Process Development for the Production of (R)-(−)-Mandelic Acid by

Article pubs.acs.org/OPRD

Process Development for the Production of (R)‑(−)-Mandelic Acid by Recombinant Escherichia coli Cells Harboring Nitrilase from Burkholderia cenocepacia J2315 Hualei Wang, Haiyang Fan, Huihui Sun, Li Zhao, and Dongzhi Wei* State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, People’s Republic of China ABSTRACT: (R)-(−)-Mandelic acid is an important chiral building block that is widely used in pharmacy and the production of fine chemicals. A more advanced method for obtaining (R)-(−)-mandelic acid is direct hydrolysis of the corresponding racemic mandelonitrile. In order to develop a cost-effective process, a highly efficient enantioselective nitrilase BCJ2315 from Burkholderia cenocepacia J2315 was used for the biotransformation of mandelonitrile to (R)-(−)-mandelic acid. The recombinant Escherichia coli M15/BCJ2315 showed high substrate tolerance and could completely hydrolyze up to 250 mM of mandelonitrile. A fed-batch reaction was performed by periodically or continuously dosing the substrate into the reactor to alleviate substrate inhibition in a monophasic buffer system. Finally, the highest substrate loading (2.9 M) was achieved in the continuous fed batch reaction mode, giving (R)-(−)-mandelic acid at the highest concentration (2.3 M, 350 g/L) with 97.4% ee ever reported. The hydrolysis process was easily scaled up to 2 and 10 L, indicating the potential for the industrial production of optically pure (R)(−)-mandelic acid.

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INTRODUCTION (R)-(−)-Mandelic acid, which is widely used in pharmacy and the production of fine chemicals, has attracted more attention recently.1 It is widely used in the synthesis of antibiotics,2 antiobesity drugs,3 and antitumor agents.4 Its application in the synthesis of cephalosporins antibiotics has been conducted on an industrial scale. This acid also acts as an important chiral resolving agent to resolve racemic alcohols and amines in the industry.5 In the past decade, many enzymatic approaches have been developed to produce optically pure (R)-(−)-mandelic acid.1a Among them, nitrilase-mediated enantioselective hydrolysis of racemic mandelonitrile is a more promising method for producing optically pure (R)-(−)-mandelic acid, due to the excellent enantioselectivity, inexpensive starting material (mandelonitrile), noninvolvement of expensive cofactors, and 100% theoretical product yield (Scheme 1). The nitrilasemediated pathway has been successfully applied in industry by Lonza, BASF, and Mitsubishi Rayon to produce (R)(−)-mandelic acid from mandelonitrile in amounts of at least several tons per year. Many high enantioselective nitrilases have been discovered and characterized to give potential excellent biocatalysts for the production of optically pure (R)-(−)-mandelic acid. Nitrilase from an uncultured organism,1c,6 Alcaligenes sp.,7 Alcaligenes faecalis ATCC8750,8 A. faecalis ZJUTB10,9 Alcaligenes sp. MTCC 10675,10 and Pseudomonas putida11 produced (R)(−)-mandelic acid with ee values of 98%, 97%, 100%, 99%, 99%, and 99%, respectively. However, the low solubility in aqueous environments and the high toxicity of mandelonitrile significantly constrains the industrial application of this biocatalytic process. To overcome these limitations, an aqueous−organic biphasic reaction system,12 enzyme immobilization,13 fed-batch reaction,14 or a combination of these methods15 has been successfully used to achieve high substrate © XXXX American Chemical Society

loading and to produce (R)-(−)-mandelic acid at high concentration by alleviating the inhibition of the highly toxic mandelonitrile. In our previous study, a highly efficient enantioselective nitrilase BCJ2315 toward mandelonitrile was identified and characterized in detail.16 Using a combination of immobilization and the ethyl acetate−water biphasic reaction system, as high a concentration as 1 M of (R)-(−)-mandelic acid was achieved with recombinant Escherichia coli M15/BCJ2315,15 demonstrating the potential to produce optically pure (R)(−)-mandelic acid in industry. However, the addition of the organic solvent to the reaction mixture negatively affected the enantioselectivity of nitrilase, as the ee value further reduced to 90% when ethyl acetate content reached 50%. To develop a more cost-effective biocatalytic process suitable for use in industry, we conducted a fed-batch reaction by periodically or continuously dosing substrate into the reactor to alleviate substrate inhibition in a monophasic buffer system using the recombinant E. coli M15/BCJ2315 in this study. High substrate loading (2.9 M) was achieved, and up to 2.3 M of (R)(−)-mandelic acid with 97.4% ee was obtained because of the dilution effect. Scale-up of the developed process was carried out to verify its potential in industry application.

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RESULTS AND DISCUSSION Cultivation of the Recombinant E. coli M15/BCJ2315. The use of easily available and low-cost whole cell biocatalysts is a much more economical alternative compared to the use of cell-free extracts, purified enzyme, immobilized cells, or enzyme. In order to obtain a low-cost and efficient biocatalyst, we cultured recombinant E. coli M15/BCJ2315 to produce

Received: August 20, 2015

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DOI: 10.1021/acs.oprd.5b00269 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

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Scheme 1. Enantioselective Hydrolysis of Racemic Mandelonitrile to Produce Optically Pure (R)-(−)-Mandelic Acid through Nitrilase

hydrolyzing 500 mM of mandelonitrile with three fungal nitrilases, including NitAn, NitAb, and NitAc.19 The optical purity of mandelic aicd correlated inversely with the mandelonitrile concentration for nitrilase BCJ2315. The ee value decreased with increasing substrate concentration. The highest ee value (99.1%) was observed for the lowest concentration of substrate (30 mM) in the reaction medium (Table 1). Therefore, high concentration and high enantioselectivity were inversely correlated. To solve this contradiction problem, the reaction mode must be well-designed. Fed-Batch Production of (R)-(−)-Mandelic Acid (100 mL Scale). To implement a cost-effective process, a high product concentration (gproduct/L) with a simple downstream process is required to meet the process economic requirements of the industry.20 Based on the negative effect of high concentration of mandelonitrile on BCJ2315 activity and enantioselectivity, a fed-batch mode was designed for BCJ2315 that included periodically or continuously dosing of substrate into the reactor to alleviate substrate inhibition. Controlling mandelonitrile concentration below the inhibitory concentration in the biocatalytic medium is essential for maintaining enzyme activity. Additionally, a relatively low substrate concentration is beneficial for improving the enantiomeric purity of the products.21 In the periodical fed-batch process, 100 mM of mandelonitrile was fed into the reaction system when the substrate was completely hydrolyzed at each batch. Finally, the reaction was performed for 11 batches, and as high as 1.1 M mandelonitrile were completely hydrolyzed in 23 h (see Figure 1). However, during the 12th feeding of mandelonitrile, only 54 mM of mandelonitrile was hydrolyzed after a long incubation time of 6 h. Finally, as high as 0.96 M of (R)-(−)-mandelic acid was obtained because of the increased reaction volume caused by serial mandelonitrile feeding for 11 batches, affording a concentration of 146 g/L product. The optical purity of the (R)-(−)-mandelic acid was determined to be 97.6% ee by chiral HPLC analysis. However, when 200 mM of mandelonitrile was fed at once, the substrate could not be completely hydrolyzed even with an extended reaction time at the fourth feeding. This may be because of enzyme inactivation over the period of hydrolysis. In the continuous fed-batch process, mandelonitrile was kept at optimal concentrations by adjusting the feeding rate, which could avoid the inactivation of the enzyme at high substrate concentration conditions (see Figure 2). At the first feeding stage, the mandelonitrile was fed at a rate of 24 g/L·h, which could maintain relatively high hydrolysis rate. This process lasted for 11 h of reaction time, and up to 1.67 M (R)-

whole cell biocatalysts with high density and high expression levels in a stirred bioreactor (7 L). The cultures were induced with isopropyl-β-D-thiogalactopyranoside (IPTG) (final concentration of 0.1 mM) when the OD600 reached 20 and the cells were harvested when the specific activity reached a maximum of 3418 U/g DCW at an OD600 of 70 after over 24 h of fermentation. A biomass of 22.8 g DCW/L and a total nitrilase activity of 77 930 U/L were finally obtained, which are higher than those of the A. faecalis ATCC8750,8 A. faecalis MTCC 10757,17 and A. faecalis ZJUTB10.18 This culture process offers an easily available, highly active, and low-cost catalyst that could be used to produce (R)-(−)-mandelic acid in industry. Effect of Mandelonitrile Concentrations on Nitrilase Activity and Enantioselectivity. High substrate loadings are generally highly desirable for practical applications in biocatalytic processes. Mandelonitrile is well-known to be highly detrimental to nitrilase, leading to a low substrate concentration for biotransformation.14 To study the effect of mandelonitrile concentration on activity and enantioselectivity of the recombinant E. coli M15/BCJ2315, we performed the hydrolysis under different concentrations of mandelonitrile (30−300 mM) with 10% methanol as a cosolvent to increase the solubility of the substrate in the aqueous reaction mixture. Nitrilase BCJ2315 could tolerate as high as 250 mM mandelonitrile (Table 1). Only 75% conversion was achieved Table 1. Effect of Mandelonitrile Concentrations on (R)(−)-Mandelic Acid Production substrate concentration (mM)

reaction time (min)

product concentration (mM)

ee value of acid (%)

30 50 100 150 200 250 300

15 25 45 70 100 180 300

30 50 100 150 200 250 225

99.1 98.8 98.1 97.5 96.7 95.3 92.1

for the hydrolysis of 300 mM mandelonitrile after 5 h of biotransformation. The tolerance of BCJ2315 toward mandelonitrile was slightly higher than that of nitrilase from Alcaligenes sp., which could hydrolyze as high as 200 mM mandelonitrile.14 Both nitrilases were unable to transform mandelonitrile at concentrations of 300 mM or higher. Compared with bacterial nitrilases, fungal nitrilases performed better in the hydrolysis of high concentrations of mandelonitrile. High conversions (75−93%) were achieved when B

DOI: 10.1021/acs.oprd.5b00269 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

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Figure 1. Time course of the hydrolysis reaction in periodical fedbatch mode (100 mL scale).

Figure 3. Time course of the hydrolysis reaction in continuous fedbatch mode (10 L scale).

follows: [α]D25 = −153.1 (c 1.0, H2O) (literature: [α]D25 = −155 (c 1.0, H2O)8); 1H NMR (400 MHz, DMSO): δ12.62 (brs, 1H), 7.45−7.39 (m, 2H), 7.36−7.31 (m, 2H), 7.31−7.26 (m, 1H), 5.84 (brs, 1H), and 5.01 (s, 1H) ppm. The success of the scale-up made this process a preferable route for large-scale production of (R)-(−)-mandelic acid in industry. Veselá et al. compared the yield (%), product concentration (g/L), volume productivity (g/L·d), and ee value (%) of the product and catalyst productivity (g/g·dcw) of nitrilase from different sources in various biocatalytic process types (batch, fed-batch, and biphasic system).19 The potential of nitrilases from Alcaligenes sp.,14,22 A. faecalis,23 B. cenocepacia J2315,15 and Aspergillus niger19 as biocatalysts in the form of recombinant whole cells or immobilized recombinant cells were compared in detail. All compared nitrilases showed an acceptable enantioselectivity of ee > 94.5%. The highest volumetric productivity and catalyst productivity were observed to be 982 g/L·d and 156 g/g·dcw, respectively, observed from the immobilized nitrilase from B. cenocepacia J2315 after 6 rounds of recycling in batch hydrolysis of 500 mM mandelonitrile in our previous study.15 Additionally, the highest product concentration was observed to be 150 g/L by completely hydrolyzing 1 M mandelonitrile in a biphasic system using the immobilized recombinant E. coli M15/BCJ2315 in the same study.15 Recently, a higher product concentration of (R)-(−)-mandelic acid was reported by Zhang et al., using immobilized recombinant E. coli cells expressing Alcaligenes sp. nitrilaseas biocatalyst.24 In a repeated fed batch hydrolysis, a total of 1.6 M mandelonitrile was completely hydrolyzed after 15 rounds of substrate dosing and 7 rounds of biocatalyst recovery, affording a product concentration of 243 g/L. However, these abovementioned higher parameters were only obtained when the catalyst was repeatedly recycled. The immobilization of nitrilase and repeated recovery of the catalysts are complex and timeconsuming, limiting industrial application. Compared with these processes, our continuous fed-batch process using recombinant E. coli M15/BCJ2315 is simple and cost-effective and shows high-volume productivity (350 g/L·d), a high ee value (97.4%) of the product, the highest concentration of product (350 g/L), and high catalyst productivity (115 g/g· dcw, the biocatalysts loading was 3.85 g dcw/L, equal to 10 g/L of wet cells)), demonstrating the potential of this method for industrial production of (R)-(−)-mandelic acid.

Figure 2. Time course of the hydrolysis reaction in continuous fedbatch mode (100 mL scale).

(−)-mandelic acid was obtained by that time. At the second feeding stage, the hydrolysis rate decreased greatly, probably due to the leakage of the E. coli cells and inactivation of the enzyme over the period of hydrolysis. Thus, the mandelonitrile feeding rate was adjusted to 12 g/L·h and this process lasted for another 11 h of reaction time. At the final stage, the substrate feeding was stopped and an additional 4 h of hydrolysis was performed to guarantee a complete hydrolysis of all the added substrate. Finally, a total of 2.9 M of mandelonitrile was completely hydrolyzed to produce as high as 2.3 M of (R)(−)-mandelic acid because of the increased reaction volume caused by continuous mandelonitrile feeding and pH control, affording 350 g/L product with 97.4% ee. Scale-up of Continuous Fed-Batch Reaction to 2 and 10 L. To explore the potential of the continuous fed-batch method for industrial large-scale production of (R)-(−)-mandelic acid, this hydrolysis process was scaled up to 2 and 10 L. Both processes were very stable during scale-up, and approximately 100% of the added mandelonitrile was completely hydrolyzed to form (R)-(−)-mandelic acid within 24 h. The time course of the hydrolysis on the 10-L scale was shown in Figure 3. The product was finally obtained as white powdery solid in 93% isolate yield and 99.5% ee after recrystallization in toluene. The product was characterized as C

DOI: 10.1021/acs.oprd.5b00269 Org. Process Res. Dev. XXXX, XXX, XXX−XXX

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CONCLUSIONS In summary, a continuous fed-batch process was developed to completely hydrolyze as high as 2.9 M mandelonitrile using the E. coli whole-cell system harboring an efficient nitrilase as biocatalyst, giving (R)-(−)-mandelic acid at the highest concentration (350 g/L) with 97.4% ee. The scale-up of this hydrolysis process was easy and stable, demonstrating great potential on large-scale production of (R)-(−)-mandelic acid in industry.

continuously supply the substrate, mandelonitrile was fed into the system after 10 min of hydrolysis at a feeding rate of 24 g/ L·h. Real-time conversion rate was monitored by HPLC, and the feeding rate was adjusted to keep the conversion above 80%. To achieve a complete hydrolysis of the added mandelonitrile, the reaction lasted for an additional 4 h after the feeding was stopped. Scale-up of Continuous Fed-Batch Reaction to 2 and 10 L. The continuous fed-batch process was scaled up to 2 and 10 L at 30 °C under mechanical agination of 250 rpm in 5 L and 10 L double jacket glass reactors (ShenSheng, Shanghai, China), respectively. The reaction mixture contained 100 mM of mandelonitrile, 100 mM of phosphate sodium buffer (pH 8.0), 10 g/L of E. coli M15/BCJ2315 (wet cells), and 10% methanol. The control of the reaction temperature and pH, agitation of the reaction mixture, and substrate feeding were performed as described previously.25 The substrate feeding was conducted as it is on the 100 mL scale. Purification of the Product. After all of the mandelonitrile in the reaction mixture was hydrolyzed completely, the biocatalysts were removed through centrifugation. Methanol, which was used as a latent solvent in the reaction, was then removed from the supernatant using a rotary evaporation apparatus. The supernatant was then acidified to pH 1.0 with HCl 11% (v/v), and then the mandelic acid of approximately 54% total mass was separated by crystallization. After crystal collection by suction filtration, the remaining solution was condensed to 30% volume by rotary evaporation. The mandelic acid of 43% total mass was separated by crystallization when cooled to 25 °C. All crystals were collected and dissolved in toluene for recrystallization. The refined mandelic acid was detected by HPLC and alkalimetric titration. Analytical Methods. TLC was used for monitoring the mandelonitrile hydrolysis in real time. The developing solvents were n-hexane/ethyl acetate/acetic acid (5:1:0.1). The retention factor value (Rf value) was 0.05 (mandelic acid), 0.7 (mandelonitrile), and 0.4 (benzaldehyde) detected at 254 nm wavelength. HPLC was used to quantitatively analyze the decrease of mandelontirle, formation, and optical purity of the (R)(−)-mandelic acid in the reaction mixture as described previously.15

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EXPERIMENTAL SECTION Materials. Mandelonitrile was purchased from Guangde Chemicals Co., Ltd. (Anhui, China). The (R)-(−)-mandelic acid and (S)-(+)-mandelic acid were purchased from SigmaAldrich (Milwaukee, USA). Thin-layer chromatography (TLC) silica gel glass sheets used for TLC analysis were purchased from Yantai Jiangyou Co., Ltd. (Shandong, China). The recombinant E. coli M15/BCJ2315 harboring the nitrilase BCJ2315 (GenBank accession number: WP_012492804) from B. cenocepacia J2315 was reported previously.16 Cultivation of the Recombinant E. coli M15/BCJ2315. The cultivation of recombinant E. coli M15/BCJ2315 was performed in a 7-L fermenter (BIOTECH, Shanghai, China) as described previously,15 with modified fermentation medium (12 g/L tryptone, 16 g/L yeast extract, 1.26 g/L glycerol, 24.75 g/L K2HPO4, 3.47 g/L KH2PO4, and 1 g/L MgSO4) and supplementary medium (40 g/L tryptone, 30 g/L yeast extract, 50% glycerol). Supplementary medium was fed at a rate of 1 mL/min after 2 h of fermentation and induction of the cells was initiated by adding IPTG at a final concentration of 0.1 mM to the fermenter when the culture density reached an OD600 of 20. Effect of Mandelonitrile Concentrations on Nitrilase Activity and Enantioselectivity. To study the effect of substrate concentration on activity and enantioselectivity of the nitrilase, the hydrolysis reaction was performed with varied substrate concentrations (30−300 mM) in a 5 mL reaction system containing 100 mM of sodium phosphate buffer (pH 8.0), 50 mg of E. coli M15/BCJ2315 wet cells, and 10% of methanol (v/v). Samples (100 μL) were periodically withdrawn, and the conversion of the reaction and optical purity of the product were determined through high-performance liquid chromatography (HPLC) analysis. Fed-Batch Production of (R)-(−)-Mandelic Acid (100 mL Scale). The fed-batch process was performed by periodically or continuously feeding the substrate into the reaction system. The hydrolysis was performed in a 250 mL three-necked round-bottom flask on 100 mL scale. The control of temperature, pH, and agitation of the reaction mixture were performed as described previously.25 The temperature of the reaction was set to 30 °C. The pH of the reaction mixture was maintained at 7.5−8.0 by adding sodium hydroxide (6 M) for pH adjustment. The agitation speed was set to 250 rpm. The reaction mixture containing 90 mL of sodium phosphate buffer (100 mM, pH 8.0) and 1 g of the E. coli M15/BCJ2315 wet cells was first incubated at 30 °C for 30 min. Next, 10 mL of mandelonitrile (solubilized in methanol, 1 M) was added to initiate the hydrolysis. Samples (100 μL) were periodically withdrawn to monitor the reaction process by TLC and HPLC analysis. To periodically supply the substrate, mandelonitrile (100 mM) was fed into the system when it was completely hydrolyzed as revealed by TLC and HPLC analysis. To

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +86-21-64252078. Fax: +86-21-64250068. Funding

This work was supported by the National Natural Science Foundation of China (No. 21406068/B060804), China Postdoctoral Science Foundation funded project (No. 2014M560308), and the National Basic Research Program of China (No. 2012CB721103). Notes

The authors declare no competing financial interest.

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DOI: 10.1021/acs.oprd.5b00269 Org. Process Res. Dev. XXXX, XXX, XXX−XXX