Abstract
Rhodococcus sp. NDB 1165, a nitrile-transforming organism was isolated from temperate forest soil of Himalayas. The nitrilase (EC 3.5.5.2) activity of this organism had higher substrate specificity toward aromatic nitriles (benzonitrile, 3-cyanopyridine and 4-cyanopyridine) and unsaturated aliphatic nitrile (acrylonitrile) in comparison to saturated aliphatic nitriles (acetonitrile, propionitrile, butyronitrile and isobutyronitrile) nitrile and arylacetonitrile (phenylacetonitrile and indole-3-acetonitrile). The nitrilase of Rhodococcus sp. NDB 1165 was inducible in nature and propionitrile proved to be an efficient inducer. However, the salts of ferrous and cobalt ions had an inhibitory effect. Under optimized reaction conditions (pH 8.0 and temperature 45°C) the nitrilase activity of this organism was 2.39 ± 0.07 U/mg dry cell mass (dcm). The half-life of this enzyme was 150 min and 40 min at 45°C and 50°C respectively. However, it was quite stable at 40°C and around 58 % activity was retained even after 6 h at this temperature. The V max and K m value of this nitrilase were 1.67 µmol/ml min and 0.1 M respectively using 3-cyanopyridine as substrate. However, the decrease in V max and K m values (0.56 µmol/ml min and 0.02 M, respectively) were ␣observed at >0.05 M 3-cyanopyridine which revealed that this enzyme experienced uncompetitive inhibition at higher substrate concentrations. Under optimized reaction conditions, 1.6 M 3-cyanopyridine was successfully converted in to nicotinic acid using 2.0 mg resting cells (dcm)/ml reaction mixture in 11 h. This is the highest production of nicotinic acid i.e. 8.95 mg/mg resting cells (dcm)/h as compared to nitrilase systems reported hitherto.
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References
Almatawah QA, Cowan DA (1999) Thermostable nitrilase catalyzed production of nicotinic acid from 3-cyanopyridine. Enzyme Microb Technol 25:718–724
Almatawah QA, Cramp R, Cowan DA (1999) Characterization of an inducible nitrilase from a thermophilic bacillus. Extremophiles 3:283–291
Banerjee A, Sharma R, Banerjee UC (2003) The nitrile-degrading enzyme: current status and future prospects. Appl Microbiol Biotechnol 60:33–44
Belitz H-D, Grosch W (1999) Food addatives. In: Food chemistry. Springer-Verlag Berlin Heidelberg, Germany, pp 423–424, ISBN 3-540-64704-x
Bhalla TC, Kumar H (2005) Nocardia globerula NHB-2: a versatile nitrile-degrading organism. Can J Microbiol 51:705–708
Bhalla TC, Mimura A, Wakamoto A, Ohba Y, Furuhashi K (1992) Asymmetric hydrolysis of α-aminonitrile to optically active amino acids by a nitrilase of Rhodococcus rhodochrous PA 34. Appl Microbiol Biotechnol 37:184–190
Blakey AJ, Colby J, Williams E, O’Reilly C (1995) Regio- and stereo-specific hydrolysis by the nitrile hydratase from Rhodococcus AJ 270. FEMS Microbiol Lett 129:57–62
Bollag, DM, Edelstein, SJ (1991) Preparation of protein isolation. In: Protein methods. A John Wiley & Sons, New York, pp 1–24, ISBN 0-471-56871-6
Chaplin, M, Bucke, C (1990) Fundamental of enzyme kinetics. In: Enzyme technology. Cambridge University Press, pp 1–39, ISBN 0 521 34429 8
Cowan D, Cramp R, Pereira R, Graham D, Almatawah Q (1998) Biochemistry and biotechnology of mesophilic and thermophilic nitrile metabolizing enzymes. Extremophiles 2:207–216
Finar IL (1997) Alkaloids. In: Organic chemistry vol 2: stereochemistry and the chemistry of natural products. Addison Wesley Longman Limited, England, pp 702–768
Gavagan JE, Di-Cosimo R, Eisenberg A, Fager SK, Folsom PW, Hann EC, Schneider KJ, Fallon RD (1999) A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitrile. Appl Microbiol Biotechnol 52:654–659
Goldlust A, Bohak Z (1989) Induction, purification and characterization of the nitrilase of Fusarium oxysporum f. sp. Melonis. Biotechnol Appl Biochem 11:581–601
Jallageas JC, Arnaud A, Galzy P (1980) Bioconversion of nitriles and their applications. Advance Biochemical Engineering 14:1–32
Kato Y, Ooi R, Asano Y (1998) Isolation and characterization of a bacterium possessing a novel aldoxime-dehydration activity and nitrile-degrading enzymes. Arch Microbiol 170:85–90
Kirschmann GJ, Kirschmann JD (1996) Nicotinic acid. In: Nutrition Almanac. McGraw Hill publication, New York, pp 88–99, ISBN 0-443-043051
Kobayashi M, Nagasawa T, Yamada H (1992) Enzymatic synthesis of acrylamide: a success story not yet over. Trends Biotechnol 10:402–408
Kopf MA, Bonnet D, Artaud I, Petre D, Mansuy D (1996) Key role of alkanoic acids on the spectral properties, activity and active site stability of iron containing nitrile hydratase from Brevibacterium sp. R 312. Euro J Biochem 240:239–244
Mathew CD, Nagasawa T, Kobayashi M, Yamada H (1988) Nitrilase-catalyzed production of nicotinic acid from 3-cyanopyridine in Rhodococcus rhodochrous J1. Appl Environ Microbiol 54:1030–1032
Nagasawa T, Kobayashi M, Yamada H (1988) Optimum culture conditions for the production of benzonitrilase by Rhodococcus rhodochrous J1. Arch Microbiol 150:89–94
Nagasawa T, Mauger J, Yamada H (1990) A novel nitrilase, arylacetonitrilase of Alcaligenes faecalis JM3 – purification and characterization. Euro J Biochem 194:765–772
Nagasawa T, Shimizu H, Yamada H (1993) The superiority of the third-generation catalyst, Rhodococcus rhodochrous J1 nitrile hydratase, for industrial production of acrylamide. Appl Microbiol Biotechnol 40:189–195
O’Reilly C, Turner PD (2003) The nitrilase family of CN hydrolyzing enzymes – a comparative study. J Appl Microbiol 95:1161–1174
Prasad S, Sharma DR, Bhalla TC (2005) Nitrile- and amide-hydrolysing activity in Kluyveromyces thermotolerans MGBY 37. World J Microbiol Biotechnol 21:1447–1450
Ramakrishna C, Dave H, Ravindranathan M (1999) Microbial metabolism of nitriles and its biotechnological potential. J␣Scientific Ind Res 58:925–947
Tipton KF (1998) Principal of enzyme assay and kinetic studies. In: Eisenthal R, Danson MJ (eds) Enzyme assays–A practical approach. Oxford University Press, New York, pp 25–28, ISBN 0-19-963143-3
Vaughan PA, Knowles CJ, Cheetham PSJ (1989) Conversion of 3-cyanopyridine to nicotinic acid by Nocardia rhodochrous LL100-21. Enzyme Microb Technol 11:815–823
Yamamoto K, Komatsu K (1991) Purification and characterization of nitrilase responsible for the enantioselective hydrolysis from Acinetobacter sp. AK 226. Agricult Biol Chem 55:1459–1466
Acknowledgement
Authors gratefully acknowledge the gift of 3-cyanopyridine from Dr. S. K. Mishra, Associate Head-Biotechnology, Jubliant Organosys Ltd., Jyotiba Phoolay Nagar, India.
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Prasad, S., Misra, A., Jangir, V.P. et al. A propionitrile-induced nitrilase of Rhodococcus sp. NDB 1165 and its application in nicotinic acid synthesis. World J Microbiol Biotechnol 23, 345–353 (2007). https://doi.org/10.1007/s11274-006-9230-5
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DOI: https://doi.org/10.1007/s11274-006-9230-5