Tian et al. 2011), and the asymmetric oxidation of racemic PED (Ni and Xu, 2012). Among these approaches, asymmetric oxidation of racemic PED for the production of

(S)-PED is a common approach via a biocatalytic reaction. The use of oxidationreduction enzyme is often confronted with the problems in cofactor regeneration,

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instability in harsh medium and difficult re-usability, etc. (Velasco‐Lozano et al. 2017;

Rueda et al. 2016). Although immobilization technology of enzyme may be beneficial

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for addressing the above issues, the employment of immobilized enzyme could be a more expensive and complex than the use of free enzyme. In comparison to enzymatic

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catalysis, the use of a whole cell biocatalyst has advantages, including cofactor regeneration and the protective effect of the cell membrane (Ni and Xu, 2012;

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Wachtmeister and Rother, 2016). Many studies have reported that the oxidation product of PED, 2-hydroxyacetophenone (HAP), is highly toxic, which reduces the biocatalytic efficiency (Liese et al. 1996; Nie et al. 2009; Wang et al. 2012). In a similar

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manner, our previous report also found that using Kurthia gibsonii SC0312 (K. gibsonii SC0312) to prepare enantiopure PED by asymmetric oxidation was complicated by the

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inhibition of oxidation products (Peng et al. 2019). To further improve substrate levels, relief from the inhibition of oxidation products is needed. The use of a biphasic reaction system, such as introducing organic solvents as the

second phase (Woodley, 2008), has been considered as an efficient way to overcome the toxicity of substrates and products. Since the first study using organic solvents as a reaction solvent in biocatalytic reaction was reported (Aleksey and Alexander, 1984), successive studies have discussed the active role of organic solvents during the process

of biotransformation. For example, Geotrichum candidum retained good half-lives in a system containing benzyl alcohol, which was capable of reducing more than 98% of 1acetonapthone in the two-phase reaction system comprised of buffer and organic solvent (Bhattacharyya et al. 2012). Previous studies also reported that the addition of dibutyl phthalate significantly improved the substrate concentration and the yield of the biocatalytic synthesis of (R)-2-hydroxy-4-phenylbutyrate (Dirk, 2007). Nevertheless, organic solvents fail to avoid their inherent shortcomings involving inactivation of biocatalysts, which restricts their general utilization in biocatalysis (Kansal and

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Banerjee, 2009). In addition, diverse organic solvents have varied impacts on different biocatalysts (Illanes et al. 2012; Kumar et al. 2016). For example, the lipases from

Bacillus megaterium, Penicillium aurantiogriseum, and Penicillium corylophilum showed significantly different stabilities in organic solvents under the same conditions

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(Lima et al. 2004). It is therefore essential, in different biocatalytic reactions, to

investigate the effects of organic solvents on the biocatalyst to choose the best organic

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solvent to optimize the reaction efficiency. Moreover, little information about the impacts of organic solvents on the catalytic properties of K. gibsonii has been reported.

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This work aimed to investigate the changes of catalytic performances of K. gibsonii SC0312 in various organic solvent-containing systems, and to subsequently

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develop a biphasic system of an organic solvent and aqueous phosphate buffer to produce enantiopure PED using microorganisms (Scheme 1). We first investigated the biocompatibility of different organic solvents, as well as their effects on cell membrane

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integrity and permeability. The influence of several key variables was then systematically explored to construct a biocompatible and efficient two-phase system

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for enantiopure (S)-PED.

2 Materials and methods 2.1 Materials K. gibsonii SC0312 was isolated from soil in Guangzhou, China and stored in our laboratory (Peng et al. 2019). Racemic PED with a purity of 98% was purchased from Aladdin (Shanghai, China). The ratio of (S)-PED in the racemic PED was 51.3%,

determined by our group. The nine organic solvents mentioned in this study, including dimethyl carbonate, n-hexanol, 2-methyltetrahydrofuran, butyl acetate, tert-amyl alcohol, 2,2,4-trimethylpentane, dipentene, dibutyl phthalate, and n-decane, were also purchased from Aladdin, with a purity of 98%. All other chemicals were of analytical grade and from commercial sources.

2.2 Culturing of K. gibsonii SC0312 cells K. gibsonii SC0312 was grown in Luria-Bertani (LB) broth medium consisting of

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0.5% yeast extract, 1% tryptone and 1% NaCl. The strain was first cultured in LB broth overnight at 180 rpm and 37°С, then 0.1% (v/v) of the fermentation broth was

inoculated in fresh medium under the same culture conditions for 12–16 h. The cells were collected by centrifugation (7,000 × g, 5 min, 4°С) and washed twice with

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physiological saline, and then resuspended in aqueous phosphate buffer for

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subsequent experiments.

2.3 Asymmetric resolution of racemic PED

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In a typical experiment, the reaction system was composed of aqueous phosphate buffer (2 mL, 100 mM, pH 6.5), one organic solvent (2 mL) and wet cells (25

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mg/mL). The reaction was triggered by the addition of racemic PED, then incubated in a thermostatic shaker at 35°С and 180 rpm. The samples were regularly withdrawn to monitor the levels and enantiomeric excess (ee) of (S)-PED. Details about the content

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of the organic solvent, buffer pH, reaction temperature, and substrate concentration were specified for each experiment. After reaction at 35°C and 180 rpm for 30 min,

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the levels of HAP generated were determined, which were defined as the initial rate. The yield of (S)-PED was defined as the ratio of the measured (S)-PED concentration to the initial amount of racemic PED. The conversion was defined as the ratio of the decreased amount of racemic PED to its initial amount. The ee of (S)-PED and E values were calculated based on the following equation. 𝐶 −𝐶

𝑒𝑒 = 𝐶𝑆 +𝐶𝑅 × 100% 𝑆

𝑅

(1)

ln(1−C)(1−𝑒𝑒)

𝐸 = ln(1−C)(1+𝑒𝑒)

(2)

Where CS and CR were the concentrations of (S)-PED and (R)-PED, respectively; C was the conversion rate of racemic PED.

2.4 Measurement of partition coefficients The partition coefficients of PED and HAP were defined as the ratios of their concentrations in an organic solvent to the buffer system. Racemic PED and HAP were dissolved in the two-phase system consisting of an organic solvent and buffer (1:1, v/v), and incubated at 35°С and 180 rpm for 24 h. The samples were diluted and assayed

2.5 Determination of cell metabolic activity retention

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using high-pressure liquid chromatography (HPLC).

The impact of the none-aqueous phase on the metabolic activity of cells was

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evaluated by determining the changes of cell growth rate. In brief, the seeded cells

were cultured in LB broth overnight at 37°С and 180 rpm. Subsequently, the

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fermentation liquid was injected into flesh medium containing an organic solvent (4%, v/v) and incubated for 6 h at 37°С and 180 rpm. The levels of cells were determined at

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600 nm using an ultraviolet spectrophotometer (UV-2550; Shimazu, Kyoto, Japan).

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2.6 Cell membrane permeability measurements The cell suspension prepared above was incubated in aqueous phosphate buffer containing various organic solvents (4%, v/v) at 37°С and 180 rpm for 24 h.

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Subsequently, the samples were withdrawn at 0 h and 24 h and centrifuged at 4°С and 7,000 × g for 5 min. The absorbances of the supernatants were determined at 260 nm

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and 280 nm using an ultraviolet spectrophotometer. The increases of absorbances reflected the leakage of nucleic acids and proteins from cells, accounting for increments of membrane permeability. The integrity of the cell membrane was also analyzed using flow cytometry (FCM). Briefly, the cell suspension was incubated in physiological saline containing 4% (v/v) of organic solvent at 35°С and 180 rpm for 6 h. The absence of organic solvents was used as the control group. The cell suspension was then diluted and stained with propidium

iodide (PI; 50 µg/mL) at 4°С for 10 min in the dark. The fluorescence signal intensity was measured at 600–620 nm with an excitation wavelength of 488 nm, and the ratio of cell membrane integrity was analyzed using FACSuite software (BD Biosciences, San Jose, CA, USA).

2.7 Analytical methods The levels of PED and HAP were determined by HPLC equipped with an XBridge BEH C18 column (5 μm, 4.6 × 250 mm), at a temperature of 35°С and monitoring

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wavelengths of 215 nm and 245 nm, respectively. The mobile phase was deionized water/acetonitrile (3:2, v/v) with a flow rate of 0.5 mL/min. The ee values of (S)-PED were determined by HPLC with a chialcel OB-H column (4.6 mm × 250 mm) at 215 nm

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and 0.7 mL/min. The mobile phase was hexane: isopropanol (9:1, v/v).

3 Results and discussion

PED using K. gibsonii SC0312

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3.1 The effect of different organic solvents on the asymmetric resolution of racemic

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To improve the reaction efficiency, a biphasic system was employed to address the inhibition of HAP oxidation product. Table 1 shows that there were different effects

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of different organic solvents on this reaction. Some organic solvents with a log P between 0.40 and 1.56 were toxic to the cells, resulting in almost no catalytic activity and a poor conversion rate. Similar studies have also reported that organic solvents of

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low log P were unsuitable for biocatalysis due to severe toxicities (Jiang et al. 2002; Lanne et al. 1987; Wei et al. 2016; Zhang et al. 2009). The disruption of the free water

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molecules on the biocatalyst might be responsible for the inactivation (Yan et al. 2014). Fortunately, three organic solvents with good biocompatibility with the biocatalyst were found, including dibutyl phthalate, 2,2,4-trimethylpentane, and n-decane. Excellent yields and enantioselectivities of (S)-PED can be achieved in the systems containing the three organic solvents, respectively. The ee values of (S)-PED reached 99.9% and a 53.8 % conversion was obtained using a dibutyl phthalate/buffer biphasic system, showing a highly efficient catalytic performance of the microbial cells.

Additionally, the conversion rate > 50% also indicated that K. gibsonii SC0312 was not absolutely enantiospecific for (R)-PED. Furthermore, the addition of organic solvents affected the enantioselectivity of the cells, which presented quite differences in E values. Ionic liquids have been identified as co-solvents to stimulate the bioreaction. We selected some biocompatible ionic liquids based on previous studies regarding their applications for whole cell catalysis (Xiao et al. 2012; Yu et al. 2014). As shown in Table S1, all examined ionic liquids presented excellent biocompatibility for K. gibonii SC0312,

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with perfect initial reaction rates. The employment of some ionic liquids, such as C4MIM·BF4 and C4MIM·NO3, improved the catalytic activity of the cells. However,

compared with the free-ionic liquid system, the decreases of E values and yields of (S)PED following the addition of the tested ionic liquids indicated that co-solvents could

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significantly affect the oxidation specificity of the cells.

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3.2 The effects of different solvents on the partition coefficient of substances The partitioning behaviors of the substrate and the oxidation product are

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beneficial in decreasing inhibition to the cells, especially for HAP. Herein, organic solvents possessing a good extraction of HAP and PED are favored. Table 2 shows the

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partition coefficients of HAP and PED in biphasic systems consisting of different organic solvents and aqueous phosphate buffer. Overall, organic solvents with log P < 2 had a better extracting capacity of HAP and PED than those with log P > 2. Unfortunately, the

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tested solvents with log P < 2 were highly toxic to the cells, resulting in poor catalytic activity. Among these solvents with log P > 2, the majority of PED was distributed in

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the aqueous phase, and dibutyl phthalate was effective in the extraction of HAP, with a partition coefficient of 6.37, which decreased the inhibition of HAP. Our previous study (Peng et al. 2019) reported that the strain had good tolerance to PED, thus, compensating for the poor extraction ability of the organic solvent.

3.3 The effect of different organic solvents on the metabolic activity and membrane permeability of K. gibsonii SC0312 cells

Evaluation of the tested organic solvents acting on the metabolic activity of the cells is shown in Figure 1. As expected, most of the tested solvents were toxic to cells, with a lower biomass than the control. In particular, organic solvents with log P < 2 generally resulted in less biomass than the organic solvents with log P > 2. However, injecting dibutyl phthalate facilitated cell growth, and the biomass increased by 13% compared to the control. In contrast to prior studies, organic solvents, whether as the second phase or as the co-solvent, usually showed inhibition of the cell metabolic activity (Kansal and Banerjee, 2009; Wang et al. 2012). The results suggested that the

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organic solvent showed good biocompatibility to K. gibsonii SC0312. The higher biomass in the system containing dibutyl phthalate (4%) may stem from the

appropriate increase in membrane permeability of the strain (Tables 3 and 4).

Additionally, the membrane lipids may be broken down by dibutyl phthalate (Xu et al.

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2004). The increment of membrane permeability could facilitate material

transportation. More efforts are also needed in the further exploration for the unique

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phenomenon.

Table 3 lists the effect of organic solvents on the cell membrane by detecting the

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changes of OD260 and OD280. Compared with the control group, the increases of OD260 and OD280 after adding organic solvents were significantly higher after incubating for

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24 h. To further confirm the above results, we also used FCM to determine the membrane integrity of the cells (Table 4). The FCM results were consistent with the results of Table 3. Together, these results indicated that the tested solvents were

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beneficial for material transfer in the membrane. Comparison with the FCM data showed that the membrane integrities of cells could be related to the log P of organic

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solvents. Organic solvents with log P < 2 resulted in a higher amount of damaged cells than those with log P > 2 (6.23%–72.63% vs. 1.14%–3.88%, respectively). In addition, a moderate positive effect on the expansion of the cell membranes was found in the system containing dibutyl phthalate. Other studies have reported that organic solvents with a low log P, which possessed a high affinity for the cell surface and altered the lipid bilayer, were harmful to cell membranes (Kumar et al. 2016; Torres et al. 2011).

3.4 Construction of a biphasic reaction system for preparation of (S)-PED Figure 2 shows that there was no evident change in the initial velocity (2.15–2.34 mM/h) at a volume ratio of dibutyl phthalate to buffer of 3/7 to 6/4; nevertheless, the catalytic activity of cells was dramatically inhibited with further increases in dibutyl phthalate, such as an initial rate of 1.60 mM/h at 8/2. This phenomenon could result from the limitations of mass transfer (Doukyu and Ogino, 2010). Optimal yields (48.3%–50.9%) and ee values (>99%) of (S)-PED were obtained using various concentrations of dibutyl phthalate.

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The influence of buffer pH on the biocatalytic resolution of racemic PED is shown in Figure 3a. The reaction rate displayed a rising tendency with increases of buffer pH

from 4.5 to 8.5 and the reaction was shortened from 7 h (pH 4.5–6) to 5 h (pH 6.5–8.5) (Figure S1). Moreover, excellent yield and ee of (S)-PED were achieved using a buffer

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with different pH values. The results showed that a low pH inhibited the catalytic

activity of the cells. The buffer pH in the next experiments was therefore set at 6.5.

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Figure 3b shows the effect of temperature on the catalytic properties of the cells. With increments of temperature, an increase in the reaction rate was found, up to a

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maximum of 3.22 mM/h at 45°C. A favorable yield and optical purity of (S)-PED could therefore be achieved using a biphasic system at various examined temperatures.

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To obtain high reaction efficiency, we finally optimized the substrate concentration. Figure 4 shows that the reaction rate reached a maximum at the racemic PED concentration of 90 mM, which dramatically decreased with further

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increases in the substrate concentration. This result indicated that inhibition of PED could occur. However, the optical purities of (S)-PED were also favorable at 120 mM

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and 140 mM PED, with yields of greater than 99.0% and 94.4%, respectively. Further increasing the concentration of PED resulted in a decrease of ee, with a slight change in the reaction rate. This result suggested that the inhibition of the oxidation product might be mainly responsible for the decline. Moreover, the reaction process was prolonged with the increase of racemic PED (Figure S2). The yield of (S)-PED showed a slight change using different concentrations of PED, such as achieving 47.7% at 120 mM and 140 mM. The results in the biphasic system were better than the aqueous

phosphate buffer, which obtained a yield of 41% and optical purity of 94% at 80 mM racemic PED (Peng et al. 2019). Other researchers have also contributed to improve the reaction efficiency. Although the strategy of continuous extracting oxidation HAP by hexane enhanced the asymmetric resolution of racemic PED, the completed conversion of racemic PED at 40 mM still needed 64 h to obtain (S)-PED of ee > 99% (Liese et al. 1996). A yield of 85% with 98% ee of (S)-PED was obtained using Candida parapsilosis CCTCC M203011 selectively to oxidize racemic PED at 105 mM by the enhancement of cofactor regeneration (LÜ et al. 2007). Although a high yield of (S)-

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PED was obtained, the reaction time was prolonged to 48 h. Furthermore, some studies reported the use of recombinant strains for improving substrate concentration. For instance, a recombinant E. coli BL21 expressing an esterase was used to prepare

(S)-PED by the selective hydrolysis of PED diacetate, affording 49% yield and 95% ee

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at 100 mM (Tian et al. 2011). Compared with the asymmetric resolution system mentioned above, the biphasic system established by us showed several additional

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advantages, such as substrate loading, reaction time, and no additional need for

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external cofactor.

4 Conclusions

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We described the effects of organic solvents on the catalytic performance and cell membranes of K. gibsonii SC0312, and constructed an efficient whole cell biphasic catalytic system for the production of enantiomerically pure (S)-PED. Among all

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examined organic solvents, dibutyl phthalate, as an exception, was beneficial in improving the metabolic activity of cells. Moreover, all tested organic solvents

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expanded the membrane permeability and changed the integrity of this membrane. Overall, the influence of organic solvents on the catalytic properties of the cell was correlated with the physicochemical performances of the solvents, such as log P. K. gibsonii SC0312 showed excellent catalytic performance in a biphasic system consisting of dibutyl phthalate and aqueous phosphate buffer, with yields of 47.7% and over 99% optical purity of (S)-PED at a 120 mM substrate level under optimal conditions. In addition, further improvement of substrate concentration could possibly

be achieved by immobilization of the cells (Sheldon and Pelt, 2013). Author Contribution Statement Fei Peng: Investigation, Data curation, Writing-Original Draft, Writing-Review & Editing Yin Zhao: Investigation, Data Curation Fan-Zhou Li: Writing-Review & Editing Xiao-Yang Ou: Writing-Review & Editing Ying-Jie Zeng: Writing-Review & Editing Min-Hua Zong: Methodology, Writing-Review & Editing

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Wen-Yong Lou: Supervision, Funding acquisition Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Acknowledgement

The authors are particularly grateful to the National Natural Science Foundation

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of China (21676104; 21878105; 21908070), the National Key Research and Development Program of China (2018YFC1603400, 2018YFC1602100), the Science and

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Technology Program of Guangzhou (201904010360), the Key Research and Development Program of Guangdong Province (2019B020213001), the Fundamental Research Funds for the Central Universities (2019PY15; 2019MS100) and China

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Postdoctoral Science Foundation (BX20180102) for partially funding this work.

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Figure captions Scheme 1 The preparation of (S)-PED using K. gibsonii SC0312 as a biocatalyst Fig. 1 Effect of different organic solvents on the cell metabolic activity retention of K. gibsonii SC0312 cells. The experiments were performed at triple and all data was expressed at mean ± standard deviation. Fig. 2 Effect of the volume ratio of organic solvent to buffer on the asymmetric resolution racemic PED by K. gibsonii SC0312 cells. Reaction conditions: aqueous phosphate buffer (100 mM, pH6.5), 20 mM PED, 25 mg/mL microbial cells, 35 °С, 180

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rpm, 6h. The experiments were performed at triple and all data was expressed at mean ± standard deviation.

Fig. 3 Effect of buffer pH and reaction temperature on the asymmetric resolution of

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racemic PED with K. gibsonii SC0312. Reaction conditions: dibutyl phthalate (2mL), 20

mM PED, 25 mg/mL wet cells, 180 rpm, a) aqueous phosphate buffer (100 mM, 2mL),

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35 °С; b) aqueous phosphate buffer (100 mM, pH6.5, 2mL), 6 h. The experiments were performed at triple and all data was expressed at mean ± standard deviation.

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Fig. 4 Effect of substrate concentration on the asymmetric resolution of racemic PED with K. gibsonii SC0312. Reaction conditions: 2 mL aqueous phosphate buffer (100 mM, pH 6.5), 2 mL dibutyl phthalate, 25 mg/mL wet cells, 35 °С, 180 r/min. The experiments

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Scheme 1

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were performed at triple and all data was expressed at mean ± standard deviation.

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Figure 4

Table 1 Effect of various organic solvents on the asymmetric oxidation of PED catalyzed

Initial log P

Yield

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reaction System

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by K. gibsonii SC0312

rate

(%)

Conversion

PED

(%)

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(mmol/L/h) Buffer

(S)E values

(%)

/

2.2

48.0

51.8

99.9

133

0.40

0.0

48.8

4.2

2.0

n.d.

1.17

0.0

n.d.

n.d.

4.8

n.d.

2-Methyltetrahydrofuran/buffer

1.19

0.0

n.d.

n.d.

4.2

n.d.

Butyl acetate/buffer

1.35

2.0

43.3

43.8

53.9

n.d.

n-Hexanol/buffer

1.56

0.0

n.d.

n.d.

2.6

n.d.

2,2,4-Trimethylpentane/buffer

3.08

3.1

41.8

51.0

99.8

40

Dipentene/buffer

3.31

0.3

43.5

9.5

3.9

n.d.

Dibutyl phthalate/buffer

3.60

2.3

48.2

53.8

99.9

208

n-Decane/buffer

4.15

3.5

47.3

51.8

99.6

113

Dimethyl carbonate/buffer

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Tert-amyl alcohol/bufffer

Reaction conditions: 2 mL phosphate buffer (100 mM, pH 6.5), 2 mL organic solvents, 20 mM PED, 25 mg/mL wet cells, 35 ºС, 180 r/min. The experiments were performed twice.

Table 2 Partition coefficients of PED and HAP bcetween two phase systems Partition coefficients between the two phases Media HAP

Dimethyl carbonate/buffer

1.67

11.09

Tert-amyl alcohol/bufffer

5.42

7.28

2-Methyltetrahydrofuran/buffer

4.63

10.92

Butyl acetate/buffer

1.73

14.32

n-Hexanol/buffer

3.98

7.35

2,2,4-Trimethylpentane/buffer

0.00

0.36

Dipentene/buffer

0.00

1.60

Dibutyl phthalate/buffer

0.51

6.37

n-Decane/buffer

0.01

0.58

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PED

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Reaction conditions: 2 mL phosphate buffer (100 mM, pH 6.5), 2 mL organic solvents, 20 mM PED,

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20 mM HAP, 35 ºС, 180 r/min, 24 h. The experiments were performed twice.

SC0312

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Table 3 Effect of different organic solvents on the cell permeability of K. gibsonii

OD260a

OD280a

0.020

0.005

Dimethyl carbonate/buffer

0.320

0.204

Tert-amyl alcohol/buffer

0.261

0.175

2-Methyltetrahydrofuran/buffer

0.164

0.067

Butyl acetate/buffer

1.071

0.975

n-Hexanol/buffer

0.478

0.333

2,2,4-Trimethylpentane/buffer

0.786

0.487

Dipentene/buffer

1.099

1.130

Dibutyl phthalate/buffer

0.363

0.374

n-Decane/buffer

0.220

0.126

System

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Control

a: The increase of OD260 and OD280 in medium after 24 h. The experiments were performed twice.

Table 4 Effect of different organic solvents on the cell membrane integrity of K. gibsonii SC0312 Damaged cell * System

Dimethyl carbonate

72.63

Tert-amyl alcohol

7.10

2-Methyltetrahydrofuran

16.04

Butyl acetate

3.88

n-Hexanol

6.23

2,2,4-Trimethylpentane

1.14

Dipentene

3.74

Dibutyl phthalate

1.80

n-Decane

1.75

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*The experiments were performed twice.

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0.23

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Control

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(%)