Continuous production of aprepitant chiral intermediate by immobilized amidase in a packed bed bioreactor

Accepted Manuscript Continuous production of aprepitant chiral intermediate by immobilized amidase in a packed bed bioreactor Chao-Ping Lin, Zhe-Ming Wu, Xiao-Ling Tang, Chang-Ling Hao, Ren-Chao Zheng, Yu-Guo Zheng PII: DOI: Reference:

S0960-8524(18)31649-3 https://doi.org/10.1016/j.biortech.2018.12.006 BITE 20762

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

7 October 2018 2 December 2018 3 December 2018

Please cite this article as: Lin, C-P., Wu, Z-M., Tang, X-L., Hao, C-L., Zheng, R-C., Zheng, Y-G., Continuous production of aprepitant chiral intermediate by immobilized amidase in a packed bed bioreactor, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.12.006

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Title: Continuous production of aprepitant chiral intermediate by immobilized amidase in a packed bed bioreactor Authors and Affiliation: Chao-Ping Lin1,2, Zhe-Ming Wu1,2, Xiao-Ling Tang1,2, Chang-Ling Hao1,2, Ren-Chao Zheng 1,2*, Yu-Guo Zheng 1,2 1

Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and

Bioengineering, Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China 2

Engineering Research Center of Bioconversion and Biopurification of Ministry of Education,

Zhejiang University of Technology, Hangzhou 310014, People’s Republic of China

*Corresponding author: Tel: +86-571-88320391, Fax: +86-571-88320884, E-mail: [email protected]

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ABSTRACT To

develop

a

highly

efficient

method

for

aprepitant

chiral

intermediate

(S)-4-fluorophenylglycine, a continuous reaction system was established in packed bed bioreactor using amidase covalently immobilized on epoxy resin as biocatalyst. The epoxy resin was firstly modified by metal-chelate method and functional groups (Cu2+-IDA) generated were able to rapidly adsorb amidases, which were further covalently bound onto the modified resin with 90.1% immobilization yield and 80.2% activity recovery. The immobilized amidase exhibited excellent thermal stability with the longest half-life of 1456.8 h at 40

o

C ever reported.

(S)-4-fluorophenylglycine was continuously produced using the reaction system with 49.9% conversion, 99.9% ee, and an outstanding space-time yield of 5.29 kg L-1 d-1. Moreover, the efficient reaction system exhibited a high operational stability and retained 86.3% catalytic activity after 25-day continuous operation. This efficient continuous bioprocess presents great industrial potential for large-scale production of (S)-4-fluorophenylglycine.

Keywords: (S)-4-fluorophenylglycine, immobilized amidase, kinetic resolution, continuous production, packed bed bioreactor

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1. Introduction (S)-4-fluorophenylglycine is an important chiral intermediate for potent antiemetic drugs including aprepitant and fosaprepitant, which are human neurokinin-1 (NK-1) receptor antagonist, and act as neurotransmitters and neuromodulators in the peripheral and central nervous system (Brands et al., 2003; Naveenkumar Kolla et al., 2007). Traditional chemical routes for (S)-4-fluorophenylglycine involved expensive chiral reagents, large amount of organic solvents, and tedious procedures, resulting in high cost and environmental pressure (Anakabe et al., 2010; Truppo et al., 2010; Wang et al., 2011b; Wilmink et al., 2015). By contrast, biocatalysis provides a promising alternative with advantages of mild reaction condition, high atom economy, and environmental friendliness. The enzymes including transaminase, R-amino acid oxidase and (S)-amino acid transferase have been reported for synthesis of (S)-4-fluorophenylglycine (Cameron et al., 2001). However, their industrial application suffered from the problems of reusability, stability, and productivity (Chen et al., 2015; Liu et al., 2017). Immobilized enzymes exhibit better operational stability, higher resistance, simple procedure of separation and purification (Eş et al., 2015; Chen et al., 2016). Various methods including physical adsorption, encapsulation, cross-linking, and covalent binding have been established for enzyme immobilization (Magner, 2013; Hartmann & Kostrov, 2013). Covalent binding interactions between enzyme and epoxy carrier can remarkably enhance the stability of free enzyme and are employed as one of the most popular immobilization methods (Guisán et al., 2010; Hassan, 2016). However, high concentration of sodium phosphate buffer is required for physical hydrophobic adsorption of protein onto the carriers prior to covalent binding, which causes

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enzyme inactivation during the immobilization process. Moreover, it is difficult for the highly hydrophilic proteins to be immobilized onto the carriers. To overcome these limitations of the traditional carrier, multifunctional carriers were designed by kinds of modification including carboxy-epoxy, amino-epoxy, thiol-epoxy and disulfide-epoxy (Mateo et al., 2007a). Packed bed bioreactors (PBBR) have received considerable attention as a practical method of process convenience with advantages of avoiding additional separation steps and continuous operation (Wang et al., 2014). In addition, it has the advantage of avoiding lost of enzyme and preventing deactivation of the enzyme caused by mechanical damage compared with stirring tank reactor (Feng et al., 2011; Terán-Hilares et al., 2016). The packed bed bioreactors have been widely applied in biotransformations, biorefineries, and micro- pollutants treatment bioprocesses (Zheng et al., 2017; Nguyen et al., 2016; Ren et al., 2011). Penicillin amidases have been widely used for semi-synthesis of β-lactam antibiotics and kinetic resolutions of racemic mixtures of α-amino acids and their derivatives (Grulich et al., 2015; Illanes et al., 2012; Xue et al., 2013). In our previous study, penicillin amidase from Bacillus megaterium was found to exhibit high activity and enantioselectivity towards racemic N-phenylacetyl-4-fluorophenylglycine, producing (S)-4-fluorophenylglycine in 49.9% conversion and 99.9% enantiometric excess (ee) (Lin et al., 2018). In order to develop an efficient reaction system with excellent operation stability and high productivity, immobilized enzyme was suggested to overcome the difficulties of stability, separation and recycling when using free enzyme as biocatalyst. In summary, development of a robust biocatalyst with excellent operation stability, and a reaction system with high productivity is of great importance for industrial production of (S)-4-fluorophenylglycine.

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In this study, more than twenty functional resins were screened and subsequently modified for immobilization. The amidase was immobilized onto modified epoxy resin by covalent binding and the properties of immobilized amidase were investigated. Furthermore, an efficient reaction system

was

established

for

continuous

kinetic

resolution

of

racemic

N-phenylacetyl-4-fluorophenylglycine by the immobilized amidase in packed bed bioreactor, providing an efficient approach for industrial production of (S)-4-fluorophenylglycine with high productivity. 2. Materials and Methods 2.1. Materials 4-Fluorophenylglycine was purchased from Shanghai Rongli Chemical Technology Co., Ltd (Shanghai, China). (S)-4-fluorophenylglycine was purchased from J&K Scientific Co., Ltd (Shanghai, China). The resins used for enzyme immobilization were purchased from Tianjin Nankai Hecheng Science and Technology Co., Ltd. (Tianjin, China), Purolite Co., Ltd. (Bala cynwyd, PA, USA), and donated by Xian Lanxiao Technology Co., Ltd. (Xian, China) respectively. BCA Protein Assay Kits were purchased from Jiangsu KeyGEN Biotechnology Co., Ltd (Jiangsu, China). The crude penicillin amidase was prepared in our laboratory (Xue et al., 2014). All other chemicals and reagents used were of analytical reagent grade and commercially available. 2.2. Screening of resin carriers To select the proper carrier for enzyme immobilization, immobilization efficiency of different resins was investigated. Twenty-four resins including epoxy resins, amino resins, and macroporous resins were tested for immobilization based on their properties. Amino resins were pre-activated by stirring for 12 h in 2% glutaraldehyde (w/v) solution to obtain the aldehyde

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groups as the functional group. The amidase was immobilized onto epoxy reins, activated amino resins, and macroporous resins, respectively, according to the following procedure: resins (0.5 g) were washed three times with 5 mL of phosphate buffer (0.5 M, pH 7.0). The cleaned resins were added to 5 mL of phosphate buffer (0.5 M, pH 7.0) containing 10 mg crude amidase. The mixtures were shaken at 120 rpm for 18 h at 25 oC. The immobilized enzymes were obtained after filtered and washed with 2 mL of phosphate buffer (0.2 M, pH 7.0) until no protein was detected in the supernatant. Protein concentrations were assayed with the BCA Protein Assayed Kit using bovine serum albumin as standard. 2.3. Preparation of Cu2+-IDA-functionalized epoxy resin The selected epoxy-resin was modified according to the following procedure (Mateo et al., 2007). The epoxy-resin (2 g) was added to 5 mL of iminodiacetic acid (IDA) (2 M, pH 10.0) and stirred with 120 rpm at 25 oC for 30 minutes. The epoxy-resin was filtered after washing three times with 5 mL distilled water. The IDA-resin (2 g) was added into 30 mL cupric sulfate solution (30 mg/mL) and stirred at 120 rpm for 3 h. The modified resin carriers were then washed with excess of distilled water and harvested for further study. 2.4. Immobilization of enzyme on Cu2+-IDA-functionalized resin carriers The amidase was immobilized on modified epoxy resin as follow procedure: 1.0 g modified resin was added to 10 mL of phosphate buffer (0.5 M, pH 7.0) containing 60 mg crude amidase. The mixture was stirred at 25 oC for 18 h with 120 rpm. The immobilized enzymes were filtered and incubated at phosphate buffer (0.5 M, pH 10.0) containing 20% glycerol and 10% fructose without stirring. The immobilized enzymes were then washed with phosphate buffer (0.2 M, pH 8.5) and harvested for further use.

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Immobilization parameters such as temperature (15-40 oC), pH (5.0-9.0), duration of immobilization (8-48h) and enzyme loading (50-120 mg/g resin) were optimized. Meanwhile, the incubation time (1-5 d) at alkaline circumstance affecting the stability of the immobilized enzyme was investigated. Relative stability was given as the ratio of the half-life of the immobilized enzyme incubated for period time to that of just immobilized enzyme without incubation. 2.5. Enzyme activity assay The substrate was dissolved in water and the pH was adjusted to 9.0 with 5% ammonia solution.

The

activity

of

the

immobilized

amidase

was

assayed

with

N-phenylacetyl-4-fluorophenylglycine as substrate in a reaction mixture containing 50 mM substrate and 10 g/L immobilized amidase. After performing the reaction in a shaking water bath at 40 oC for 3 min, the immobilized amidase was separated from the mixture and concentration of (S)-4-fluorophenylglycine was analyzed with high performance liquid chromatography (HPLC). One unit (U) of immobilized amidase activity was defined as the amount of immobilized enzyme that produces 1 μmol of (S)-4-fluorophenylglycine per minute under the standard activity assay conditions. The specific activity, activity recovery (%), immobilization yield (%) and relative activity (%) were calculated using Eqs. (1)-(4): Specific activity (U/g carrier) = Activity of immobilized enzyme/amount of carrier

(1)

Activity recovery (%) = Total activity of immobilized enzyme/total activity of free enzyme × 100%

(2)

Immobilization yield (%) = Immobilized protein/total loading protein × 100%

(3)

Relative activity (%) = Residue activity/original activity × 100%

(4)

2.6. Characterization of immobilized amidase

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To further explore the properties of the immobilized enzyme, the effects of temperature and pH on the activity of immobilized enzyme were examined under standard activity assay conditions. The optimal temperature and pH for the immobilized amidase activity were examined at different temperatures (20-60 oC) and various pHs (4.0-11.0), respectively. Meanwhile, thermal stability of the immobilized and free enzymes was determined by incubating the enzymes in glycine-sodium hydroxide buffer (100 mM, pH 9.0) for 1 to 15 days at different temperatures of 30, 40, 50 oC, respectively. The residual activities of free and immobilized amidase were measured by HPLC. The initial enzyme activity (without incubation) was defined as 100% relative activity. Moreover, the storage stability of the immobilized amidase was also investigated by incubation of the immobilized amidase in glycine-sodium hydroxide buffer (100 mM, pH 9.0) at 4 oC for several weeks. The residual activities of immobilized amidase were assayed under the standard assay conditions. 2.7. Construction and optimization of continuous packed bed bioreactor The immobilized amidase (5.0 g) was packed into the water-jacketed glass column and the column temperature was maintained at 30

o

C using circulating constant water by outer

water-jacket. The substrate solution reservoir containing different substrate concentrations (20-80 g/L) was also kept at 30 oC. The substrate solution was continuously fed into the packed bed filled with immobilized amidase from bottom to top using a peristaltic pump. The eluate from the outlet of packed bed bioreactor was periodically withdrawn to monitor the conversion and ee by HPLC. The critical process parameters including height to diameter ratios (H/D, 6:1-14:1) and flow rates (0.5-3.0 mL/min) were examined for their optimal mass transfer and efficiency. 2.8. Analytical methods and statistical analysis

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The concentrations of N-phenylacetyl-4-fluorophenylglycine, 4-fluorophenylglycine was measured by HPLC (Shimadzu Co., Kyoto, Japan) with a Diamonsil C18 column (5 μm × 250 mm × 4.6 mm, Diamonsil Technologies Co., Beijing, China) equipped with an ultraviolet detector (SPD-10A VP Plus, Shimadzu, Japan) at 220 nm. The mobile phase consisted of acetonitrile and 0.1% perchloric acid at a ratio of 50:50 (v/v), and the flow rate was set to be 0.7 ml min-1. The retention times of N-phenylacetyl-4-fluorophenylglycine and 4-fluorophenylglycine were 8.20 min and 3.74 min, respectively. The ee values of (S)-4-fluorophenylglycine were determined on a chiral column (ChirobioticTM R 5 μm × 250 × 4.6 mm, Sigma, USA) using acetonitrile and 0.5% acetic acid (80:20, v/v) as the mobile phase. The eluent was monitored at 220 nm with a flow rate of 1.0 ml min-1. The retention times of (S)-4-fluorophenylglycine and (R)-4-fluorophenylglycine were 13.02 min and 14.84 min, respectively. All trials were performed at least in triplicate in this study unless otherwise noted. The datas were analyzed using SAS program version 8.1 (SAS Institute Inc., Cary, NC, USA). All the graphs in this study were constructed using the Origin software version 8.0 (Origin Lab Corp., Northampton, MA, USA). 3. Results and discussion 3.1. Immobilization of amidase with different resin carriers The recombinant amidase was secreted by Bacillus subtilis WB800 with high efficiency and comprised more than 80% of the total proteins in the fermentation broth (Xue el al., 2014). What’s more, immobilization of crude enzyme shows advantages of easy separation and high activity recovery compared with purified enzyme. Therefore, the crude amidase with relative high purity

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(>80%) was used for immobilization. To obtain the appropriate resin carrier with high immobilization efficiency for amidase, three kinds of typical resin carriers including epoxy resins, amino resins and macroporous resins with various functional groups were investigated. Amino resins had to be activated with glutaraldedyde to obtain functional groups prior to covalent immobilization process. Epoxy resins could be directly used for immobilization via covalent binding between enzyme and epoxy groups on their surface, while macroporous resins were used for immobilization by physical adsorption. The immobilization efficiency of those resins was shown in Fig. 1. The immobilization yields with epoxy resins LX-103B, LX-1000EP, ES-103B (a), ES-103B (b), ECR8214, and amino resin ECR8319 as carriers reached more than 80%, whereas that with macroporous resins as carrier were below 63%. Since the specific activity and activity recovery of amidase immobilized on ES-103B (a) were higher than others, it was chosen as the immobilization carrier for further study. ES-103B (a) is a kind of epoxy porous resin with high hydrophobicity, which is beneficial to hydrophobic interaction between enzyme and carrier compared with hydrophilic carrier (ES-1). In addition, rich epoxy groups of ES-103B (a) led to abundant covalent binding sites for immobilization. Moreover, the large pore (pore diameter 10-50 nm) and high pore volume (0.46 cm2 g-1) of the resin promote the accessibility of substrate to the active site of enzyme. 3.2. Immobilization of amidase using modified resin carrier The resin ES-103B (a) was modified to further improve immobilization efficiency. Although three modification methods including aminated-epoxy support (Desmet & Marquette, 2016), boronate-epoxy carrier and carboxy-epoxy carrier (Barbosa et al., 2013; Mateo et al., 2007b) were tested, the immobilization efficiency were not significantly improved (data not shown). The

10

metal-chelate method was finally investigated to modify ES-103B (a). The epoxy resin was treated with IDA, given the carboxyl groups on the surface of epoxy resin. Subsequently, the IDA-resins were combined with copper ions, and then the bifunctional carriers were formed (Fig. 2). The additional group (Cu2+-IDA) generated on the carrier surface was able to rapidly adsorb proteins in mild conditions. Thus, immobilized yield of enzyme was guaranteed and biological activity of proteins was retained. The immobilized amidase exhibited excellent immobilization yield (85.2%) and activity recovery (67.7%), which was much higher than that of unmodified ES-103B (a). Therefore, the modified resin (Cu2+-IDA-ES-103B) was employed as carrier for immobilization. The modified resin promoted the attachment speed of protein onto the carrier and retained biological activity of proteins, which improved the following intramolecular multipoint binding between the epoxy groups of the carrier and nucleophiles on the surface of the protein participation. In addition, this method provides a possibility for covalent immobilization of enzyme through epoxy groups at low ionic strength or on hydrophilic carriers. 3.3. Optimization of immobilization process 3.3.1 Effects of immobilization temperature and pH The immobilization temperature and pH were further investigated. The activity recovery and immobilization yield did not change much at temperatures ranging from 15 to 40 oC, and the specific activity reached the maximum at 25 oC (Fig. 3a). The influence of pH on the immobilization efficiency was examined at pH ranging from 5.0 to 9.0 (Fig. 3b). The specific activity of the immobilized amidase gradually increased with the increase of pH from 5.0 to 7.0. The maximum specific activity of the immobilized amidase was achieved at pH 7.0, and a

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remarkable decrease was observed by further increasing the pH, while the immobilization yield was almost not affected except at extreme pH of 4.0 and 11.0. As such, pH 7.0 was chosen as the optimum pH value for further optimization. 3.3.2 Effect of enzyme loading The immobilization yield, activity recovery, and specific activity were investigated at different enzyme loading (50-120 mg/g resin). As shown in Fig. 3c, the specific activity increased with the increase of enzyme loading and reached the maximum at 90 mg/g resin, while the immobilization yield and activity recovery decreased. Thus, 90 mg/g resin was selected as the optimum enzyme loading for further study. Meanwhile, the duration of immobilization was further optimized to be 18 h. 3.3.3 Effect of incubation time Incubation at alkaline circumstance could enhance the reactivity of nucleophilic groups of the enzyme surface, obtain multipoint covalent attachment between enzyme and carrier, and then increase the stability by improving the rigidity of enzyme (Guerrero et al., 2017). When moderate concentration of glycerol and fructose was added to incubation buffer, hydrophilic functional group of glycerol and fructose dramatically affected water content surrounding enzyme surface and active site region, and a stable water layer around enzyme surface was formed, thereby reducing structural disturbance of enzyme in the incubation process. To further improve the stability of immobilized amidase, the effect of incubation time (0-5 d) on relative stability and residual activity of the immobilized amidase were investigated (Fig. 3d). The relative stability of the immobilized amidase rapidly improved with the increase of incubation time from 1 to 3 days and then it tended to increase slowly. Unfortunately, the residual activity gradually declined as

12

incubation time was prolonged. To take residual activity of immobilized amidase, incubation for 3 days at pH 10.0 was chosen as optimum incubation time. 3.4. Characterization of immobilized amidase 3.4.1 Effects of temperature and pH on immobilized amidase activity The effect of temperature on the immobilized enzyme, as well as that on free amidase activity was investigated over a temperature range from 20 to 60 oC. The immobilized amidase reserved more than 60% of its original activity in the temperature range of 20-60 oC and reached the maximum at 40 oC, while the free amidase obtained the highest activity at 30 oC and less than 50% was retained above 50 oC (Fig. 4a). In terms of pH, both the immobilized and free amidase reached their maximum activity at pH 9.0. The immobilized amidase retained over 80% of its initial activity at pH 4.0-11.0, while the free amidase lost 50% of its activity below pH 6.0 or above pH 10.0 (Fig. 4b). Although the optimum pH of immobilized enzyme is consistent with the free form, the immobilized amidase was found to be less sensitive to a broad pH scope, which might be due to the increased stability of immobilized amidase and the changes of the microenvironment surrounding enzyme (Yong et al., 2008). The optimum temperature was slightly shifted to 40 oC, which was higher than that of the free amidase. Similar shifts in the optimum temperature were also observed for the Candida boidinii formate dehydrogenase immobilized on the amino-epoxy carrier (Bolivar et al., 2007), and Pseudomonas putida esterase immobilized on epoxy resin (Ma et al., 2016). This shift of tending to higher optimum temperature could be attributed to enhanced thermal stability and adaptability of immobilized amidase (Wang et al., 2011a). Moreover, the acceleration of external mass transfer for the substrate at higher temperature might be another crucial factor (Chen et al., 2012).

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3.4.2 Thermal stability To investigate the thermal stability, the immobilized amidase was incubated at different temperature (30-50 oC) and its residual activity was detected. As shown in Fig. 4c, the immobilized amidase showed the residual activity of 85.1% over a period of 15 days incubation ranging from 30-40 oC. However, the free enzyme retained only 50.2% of its original activity following incubation at 40 oC for 15 d (Fig. 4d). The half-lives of immobilized enzyme (t1/2) at 30 o

C, 40 oC, and 50 oC were 2409.6 h, 1456.8 h, and 422.4 h, with the inactivation rate (kd) of

0.00028, 0.00047, and 0.00164, respectively. Moreover, the half-lives of immobilized amidases using different carriers and immobilization methods reported were compared (Torres-Bacete et al., 2001; Hormigo et al., 2009; Xue et al., 2013; Kneževićjugović et al., 2015). Obviously, the amidase immobilized on modified ES-103B of this study exhibited the highest thermal stability. The improved thermal stability of the immobilized enzyme might be due to the increasing rigidity of the enzyme conformation by multipoint covalent attachment (Mihailović et al., 2014). The noticeable thermal stability of the immobilized amidase indicated its potential for long-term performance in industrial applications. 3.4.3 Storage stability The storage stability of immobilized amidase is a key parameter for its industrial application. Good storage stability could decrease the cost of enzyme and cut the time required to immobilize enzyme off (Fernandes et al., 2009). The immobilized amidase was stored at 4 oC for 4 weeks, and more than 99.1% of its activity was retained, suggesting the significant storage stability. 3.5. Synthesis of (S)-4-fluorophenylglycine in packed bed bioreactor 3.5.1 Optimization of operation parameters

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Enzymatic synthesis of (S)-4-fluorophenylglycine by the immobilized amidase was carried out in packed bed bioreactor. The substrate solution was continuously fed to the packed bed bioreactor and the eluent containing (S)-4-fluorophenylglycine and the unreacted substrate was collected in the product reservoir. Five grams of immobilized amidase (bed volume 8 mL) was added into the packed bed bioreactor and the effect of the height/diameter ratio (H/D) of the packed bed bioreactor on the enzymatic resolution was evaluated at the same flow rate (3.0 mL/min). The conversion of substrate was improved with the increase of the H/D ratio (6:1-10:1), and then was basically flat with H/D ratio from 10:1 to 14:1, which was probably caused by partially fluidized state of the packed bed and external mass transfer of substrate. Moreover, at the same flow rate of substrate supply, it is hard to pump the solution with high H/D ratio because of the high pressure drop of the packed bed, and there is possibility of clogging the enzyme packed bed (Ma et al., 2016; Rakmai & Cheirsilp, 2016). Thus, the H/D ratio of 10:1 was chosen for further study. Furthermore, the effect of the flow rate on substrate conversion was investigated. The conversion reached 49.9% at the flow rate from 0.5 to 2.0 mL/min, and then gradually decreased with further increased flow rate (2.0-4.0 mL/min). Thus, the optimum flow rate of 2.0 mL/min was chosen for further study. The effect of substrate concentration was also investigated as high substrate concentration could improve the volumetric efficiency and decrease the cost for industrial application. When the flow rate was set at 2.0 mL/min, the conversion could reach 49.9% under the substrate concentration from 20 to 50 g/L and gradually decrease when the substrate concentration higher than 50 g/L. However, a higher substrate concentrate (60 g/L) was introduced to the packed bed bioreactor at flow rate of 1.0 mL/min, the conversion of 49.9% was also achieved and the

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space-time yield was calculated to be 3.17 kg L-1 d-1. Comparatively, the space-time yield of 5.29 kg L-1 d-1 was achieved with 50 g L-1 substrate loading, which markedly exceeded the average space-time yield of industrial bioprocess (0.372 kg L-1 d-1) (Bommarius & Riebel, 2004; Xue et al., 2015). Moreover, the specific productivity for (S)-4-fluorophenylglycine was further calculated to be 8.46 g gimmobilized enzyme-1 d-1 with the PBBR implemented with immobilized amidase at 1.0 mL/min flow rate, which was higher than that reported by Xue et al. (0.048 g gimmobilized enzyme -1 d-1) and Ma et al. (3.711 g gimmobilized enzyme-1 d-1), exhibiting efficient catalytic performance (Xue et al., 2013; Ma et al., 2016). This PBBR implemented with immobilized amidase on Cu2+-IDA functional epoxy resin was proved to be a powerful tool for continuous biosynthesis of (S)-4-fluorophenylglycine. 3.5.2 Continuous synthesis of (S)-4-fluorophenylglycine by immobilized amidase in PBBR To demonstrate the feasibility of continuous biosynthesis of (S)-4-fluorophenylglycine, a PBBR packed with immobilized amidases was operated for 25 days under the optimum conditions. Meanwhile, the operational life span of the PBBR was investigated. As shown in the Fig. 5, a conversion of 49.9% was achieved and 86.1% of initial activity was kept even after 25-day continuous operation. Moreover, (S)-4-fluorophenylglycine was collected with 99.9% ee. during the whole process and the space-time yield of the packed bed bioreactor was calculated to be 5.29 kg L-1 d-1. Meanwhile, using immobilized amidase for (S)-4-fluorophenylglycine synthesis is much convenient than that using free enzyme or cells (Cameron et al., 2001; Xia et al., 2015). This efficient continuous reaction system showed excellent properties in the achievement of high productivity, high stability and simplification of the operations. 4. Conclusion

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In this study, the amidase was immobilized on epoxy resin modified by the metal-chelate method. The immobilized amidase showed significant thermal stability with the half-life of 1456.8 h at 40 oC. Continuous biosynthesis of (S)-4-fluorophenylglycine catalyzed by the immobilized amidase was carried out in a PBBR with 49.9% conversion and 99.9% ee, and space-time yield of 5.29 kg L-1 d-1. Moreover, the bioprocess exhibited great operational stability, which retained 86.3% of initial activity after continuous operation of 25 days. These results obtained here showed great industrial potential of the efficient reaction system for (S)-4-fluorophenylglycine production. Acknowledgments This work was financially supported by the National High Technology Research and Development Program of China (No. 2012AA022201) and the Natural Science Foundation of Zhejiang Province (No. LQ18B06007). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/. Reference 1. Anakabe, E., Vicario, J.L., Badia, D., Carrillo, L., Yoldi, V., 2010. Asymmetric synthesis of arylglycines and their use as chiral templates for the stereocontrolled synthesis of 7,8-disubstituted 3-aryl-1,2,3,4-tetrahydroisoquinolin-4-ols. Eur. J. Org. Chem. 33, 4343-4353. 2. Barbosa, O., Torres, R., Ortiz, C., BerenguerMurcia, Á., Rodrigues, R.C., Fernandezlafuente, R., 2013. Heterofunctional supports in enzyme immobilization: From traditional immobilization protocols to opportunities in tuning enzyme properties. Biomacromolecules 14, 2433-2462. 3. Bolivar, J.M., Wilson, L., Ferrarotti, S.A., Fernandez-Lafuente, R., Guisan, J.M., Mateo, C.,

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2007. Evaluation of different immobilization strategies to prepare an industrial biocatalyst of formate dehydrogenase from Candida boidinii. Enzyme Microb. Technol. 40, 540-546. 4. Bommarius, A.S., Riebel, B.R., 2004. Biocatalysis: fundamentals and applications. John Wiley & Sons. pp. 1-2. 5. Brands, K.M.J., Payack, J.F., Rosen, J.D., Nelson, T.D., Candelario, A., Huffman, M.A., Zhao, M.M., Li, J., Craig, B., Song, Z.J., 2003. Efficient synthesis of NK1 receptor antagonist aprepitant using a crystallization-induced diastereoselective transformation. J. Am. Chem. Soc. 125, 2129-2135. 6. Cameron, M., Cohen, D., Cottrell, I.F., Kennedy, D.J., Roberge, C., Chartrain, M., 2001. The highly stereospecific enzyme catalysed transamination of 4-fluorophenylglyoxylic acid to 4-(S)-fluorophenylglycine. J. Mol. Catal. B Enzym. 14, 1-5. 7. Chen, B., Yin, C., Cheng, Y., Li, W., Cao, Z.A., Tan, T., 2012. Using silk woven fabric as support for lipase immobilization: The effect of surface hydrophilicity/hydrophobicity on enzymatic activity and stability. Biomass Bioenergy, 39, 59-66. 8. Chen, X., Liu, Z.Q., Huang, J.F., Lin, C.P., Zheng, Y.G., 2015. Asymmetric synthesis of optically active methyl-2-benzamido-methyl-3-hydroxy-butyrate by robust short-chain alcohol dehydrogenases from Burkholderia gladioli. Chem. Commun. 51, 12328-12331. 9. Chen, X., Liu, Z.Q., Lin, C.P., Zheng, Y.G., 2016. Efficient biosynthesis of ethyl (R)-4-chloro-3-hydroxybutyrate using a stereoselective carbonyl reductase from Burkholderia gladioli. BMC Biotechnol. 16, 1472-6750. 10. Desmet, C., Marquette, C.A., 2016. Surface functionalization for immobilization of probes on microarrays. Microarray Technology. Human Press, Springer New York 1368, 7-23.

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enzymes.

Figure and table captions Fig. 1. The immobilization efficiency of different resin carriers Fig. 2. The illustration of the modify process of resin and immobilization mechanism Fig. 3. Optimization of immobilization process. (a). Effect of temperature on immobilization; (b). Effect of pH on immobilization; (c). Effect of enzyme loading on immobilization; (d). Effect of duration of immobilization Fig. 4. Characterization of immobilized and free amidase. (a). Effect of temperature on immobilized amidase activity; (b). Effect of pH on immobilized amidase activity; (c). Thermal stability of immobilized amidase; (d). Thermal stability of free amidase Fig. 5. Operation life of continuous packed bed bioreactor

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Activity recovery (epoxy resin) Activity recovery (amino resin) Activity recovery (macroporous resin)

Specific activity (epoxy resin) Specific activity (amino resin) Specific activity (macroparous)

200

200

180

180

160

160

140

140

120

120

100

100

80

80

60

60

40

40

20

20

0

0

) ) ) ) 5 7 8 1 2 3 1 4 4 0 9 P B A A A H SSBH 103 P(F 0E HF B(a B(b ES- 821 420 SR- SR- SR- 831 831 A(A 0H 0N A AD AD 0 3 3 00 0 0 E 0 R R R R E E E 0 0 H 0 0 0 0 0 0 0 -1 LX 100 X-1 -10 S-1 S-1 EC EC EC EC 00 X-1 X-1 E E L X -1 LX L L X L X Resin type L L

Fig. 1

25

4 6 -5 X 880 880 R R EC EC

Specific activity

Activity recovery Immobilization yield

Immobilization yield (epoxy resin) Immobilization yield (amino resin) Immobilization yield(macroporous)

Fig. 2

26

100

300

Activity recovery (%) Immobilization yield (%)

200 60 150 40

Immobilization yield Activity recovery Specific activity

100

20

Specific activity (U/g)

250

80

50

0

0 15

20

25

30 Temperature (oC)

35

40

Fig. 3a

100

80

250

60

200 150

40

Immobilization yield Activity recovery Specific activity

20

100 50

0

0 5

6

7 pH

Fig. 3b

27

8

9

Specific activity (U/g)

Activity recovery (%) Immobilization yield (%)

300

600 500

80

400

60

300 40 Immobilization yield Activity recovery Specific activity

20

200 100

0 40

50

60

70

80

90

100

110

Specific activity (U/g)

Activity recovery (%) Immobilization yield (%)

100

120

0 130

Enzyme loading (mg/g)

Fig. 3c

500

Specific activity (U/g)

400

4

350 3

300 250

Specific activity Relative stability

200

2 1

150 100

0 0d

1d

2d

3d

Time (d)

Fig. 3d

28

4d

5d

Relative stability (%)

5

450

Relative activity (%)

100 80 60 40 Immobilized amidase Free enzyme

20 0 20

30

40

50

60

Temperature (oC)

Fig. 4a

100

Relative activity (%)

80 60 40 Immobilized amidase Free enzyme

20 0 3

4

5

6

7

8 pH

Fig. 4b

29

9

10

11

12

120

Relativc activity (%)

100 80 60 30 oC 40 oC

40

50 oC

20 0 0

2

4

6

8

10

12

14

16

Time (d)

Fig. 4c

Relative activity (%)

100 80 60 40 O

30 C O 40 C O 50 C

20 0 0

2

4

6

8 Time (d)

Fig. 4d

30

10

12

14

16

60

120 Relative activity 100

40

80

30

60

20

40

10

20

0

0 0

5

10

15

Time (d)

Fig. 5

31

20

25

Relative activity (%)

Conversion (%)

Conversion 50

Continuous production of aprepitant chiral intermediate by immobilized amidase in a packed bed bioreactor Graphical abstract

32

Continuous production of aprepitant chiral intermediate by immobilized amidase in a packed bed bioreactor

Highlights 1. Amidase was covalently immobilized on epoxy resin modified by metal-chelate method. 2. Immobilized amidase exhibited high thermal stability with half-life of 1456.8 h at 40 oC. 3. Continuous PBBR was first constructed for (S)-4-fluorophenylglycine production. 4. High space-time yield of 5.29 kg L-1 d-1 was obtained in continuous PBBR.

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