Improving Pseudomonas sp. esterase performance by engineering approaches for kinetic resolution of 2-acetoxyphenylacetic acids

Improving Pseudomonas sp. esterase performance by engineering approaches for kinetic resolution of 2-acetoxyphenylacetic acids

Biochemical Engineering Journal 57 (2011) 63–68 Contents lists available at SciVerse ScienceDirect Biochemical Engineering Journal journal homepage:...

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Biochemical Engineering Journal 57 (2011) 63–68

Contents lists available at SciVerse ScienceDirect

Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej

Improving Pseudomonas sp. esterase performance by engineering approaches for kinetic resolution of 2-acetoxyphenylacetic acids Xin Ju, Jiang Pan, Hui-Lei Yu, Chun-Xiu Li, Jian-He Xu ∗ Laboratory of Biocatalysis and Synthetic Biotechnology, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China

a r t i c l e

i n f o

Article history: Received 4 May 2011 Received in revised form 18 July 2011 Accepted 12 August 2011 Available online 22 August 2011 Keywords: Biocatalysis Bioprocess design Enantioseparation Pseudomonas sp. Esterase Immobilized cells Mandelic acid o-Chloromandelic acid

a b s t r a c t The catalytic performance of Pseudomonas sp. ECU1011 esterase (PsE) in the kinetic resolution of (R,S)2-acetoxyphenylacetic acid (APA) was significantly improved by substrate modification, biocatalyst permeabilization and immobilization. The reaction system was modified, and the sodium salt of the substrate (APA Na), instead of APA, was hydrolyzed in aqueous phase without buffer. Considering the improved substrate solubility and the decreased biocatalyst inactivation, the reaction rate increased 3.7folds and the spontaneous hydrolysis of the substrate reduced by 48%. During the cell permeabilization, the hydrolytic activity of the whole-cell biocatalyst was increased by 2.3-fold after 2 h of pretreatment with 10% (v/v) toluene. The permeabilized cells were further entrapped in calcium alginate, resulting in 171% activity recovery with a half-life of 123 h at 30 ◦ C. Using the modified reaction system with high reaction rates and the modified biocatalyst with high activity and stability, this biocatalytic process can be transformed into a practical and environmentally friendly bioprocess for the efficient production of (S)-mandelic acid and (S)-o-chloromandelic acid. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Esterases have been widely used in pharmaceutical and chemical industries because of their advantageous features like coenzyme independence, organic solvent tolerance and high enantioselectivity [1]. There are many successful instances employing esterases in the enantioselective hydrolysis of chiral and prochiral esters to produce useful chiral alcohols and carboxylic acids [2]. We have reported the biocatalytic resolution of racemic 2acetoxyphenylacetic acids (APA) via enantioselective hydrolysis employing a novel Pseudomonas sp. esterase (PsE) (Fig. 1A) [3]. The remaining R-APA and the product, S-mandelic acid (S-MA) are important prodrugs and chiral synthons of wide-spectrum applications [4,5]. Compared with the traditional resolution of mandelate esters [6,7], this route has interesting properties such as an aqueous monophase reaction system and a wide substrate spectrum. However, its potential has not been fully developed due to the problems with catalyst stability and catalytic efficiency. The transformation of a laboratory-scale bioprocess into an industrially feasible process needs to overcome two main obstacles: economic assessment and downstream processing. Reducing the reaction expense requires biocatalysts with high activity, stability and low cost [8]. In terms of separation, bioprocesses should

∗ Corresponding author. Fax: +86 21 6425 2250. E-mail address: [email protected] (J.-H. Xu). 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2011.08.009

have the ingredients of higher product concentrations (>10%, typically), lower catalyst addition, and catalyst granules that are easier to separate. This also means that a high ratio of substrate to catalyst (S/C, >5) is favorable [9]. For these reasons, catalyst and substrate engineering are considered as agile and economical ways to improve the practical performance of bioprocesses. In catalyst engineering, improvement of biocatalysts could be realized through immobilization and permeabilization of microbial cells. Immobilization can improve the stability of biocatalysts and benefit the continuous production [10]. Three main methodologies have been employed for esterase immobilization, including carrier adsorption [11], gel entrapment [12], and covalent cross-linking or modification [13–16]. Cross-linked enzyme aggregates (CLEAs) could generate biocatalysts with high specific activity because they need little or no carriers [17], but the enzyme activity recoveries of covalent modification are varied [18,19]. On the other hand, permeabilization of cells using organic solvents or surfactants has also been found as an efficient way to improve the catalytic efficiency of whole-cell biocatalysts [20,21]. Overcoming permeability barriers could improve the apparent activity and product tolerance of biocatalysts [22]. In another aspect, substrate and reaction system engineering play key roles in the production of chiral alcohols and carboxyl acids employing esterases. Regarding substrates, previous research has mainly focused on the ester substrate derivatives with different leaving and derivative groups [7,23]. However, there are few studies on the enzymatic hydrolysis of acetylated mandelic acids,

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Fig. 1. Kinetic resolution of 2-acetoxyphenylacetic acids catalyzed by Pseudomonas sp. esterase (PsE) with a wide spectrum of substrates, either in the form of organic acids (A) or in the form of organic salts after a preliminary reformation of the substrate with NaHCO3 (B).

such as APA and 2-butyryloxyphenylacetic acid. In addition, the substrates are generally hydrolyzed at low concentrations in buffer solution [24,25]. Given the poor substrate solubility in aqueous phase, the reaction rate and substrate concentration could not meet the requirements of practical production. Several methods such as the biphasic system [26] and in situ product removal [27] have been used to increase substrate concentrations to 100–500 mM in the production of chiral mandelic acid, with respect to the modification of the reaction system. But the issues of improving substrate solubilization and dispersion have not been studied in depth. This study aims to modify the PsE biocatalyst and to find a proper form of reaction system to make the bioprocess more efficient and practical. Methods for catalyst engineering including immobilization and permeabilization, have been studied, and the reaction systems with different substrate solubility conditions have also been founded, with a version that may provide a practical sample for laboratory-scale bioprocess engineering.

2. Materials and methods

diisopropyl ether by adding an equimolar amount of acetyl chloride for 2 h at room temperature. 2.2. Modification of the reaction system The initial reaction system used in this work was APA dissolved in an aqueous monophase of KPB. The pH of the initial system was 7.0 and the phosphate concentration was 100 mM. The substrate was then changed to APA Na and the reaction medium was changed to an aqueous monophase without buffer, by adding solid salt of NaHCO3 into the APA aqueous suspension and adjusting the pH to 7.0. The enzymatic hydrolysis reaction in different reaction systems was generally performed as follows: 0.5 g of lyophilized cells was suspended in 10 ml reaction medium containing 20 mM substrate at 30 ◦ C and 200 rpm, after which the corresponding reaction rate and the enantiomeric excess of product (eep ) were analyzed at different intervals. The change in pH in the reaction medium was monitored with a pH meter (Mettler-Toledo, Switzerland).

2.1. Bacterial strain and materials

2.3. Immobilization of whole-cell biocatalyst

The bacterial strain used in this study was isolated from soil samples and is currently stored in the China General Microbiological Culture Collection Centre (CGMCC, Beijing, China) under accession number of 2872. The cultivation medium was composed of 15 g of glycerol, 5 g of peptone, 5 g of yeast extract, 1 g of NaCl, 0.2 g of MgSO4 , 0.5 g of KH2 PO4 , and 0.5 g of K2 HPO4 in 1000 ml tap water at pH 7.0. The cultivation of Pseudomonas sp. ECU1011 was conducted as described in a previous paper [3]. Whole-cell biocatalysts were harvested after 14 h of cultivation, washed and lyophilized by vacuum-freezing process, with an apparent esterase activity of 3717 U/g using APA as a standard substrate in potassium phosphate buffer solution (KPB). The (R,S)-mandelic acids were from Guangde Chemicals Co. Ltd. (Anhui, China). The components of microbial medium were from Sino Pharm Co. Ltd. (Shanghai, China). All other chemicals and solvents were obtained commercially with the purity of analytical grade. The (R,S)-APA was prepared from (R,S)-MA dissolved in an equal molar amount of acetyl chloride for 15 min at room temperature. The (R,S)-o-chloroacetoxyphenylacetic acid (o-Cl-APA) was prepared from (R,S)-o-chloromandelic acid (o-Cl-MA) dissolved in

The lyophilized cells of Pseudomonas sp. ECU1011 were immobilized following the instructions from a series of articles and necessary modifications were made in the current study [11,12]. The lyophilized cells were immobilized through calcium alginate gel entrapment; 2.5 g of lyophilized cells were added into 10 ml of deionized water containing 3% (w/v) sodium alginate and stirred for 10 min at 4 ◦ C. The mixture was extruded dropwise through a nozzle into 100 ml of 2% (w/v) calcium chloride solution to form beads with the particle size of 2–4 mm in diameter. The immobilized beads were washed with deionized water and kept in 0.2% (w/v) calcium chloride solution at 4 ◦ C. 2.4. Treatment of the whole cell biocatalysts for permeabilization To conduct the cell permeabilization, 2.5 g of lyophilized cells were suspended in 10 ml KPB (100 mM, pH 7.0). Then 1 ml of toluene was added into the solution to a final volume ratio of 1:10 and the mixtures were kept at 200 rpm at 30 ◦ C for 2 h. The permeabilization mixtures were centrifuged and the precipitates were washed and resuspended in KPB.

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For the permeabilization and entrapment of the PsE lyophilized cells, 0.1 g of cells were suspended in 10 ml of deionized water and then mixed with 1 ml of toluene. The mixture was incubated at 30 ◦ C for 2 h and then centrifuged. The precipitates were washed and then immobilized as described in Section 2.3. 2.5. Evaluation of enzyme stability For evaluation of preservative stability, three biocatalysts including lyophilized cells, immobilized cells and immobilized permeabilized cells were suspended in water and preserved at 30 ◦ C and 200 rpm for certain periods. Then the residual activity of biocatalysts was assayed. Subsequently, the relative activity was calculated by the corresponding residual activities. The half-lives of the biocatalysts were calculated by modeling the inactivation curves by linear fitting. The repetitive resolution of APA was performed in a 100 ml stirring tank reactor containing 20 ml of the reaction mixture to evaluate the operational stability of the biocatalysts. The wholecell catalyzed resolution reactions were performed in KPB with a biocatalyst load of 10% (w/v, dry weight). The immobilized cell-catalyzed resolution reactions were performed in water with biocatalyst loads of 25% and 50% (w/v, wet weight). The concentration of the salt form of APA was 40 mM. The reaction was terminated when the conversion was higher than 40% after intermittent sampling followed by high-performance liquid chromatography (HPLC) analysis or the batch reaction time was more than 12 h. 2.6. Analysis The specific activities of the biocatalysts were assayed by reactions initiated with certain amounts of whole cells into 1 ml of KPB (100 mM, pH 7.0) and immobilized cells into 1 ml water containing 10 mM APA with shaking on a mixer (Thermomixer Compact, Eppendorf) at 30 ◦ C and 1000 rpm for 1 h. The activity of biocatalysts was calculated based on the specific activity tested and the biocatalyst weight. The general procedures for the hydrolysis reaction analysis are as follows. The reaction mixtures were acidified with H2 SO4 and extracted with an equal volume of ethyl acetate. The organic layer was analyzed using HPLC (Shimadzu SPD-10A) equipped with a chiral column (Chiracel OD-H, Daicel Co., Japan) to determine the activity and enantioselectivity quantitatively. The conversion (c) of the substrate and the enantiomeric excess of the product (eep ) were calculated as the method described in our previous paper [3]. The activity was calculated based on the conversion of the substrate.

Table 1 Effect of different substrate forms on the PsE-catalyzed reactions. Substrate

APA in KPB (pH 7.0)

Solubility (g/100 ml)a Reaction time (h)b Reaction rate (mM/h) eep (%)

0.238 ± 0.002 25 0.93 ± 0.30 98.7

(Tables S1, S2 and Fig. S1). When APA was transformed into its sodium salt, sodium acetoxyphenylacetate (APA Na), the resolution reaction rate increased by 375% in the aqueous phase without KPB. The comparison of different reaction systems is shown in Table 1 and the time courses are shown in Fig. 2. Another interesting phenomenon could be observed in the results: the APA Na showed a lower rate of spontaneous hydrolysis as compared with APA (Fig. 2). This probably means that the substrate in salt form is more stable in the aqueous phase than the organic acid form. Although there was no buffer solution in the modified reaction system, the change in pH was monitored and it did obviously affect the enzyme hydrolysis. The improved reaction efficiency and substrate spontaneous hydrolysis might be accounted for by the following reasons. One explanation is that the change in solubility of APA decreased the inactivation of organic substrate on the biocatalyst. The residual activity of the whole-cell PsE after reaction under different kinds of substrate is compared in Fig. 3. The inactivation of the wholecell PsE decreased by more than 50% in the modified reaction systems. Another possible reason may be that the substrate dispersion/solubilization was improved. The solubility of APA Na was improved greatly compared with the substrate of insoluble acid (Table 1). The improved mass transfer in the modified reaction system might help to achieve higher reaction rates. 3.2. Permeabilization of whole cell biocatalyst to break mass transfer barriers Modification of biocatalysts is another important aspect of whole-cell catalyzed enzymatic reactions. Permeabilization reportedly enhances the permeability of cell membranes. Surfactants [20,21] and organic solvents [22] were both proved to be able to break the permeability barriers successfully and to accelerate biocatalytic reactions.

50

Conv. of substrate (%)

The three systems most frequently used in kinetic resolution reactions are organic solvent, organic–aqueous biphase, and buffer solution [3,6,7,23–25]. The organic phase and biphase systems have advantages of relatively high substrate concentrations and easy to separate, but organic solvents also entail potential environmental and safety risks [28]. Although the aqueous phase could reduce environmental hazards, concentrations of organic substrate in aqueous phase systems are generally low. The acylation products of MA have been considered as organic acids for the carboxyl group, and they were dissolved in buffer for aqueous reactions in previous instances [3,23–25]. The modification of the substrate (APA) to obtain better reaction features was carried out (Fig. 1B) after screening other methods, such as changing the leaving groups and the reaction media

APA Na in water (pH 7.0) >15 8 3.82 ± 0.56 96.9

a Solubility was tested in KPB or water respectively. The substrate concentration was calculated by HPLC calibration curves. b The reaction was performed at 30 ◦ C and 200 rpm. Lyophilized cells (0.2 g) were added into 10 ml reaction system containing 20 mM substrate.

3. Results and discussion 3.1. Modification of the reaction system to improve the reaction efficiency

65

40

enzymatic hydrolysis of APA Na enzymatic hydrolysis of APA spontaneous hydrolysis of APA spontaneous hydrolysis of APA Na

30

20

10

0 0

12

24

36

48

Reaction time (h) Fig. 2. Enzymatic and spontaneous hydrolysis of APA or APA Na. Enzymatic reaction was conducted by adding 0.2 g lyophilized cells into 10 ml reaction medium containing 20 mM substrate at 30 ◦ C, 200 rpm. Spontaneous hydrolysis was conducted without cells addition.

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(A)

4000

Conv. & eep (%)

Residual activity (U/g)

100

3000

2000

1000

80 60 40 20 0 1

2

3

APA

APA Na

Blank

5

6

(B)

Initial

100

After screening for permeabilization methods, toluene was found to be the most effective enhancer: it could enhance the apparent activity of whole cells from 3717 U/g to 8475 U/g (Figs. S2 and S3). The process parameters of the toluene pretreatment were then optimized. The appropriate toluene content was 1:10 (v/v), the proper temperature was 30 ◦ C and the best results were achieved after 3 h. After the optimization, the specific activity of the whole cell biocatalyst was 8904 U/g dry cell weight, about 2.4-fold that of the untreated cells.

Conv. & eep (%)

Fig. 3. Residual activities of whole-cell PsE by using APA or APA Na as the substrate. Blank: after 24 h reservation in water; initial: initial activity of whole cells.

80 60 40 20 0 1

2

3

4

5

6

7

8

9

10

11

8

9

12

Batch

(C)

3.3. Entrapment of whole cells to obtain the immobilized biocatalyst

80 60 40 20 0 1

(D) Conv. & eep (%)

100

Conv. & eep (%)

100

For the immobilization of biocatalysts, various methods had been screened and entrapment in calcium alginate gel was selected in this work because of its good biocompatibility and high activity recovery (Tables S3–S5). The cell load in alginate gel entrapment was then optimized. The results showed that a 5% cell load led to an activity of 185 U/g and the highest activity recovery at 76%. In contrast, a 20% cell load resulted in the highest specific activity at 243 U/g, but with only 44% activity recovery. A higher specific activity was expected at 25% load of cells but it was not observed, which was possibly due to the diffusion factor [29]. For the main drawback of this method is the relatively low specific activity, the 20% cell load was applied in the following research.

Residual activity (%)

4

Batch

0

2

3

4

5

6

7

10

Batch 120 100 80 60 40 20

80

0 1

2

3

4

5

6

7

Batch

60

Fig. 5. Operational stability of biocatalysts in repeated resolution of APA Na and o-Cl APA Na. The reactions were conducted at 30 ◦ C, 200 rpm with 20 mM substrate concentration and 5% biocatalyst load. A, B and C: repeated resolution APA Na by lyophilized cells, immobilized cells and immobilized permeabilized cells; D: resolution o-Cl APA Na by permeabilized immobilized cells. Symbols: filled bars, conversion; unfilled bars: eep .

immobilized cells lyophilized cells

40

permeabilized immobilized cells

20

0 0

24

48

72

96

120

Preservation time (h) Fig. 4. Preservation stability of immobilized biocatalysts compared with that of lyophilized cells. The catalysts were kept at 30 ◦ C, 200 rpm for certain intervals. Symbols: diamonds, immobilized cells; triangles, lyophilized cells; squares, permeabilized immobilized cells.

Using a combination of the two methods, permeabilized and immobilized cells were obtained through further entrapment of the toluene-permeabilized cell with calcium alginate gel, yielding a 171% activity recovery and a specific activity of 489 U/g.

X. Ju et al. / Biochemical Engineering Journal 57 (2011) 63–68

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Table 2 Comparison of repeated bioresolution processes. Biocatalyst

Substrate

Biocatalyst load (%)a

Lyophilized cells Immobilized cells Immobilized permeabilized cells Immobilized permeabilized cells

APA Nac APA Na APA Na o-Cl APA Nad

10 50 25 25

a b c d

Operational time (h)b 32 140 120 70

Batch completed 4 12 10 7

Substrate transformed (g)

Productivity (g/l/h)

0.274 0.886 0.814 0.644

0.348 0.323 0.339 0.191

Wet weight when immobilized cells were used. Reactions were carried out in 100-ml stirring tank reactor containing 20 ml reaction mixture at 30 ◦ C and 200 rpm. Time used for renewing reaction medium was neglected. Substrate concentration was 40 mM in the APA Na reactions. Substrate concentration was 20 mM in the o-Cl APA Na reactions.

3.4. Evaluation of modified biocatalysts and reaction systems

Appendix A. Supplementary data

The thermal stabilities of the biocatalysts were examined and the results were shown in Fig. 4. Considering that the optimized temperature of the APA resolution reaction was 30 ◦ C [3], the test of thermal stability was conducted at 30 ◦ C. The best results were achieved using immobilized cells, which remained 84.4% activity after 108 h at 30 ◦ C and 200 rpm, whereas the lyophilized cells could only maintain 4.8% activity after 60 h. The half-life of the former (313 h) is 12.6-fold of the latter (24.7 h). The half-life of the permeabilized cells entrapped in alginate gel was 123 h, 5-fold of the lyophilized cells. The reusability of the lyophilized cells and the immobilized cells is compared in Fig. 5. The lyophilized cells could not be reused more than five reaction batches (Fig. 5A), where as the immobilized cells and immobilized permeabilized cells could be reused for more than 10 reaction batches (Fig. 5B and C). The reaction time of each batch was about 8–12 h, i.e., the total operational time of the immobilized biocatalysts could reach more than 150 h. To evaluate the applicability of the modified biocatalysts and reaction systems, the immobilized and permeabilized cells were further employed to resolve the (R,S)-o-Cl-APA Na (Fig. 5D), another important substrate for the production of Clopidogrel. After a 70 h reaction, 7 repeated reactions were completed and enantiopure So-Cl-MA was obtained. This repeated resolution reactions showed the practicability of this work. The four repeated resolution reactions in this work are compared in Table 2. Based on the total operational time, the stability of the immobilized biocatalysts was improved significantly. In addition, the modified reaction system helped to transform more substrate than the lyophilized free cells. During the biotransformation process, immobilized granules also have advantages of separation which was not described using data here.

Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bej.2011.08.009.

4. Conclusions The two main methods for improving the bioreaction performance, catalyst engineering and substrate engineering, were integrated. By combining these two strategies, the bioprocess of kinetic resolution of APA Na using permeabilized and immobilized whole cells showed good practical prospects. Therefore the present work, together with other successful cases, could make the bioprocess for production of enantiopure chiral chemicals more economic, efficient and environmental friendly.

Acknowledgements This research was financially supported by the National Natural Science Foundation of China (Nos. 20902023 and 31071604), the Ministry of Science and Technology (Grant No. 2009CB724704), and China National Special Fund for State Key Laboratory of Bioreactor Engineering (Grant No. 2060204).

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