Antonie van Leeuwenhoek

, Volume 74, Issue 1–3, pp 89–97 | Cite as

Biotransformation of nitriles by rhodococci

  • Alan William Bunch


Rhodococci have been shown to be capable of a very wide range of biotransformations. Of these, the conversion of nitriles into amides or carboxylic acids has been studied in great detail because of the biotechnological potential of such activities. Initial investigations used relatively simple aliphatic nitriles. These studies were quickly followed by the examination of the regio- and stereoselective properties of the enzymes involved, which has revealed the potential synthetic utility of rhodococcal nitrile biotransforming enzymes. Physiological studies on rhodococci have shown the importance of growth medium design and bioreactor operation for the maximal conversion of nitriles. This in turn has resulted in some truly remarkable biotransformation activities being obtained, which have been successfully exploited for commercial organic syntheses (e.g. acrylamide production from acrylonitrile).

The two main types of enzyme involved in nitrile biotransformations by rhodococci are nitrile hydratases (amide synthesis) and nitrilases (carboxylic acid synthesis with no amide intermediate released). It is becoming clear that many rhodococci contain both activities and multiple forms of each enzyme, often induced in a complex way by nitrogen containing molecules. The genes for many nitrile-hydrolysing enzymes have been identified and sequenced. The crystal structure of one nitrile hydratase is now available and has revealed many interesting aspects of the enzyme structure in relationship to its catalytic activity and substrate selectivity.

nitriles nitrile hydratase nitrilase biotransformations 


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  1. Ahmed A E & Trieff N M (1983) Aliphatic nitriles; metabolism and toxicity. In: Bridges J W & Chasseaud L F, (Eds) Progress in Drug Metabolism, Vol. 7 (pp 230–294) John Wiley and Son Ltd. UK.Google Scholar
  2. Bandyopadhyay A K, Nagasawa T, Asano Y, Fujishiro K, Tani Y & Yamada H (1986) Purification and characterisation of benzonitrilases from Arthrobacter sp. strain J-1. Appl. Environ. Microbiol. 51: 302–306.Google Scholar
  3. Blakey A J, Colby J, Williams E & O & #x2019;Reilly C (1995) Regioand stereo-specific hydrolysis by the nitrile hydratase from Rhodococcus AJ270. FEMS Microbiol. Lett. 129: 57–62.Google Scholar
  4. Bork P & Koonin E V (1994) A new family of carbon-nitrogen hydrolases. Protein Sci. 3: 1344–1346.Google Scholar
  5. Brennan B A, Alms G, Nelson M J, Durney L T & Scarrow R C (1996) Nitrile hydratase from Rhodococcus rhodochrous J1 contains a non-corrinoid cobalt with two sulphur ligands. J. Am. Chem. Soc. 118: 9194–9195.Google Scholar
  6. Cramp R, Gilmour M & Cowan D A (1997) Novel thermophilic bacteria producing nitrile-degrading enzymes. Microbiology 143: 2313–2320.Google Scholar
  7. Crosby J, Moilliet J, Parratt J S & Turner N J (1994) Regioselective hydrolysis of aromatic dinitriles using a whole cell catalyst. J. Chem. Soc. Perkin Trans. 1: 1679–1687.Google Scholar
  8. Dufour E, Storer A C & Menard R (1995) Engineering nitrile hydratase activity into a cysteine protease by a single mutation. Biochemistry 34: 16382–16388Google Scholar
  9. Duran R, Nishiyama M, Horinouchi S & Beppu T (1993) Characterisation of nitrile hydratase genes cloned by DNA screening from Rhodococcus erythropolis. Biosci. Biotechnol. Biochem. 57: 1323–1328Google Scholar
  10. Endo T & Watanabe I (1989) Nitrile hydratase of Rhodococcus sp.N-774-purification and amino acid sequences. FEBS Lett. 243: 61–64Google Scholar
  11. Faber K (1994) Biotransformations in Organic Chemistry. 2nd Ed. Springer-Verlag, Berlin.Google Scholar
  12. Goodfellow M, Alderson G & Chun J (1998) Rhodococcal systematics: problems and developments. Antonie van Leeuwenhoek 74: 3–20Google Scholar
  13. Gradley M L & Knowles C J (1994) Asymmetric hydrolysis of chiral nitriles by Rhodococcus rhodochrous NCIMB 11216 nitrilase. Biotech. Lett. 16: 41–46Google Scholar
  14. Harper D B (1977) Microbial metabolism of aromatic nitriles– Enzymology of C–N cleavage by Nocardia sp. (Rhodococcus group) NCIB 11216. Biochem. J. 165: 309–319Google Scholar
  15. Hashimoto Y, Nishiyama M, Yu F, Watanabe I, Horinouchi S & Beppu T (1992) Development of a host-vector system in a Rhodococcus strain and its use for expression of the cloned nitrile hydratase gene cluster. J. Gen. Microbiol. 138: 1003–1010Google Scholar
  16. Hashimoto Y, Nishiyama M, Horinouchi S & Beppu T (1994) Nitrile hydratase gene from Rhodococcus sp-774 requirement for its down stream region for efficient expression. Biosci. Biotech. Biochem. 58: 1859–1865Google Scholar
  17. Huang W, Jia J, Nelson M, Schneider G & Lindqvist Y (1997) Crystal structure of nitrile hydratase reveals a novel iron centre in a novel fold. Structure 5: 691–699Google Scholar
  18. Hughes J, Armitage Y C & Symes K (1998) Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture. Antonie van Leeuwenhoek (in press)Google Scholar
  19. Ikehata O, Nishiyama M, Horinouchi S & Beppu T (1989) Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus species and its expression in Escherichia coli. Eur. J. Biochem. 181: 563–570Google Scholar
  20. Kakeya H, Sakai N, Sugai T & Ohta H (1991) Microbial hydrolysis as a potent method for the preparation of optically active nitriles, amides and carboxylic acids. Tetrahedron Lett. 32: 1343–1346Google Scholar
  21. Knowles C J & Bunch A W (1986) Microbial cyanide metabolism. Adv. Microbial Phys. 27: 73–111Google Scholar
  22. Kobayashi M, Nagasawa T, Yanaka N & Yamada H (1989) Nitrilase-catalysed production of p-aminobenzoic acid from p-aminobenzonitrile with Rhodococcus rhodochrous J1. Biotech. Lett. 11: 27–30Google Scholar
  23. Kobayashi M, Yanaka N, Nagasawa T & Yamada H (1990) Monohydrolysis of an aliphatic dinitrile compound by nitrilase from Rhodococcus rhodochrous K22. Tetrahedron 46: 5587–5590Google Scholar
  24. Kobayashi M, Nishiyama M, Nagasawa T, Horinouchi S, Beppu T & Yamada H (1991) Cloning, nucleotide sequence and expression in Escherichia coli of two cobalt-containing nitrile hydratase genes from Rhodococcus rhodochrous J1. Biochim. Biophys. Acta 1129: 23–33Google Scholar
  25. Kobayashi M, Nagasawa T & Yamada H (1992a) Enzymatic synthesis of acrylamide: a success story not yet over. Trends Biotech. 10: 402–408Google Scholar
  26. Kobayashi M, Yanaka N, Nagasawa T & Yamada H (1992b) Primary structure of an aliphatic nitrile-degrading enzyme, aliphatic nitrilase, from Rhodococcus rhodochrous K22 and expression of its gene and identification of its active site residue. Biochemistry 31: 9000–9007Google Scholar
  27. Langdahl B R, Bisp P & Ingvorsen K (1996) Nitrile hydrolysis by Rhodococcus erythropolis BL1, an acetonitrile-tolerant strain isolated from a marine sediment. Microbiology 142: 145–154Google Scholar
  28. Layh N, Hirrlinger B, Stolz A & Knackmuss H-J (1997) Enrichment strategies for nitrile hydrolysing bacteria. Appl. Microbiol. Biotech. 47: 668–674Google Scholar
  29. Legras J L, Chuzel G, Arnuad A & Galzy P (1990) Natural nitriles and their metabolism. World J. Microbiol. Biotech. 6: 83–108Google Scholar
  30. Maier-Greiner U H, Obermaier-Skrobranek B M M, Estermaier LM, Kammerloher W, Freund C, Wulfing C, Burkert U I, Matern D H, Breuer M, Eulitz M, Kufrevioglu I & Hartman G R (1991) Isolation and properties of a nitrile hydratase from the soil fungus Myrothecium verrucaria that is highly specific for the fertilizer cyanamide and cloning of its gene. Proc. Natl. Acad. Sci. USA 88: 4260–4264Google Scholar
  31. Nagasawa T & Yamada H (1995) Interrelations of chemistry and biotechnology. 6. Microbial production of commodity chemicals. Pure Appl. Chem. 67: 1241–1256Google Scholar
  32. Nagamune T, Kurata H, Hirata M, Honda J, Koike H, Ikeuchi M, Inoue Y, Hirata A & Endo I (1990a) Purification of inactivated photoresponsive nitrile hydratase. Biochem. Biophys. Res. Commun. 168: 437–442Google Scholar
  33. Nagamune T, Kurata H, Hirata M, Honda J, Hirata A & Endo I (1990b) Photosensitive phenomena of nitrile hydratase of Rhodococcus sp N-771. Photochem. Photobiol. 51: 87–90Google Scholar
  34. Nagasawa T, Nanba H, Ryuno K, Takeuchi K & Yamada H (1987) Nitrile hydratase of Pseudomonas chlororaphis B23: Purification and characterisation. Eur. J. Biochem. 162: 691–698Google Scholar
  35. Nagasawa T, Mathew C D, Mauger J & Yamada H (1988a) Nitrile hydratase catalysed production of nicotinamide from 3-cyanopyridine in Rhodococcus rhodochrous J1. Appl. Environ. Microbiol. 54: 1766–1769Google Scholar
  36. Nagasawa T, Takeuchi K & Yamada H (1988b) Occurrence of a cobalt-induced and cobalt containing nitrile hydratase in Rhodococcus rhodochrous J1. Biochem. Biophys. Res. Commun. 155: 1008–1016Google Scholar
  37. Nakajima Y, Doi T, Satoh Y, Fujiwara A & Watanabe I (1987) A photoresponsive nitrile hydratase from Rhodococcus N-771. Chem. Lett. 9: 1767–1770Google Scholar
  38. Odaka M, Fujii K, Hoshino M, Noguchi T, Tsujimura M, Nagashima S, Yohda M, Nagamune T, Inoue Y & Endo I (1997) Activity regulation of photoreactive nitrile hydratase by nitric oxide. J. Am. Chem. Soc. 119: 3785–3791Google Scholar
  39. Roberts S M, Turner N J, Willets A J & Turner M K (1995) Introduction to Biocatalysis using Enzymes and Microorganisms. Cambridge University Press, Cambridge, UK.Google Scholar
  40. Stevenson D E, Feng R & Storer A C (1990) Detection of covalent enzyme–substrate complexes of nitrilase by ion-spray mass spectroscopy. FEBS Lett. 277: 112–114Google Scholar
  41. Thimann KV & Mahadevan S (1964) Nitrilase I. Occurrence preparation and general properties of the enzyme. Arch. Biochem. Biophys. 105: 133–141Google Scholar
  42. Watanabe I, Yoshiaki S & Enomoto K (1987a) Screening, isolation and taxonomical properties of microorganisms having acrylonitrile-hydrating activity. Agric. Biol. Chem. 51: 3193–3199Google Scholar
  43. Watanabe I, Satoh Y, Enomoto K, Seki S & Sakashita K (1987b) Optimal conditions for cultivation of Rhodococcus sp. N-774 and for conversion of acrylonitrile to acrylamide by resting cells. Agric. Biol. Chem. 51: 3201–3206Google Scholar
  44. Warhurst A M & Fewson C A (1994) Biotransformations catalysed by the genus Rhodococcus. Crit. Rev. Biotech. 14: 29–73Google Scholar
  45. Yamada H & Kobayashi M (1996) Nitrile hydratase and its applications to the industrial production of acrylamide. Biosci. Biotech. Biochem. 60: 1391–1400Google Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Alan William Bunch
    • 1
  1. 1.Research School of BiosciencesThe University of KentCanterbury, KentUK

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