A Novel Integrated Bioprocess for Efficient Production of (R

Article pubs.acs.org/OPRD

A Novel Integrated Bioprocess for Efficient Production of (R)‑(−)Mandelic Acid with Immobilized Alcaligenes faecalis ZJUTB10 Ya-Ping Xue,†,‡ Ming Xu,§ Hong-Sheng Chen,†,‡ Zhi-Qiang Liu,†,‡ Ya-Jun Wang,†,‡ and Yu-Guo Zheng*,†,‡ †

Institute of Bioengineering, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China Engineering Research Center of Bioconversion and Biopurification of Ministry of Education, Zhejiang University of Technology, Hangzhou 310014, Zhejiang, China § Zhejiang Laiyi Biotechnology Co., Ltd., Shengzhou 312400, Zhejiang, China ‡

ABSTRACT: An integrated bioprocess for the enantioselective hydrolysis of mandelonitrile to (R)-(−)-mandelic acid (R-MA) with immobilized Alcaligenes faecalis ZJUTB10 cells was constructed. Production of A. faecalis ZJUTB10 nitrilase in a pilot-scale fermenter (700 L) with high activity was achieved after optimizing cultivation conditions. A. faecalis ZJUTB10 cells were then immobilized in Ca-alginate. Efficient reusability of the biocatalyst up to 9 batches was obtained by immobilization, and treatment with polyethyleneimine (PEI) and glutaraldehyde (GA) further extended the longevity to 19 batches. The immobilized cells showed maximum activity at 40 °C and pH 8.0. A method for in situ product recovery (ISPR) based on an external extraction loop was established to overcome product inhibition. Anion-exchange column containing resin HZ202 was coupled to the packed bed bioreactor and enabled product recovery by continuously recirculating reaction mixture through the ISPR unit. This integrated bioprocess led to a high productivity of 8.87 mM/h after 16 h of reaction. The productivity of R-MA did not drop significantly even after 80 h of reaction, and the accumulative R-MA amount reached a final value of 550 mmol with excellent enantiomeric excess (>99%). The present studies demonstrated the potential of using the integrated bioprocess for continuous production of R-MA on an industrial scale. and Alcaligenes sp. ECU040115 have been reported to hydrolyze mandelonitrile to R-MA. Recently, the strain A. faecalis ZJUTB10 with high nitrilase activity and enantioselectivity for mandelonitrile was newly isolated in our laboratory by using screening and mutagenesis methods.22 Although the production of R-MA by this method is very attractive, hydrolysis of nitriles via whole cell biocatalysis has been shown to be susceptible to inhibition associated with the substrate and/or product. Toxic effects have resulted in low productivity.23−25 Recent years have seen a rise in economic pressure on the production of biotechnological products. Thus, bioprocesses are faced with a strong demand for intensification and integration of process steps to increase productivity, to reduce process time, and to cut down in running costs and capital expenditure.26 An integrated bioprocess, in which a potentially inhibitory product is continuously removed from the reaction mixture as it is produced, has important advantages in improving productivity relative to conventional processes.27 The productivity of the bioprocess for the production of R-MA by A. faecalis ZJUTB10 may be considerably improved by process integration. Based on the above considerations, we developed an integrated bioprocess for the continuous production of the RMA in a packed bed reactor. First, production of enantioselective A. faecalis ZJUTB10 nitrilase in a pilot-scale fermenter was performed. A. faecalis ZJUTB10 cells were then immobilized on cheaply available supports. The enzymatic

1. INTRODUCTION (R)-(−)-Mandelic acid (R-MA) is both a versatile intermediate for pharmaceuticals (e.g., semisynthetic β-lactam antibiotics) and a resolving agent in chiral resolution processes.1,2 Due to the importance of enantiomerically pure R-MA, it is not surprising that tremendous efforts have been made to establish enantioselective routes for its production. Asymmetric methods to R-MA have been realized using organic catalysts or metalbased complexes, respectively. In addition, the asymmetric formation of R-MA can be also realized by choosing a biocatalytic process as a synthetic key step.3 Enzymes are highly valuable catalysts and allow the manufacture of chiral chemicals on an industrial scale with high enantioselectivity, yield, volumetric productivity, and little waste. Many enzymatic approaches to R-MA have been developed: hydrolysis of methyl mandelate with lipase,4 asymmetric reduction of benzoylformic acid with microorganisms,5 microbial oxidation of 1-phenyl-1,2-ethanediol,6 enzymatic asymmetric synthesis from α-keto aldehyde,7 microbial oxidation of racemic mandelic acid,8,9 and biotransformation of mandelonitrile with nitrilase.10 The nitrilase-mediated pathway offers significant advantages over other routes because of the absence of cofactor involvement, reaction in an aqueous medium, cheap starting material in the form of racemic mandelonitrile, and above all the possibility of carrying out a dynamic kinetic resolution which provides theoretically a 100% yield of the product.10,11 Many studies on the biosynthesis of R-MA from mandelonitrile with nitrilase have been published.10−21 The nitrilases from Alcaligenes faecalis ATCC 8750,10,11,14 Pseudomonas putida MTCC 5110, Microbacterium paraoxydans, M. liquefaciens,13,18 © 2013 American Chemical Society

Received: July 20, 2012 Published: January 15, 2013 213

dx.doi.org/10.1021/op3001993 | Org. Process Res. Dev. 2013, 17, 213−220

Organic Process Research & Development

Article

and trifluoroacetic acid (90:10:0.1, v/v). A detection wavelength was set at 228 nm. Prior to optical purity determination, pretreatment steps were performed on the reaction mixture. The pH of the reaction mixture was adjusted to 1.5 with 6.0 M HCl, and then the enantiomers were extracted with an equal volume of ethyl acetate. The extract obtained was concentrated, and the residual solid was dissolved in the mobile phase for determination. The enantiomeric excess (ee, %) was calculated as follows: ee = |f R − f S| × 100, where f R and f S are the mole fractions of the R and S enantiomers in the product such that f R + f S = 1. Enzyme Assays. In the case of free cells, the standard reaction mixture (10 mL) consisted of resting cells (10 mg DCW) suspended in Tris-HCl buffer (100 mM, pH 8.0). Mandelonitrile (20 mM) was added to initiate the reaction, and the mixture was incubated in a rotary shaker at 40 °C, 150 rpm for 30 min. Similar conditions were maintained for the immobilized cells. The reaction was stopped by centrifugation (9000 × g, 4 °C for 10 min). The product was isolated and analyzed as described above. One unit of enzyme activity (U) was defined as the amounts of enzyme that produce 1.0 μmol of mandelic acid per minute under standard assay conditions. Immobilization of Whole Cells. For alginate entrapment, cells were mixed with 0.9% (w/v) sodium chloride and added to a 2.0% (w/v) sodium alginate solution. The mixture was dropped into the solution composed of 1.0% (w/v) calcium chloride by a syringe to form beads of 3.0 mm diameter. After being entrapped for 12 h, the beads were washed with 0.9% (w/ v) sodium chloride three times and stored for further use. When chitosan was used for entrapment, 2.0% (w/v) chitosan was first dissolved in 1.0% (w/v) acetic acid solution. The mixture of cells and chitosan was dropped into a solution of 1.5% (w/v) sodium triphosphate. After immobilization for 1 h, the beads were washed with 0.9% (w/v) sodium chloride three times and stored for further use. When agar was used for entrapment, the harvested cells were resuspended in 0.9% (w/ v) sodium chloride and then mixed with 4.0% (w/v) agar at 45 °C. The obtained gel was cut into cubes (about 3 mm × 3 mm × 3 mm) after cooling for use. For polyacrylamide immobilization, the gel consisted of acrylamide, cross-linking agent N,N′-methylene-bisacrylamide, dimethylaminopropionitrile, and ammonium persulfate. The final concentration of polyacrylamide gel was 15.0% (w/v). The polymerization of the gel was finished in 10 min. The gels were sliced into particles of average size 3 mm × 3 mm × 3 mm. The beads were washed three times and stored for later use. In order to stabilize the biocatalyst beads, the Ca-alginate beads were treated with polyethyleneimine (PEI) and subsequent glutaraldehyde (GA). First, the beads were suspended in 0.2% (w/v) PEI-HCl solution containing 30 mM CaCl2 (pH 7.0) and stirred for 12 h in a water bath at 30 °C. The gel beads were washed briefly with water and subsequently incubated in sodium phosphate (100 mM, pH 7.0) containing 1.0% (v/v) GA, at room temperature for 30 s with stirring. The treated beads were washed twice with water and stored at 4 °C in a minimum amount of water until used.29 Effect of pH and Temperature on the Activity of the Immobilized Cells. The optimum pH was assayed as a relative activity after incubation at 40 °C for 30 min. The reaction mixture (10 mL) consisted of 1.0 g of the immobilized cells in 100 mM of buffer at various pH values (pH 4.9−8.0, phosphate buffer; pH 8.0−9.0, Tris-HCl buffer) and 20 mM of mandelonitrile. The optimum temperature was assayed as a

reaction for the stereoselective hydrolysis of mandelonitrile was carried out with the immobilized cells in a packed bed reactor. By combining an in situ product recovery (ISPR) process with the bioconversion process, a potentially inhibitory product is continuously removed from the reaction mixture as it is produced.

2. EXPERIMENTAL METHODS Chemicals. Mandelonitrile, R-MA, and S-MA were purchased from J&K Chemical Co., Ltd. (Shanghai, China). Anion resin HZ202 was provided by Shanghai Huazhen Sci. & Tech. Co., Ltd. (Shanghai, China). Prior to use, the resin was converted to the OH− form by repeated treatments with 1.0 M HCl and 1.0 M NaOH and rinsed thoroughly with deionized water until the conductivity is lower than 1 μS/cm. Properties of HZ202 have been given in our previous work.28 All the other chemicals were of reagent grade and obtained from commercial sources. Microorganism, Medium, and Cultivation Conditions. A. faecalis ZJUTB10, which was screened by our lab and previously deposited at the China Center for Type Culture Collection (Wuhan, China) as CCTCC M 208168, was used in this work. This strain was maintained at 4 °C on agar slants with the following composition (g/L): glucose, 10; yeast extract, 5; peptone, 5; K2HPO4, 5; MgSO4, 0.2; FeSO4, 0.03; NaCl, 1.0; and agar, 20 (pH 7.0). It was transferred every three months. The seed medium was composed of the following (g/ L): ammonium acetate, 10, yeast extract, 6; K2HPO4, 5; MgSO4, 0.2; and NaCl, 1.0. The initial pH was adjusted to ∼7.2. The fermentation medium was composed of the following (g/L): ammonium acetate, 12.14; yeast extract, 7.79; K2HPO4, 5; MgSO4, 0.2; and NaCl, 1.0. The initial pH was adjusted to ∼7.5. To prepare the pure seed culture for pilot-scale fermentation, cells were first transferred to 500-mL flasks containing 100 mL of the fresh seed medium from the colony using an inoculating loop and incubated at 30 °C, 180 rpm. When cells were grown to the end of the exponential growth phase, 300 mL of the culture broth were transferred to a 15-L fermenter containing 8 L of the fresh seed medium. Cells in the reactor were cultivated at 30 °C for 20 h with aeration at 0.4 vvm (air volume/culture volume/minute) and agitation at 150 rpm. An 8 L volume of the culture broth was then transferred to a 700-L fermenter containing 400 L of fermentation medium. Then, nbutyronitrile (3.29 g/L) was added to induce the nitrilase activity. Fermentation was carried out at 30 °C with aeration at 0.36 vvm and agitation at 120 rpm. A. faecalis ZJUTB10 cells employed in reactions, immobilizations, or assays were obtained from cell pellets prepared by centrifugation. Analytical Methods. Cell mass was measured by dry cell weight (DCW). A 5 mL culture was centrifuged at 9000 × g for 10 min, and then the pellets were washed twice with distilled water and dried at 105 °C until a constant weight was achieved. The amounts of mandelonitrile and R-MA were assayed by HPLC (LC-10AS, Shimadzu, Japan) equipped with an ODS column (250 mm × 4.6 mm) (Elite Analytical Instruments Co., Ltd., China) at a flow rate of 1.0 mL/min with a solvent system composed of 10 mM NH4H2PO4 and methanol (4:1, v/v). The detection wavelength was set at 228 nm. The optical purity of R-MA was determined by analysis of the enantiomers on a Chiralcel-OD-H column (250 mm × 4.6 mm) (Daicel Chemical Industries, Japan) at a flow rate of 0.8 mL/min with a mobile phase containing hexane, isopropanol, 214



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amount of R-MA adsorbed by HZ202. The column was washed first by deionized water and then eluted by a 1.0 M HCl solution; the flow rate was 50 mL/h. Finally, all of the eluate was collected and the concentration of the R-MA solution was determined by HPLC.

relative activity after incubation for 30 min at various temperatures (20−50 °C, step 5 °C). The reaction mixture (10 mL) consisted of 1.0 g of the immobilized cells and 20 mM of mandelonitrile in 100 mM Tris-HCl buffer (pH 8.0). Determination of Kinetic Parameters. The experiments were carried out at 40 °C in 100 mM Tris-HCl buffer (pH 8.0) in the batch reactor to estimate kinetic parameters at substrate concentrations over a range of 1−27 mM. The initial reaction rates were evaluated by linear regression of the experimental data from a plot of R-MA concentration versus time. The slope of the curve during the first 10 min was defined as the initial reaction rate. The apparent kinetic constants (Vmax and Km) of the free cells and immobilized cells were calculated by fitting the initial reaction rate versus initial mandelonitrile concentration data to the Michaelis−Menten kinetics equation using Origin 6.0 software (Microcal Software, Inc.). The product inhibition for the free cells and immobilized cells were assessed by determination of the initial reaction rates in the presence of 10, 20, 30, 40, and 50 mM R-MA in the reaction system at substrate concentrations of 20 and 30 mM. The product inhibition constants of the free cells and immobilized cells were determined by Dixon plots.30 Biotransformation of Mandelonitrile to R-MA with Immobilized Cells in a Packed Bed Reactor. A packed bed reactor with a jacket for constant temperature operation was used for the production of R-MA with immobilized cells. Prior to the reaction, 120 g of immobilized beads were packed into a glass column (300 mm × 30 mm). 1.0 L of substrate solution (20 mM racemic mandelonitrile in 100 mM Tris-HCl buffer at pH 8.0) was recirculated through a packed bed reactor from the bottom by operating a peristaltic pump to contact these immobilized cells at 15 mL/min. The column temperature was maintained at 40 °C using a circulator, and the vessel was at the same temperature using a water bath. For semicontinuous production of R-MA in batch mode, when the substrate was almost converted to product, the substrate solution was withdrawn by operating a peristaltic pump in the opposite direction. The next enzyme reaction was started by supplying a fresh substrate solution into the vessel. For fed-batch mode, when the substrate was below 2 mM, the next enzyme reaction was started by supplying 20 mM of racemic mandelonitrile in the substrate reservior. Continuous Production of R-MA with Immobilized Cells in an Integrated Bioprocess. Continuous production of R-MA in an integrated bioprocess was performed in a packed bed reactor equipped with an external loop for ISPR by an anion-exchange column packed with HZ202, a strong basic anion exchange resin. The system was set up and operated in an identical fashion as that described in the previous section for the production of R-MA without ISPR. The anion-exchange column consisted of a glass cylinder with barbed hose fittings on either end. HZ202 (60 mL) was loaded through the top of the column, and a glass mesh at the bottom of the column prevented the beads from exiting the bottom. The reaction mixture (1.0 L) was circulated through the top of the anionexchange column at a rate of 15 mL/min via a peristaltic pump, and the effluent was returned to the substrate reservior. In the integrated bioprocess, only the hydroxyl of the resin exchanged with mandelic acid. When the substrate was below 2 mM, 20 mM of mandelonitrile was added to the substrate reservior. Multicolumns were assembled and were used sequentially. After removing each column from the extraction circuit, a desorption was performed on the anion-exchange column to determine the

3. RESULTS AND DISCUSSION Production of Enantioselective Nitrilase of A. faecalis ZJUTB10 in a 700-L Fermenter. In spite of the synthetic potential of nitrilases for the production of optically active carboxylic acids, their utilization as a versatile biocatalyst is largely unexploited, as compared to the successful utilization of lipases and esterases in enantioselective synthesis.31 Although commercial nitrilases are available from Novus Biologicals, Prozomix, and Syncozymes, the high price of nitrilases limits the practical applicability of this useful technology. As a consequence, there is a need for the preparation of low-cost and efficient nitrilases. In our laboratory, the strain A. faecalis ZJUTB10 with high nitrilase activity and enantioselectivity for the production of R-MA from mandelonitrile was newly isolated by using screening and mutagenesis methods.22 The successful utilization of A. faecalis ZJUTB10 nitrilase for industrial scale production requires optimized production technology. The cultivation conditions for the production of nitrilase were optimized by a single-factor method and response surface methodology in 500-mL flasks (data no shown). The optimized medium composition was as follows (g/L): ammonium acetate, 12.14; yeast extract, 7.79; K2HPO4, 5; MgSO4, 0.2; NaCl, 1; and n-butyronitrile, 3.29. The optimum conditions for cell growth and nitrilase production were as follows: 30 °C, pH 7.5. Nitrilase production was then carried out in a 700-L fermenter with a working volume of 400 L. Samples were withdrawn regularly from the fermenter under aseptic conditions and analyzed for biomass content and nitrilase activity. The time course of the cultivation is shown in Figure 1. A lag phase of 3 h followed by the exponential cell

Figure 1. Time course of nitrilase production in a 700-L fermenter with working volume of 400 L. Symbols: (■) DCW; (□) specific activity; (○) enzyme activity. Experiments were carried out at 30 °C with aeration of 0.36 vvm and agitation speed of 120 rpm.

growth was typically observed. The maximum growth rate was 0.21 h−1. The growth rate slowed significantly after 12 h of culturing, and the stationary phase was reached at 20 h and lasted 10 h. The specific activity of nitrilase increased rapidly within 16−24 h. The peak occurred at 24 h with a specific activity of 454.80 U/g DCW. At the time of maximum specific activity, the cultivation process yielded a biomass of 3.45 g 215



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The effects of sodium alginate concentration, CaCl 2 concentration, and bead diameter on the immobilization of whole cells of A. faecalis ZJUTB10 were examined. Maximum activity was achieved when the above parameters were set as 3.0% (w/v) sodium alginate, 1.0% (w/v) CaCl2, and a bead diameter of 2.0 mm. Reinforcement of the Intensity of Ca-Alginate Beads. The susceptibility of alginate matrix to cation-chelating agents, which can cause bead disruption or dissolution, is a major limitation in the bioconversion of nitriles. In order to further stabilize the biocatalyst beads, chemical cross-linking of the alginate beads was attempted using a combination of PEI and GA. It was found that treatment with a combination of PEI and GA resulted in greater stabilization of the biocatalyst at the cost of escalation of the mass-transfer problem. Hence, the stabilized beads showed minimally lowered activity. When the immobilized cells were cross-linked by PEI and GA, the activity was decreased from 217.04 to 200.11 U/g DCW. No significant change in the ee (%) values of the R-MA was observed. The reusability of the beads with PEI/GA treatment and without treatment was compared in Figure 2. There was a phenomenon

DCW/L and a total nitrilase activity of 1569.06 U/L, which is higher than that of A. faecalis ATCC 8750,10 A. faecalis MTCC 10757,32 and Streptomyces sp. MTCC 7546.33 The enantioselectivity of nitrilase at different culture stages was determined by analyzing the optical purity of the product on a Chiralcel OD-H column. The ee (%) values of R-MA were all maintained at above 99%. We did not observe any significant change in the ee (%) values of the R-MA formed due to variation in culture time. Although a number of microbes have been reported in literatures to have the ability to produce the enantioselective nitrilase, there are only a few reports on the production of enantioselective nitrilase based on the laboratory scale fermenter. We presently reported the production of enantioselective nitrilase with high activity and enantioselectivity in the pilot-scale fermenter (700 L). Immobilization of A. faecalis ZJUTB10 Cells. Although the biotransformation mediated by freely suspended cells of A. faecalis ZJUTB10 proceeds effectively, the reuse or recycle of the biocatalyst is rather difficult.22 Cell immobilization may alleviate this problem by offering numerous technical and economical advantages, such as feasibility of continuous processing, high cell concentration, and lower costs of recovery, recycling, and downstream processing.12,34,35 Entrapment is one of the simplest and more important methods currently employed in cell immobilization. Encapsulated cells often have higher operation stability due to the protection from direct exposure to toxic compounds in the environment; in particular, it is rather beneficial in the case of nitriles as the substrate.35 Therefore, we have attempted to develop and characterize the most suitable matrix for entrapment of whole cells of A. faecalis ZJUTB10 for production of R-MA. Various inexpensive entrapment matrices such as Ca-alginate, chitosan, agar, and polyacrylamide were chosen to investigate their effects on both enzyme activity and reusability of the beads. The results revealed that the Ca-alginate carrier exhibited the highest immobilization yield among the materials tested (Table 1).

Figure 2. Repetitive batch bioconversions of mandelonitrile to R-MA by Ca-alginate immobilized A. faecalis ZJUTB10 cells without (A) and with (B) treatment by PEI and GA.

Table 1. Screening of entrapment matrices based on immobilization yield, activity after immobilization, and residual activity after 5 recycles matrix

immobilization yield (%)a

activity after immobilization (U/g DCW)

residual activity after 5 recycles (U/g DCW)

Ca-alginate chitosan agar polyacrylamide

45.76 32.97 41.59 35.18

208.12 149.94 189.15 160.00

204.09 141.05 130.16 142.91

of expansion for beads without treatment by PEI and GA in the process of the reusability test. A significant increase in the mean diameter of beads was observed after 9 runs of reuse. The decline of activity of the beads after 12 batches could be attributed to the beads being cracked, leading to leakage of cells from the gel or the loss of beads during repeated use. As Caalginate beads were reinforced with PEI and subsequently GA treatment, reusability was considerably improved. High retention of enzyme activity (about 90%) was detected even after 19 consecutive batches while no apparent cell release occurred. Stabilization with PEI and subsequently GA could avoid the problem of swelling, as it formed an alkaline membrane in the surface of the sphere.36 Properties of the Immobilized A. faecalis ZJUTB10 Cells. Effect of Temperature and pH. The immobilized A. faecalis ZJUTB10 cells were assayed at various temperatures and pH values. The effects of pH on the activity and enantioselectivity of immobilized A. faecalis ZJUTB10 were determined at pH values ranging from 4.9 to 9.0. The pH of the reaction mixture had a significant effect not only on enzyme activity but also on the optical purity of R-MA. The maximum activity was observed at pH 8.0. The surface of the beads has been reported to have a cationic or anionic nature. The charged

a

Defined as the ratio of the activity of the immobilized cells to the activity of the whole cells put in contact with the matrix.

However, other materials such as polyarylamide, as a synthetic compound, afforded lower activity. Cells entrapped in agar could easily leak out of the matrix, and the nitrilase activity decreased from 189.15 to 130.16 U/g DCW after five batch reactions. Similar results have been reported during the immobilization of other nitrilase-producing strains such as Bacillus subtilis CCTCCM 206038,35 Arthrobacter nitroguajacolicus ZJUTB06-99,36 and A. faecalis MTCC 126.12 Ca-alginate as a low-cost and low-toxicity support is considered to be a desirable material for the immobilization of A. faecalis ZJUTB10 cells in view of the nitrilase activity and the reusability of the beads. 216



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surface of beads and the enzyme produces a charged microenvironment, which might affect the nature of the active enzyme and alter the pH of the entrapped enzyme.37 Nonetheless, in this study, the surface of beads had no effect on the active enzyme; therefore, the optimum pH of free cells and immobilized cells remained the same.22 The optical purity of the product increased with the increase of pH from acidic to slightly alkaline (Figure 3A). The effect of temperature on the

Figure 4. Michaelis−Menten kinetics of free cells (●) and immobilized cells (■). Solid lines () indicated the fitting curves. The experiments were carried out at 40 °C in 10 mL of reaction mixture at various substrate concentrations (1−27 mM) in 100 mM Tris-HCl buffer (pH 8.0).

min/g DCW and 3.43 mM, 0.79 mM/min/g DCW, respectively. The Michaelis−Menten kinetics fitted well to the experimental data. The immobilized A. faecalis ZJUTB10 cells exhibit a higher apparent Km and a lower apparent Vmax in comparison with the free cells. The higher apparent Km suggests a lower affinity for the substrate by the immobilized enzyme, so a higher substrate concentration is needed to achieve a given enzyme activity. Diffusional limitations may contribute to the increased apparent Km value due to a decrease in the accessibility of substrate to the enzyme active site. Structural and conformational changes in the enzyme after immobilization could also cause a change in apparent Km value. The effect of product on the initial reaction rates of free cells and immobilized cells was assessed by determination of initial reaction rates in the presence of 10 to 50 mM R-MA in the reaction mixture at substrate concentrations of 20 and 30 mM. The inhibition constants were determined by a Dixon plot as described by Wang et al.30 In a plot of 1/v0 against R-MA concentrations, the product inhibition constants of the free cells and immobilized cells were calculated to be 10.9 mM and 28.2 mM, respectively. After immobilization, the product inhibition constant was increased by 159%. The higher product inhibition constant suggested a lower product inhibition for the immobilized cells. Storage Stability. The storage stability of immobilized cells was investigated by storing the whole cells at 0 and 4 °C, and the activity was monitored at different time periods. The initial activity of the immobilized cells was 200.11 U/g DCW. The activities of the immobilized cells stored at 0 and 4 °C for 100 days were 197.51 and 193.79 U/g DCW, respectively. The immobilized cells retained >96% of the initial activity and exhibited excellent storage stability. Biotransformation of Mandelonitrile to R-MA by Immobilized Cells in a Packed Bed Reactor. For efficient use of the biocatalyst, biotransformation of mandelonitrile to RMA was performed using the immobilized A. faecalis ZJUTB10 cells in a packed bed reactor which was operated at 40 °C (Figure 5A). The semicontinuous production procedure in batch mode, that is, repeated batch production of R-MA from the substrate solution (20 mM), was performed using the immobilized A. faecalis ZJUTB10 cells. A batch conversion process in the semicontinuous procedure was shown in Figure 5B. The substrate was almost converted to product within 2 h. The product concentration reached 18.4 mM at 2 h, which

Figure 3. Effects of pH (A) and temperature (B) on the activity and selectivity of immobilized cells. pH 4.9−8.0, phosphate buffer; pH 8.0−9.0, Tris-HCl buffer. Symbols: (●) relative activity; (■) ee. Reactions were carried out for 30 min with 1.0 g of the immobilized cells in 10 mL of reaction mixture.

activity of immobilized A. faecalis ZJUTB10 cells was investigated at various temperatures (25−50 °C, step 5 °C). The maximum activity was observed at 40 °C, higher than that of free cells (35 °C). The ee (%) values of the product were all kept above 99% and showed little variation in the temperature range from 25 to 50 °C (Figure 3B). The result was different from that of immobilized A. faecalis MTC 126 cells. As the temperature was increased, there was a slight decrease in enzyme enantioselectivity.12 Kinetic Parameters. Kinetic parameters for free cells and immobilized A. faecalis ZJUTB10 cells were determined. The initial reaction rates (v0) were evaluated during the first 10 min. Because the R-MA concentration in the reaction mixture was low, the effect of R-MA on the activity of free cells and immobilized cells can be negligible. As shown in Figure 4, the initial reaction rates at lower substrate concentrations were directly proportional to the substrate concentrations, while at higher substrate concentrations the initial reaction rates tended towards a maximum value. The results indicated that the reaction obeyed Michaelis−Menten kinetics. Thus, it can be deduced that the substrate inhibition did not appear in the concentration range examined (1−27 mM). The data collected were fitted to the Michaelis−Menten equation to determine the apparent kinetic constants (Km and Vmax) (Figure 4). The values of apparent Km and apparent Vmax for free cells and immobilized cells were calculated to be 1.39 mM, 1.23 mM/ 217



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the optimal reaction time. Therefore, the substrate solution was withdrawn every 2 h and then a fresh substrate solution was supplied. After 16 h of operation, the conversion of substrate was 90.5% (average of all 8 runs). The results showed that there was little leakage of the cells and inactivation of enzyme over the period of the semicontinuous operation. The semicontinuous biotransformation in batch mode was conducted at a low product concentration, and therefore it required tedious procedures for R-MA recovery from a huge volume of the dilute reaction mixture. In our previous work, we had developed an ion-exchange process in a fixed bed packed with HZ202 for the separation of R-MA biosynthesized from mandelonitrile by nitrilase.28 The results indicated that the recovery yield of R-MA was above 90%. The disadvantage of this process is that it will produce a large amount of wastewater due to the low concentration of R-MA, which is unfavorable for industrial application. In order to obtain a high concentration of the product in the reaction mixture while simplifying the downstream process and reducing wastewater, the semicontinuous production of R-MA in a packed bed reactor was alternatively operated in a fed-batch mode. Consequential addition of pure mandelonitrile (20 mmol) in the substrate reservior was performed when the substrate was below 2 mM. Three mandelonitrile additions were carried out over the course of the 16 h reaction (Figure 5C). The concentration of R-MA reached 61.0 mM, which is ∼3.3 times that of the semicontinuous operation in batch mode. However, the productivity decreased with the increase of accumulative RMA concentration. As the product concentration in the reaction mixture was increased from 18.4 mM (at 2 h) to 61.0 mM (at 16 h), the productivity decreased from 9.2 to 3.81 mM/h (Figure 5C). This phenomenon was expected, as the above studies showed the inhibitory behavior of R-MA on the activity of A. faecalis ZJUTB10 cells. Employing the ISPR method during biotransformation may avoid product inhibition and maintain high productivity.38 Continuous Production of R-MA by Immobilized Cells in an Integrated Bioprocess. In integrated processes for continuous production of R-MA, the substrate and product concentrations may be maintained at optimal levels for high productivity simply by continuous feeding and recovery of product. We have previously demonstrated that HZ202, a strong basic anion exchange resin, is the suitable resin for ISPR development of biotransformation of mandelonitrile to R-MA by nitrilase.23 In this work, the integrated bioprocess for the continuous biotransformation of mandelinitrile was performed in a packed bed reactor equipped with an external loop for ISPR using extraction columns loaded with resin HZ202 (Figure 6A). By this means, mandelonitrile (20 mM) was fed to the reservior when the substrate was below 2 mM, R-MA was extracted continuously from the reaction mixture, and replenishment with fresh resin was easily possible when needed. The void volume of the recovery loop (including the column with the resin and the tubing) did not exceed 5% of the total volume of the reaction mixture (1.0 L). Circulation of the reaction mixture through the extraction column was carried out at 15 mL/min, which corresponds to a residence time of 2.8 min. As shown in Figure 6B, the R-MA concentration in the aqueous medium approached ∼11 mM after 4 h of reaction. This product accumulation in the aqueous phase suggests that the resin became saturated. The recirculation pump was stopped, the extraction column was removed and exchanged with a fresh column packed with the same amount of HZ202,

Figure 5. Fed-batch biotransformation of mandelonitrile to R-MA with immobilized cells in a packed bed reactor. The packed bed reactor was operated at 40 °C. (A) Schematic diagram. (B) A batch biotransformation in semicontinuous production procedure. (C) A fed-batch biotransformation in semicontinuous production procedure. The arrows indicated the addition of 20 mM mandelonitrile to the substrate reservior each time when the substrate concentration was below 2 mM.

corresponds to 92% conversion and a productivity of 9.2 mM/ h. After 2 h, the product concentration increases more and more slowly and tended towards a maximum value. The substrate can be completely converted to product at 3 h. From an economic point of view, the experiments for the semicontinuous production procedure in batch mode adopted 2 h as 218



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MA reached a final value of 550 mmol. The ee (%) values of RMA were all kept above 99% (Figure 6B). The integrated bioprocess described here proposed an alternative system where biotransformation was combined with adsorptive recovery of the product to allow the fed batch transformation to proceed efficiently and to obtain the product continuously. The process can be considered to be one of the most feasible modes for the long-term, large-scale production of R-MA. Upon comparison with the method for R-MA production by immobilized cells reported previously,12,16,40 the merits of the integrated process reported in this paper are obvious: better operation stability with high productivity, and a simpler production process (one step, in contrast to two steps in the literature).

4. CONCLUSIONS A. faecalis ZJUTB10 cells were immobilized with Ca-alginate for the biotransformation of mandelonitrile to R-MA. In order to further stabilize the biocatalyst beads, chemical cross-linking with PEI and subsequent GA was used to reinforce the beads’ mechanical strength and further extend the reuse batches to 19 times. A novel integrated bioprocess combining enzyme reaction by immobilized A. faecalis ZJUTB10 cells in a packed bed reactor with product recovery using an exchange column packed with HZ202 was constructed. This technology has the ability to continuously remove R-MA from the reaction mixture. So it avoids product inhibition on A. faecalis ZJUTB10 nitrilase, reduces the process time, and maintains high productivity for R-MA. The accumulative R-MA amount reached a final value of 83.7 g with excellent ee (>99%) after 80 h of reaction. The results of the continuous biotransformation in a novel integrated bioprocess successfully illustrated the potential of the mode. The selective in situ solid-phase adsorption of the product also enables considerably simplified downstream processing.41 This work may have model character for other biotransformations of nitriles to carboxylic acids.

Figure 6. Continuous production of R-MA with immobilized cells in a packed bed reactor coupled to an anion exchange-based ISPR. The packed bed reactor was operated at 40 °C. (A) Schematic diagram. (B) Time course profiles. The arrows indicated the exchange of the ion exchange column with a fresh column packed with the same amount of HZ202. Consequential adding of mandelonitrile (20 mM) in the substrate reservior was performed when the substrate was below 2 mM.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Telephone: +86-571-88320630. Fax: +86-571-88320630.

the pump was restarted, and biotransformation was allowed to continue. Immediately upon exchange of the column, the system experienced a decrease in R-MA concentration. The extraction column was again replaced with a fresh column as the R-MA concentration in the aqueous phase increased and behavior similar to that for the previous column was observed. After removing each column from the extraction circuit, the loaded resin was washed and eluted with 1.0 M HCl for the recovery of R-MA. HPLC analysis revealed traces of mandelonitrile in the eluent. The amount of R-MA produced during the 16 h of reaction was 142 mmol, 130 mmol of which were adsorbed to 4 × 60 mL of HZ202 resin, corresponding to an average capacity of 3.56 mmol/g dry resins. The eluate was concentrated. The desired product R-MA with >98.5% purity and >99% ee by HPLC analysis was obtained by cooling crystallization as described by Mao et al.39 As illustrated in Figure 6B, the accumulative R-MA rose linearly to a final value of 142 mmol after 16 h. The productivity of the system was calculated to be 8.87 mM/h. More importantly, placement of resin with an external extraction column enables continuous product removal through multiple column exchanges thus extending the life of normal system operation and providing enhanced performance. The productivity of R-MA did not drop significantly even after 80 h of reaction. The accumulative R-

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of Zhejiang Province (No. Y4110409), National Natural Science Foundation of China (No. 31170761), National High Technology Research and Development Program of China (863 Program) (No. 2011AA02A210), and Major Basic Research Development Program of China (973 Project) (No. 2011CB710806).

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ABBREVIATIONS R-MA (R)-(−)-Mandelic acid ISPR In situ product recovery DCW Dry cell weight PEI Polyethyleneimine GA Glutaraldehyde ee Enantiomeric excess vvm Air volume/culture volume/minute, L/L/min Km Michaelis constant, mM 219


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Maximum reaction rate, mM/min Initial reaction rate, mM/min

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A Novel Integrated Bioprocess for Efficient Production of (R

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