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Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli
Process Biochemistry 49 (2014) 655–659
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli Omkar Pai a , Linga Banoth a , Saptarshi Ghosh a , Yusuf Chisti b , Uttam Chand Banerjee a,∗ a Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar 160 062, Punjab, India b School of Engineering, Massey University, Private Bag 11 222, Palmerston North, New Zealand
a r t i c l e
i n f o
Article history: Received 5 October 2013 Received in revised form 20 December 2013 Accepted 20 January 2014 Available online 28 January 2014 Keywords: Biotransformation 3-Cyanopyridine Nicotinic acid Alcaligenes faecalis
a b s t r a c t An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37 ◦ C, 100 mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1 M 3-cyanopyridine to nicotinic acid within 5 h at a cell mass concentration (fresh weight) of 500 mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Nicotinic acid (vitamin B3 , niacin) is widely added to foodstuff and has pharmaceutical applications. Global production of nicotinic acid and its amide (nicotinamide) is at least 22,000 tons annually [1]. Nicotinic acid is used as a vitamin supplement and has antilipidemic properties [2] helping to lower the risk of heart disease and atherosclerosis. Nicotinic acid is incorporated in the body in nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) which are important cofactors in numerous biochemical reactions relating to metabolism of fats, carbohydrates and proteins. Incorporation of nicotinic acid in fodder may be used to modify the milk fat composition of dairy cows [3]. Several biocatalytic processes for the production of nicotinic acid [4–9] and its derivatives [10] have been reported. These processes generally require an extended reaction period to achieve quantitative conversion [11], or the use of multiple reaction steps [12]. The substrate conversion may be incomplete and unwanted byproducts may be produced. The process apparently used for the commercial production of nicotinic acid from 3-cyanopyridine involved the use of resting cells of Rhodococcus rhodochrous
∗ Corresponding author. Tel.: +91 172 2214682/2214683/2214684/2214685/ 2214686/2214687x2142; fax: +91 172 2214692. E-mail address: [email protected] (U.C. Banerjee). 1359-5113/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2014.01.023
[11]. An initial concentration up to 1.4 M 3-cyanopyridine was converted to nicotinic acid, but both the substrate and the product of the reaction inhibited the biocatalytic activity. Substrate inhibition was observed at a 3-cyanopyridine concentration of ≥200 mM. To reduce inhibition, the substrate was fed in seven stages such that at the end of each feeding the concentration was 200 mM. Feeding was given over a period of up to 26 h and the conversion to nicotinic acid was nearly 100%. Total bioconversion (100%) at 200 mM substrate level was reported using whole cells of Bacillus pallidus whereas substrate inhibition was found at 300 mM 3-cyanopyridine [4]. Bioconversion using whole cells of Microbacterium imperiale in continuous stirred tank membrane reactor (CSMR) was also reported with a yield of 200 mM nicotinic acid [5,6,7,12] in 24 h. Using whole cells of Nocardia rhodochrous, nicotinic acid yield of 0.5 M was reported in literature [13]. A nicotinic acid yield of 40 mM was also mentioned in literature using whole cells of Nocardia globerula [8]. Recombinant Escherichia coli harboring the Gibberella intermedia nitrilase gene was also reported to transform 100 mM 3-cyanopyridine to nicotinic acid with 100% conversion efficiency [14]. According to the reports discussed above, a maximum of 200 mM 3-cyanopyridine was used for the transformation and its higher concentration caused substrate inhibition as well as by-product formation. The present study was aimed at developing a relatively rapid, low-cost and single-step process for the biotransformation of 3cyanopyridine to nicotinic acid. The new process involved, the use of recombinant whole cells of E. coli JM109 harboring the nitrilase
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gene from Alcaligenes faecalis MTCC 126. The enzyme responsible for the biotransformation was A. faecalis nitrilase. This enzyme was first reported by Yamamoto et al. [15] for the enantioselective conversion of racemic mandelonitrile to R-mandelic acid. The enzyme was purified and characterized by Yamamoto et al. [16]. Studies on other microbial nitrilases are also reported [17–19] in literature. The nitrilase-containing immobilized whole cells of A. faecalis have been used for the biotransformation of racemic mandelonitrile [20]. The cloning of nitrilase gene form A. faecalis into a vector pJOE2775 and its subsequent transformation into E. coli JM109, production and purification of nitrilase and the formation of cross-linked enzyme aggregates (CLEAs) have been reported earlier [21]. In the present work, whole cells of the nitrilase-containing recombinant E. coli JM109 were used for the biotransformation of 3-cyanopyridine to nicotinic acid. Here 100% conversion of 1 M 3cynopyridine to nicotinic acid with 93% yield was achieved. In this method, we have shown higher substrate utilization (up to 1 M) with no signs of substrate or product inhibition, thus making it better approach for the production of nicotinic acid. 2. Materials and methods 2.1. Materials Nicotinic acid, 3-cyanopyridine and nicotinamide were purchased from Sigma–Aldrich (www.sigmaaldrich.com). Medium components and ampicillin were obtained from Hi-Media, Inc. (Mumbai, India). The solvents used for HPLC were procured from Mallinckrodt Baker, Inc. (Phillipsburg, USA). All other reagents used were of analytical grade. 2.2. Microorganism Recombinant E. coli JM109 cells harboring nitrilase gene from A. faecalis MTCC 126, cloned in our laboratory were used. pJOE2775 vector was used for the expression. 2.3. Growth conditions The recombinant E. coli was revived from a glycerol stock in a 20 mL starter culture in Luria Bertani (LB) broth of the following composition (g/L): tryptone 10; yeast extract 5; NaCl 10, and ampicillin at a final concentration of 0.1 mg/mL. Master plates were then prepared by streaking from this revived culture on LB–ampicillin agar. The agar medium had the above specified composition supplemented with agar (15 g/L). A single colony of the bacterial cells from a freshly prepared agar plate was used to inoculate the starter culture. The starter was grown overnight in the above specified liquid medium at 37 ◦ C, 250 rpm, in an incubator shaker. The starter was transferred to the production medium as above, to achieve a final starter concentration of 2% by volume. This production culture was incubated (37 ◦ C, 250 rpm) for 2 h and then rhamnose was added to a concentration of 2 g/L for the induction of the nitrilase gene. The incubation (37 ◦ C, 250 rpm) was continued for a further 8 h. The cells were then harvested by centrifugation (10,000 × g; Sigma 6K15, GmbH, Germany) at 4 ◦ C for 10 min. The cells were washed with 10 mM sodium phosphate buffer (pH 7.5). 2.4. Analytical procedures 2.4.1. Nitrilase activity The nitrilase activity in the cells was assayed using 3-cyanopyridine (12.5 mM) as substrate. A suspension (20 mg/mL fresh weight concentration) of cells in 10 mM sodium phosphate buffer (pH 7.5) was incubated with the substrate at 37 ◦ C, 200 rpm for 20 min. The reaction was quenched by adding 200 L HCl (1 M). The cells were then removed from the reaction mixture by centrifugation at 10,000 × g for 10 min. The supernatant thus obtained was used to measure the concentrations of the residual substrate and the product by reversed phase HPLC. One unit of nitrilase activity was defined as the amount of the enzyme required to produce 1 mole of the product per minute from 3-cyanopyridine. 2.4.2. Nicotinic acid and 3-cyanopyridine The residual substrate 3-cyanopyridine and the product nicotinic acid in suitably diluted reaction mixture were quantified by analytical high performance liquid chromatography (Shimadzu 10AD VP; Kyoto, Japan). A Symmetry® RP-18 column (250 mm × 4.6 mm, 5 m) (Waters; MA, USA) was used. The mobile phase was a mixture of acetonitrile and sodium phosphate buffer (pH 4.5) in the volume ratio of 55:45. The flow rate of the mobile phase was 0.7 mL/min. The elution of the substrate and the product was detected using a UV detector at a wavelength of 217 nm.
2.5. Immobilization of whole cells Freshly harvested cells were weighed and suspended in 100 mM Tris buffer (pH 7.5) to achieve the desired fresh weight concentration. An amount of sodium alginate was dissolved separately by heating in the above specified buffer to obtain a concentration of 2.5% (w/v). Once the alginate solution had cooled to 40 ◦ C, the cell slurry was added and mixed to obtain a uniform dispersion. This suspension of cells was added dropwise via a syringe to an ice cold solution of calcium chloride (20 g/L) to produce beads that were 2–3 mm in diameter. The alginate beads formed were held in a refrigerator (4 ◦ C) overnight for hardening. These hardened beads containing the immobilized cells were washed with distilled water and used for the biotransformation reaction. 2.6. Optimization of reaction parameters In separate experiments, the biotransformation of 3-cyanopyridine to nicotinic acid was carried out using different specified temperatures, pH values, buffers, cosolvents, biocatalyst concentration, and the initial substrate concentration [22–24]. 2.7. Downstream of nicotinic acid To isolate nicotinic acid from the reaction mixture, the crystals formed were completely dissolved in distilled water and the immobilized enzyme beads were separated by filtration and thoroughly washed. The combined wash out was centrifuged at 12,000 × g for 15 min to remove any insoluble matter and concentrated at 45 ◦ C in vacuum. White crystals of nicotinic acid were obtained. Re-crystallization was carried out by dissolving the crystals in hot distilled water and allowing it to cool overnight in an ice-water bath. Fine needle shaped crystals of nicotinic acid thus obtained, were separated by filtration.
3. Results and discussion 3.1. Effect of reaction temperature For the reaction carried out at different incubation temperatures and a fixed reaction period of 0.5 h (Fig. 1a), both the freely suspended cells and the immobilized cells had an optimal conversion temperature of 50 ◦ C, but the maximum conversion did not exceed above 90%. At 50 ◦ C, the biotransformation reaction was rapid, but so was the rate of denaturation of nitrilase in the cells. Therefore, the cells used at this temperature could not be reused. For a longer fixed reaction period of 2 h, the reaction carried out at different incubation temperatures had the conversion as shown in (Fig. 1b). The optimal temperature was around 37–40 ◦ C. The conversion was slower, but both the freely suspended cells and the immobilized cells retained most of their initial nitrilase activity and could therefore be reused. In addition, a substrate conversion of 100% was achieved (Fig. 1b). Considering the full conversion and reusability of the immobilized biocatalyst, a reaction temperature of 37 ◦ C was concluded to be optimal for this reaction. In view of these results, reaction time was optimized at 37 ◦ C by allowing the reaction to occur for a period of 0.5–4 h (Fig. 1c). Samples were collected at different time interval and analyzed by HPLC. For the measurements shown in Fig. 1a–c, the fresh weight concentration of the cells in all cases was 20 mg/mL. The reaction was carried out in 100 mM phosphate buffer (pH 7.5) for the freely suspended cells and 100 mM Tris buffer (pH 7.5) for the immobilized cells. The initial concentration of the substrate was always 12.5 mM. No co-solvents were used. 3.2. Effect of reaction buffer The effect of the type of buffer on the conversion was tested using phosphate, HEPES and Tris buffers. All buffers (100 mM) were used at pH 7.5. The conversion of the substrate (initial concentration 12.5 mM) after 2 h of reaction at 37 ◦ C is shown in Table 1. The data (Table 1) were measured at a cell fresh weight concentration of 20 mg/mL for both free and immobilized cells. No co-solvents were used. Nearly 100% conversion was achieved in all cases except in phosphate buffer where sodium ions leached out from the beads (Table 1). For all future experiments, phosphate buffer (100 mM,
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Fig. 2. Effect of pH (100 mM phosphate buffer for freely suspended cells; 100 mM Tris buffer for immobilized cells) on the conversion of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant E. coli.
pH 7.5) was concluded to be satisfactory for freely suspended cells and Tris buffer (100 mM, pH 7.5) for immobilized cells. 3.3. Effect of reaction pH Effect of reaction pH on the conversion of 3-cyanopyridine (12.5 mM) to nicotinic acid by free and immobilized cells was examined using 100 mM phosphate buffers for freely suspended cells and 100 mM Tris buffers for immobilized cells. The pH values ranged from 4.5 to 10.5 in different experiments. The observed conversion after 2 h at 37 ◦ C is shown in Fig. 2. For the data in Fig. 2, the fresh weight concentration of the biocatalyst was 20 mg/mL for both free as well as immobilized cells. No co-solvents were used. The activity of freely suspended cells was adversely affected by pH values less than 6.5 and greater than 7.5 (Fig. 2). The optimal pH for the activity was about 7.5. In contrast to free cells, the immobilized cells achieved a nearly 100% conversion over the entire pH range tested (Fig. 2), apparently because the immobilization matrix protected the cells from the adverse pH of the medium surrounding the beads. For long term stability and repeated use of the beads and considering the data of the freely suspended cells (Fig. 2), a biotransformation pH 7.5 was taken as optimal for the immobilized biocatalyst. All the subsequent biotransformation experiments were carried out at this pH. 3.4. Effect of co-solvent
Fig. 1. (a) Effect of incubation temperature on the conversion of 3-cyanopyridine to nicotinic acid. Incubation period was 0.5 h. (b) Effect of incubation temperature on the conversion of 3-cyanopyridine to nicotinic acid. Incubation period was 2 h. (c) Course of conversion of 3-cyanopyridine to nicotinic acid (reaction temperature was 37 ◦ C).
Table 1 Effect of different buffers (100 mM, pH 7.5) on the conversion of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant E. coli. Buffer
Phosphate Tris HEPES
Conversion (%) Free cells
Immobilized cells
99 98 91
– 100 99
Effect of seven different co-solvents on the conversion by freely suspended cells was examined (Table 2). The other reaction conditions were fixed at: 100 mM phosphate buffer, pH 7.5; 37 ◦ C; initial substrate concentration of 12.5 mM; and a cell fresh weight concentration of 20 mg/mL. The concentration of the co-solvents in the buffer was always 10% (v/v). In this study after 2 h of reaction, alcohols as co-solvents (ethanol, IPA, and methanol) showed 100% conversion, whereas acetone and DMSO showed 92% and 88% Table 2 Effect of different co-solvents on the conversion of 3-cyanopyridine to nicotinic acid by free cells of recombinant E. coli. Co-solvent (10%, v/v)
Conversion (%)
Ethanol Isopropanol Methanol Acetone Dimethyl sulfoxide Tetrahydrofuran Dimethylacetamide
100 100 100 92 88 2 4
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Fig. 3. Effect of initial substrate concentration on the conversion catalyzed by immobilized cells of recombinant E. coli. Cell mass concentration used was 20 mg/mL (fresh cell weight).
Fig. 4. Effect of cell mass (fresh cell weight) concentration on nicotinic acid production by immobilized cells of recombinant E. coli. The initial substrate concentration was 1 M.
conversion, respectively. None of the alcohols affected the conversion relative to no co-solvent being present, but the other co-solvents reduced conversion by various levels. Tetrahydrofuran (2%) and dimethylacetamide (4%) severely affected the conversion. It might be due to the reduction in enzyme activity.
achieved a 100% conversion in 5 h starting with an initial substrate concentration of 1 M (enzyme activity 6.67 U/mg wet cell weight) under exceedingly mild reaction conditions (37 ◦ C, pH 7.5).
3.5. Effect of initial substrate concentration
For commercial use, the most desired property of a biocatalyst is its operational stability and reusability. The immobilized cell beads were tested for the reusability so as to make the biocatalysts applicable for repeated cycles of production of nicotinic acid from 3-cyanopyridine. Efficient biocatalyst recycling was achieved as a result of immobilization. Free cells could be reused up to 10 batches. On the other hand immobilized cells retained 100% activity and could be used up to 25 batches (Fig. 6) making the biocatalyst useful for scale-up.
The initial concentration of 3-cyanopyridine in the reaction mixture was varied in different experiments. The fresh weight concentration of the immobilized cells remained fixed at 20 mg/mL. The reaction was carried out at 37 ◦ C, pH 7.5, using 100 mM Tris buffer. No co-solvents were used. The substrate conversion profiles are shown in Fig. 3. Up to a substrate concentration of 25 mM, complete conversion was achieved within 5 h. Further increase in the initial concentration of the substrate progressively slowed the reaction (Fig. 3). In all cases, full conversion was achieved eventually, but it took longer time with the increasing substrate concentration. For a substrate concentration of 75 mM, full conversion was achieved in 17 h whereas it took 35 h for 150 mM substrate conversion.
3.7. Reusability of the immobilized whole cell biocatalyst
3.8. Typical biotransformation procedure The immobilized beads were suspended in 250 mL (100 mM Tris–HCl, pH 7.5) buffer. The final concentration of immobilized cells was 500 mg/mL (wet weight). 3-Cyanopyridine (1 M) which is freely soluble in aqueous medium was added in the reaction
3.6. Effect of cell mass concentration As the rate of conversion of the substrate was considerably reduced at an initial substrate concentration of 100 mM (Fig. 3) at a biocatalyst concentration of 20 mg/mL, attempts were made to counter this by increasing the concentration of the catalyst. The initial substrate concentration was increased to 1 M. In different experiments, the immobilized cell fresh weight concentration was varied from 200 to 500 mg/mL. The reaction was carried out at 37 ◦ C, 200 rpm, and pH 7.5 (100 mM Tris buffer), without co-solvents. The nicotinic acid production profiles are shown in Fig. 4. A nicotinic acid concentration of 1 M equates to a 100% conversion of the substrate. As shown in Fig. 4, at a biocatalyst concentration of 500 mg/mL, complete conversion of the substrate was achieved in 6 h, despite the initial substrate concentration being high at 1 M. The experiments were carried out up to 48 h to study the effect of biocatalyst concentration on the conversion. The data obtained at a cell mass concentration of 500 mg/mL were validated by repeating the experiment with the reaction carried out for a total of 6 h. Sampling was done at 1 h interval up to 6 h and the course of nicotinic acid production with a cell mass concentration of 500 mg/mL in shown in Fig. 5 (all the supporting data are also provided in the supporting information). The other reaction conditions were exactly same as for the data in Fig. 4. Clearly, the biocatalyst
Fig. 5. Production of nicotinic acid from 3-cyanopyridine (1 M initial concentration) catalyzed by immobilized cells of recombinant E. coli. The fresh cell weight was 500 mg/mL.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.procbio.2014.01.023. References
Fig. 6. Reusability of the free and immobilized whole cell biocatalyst for the production of nicotinic acid from 3-cyanopyridine (1 M initial concentration).
mixture. The reaction mixture was incubated in shake flasks at 37 ◦ C, 200 rpm for 6 h. Formation of nicotinic acid was monitored at regular intervals. On completion of the reaction, the product formed was isolated and characterized. 3.9. Identification of nicotinic acid The accumulation of nicotinic acid under the optimized reaction conditions (according to Fig. 5) corresponded to 123 g/L. No formation of nicotinamide was detected in the reaction mixture. Needle shaped crystals appeared after the enzymatic reaction. The product was characterized as nicotinic acid by 1 H and 13 C NMR and mass spectra. Analytical data were as follows: pyridine-3carboxylic acid: white solid (93% yield, 11.43 g); 1 H NMR (400 MHz, MeOD): ı 3.31–3.32 (m, 1H), 7.56–7.59 (m, 1H), 8.40–8.43 (m, 1H), 8.73–8.75 (m, 1H), 9.12–9.13 (d, J = 1.4 Hz, 1H); 13 C NMR (100 MHz, MeOD): ı 123.85, 127.28, 137.85, 149.81, 152.25, 166.36; GC–MS (m/z) 124.01. 4. Conclusions Recombinant E. coli JM109 harboring the nitrilase gene from A. faecalis MTCC 126 was used as catalyst for the biotransformation of 3-cyanopyridine to nicotinic acid. Optimal conditions for the biotransformation using the immobilized cells were as follows: 100 mM Tris buffer, pH 7.5; 37 ◦ C; an immobilized cell fresh weight concentration of 500 mg/mL (enzyme activity 6.67 U/mg of wet cell weight); and an initial substrate concentration of 1 M. Within 5 h, 100% conversion of the substrate was achieved with a yield of 93%. Substrate inhibition was reported in literature at 200–300 mM level but in the current method, no substrate inhibition was observed up to 1 M level. Compared to the freely suspended cells; the immobilized cells were stable at a wider range of pH and were less susceptible to substrate inhibition. Immobilized cells showed good level of reusability and stability up to 25 batches compared to free cells (10 batches). In view of its simplicity, higher substrate utilization, ability to achieve full conversion relatively rapidly and higher stability, the biotransformation process developed may be suitable for large scale use.
[1] Shimizu S. Vitamins and related compounds: microbial production. Biotechnology: special processes, vol. 10. New York: Wiley; 2001. p. 318–40. [2] Bova DJ. Nicotinic acid compositions for treating hyperlipidemia and related methods therefor. US Patent 7,998,506 (2011). [3] Wagner K, Möckel P, Lebzien P, Flachowsky G. Influence of duodenal infusion of nicotinic acid on the milk fat composition of dairy cows. Arch Tierernahr 1997;50:239–44. [4] Almatawah QA, Cowan DA. Thermostable nitrilase catalysed production of nicotinic acid from 3-cyanopyridine. Enzyme Microb Technol 1999;25:718–24. [5] Cantarella M, Cantarella L, Gallifuoco A, Intellini R, Kaplan O, Spera A, et al. Amidase-catalyzed production of nicotinic acid in batch and continuous stirred membrane reactors. Enzyme Microb Technol 2008;42:222–9. [6] Cantarella L, Gallifuoco A, Malandra A, Martinkova L, Spera A, Cantarella M. High-yield continuous production of nicotinic acid via nitrile hydratase–amidase cascade reactions using cascade CSMRs. Enzyme Microb Technol 2011;48:345–50. [7] Cantarella M, Cantarella L, Gallifuoco A, Spera A, Martinkova L. Nicotinic acid bio-production by Microbacterium imperiale CBS 489-74: effect of 3-cyanopyridine and temperature on amidase activity. Process Biochem 2012;47:1192–6. [8] Sharma NN, Sharma M, Kumar H, Bhalla TC. Nocardia globerula NHB-2: bench scale production of nicotinic acid. Process Biochem 2006;41:2078–81. [9] Jin L-Q, Liu Z-Q, Xu J-M, Zheng Y-G. Biosynthesis of nicotinic acid from 3cyanopyridine by a newly isolated Fusarium proliferatum ZJB-09150. World J Microbiol Biotechnol 2013;29:431–40. [10] Jin L-Q, Li Y-F, Liu Z-Q, Zheng Y-G, Shen Y-C. Characterization of a newly isolated strain Rhodococcus erythropolis ZJB-09149 transforming 2-chloro-3cyanopyridine to 2-chloronicotinic acid. New Biotechnol 2011;28:610–5. [11] Mathew CD, Nagasawa T, Kobayashi M, Yamada H. Nitrilase-catalyzed production of nicotinic acid from 3-cyanopyridine in Rhodococcus rhodochrous J1. Appl Environ Microb 1988;54:1030–2. [12] Cantarella L, Gallifuoco A, Malandra A, Martinkova L, Pasquarelli F, Spera A, et al. Application of continuous stirred membrane reactor to 3-cyanopyridine bioconversion using the nitrile hydratase-amidase cascade system of Microbacterium imperiale CBS 498-74. Enzyme Microb Technol 2010;47:64–70. [13] Vaughan PA, Knowles CJ, Cheetham PSJ. Conversion of 3-cyanopyridine to nicotinic acid by Nocardia rhodococcus LL100-21. Enzyme Microb Technol 1989;11:815–23. [14] Gong JS, Li H, Zhu XY, Lu ZM, Wu Y, Shi JS, et al. Fungal his-tagged nitrilase from Gibberella intermedia: gene cloning heterologous expression and biochemical properties. PLoS One 2012;7(11):e50622. [15] Yamamoto K, Oishi K, Fujimatsu I, Komatsu KI. Production of R-(−)-mandelic acid from mandelonitrile by Alcaligenes faecalis ATCC 8750. Appl Environ Microbiol 1991;57:3028–32. [16] Yamamoto K, Fujimatsu I, Komatsu KI. Purification and characterization of the nitrilase from Alcaligenes faecalis ATCC 8750 responsible for enantioselective hydrolysis of mandelonitrile. J Ferment Bioeng 1992;73:425–30. [17] Banerjee A, Dubey S, Kaul P, Barse B, Piotrowski M, Banerjee UC. Enantioselective nitrilase from Pseudomonas putida: cloning, heterologous expression, and bioreactor studies. Mol Biotechnol 2009;41:35–41. [18] O’Reilly C, Turner PD. The nitrilase family of CN hydrolysing enzymes – a comparative study. J Appl Microbiol 2003;95:1161–74. [19] Singh R, Sharma R, Tewari N, Geetanjali, Rawat DS. Nitrilase and its application as a ‘green’ catalyst. Chem Biodivers 2006;3:1279–87. [20] Kaul P, Banerjee A, Banerjee UC. Stereoselective nitrile hydrolysis by immobilized whole-cell biocatalyst. Biomacromolecules 2006;7:1536–41. [21] Kaul P, Stolz A, Banerjee UC. Cross-linked amorphous nitrilase aggregates for enantioselective nitrile hydrolysis. Adv Synth Catal 2007;349:2167–76. [22] Li L, Wang Y, Zhang L, Maa C, Wanga A, Tao F, et al. Biocatalytic production of (2S,3S)-2,3-butanediol from diacetyl using whole cells of engineered Escherichia coli. Bioresour Technol 2012;115:111–6. [23] Zhang W, Gao C, Che B, Maa C, Zheng Z, Qin T, et al. Efficient bioconversion of l-threonine to 2-oxobutyrate using whole cells of Pseudomonas stutzeri SDM. Bioresour Technol 2012;110:719–22. [24] Gao C, Zhang W, Huang Y, Mab C, Xu P. Efficient conversion of 1,2-butanediol to (R)-2-hydroxybutyric acid using whole cells of Gluconobacter oxydans. Bioresour Technol 2012;115:75–8.
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Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Biotransformation of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant Escherichia coli Omkar Pai a , Linga Banoth a , Saptarshi Ghosh a , Yusuf Chisti b , Uttam Chand Banerjee a,∗ a Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and Research, Sector-67, S.A.S. Nagar 160 062, Punjab, India b School of Engineering, Massey University, Private Bag 11 222, Palmerston North, New Zealand
a r t i c l e
i n f o
Article history: Received 5 October 2013 Received in revised form 20 December 2013 Accepted 20 January 2014 Available online 28 January 2014 Keywords: Biotransformation 3-Cyanopyridine Nicotinic acid Alcaligenes faecalis
a b s t r a c t An efficient biocatalytic process for the production of nicotinic acid (niacin) from 3-cyanopyridine was developed using cells of recombinant Escherichia coli JM109 harboring the nitrilase gene from Alcaligenes faecalis MTCC 126. The freely suspended cells of the biocatalyst were found to withstand higher concentrations of the substrate and the product without any signs of substrate inhibition. Immobilization of the cells further enhanced their substrate tolerance, stability and reusability in repetitive cycles of nicotinic acid production. Under optimized conditions (37 ◦ C, 100 mM Tris buffer, pH 7.5) for the immobilized cells, the recombinant biocatalyst achieved a 100% conversion of 1 M 3-cyanopyridine to nicotinic acid within 5 h at a cell mass concentration (fresh weight) of 500 mg/mL. The high substrate/product tolerance and stability of the immobilized whole cell biocatalyst confers its potential industrial use. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Nicotinic acid (vitamin B3 , niacin) is widely added to foodstuff and has pharmaceutical applications. Global production of nicotinic acid and its amide (nicotinamide) is at least 22,000 tons annually [1]. Nicotinic acid is used as a vitamin supplement and has antilipidemic properties [2] helping to lower the risk of heart disease and atherosclerosis. Nicotinic acid is incorporated in the body in nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) which are important cofactors in numerous biochemical reactions relating to metabolism of fats, carbohydrates and proteins. Incorporation of nicotinic acid in fodder may be used to modify the milk fat composition of dairy cows [3]. Several biocatalytic processes for the production of nicotinic acid [4–9] and its derivatives [10] have been reported. These processes generally require an extended reaction period to achieve quantitative conversion [11], or the use of multiple reaction steps [12]. The substrate conversion may be incomplete and unwanted byproducts may be produced. The process apparently used for the commercial production of nicotinic acid from 3-cyanopyridine involved the use of resting cells of Rhodococcus rhodochrous
∗ Corresponding author. Tel.: +91 172 2214682/2214683/2214684/2214685/ 2214686/2214687x2142; fax: +91 172 2214692. E-mail address: [email protected] (U.C. Banerjee). 1359-5113/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.procbio.2014.01.023
[11]. An initial concentration up to 1.4 M 3-cyanopyridine was converted to nicotinic acid, but both the substrate and the product of the reaction inhibited the biocatalytic activity. Substrate inhibition was observed at a 3-cyanopyridine concentration of ≥200 mM. To reduce inhibition, the substrate was fed in seven stages such that at the end of each feeding the concentration was 200 mM. Feeding was given over a period of up to 26 h and the conversion to nicotinic acid was nearly 100%. Total bioconversion (100%) at 200 mM substrate level was reported using whole cells of Bacillus pallidus whereas substrate inhibition was found at 300 mM 3-cyanopyridine [4]. Bioconversion using whole cells of Microbacterium imperiale in continuous stirred tank membrane reactor (CSMR) was also reported with a yield of 200 mM nicotinic acid [5,6,7,12] in 24 h. Using whole cells of Nocardia rhodochrous, nicotinic acid yield of 0.5 M was reported in literature [13]. A nicotinic acid yield of 40 mM was also mentioned in literature using whole cells of Nocardia globerula [8]. Recombinant Escherichia coli harboring the Gibberella intermedia nitrilase gene was also reported to transform 100 mM 3-cyanopyridine to nicotinic acid with 100% conversion efficiency [14]. According to the reports discussed above, a maximum of 200 mM 3-cyanopyridine was used for the transformation and its higher concentration caused substrate inhibition as well as by-product formation. The present study was aimed at developing a relatively rapid, low-cost and single-step process for the biotransformation of 3cyanopyridine to nicotinic acid. The new process involved, the use of recombinant whole cells of E. coli JM109 harboring the nitrilase
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gene from Alcaligenes faecalis MTCC 126. The enzyme responsible for the biotransformation was A. faecalis nitrilase. This enzyme was first reported by Yamamoto et al. [15] for the enantioselective conversion of racemic mandelonitrile to R-mandelic acid. The enzyme was purified and characterized by Yamamoto et al. [16]. Studies on other microbial nitrilases are also reported [17–19] in literature. The nitrilase-containing immobilized whole cells of A. faecalis have been used for the biotransformation of racemic mandelonitrile [20]. The cloning of nitrilase gene form A. faecalis into a vector pJOE2775 and its subsequent transformation into E. coli JM109, production and purification of nitrilase and the formation of cross-linked enzyme aggregates (CLEAs) have been reported earlier [21]. In the present work, whole cells of the nitrilase-containing recombinant E. coli JM109 were used for the biotransformation of 3-cyanopyridine to nicotinic acid. Here 100% conversion of 1 M 3cynopyridine to nicotinic acid with 93% yield was achieved. In this method, we have shown higher substrate utilization (up to 1 M) with no signs of substrate or product inhibition, thus making it better approach for the production of nicotinic acid. 2. Materials and methods 2.1. Materials Nicotinic acid, 3-cyanopyridine and nicotinamide were purchased from Sigma–Aldrich (www.sigmaaldrich.com). Medium components and ampicillin were obtained from Hi-Media, Inc. (Mumbai, India). The solvents used for HPLC were procured from Mallinckrodt Baker, Inc. (Phillipsburg, USA). All other reagents used were of analytical grade. 2.2. Microorganism Recombinant E. coli JM109 cells harboring nitrilase gene from A. faecalis MTCC 126, cloned in our laboratory were used. pJOE2775 vector was used for the expression. 2.3. Growth conditions The recombinant E. coli was revived from a glycerol stock in a 20 mL starter culture in Luria Bertani (LB) broth of the following composition (g/L): tryptone 10; yeast extract 5; NaCl 10, and ampicillin at a final concentration of 0.1 mg/mL. Master plates were then prepared by streaking from this revived culture on LB–ampicillin agar. The agar medium had the above specified composition supplemented with agar (15 g/L). A single colony of the bacterial cells from a freshly prepared agar plate was used to inoculate the starter culture. The starter was grown overnight in the above specified liquid medium at 37 ◦ C, 250 rpm, in an incubator shaker. The starter was transferred to the production medium as above, to achieve a final starter concentration of 2% by volume. This production culture was incubated (37 ◦ C, 250 rpm) for 2 h and then rhamnose was added to a concentration of 2 g/L for the induction of the nitrilase gene. The incubation (37 ◦ C, 250 rpm) was continued for a further 8 h. The cells were then harvested by centrifugation (10,000 × g; Sigma 6K15, GmbH, Germany) at 4 ◦ C for 10 min. The cells were washed with 10 mM sodium phosphate buffer (pH 7.5). 2.4. Analytical procedures 2.4.1. Nitrilase activity The nitrilase activity in the cells was assayed using 3-cyanopyridine (12.5 mM) as substrate. A suspension (20 mg/mL fresh weight concentration) of cells in 10 mM sodium phosphate buffer (pH 7.5) was incubated with the substrate at 37 ◦ C, 200 rpm for 20 min. The reaction was quenched by adding 200 L HCl (1 M). The cells were then removed from the reaction mixture by centrifugation at 10,000 × g for 10 min. The supernatant thus obtained was used to measure the concentrations of the residual substrate and the product by reversed phase HPLC. One unit of nitrilase activity was defined as the amount of the enzyme required to produce 1 mole of the product per minute from 3-cyanopyridine. 2.4.2. Nicotinic acid and 3-cyanopyridine The residual substrate 3-cyanopyridine and the product nicotinic acid in suitably diluted reaction mixture were quantified by analytical high performance liquid chromatography (Shimadzu 10AD VP; Kyoto, Japan). A Symmetry® RP-18 column (250 mm × 4.6 mm, 5 m) (Waters; MA, USA) was used. The mobile phase was a mixture of acetonitrile and sodium phosphate buffer (pH 4.5) in the volume ratio of 55:45. The flow rate of the mobile phase was 0.7 mL/min. The elution of the substrate and the product was detected using a UV detector at a wavelength of 217 nm.
2.5. Immobilization of whole cells Freshly harvested cells were weighed and suspended in 100 mM Tris buffer (pH 7.5) to achieve the desired fresh weight concentration. An amount of sodium alginate was dissolved separately by heating in the above specified buffer to obtain a concentration of 2.5% (w/v). Once the alginate solution had cooled to 40 ◦ C, the cell slurry was added and mixed to obtain a uniform dispersion. This suspension of cells was added dropwise via a syringe to an ice cold solution of calcium chloride (20 g/L) to produce beads that were 2–3 mm in diameter. The alginate beads formed were held in a refrigerator (4 ◦ C) overnight for hardening. These hardened beads containing the immobilized cells were washed with distilled water and used for the biotransformation reaction. 2.6. Optimization of reaction parameters In separate experiments, the biotransformation of 3-cyanopyridine to nicotinic acid was carried out using different specified temperatures, pH values, buffers, cosolvents, biocatalyst concentration, and the initial substrate concentration [22–24]. 2.7. Downstream of nicotinic acid To isolate nicotinic acid from the reaction mixture, the crystals formed were completely dissolved in distilled water and the immobilized enzyme beads were separated by filtration and thoroughly washed. The combined wash out was centrifuged at 12,000 × g for 15 min to remove any insoluble matter and concentrated at 45 ◦ C in vacuum. White crystals of nicotinic acid were obtained. Re-crystallization was carried out by dissolving the crystals in hot distilled water and allowing it to cool overnight in an ice-water bath. Fine needle shaped crystals of nicotinic acid thus obtained, were separated by filtration.
3. Results and discussion 3.1. Effect of reaction temperature For the reaction carried out at different incubation temperatures and a fixed reaction period of 0.5 h (Fig. 1a), both the freely suspended cells and the immobilized cells had an optimal conversion temperature of 50 ◦ C, but the maximum conversion did not exceed above 90%. At 50 ◦ C, the biotransformation reaction was rapid, but so was the rate of denaturation of nitrilase in the cells. Therefore, the cells used at this temperature could not be reused. For a longer fixed reaction period of 2 h, the reaction carried out at different incubation temperatures had the conversion as shown in (Fig. 1b). The optimal temperature was around 37–40 ◦ C. The conversion was slower, but both the freely suspended cells and the immobilized cells retained most of their initial nitrilase activity and could therefore be reused. In addition, a substrate conversion of 100% was achieved (Fig. 1b). Considering the full conversion and reusability of the immobilized biocatalyst, a reaction temperature of 37 ◦ C was concluded to be optimal for this reaction. In view of these results, reaction time was optimized at 37 ◦ C by allowing the reaction to occur for a period of 0.5–4 h (Fig. 1c). Samples were collected at different time interval and analyzed by HPLC. For the measurements shown in Fig. 1a–c, the fresh weight concentration of the cells in all cases was 20 mg/mL. The reaction was carried out in 100 mM phosphate buffer (pH 7.5) for the freely suspended cells and 100 mM Tris buffer (pH 7.5) for the immobilized cells. The initial concentration of the substrate was always 12.5 mM. No co-solvents were used. 3.2. Effect of reaction buffer The effect of the type of buffer on the conversion was tested using phosphate, HEPES and Tris buffers. All buffers (100 mM) were used at pH 7.5. The conversion of the substrate (initial concentration 12.5 mM) after 2 h of reaction at 37 ◦ C is shown in Table 1. The data (Table 1) were measured at a cell fresh weight concentration of 20 mg/mL for both free and immobilized cells. No co-solvents were used. Nearly 100% conversion was achieved in all cases except in phosphate buffer where sodium ions leached out from the beads (Table 1). For all future experiments, phosphate buffer (100 mM,
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Fig. 2. Effect of pH (100 mM phosphate buffer for freely suspended cells; 100 mM Tris buffer for immobilized cells) on the conversion of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant E. coli.
pH 7.5) was concluded to be satisfactory for freely suspended cells and Tris buffer (100 mM, pH 7.5) for immobilized cells. 3.3. Effect of reaction pH Effect of reaction pH on the conversion of 3-cyanopyridine (12.5 mM) to nicotinic acid by free and immobilized cells was examined using 100 mM phosphate buffers for freely suspended cells and 100 mM Tris buffers for immobilized cells. The pH values ranged from 4.5 to 10.5 in different experiments. The observed conversion after 2 h at 37 ◦ C is shown in Fig. 2. For the data in Fig. 2, the fresh weight concentration of the biocatalyst was 20 mg/mL for both free as well as immobilized cells. No co-solvents were used. The activity of freely suspended cells was adversely affected by pH values less than 6.5 and greater than 7.5 (Fig. 2). The optimal pH for the activity was about 7.5. In contrast to free cells, the immobilized cells achieved a nearly 100% conversion over the entire pH range tested (Fig. 2), apparently because the immobilization matrix protected the cells from the adverse pH of the medium surrounding the beads. For long term stability and repeated use of the beads and considering the data of the freely suspended cells (Fig. 2), a biotransformation pH 7.5 was taken as optimal for the immobilized biocatalyst. All the subsequent biotransformation experiments were carried out at this pH. 3.4. Effect of co-solvent
Fig. 1. (a) Effect of incubation temperature on the conversion of 3-cyanopyridine to nicotinic acid. Incubation period was 0.5 h. (b) Effect of incubation temperature on the conversion of 3-cyanopyridine to nicotinic acid. Incubation period was 2 h. (c) Course of conversion of 3-cyanopyridine to nicotinic acid (reaction temperature was 37 ◦ C).
Table 1 Effect of different buffers (100 mM, pH 7.5) on the conversion of 3-cyanopyridine to nicotinic acid by free and immobilized cells of recombinant E. coli. Buffer
Phosphate Tris HEPES
Conversion (%) Free cells
Immobilized cells
99 98 91
– 100 99
Effect of seven different co-solvents on the conversion by freely suspended cells was examined (Table 2). The other reaction conditions were fixed at: 100 mM phosphate buffer, pH 7.5; 37 ◦ C; initial substrate concentration of 12.5 mM; and a cell fresh weight concentration of 20 mg/mL. The concentration of the co-solvents in the buffer was always 10% (v/v). In this study after 2 h of reaction, alcohols as co-solvents (ethanol, IPA, and methanol) showed 100% conversion, whereas acetone and DMSO showed 92% and 88% Table 2 Effect of different co-solvents on the conversion of 3-cyanopyridine to nicotinic acid by free cells of recombinant E. coli. Co-solvent (10%, v/v)
Conversion (%)
Ethanol Isopropanol Methanol Acetone Dimethyl sulfoxide Tetrahydrofuran Dimethylacetamide
100 100 100 92 88 2 4
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Fig. 3. Effect of initial substrate concentration on the conversion catalyzed by immobilized cells of recombinant E. coli. Cell mass concentration used was 20 mg/mL (fresh cell weight).
Fig. 4. Effect of cell mass (fresh cell weight) concentration on nicotinic acid production by immobilized cells of recombinant E. coli. The initial substrate concentration was 1 M.
conversion, respectively. None of the alcohols affected the conversion relative to no co-solvent being present, but the other co-solvents reduced conversion by various levels. Tetrahydrofuran (2%) and dimethylacetamide (4%) severely affected the conversion. It might be due to the reduction in enzyme activity.
achieved a 100% conversion in 5 h starting with an initial substrate concentration of 1 M (enzyme activity 6.67 U/mg wet cell weight) under exceedingly mild reaction conditions (37 ◦ C, pH 7.5).
3.5. Effect of initial substrate concentration
For commercial use, the most desired property of a biocatalyst is its operational stability and reusability. The immobilized cell beads were tested for the reusability so as to make the biocatalysts applicable for repeated cycles of production of nicotinic acid from 3-cyanopyridine. Efficient biocatalyst recycling was achieved as a result of immobilization. Free cells could be reused up to 10 batches. On the other hand immobilized cells retained 100% activity and could be used up to 25 batches (Fig. 6) making the biocatalyst useful for scale-up.
The initial concentration of 3-cyanopyridine in the reaction mixture was varied in different experiments. The fresh weight concentration of the immobilized cells remained fixed at 20 mg/mL. The reaction was carried out at 37 ◦ C, pH 7.5, using 100 mM Tris buffer. No co-solvents were used. The substrate conversion profiles are shown in Fig. 3. Up to a substrate concentration of 25 mM, complete conversion was achieved within 5 h. Further increase in the initial concentration of the substrate progressively slowed the reaction (Fig. 3). In all cases, full conversion was achieved eventually, but it took longer time with the increasing substrate concentration. For a substrate concentration of 75 mM, full conversion was achieved in 17 h whereas it took 35 h for 150 mM substrate conversion.
3.7. Reusability of the immobilized whole cell biocatalyst
3.8. Typical biotransformation procedure The immobilized beads were suspended in 250 mL (100 mM Tris–HCl, pH 7.5) buffer. The final concentration of immobilized cells was 500 mg/mL (wet weight). 3-Cyanopyridine (1 M) which is freely soluble in aqueous medium was added in the reaction
3.6. Effect of cell mass concentration As the rate of conversion of the substrate was considerably reduced at an initial substrate concentration of 100 mM (Fig. 3) at a biocatalyst concentration of 20 mg/mL, attempts were made to counter this by increasing the concentration of the catalyst. The initial substrate concentration was increased to 1 M. In different experiments, the immobilized cell fresh weight concentration was varied from 200 to 500 mg/mL. The reaction was carried out at 37 ◦ C, 200 rpm, and pH 7.5 (100 mM Tris buffer), without co-solvents. The nicotinic acid production profiles are shown in Fig. 4. A nicotinic acid concentration of 1 M equates to a 100% conversion of the substrate. As shown in Fig. 4, at a biocatalyst concentration of 500 mg/mL, complete conversion of the substrate was achieved in 6 h, despite the initial substrate concentration being high at 1 M. The experiments were carried out up to 48 h to study the effect of biocatalyst concentration on the conversion. The data obtained at a cell mass concentration of 500 mg/mL were validated by repeating the experiment with the reaction carried out for a total of 6 h. Sampling was done at 1 h interval up to 6 h and the course of nicotinic acid production with a cell mass concentration of 500 mg/mL in shown in Fig. 5 (all the supporting data are also provided in the supporting information). The other reaction conditions were exactly same as for the data in Fig. 4. Clearly, the biocatalyst
Fig. 5. Production of nicotinic acid from 3-cyanopyridine (1 M initial concentration) catalyzed by immobilized cells of recombinant E. coli. The fresh cell weight was 500 mg/mL.
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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.procbio.2014.01.023. References
Fig. 6. Reusability of the free and immobilized whole cell biocatalyst for the production of nicotinic acid from 3-cyanopyridine (1 M initial concentration).
mixture. The reaction mixture was incubated in shake flasks at 37 ◦ C, 200 rpm for 6 h. Formation of nicotinic acid was monitored at regular intervals. On completion of the reaction, the product formed was isolated and characterized. 3.9. Identification of nicotinic acid The accumulation of nicotinic acid under the optimized reaction conditions (according to Fig. 5) corresponded to 123 g/L. No formation of nicotinamide was detected in the reaction mixture. Needle shaped crystals appeared after the enzymatic reaction. The product was characterized as nicotinic acid by 1 H and 13 C NMR and mass spectra. Analytical data were as follows: pyridine-3carboxylic acid: white solid (93% yield, 11.43 g); 1 H NMR (400 MHz, MeOD): ı 3.31–3.32 (m, 1H), 7.56–7.59 (m, 1H), 8.40–8.43 (m, 1H), 8.73–8.75 (m, 1H), 9.12–9.13 (d, J = 1.4 Hz, 1H); 13 C NMR (100 MHz, MeOD): ı 123.85, 127.28, 137.85, 149.81, 152.25, 166.36; GC–MS (m/z) 124.01. 4. Conclusions Recombinant E. coli JM109 harboring the nitrilase gene from A. faecalis MTCC 126 was used as catalyst for the biotransformation of 3-cyanopyridine to nicotinic acid. Optimal conditions for the biotransformation using the immobilized cells were as follows: 100 mM Tris buffer, pH 7.5; 37 ◦ C; an immobilized cell fresh weight concentration of 500 mg/mL (enzyme activity 6.67 U/mg of wet cell weight); and an initial substrate concentration of 1 M. Within 5 h, 100% conversion of the substrate was achieved with a yield of 93%. Substrate inhibition was reported in literature at 200–300 mM level but in the current method, no substrate inhibition was observed up to 1 M level. Compared to the freely suspended cells; the immobilized cells were stable at a wider range of pH and were less susceptible to substrate inhibition. Immobilized cells showed good level of reusability and stability up to 25 batches compared to free cells (10 batches). In view of its simplicity, higher substrate utilization, ability to achieve full conversion relatively rapidly and higher stability, the biotransformation process developed may be suitable for large scale use.
[1] Shimizu S. Vitamins and related compounds: microbial production. Biotechnology: special processes, vol. 10. New York: Wiley; 2001. p. 318–40. [2] Bova DJ. Nicotinic acid compositions for treating hyperlipidemia and related methods therefor. US Patent 7,998,506 (2011). [3] Wagner K, Möckel P, Lebzien P, Flachowsky G. Influence of duodenal infusion of nicotinic acid on the milk fat composition of dairy cows. Arch Tierernahr 1997;50:239–44. [4] Almatawah QA, Cowan DA. Thermostable nitrilase catalysed production of nicotinic acid from 3-cyanopyridine. Enzyme Microb Technol 1999;25:718–24. [5] Cantarella M, Cantarella L, Gallifuoco A, Intellini R, Kaplan O, Spera A, et al. Amidase-catalyzed production of nicotinic acid in batch and continuous stirred membrane reactors. Enzyme Microb Technol 2008;42:222–9. [6] Cantarella L, Gallifuoco A, Malandra A, Martinkova L, Spera A, Cantarella M. High-yield continuous production of nicotinic acid via nitrile hydratase–amidase cascade reactions using cascade CSMRs. Enzyme Microb Technol 2011;48:345–50. [7] Cantarella M, Cantarella L, Gallifuoco A, Spera A, Martinkova L. Nicotinic acid bio-production by Microbacterium imperiale CBS 489-74: effect of 3-cyanopyridine and temperature on amidase activity. Process Biochem 2012;47:1192–6. [8] Sharma NN, Sharma M, Kumar H, Bhalla TC. Nocardia globerula NHB-2: bench scale production of nicotinic acid. Process Biochem 2006;41:2078–81. [9] Jin L-Q, Liu Z-Q, Xu J-M, Zheng Y-G. Biosynthesis of nicotinic acid from 3cyanopyridine by a newly isolated Fusarium proliferatum ZJB-09150. World J Microbiol Biotechnol 2013;29:431–40. [10] Jin L-Q, Li Y-F, Liu Z-Q, Zheng Y-G, Shen Y-C. Characterization of a newly isolated strain Rhodococcus erythropolis ZJB-09149 transforming 2-chloro-3cyanopyridine to 2-chloronicotinic acid. New Biotechnol 2011;28:610–5. [11] Mathew CD, Nagasawa T, Kobayashi M, Yamada H. Nitrilase-catalyzed production of nicotinic acid from 3-cyanopyridine in Rhodococcus rhodochrous J1. Appl Environ Microb 1988;54:1030–2. [12] Cantarella L, Gallifuoco A, Malandra A, Martinkova L, Pasquarelli F, Spera A, et al. Application of continuous stirred membrane reactor to 3-cyanopyridine bioconversion using the nitrile hydratase-amidase cascade system of Microbacterium imperiale CBS 498-74. Enzyme Microb Technol 2010;47:64–70. [13] Vaughan PA, Knowles CJ, Cheetham PSJ. Conversion of 3-cyanopyridine to nicotinic acid by Nocardia rhodococcus LL100-21. Enzyme Microb Technol 1989;11:815–23. [14] Gong JS, Li H, Zhu XY, Lu ZM, Wu Y, Shi JS, et al. Fungal his-tagged nitrilase from Gibberella intermedia: gene cloning heterologous expression and biochemical properties. PLoS One 2012;7(11):e50622. [15] Yamamoto K, Oishi K, Fujimatsu I, Komatsu KI. Production of R-(−)-mandelic acid from mandelonitrile by Alcaligenes faecalis ATCC 8750. Appl Environ Microbiol 1991;57:3028–32. [16] Yamamoto K, Fujimatsu I, Komatsu KI. Purification and characterization of the nitrilase from Alcaligenes faecalis ATCC 8750 responsible for enantioselective hydrolysis of mandelonitrile. J Ferment Bioeng 1992;73:425–30. [17] Banerjee A, Dubey S, Kaul P, Barse B, Piotrowski M, Banerjee UC. Enantioselective nitrilase from Pseudomonas putida: cloning, heterologous expression, and bioreactor studies. Mol Biotechnol 2009;41:35–41. [18] O’Reilly C, Turner PD. The nitrilase family of CN hydrolysing enzymes – a comparative study. J Appl Microbiol 2003;95:1161–74. [19] Singh R, Sharma R, Tewari N, Geetanjali, Rawat DS. Nitrilase and its application as a ‘green’ catalyst. Chem Biodivers 2006;3:1279–87. [20] Kaul P, Banerjee A, Banerjee UC. Stereoselective nitrile hydrolysis by immobilized whole-cell biocatalyst. Biomacromolecules 2006;7:1536–41. [21] Kaul P, Stolz A, Banerjee UC. Cross-linked amorphous nitrilase aggregates for enantioselective nitrile hydrolysis. Adv Synth Catal 2007;349:2167–76. [22] Li L, Wang Y, Zhang L, Maa C, Wanga A, Tao F, et al. Biocatalytic production of (2S,3S)-2,3-butanediol from diacetyl using whole cells of engineered Escherichia coli. Bioresour Technol 2012;115:111–6. [23] Zhang W, Gao C, Che B, Maa C, Zheng Z, Qin T, et al. Efficient bioconversion of l-threonine to 2-oxobutyrate using whole cells of Pseudomonas stutzeri SDM. Bioresour Technol 2012;110:719–22. [24] Gao C, Zhang W, Huang Y, Mab C, Xu P. Efficient conversion of 1,2-butanediol to (R)-2-hydroxybutyric acid using whole cells of Gluconobacter oxydans. Bioresour Technol 2012;115:75–8.