Antonie van Leeuwenhoek

, Volume 74, Issue 1–3, pp 107–118 | Cite as

Application of whole cell rhodococcal biocatalysts in acrylic polymer manufacture

  • Jonathan Hughes
  • Yvonne C. Armitage
  • Kenneth C. Symes


Rhodococci are ubiquitous in nature and their ability to metabolise a wide range of chemicals, many of which are toxic, has given rise to an increasing number of studies into their diverse use as biocatalysts. Indeed rhodococci have been shown to be especially good at degrading aromatic and aliphatic nitriles and amides and thus they are very useful for waste clean up where these toxic chemicals are present.

The use of biocatalysts in the chemical industry has in the main been for the manufacture of high-value fine chemicals, such as pharmaceutical intermediates, though investigations into the use of nitrile hydratase, amidase and nitrilase to convert acrylonitrile into the higher value products acrylamide and acrylic acid have been carried out for a number of years. Acrylamide and acrylic acid are manufactured by chemical processes in vast tonnages annually and they are used to produce polymers for applications such as superabsorbents, dispersants and flocculants. Rhodococci are chosen for use as biocatalysts on an industrial scale for the production of acrylamide and acrylic acid due to their ease of growth to high biomass yields, high specific enzyme activities obtainable, their EFB class 1 status and robustness of the whole cells within chemical reaction systems.

Several isolates belonging to the genus Rhodococcus have been shown in our studies to be among the best candidates for acrylic acid preparation from acrylonitrile due to their stability and tolerance to high concentrations of this reactive and disruptive substrate. A critical part of the selection procedure for the best candidates during the screening programme was high purity product with very low residual substrate concentrations, even in the presence of high product concentrations. Additionally the nitrile and amide substrate scavenging ability which enables rhodococci to survive very successfully in the environment leads to the formation of biocatalysts which are suitable for the removal of low concentrations of acrylonitrile and acrylamide in waste streams and for the removal of impurities in manufacturing processes.

amidase biocatalyst bioreactor nitrilase polyacrylamide Rhodococcus 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Acharya A & Desai AJ (1997) Studies on utilisation of acetonitrile by Rhodococcus erythropolis A10. World J. Microbiol. Biotechnol. 13: 175–178.Google Scholar
  2. Amarant T, Vered Y & Bohak Z (1989) Substrates and inhibitors of the nitrile hydratase and amidase of Corynebacterium nitrilophilus. Biotechnol. Appl. Biochem. 11: 49–59.Google Scholar
  3. Arai T, Kuroda S & Watanabe I (1981) Biodegradation of acrylamide monomer by a Rhodococcus strain. Zentralbl. Bakteriol. Mikrobiol. Hyg. Abt. I Suppl. 11: 297–307.Google Scholar
  4. Armitage YC & Hughes J (1997) Preparation of amidase or nitrilase for use in amide conversion to its acid or salt. WO patent application no. 96/01951.Google Scholar
  5. Armitage YC, Hughes J & Webster NA (1997) Enzymes, their preparation and their use in the producion of ammonium acrylate. WO patent application no. 96/03080.Google Scholar
  6. Arnaud A, Galzy P & Jallageas JL (1976) Etude de l & #x2019;activite nitrilasique de quelques bacteries. Rev. Ferment. Indust. Aliment. 31: 39–44.Google Scholar
  7. Asano Y, Yasuda T, Tani Y & Yamada H (1982) A new enzymatic method of acrylamide production. Agric. Biol. Chem. 46: 1183–1189.Google Scholar
  8. Bandyopadhyay AK, 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
  9. Bengis-Garber C & Gutman AL (1989) Selective hydrolysis of dinitriles into cyano-carboxylic acids by Rhodococcus rhodochrous NCIB 11216. Appl. Microbiol. Biotechnol. 32: 11–16.Google Scholar
  10. Brennan MR, Armitage YC, Mortimer MG, Hughes J & Ramsden DK (1995) Amidase active whole cells of Corynebacterium nitrilophilus for ammonium acrylate production. Biotechnol. Lett. 17: 513–518.Google Scholar
  11. Bui K, Maestracci M, Thiery A, Arnaud A & Galzy P (1984) A note on the enzymic action and biosynthesis of a nitrile-hydratase from a Brevibacterium sp. J. Appl. Bacteriol. 57: 183–190.Google Scholar
  12. Cavins JF & Friedman M (1968) Specific modification of protein sulfhydryl groups with & #x03B1;-& #x03B2;-unsaturated compounds. J. Biol. Chem. 243: 3357–3360.Google Scholar
  13. Collins PA & Knowles CJ (1983) The utilisation of nitriles and amides by Nocardia rhodochrous. J. Gen. Microbiol. 129: 711–718.Google Scholar
  14. Commeyras A, Arnaud A, Galzy P & Jallageas J-C (1973) Demande de brevet D & #x2019;invention. US patent no. 3,940,316.Google Scholar
  15. Commeyras A, Arnaud A, Galzy P & Jallageas J-C (1977) Process for the preparation of amides by biological hydrolysis. US patent no. 4,001,081.Google Scholar
  16. Croll BT, Arkell GM & Hodge RPJ (1974) Residues of acrylamide in water. Water Res. 8: 989–993.Google Scholar
  17. DiGeronimo MJ & Antoine AD (1976) Metabolism of acetonitrile and propionitrile by Nocardia rhodochrous LL100–21. Appl. Environ. Microbiol. 31: 900–906.Google Scholar
  18. Farrar D & Flesher P (1989) Polymeric compositions and their production. EP patent no. 0329325.Google Scholar
  19. Firmin JL & Gray DO (1976) The biochemical pathway for the breakdown of methyl cyanide (acetonitrile) in bacteria. Biochem. J. 158: 223–229.Google Scholar
  20. Harper DB (1977a) Microbial metabolism of aromatic nitriles. Biochem. J. 165: 309–319.Google Scholar
  21. Harper DB (1977b) Fungal degradation of aromatic nitriles. Biochem. J. 167: 685–692.Google Scholar
  22. Harper DB (1985) Characterisation of a nitrilase from Nocardia sp. (rhodochrous group) NCIB 11215, using p-hydroxybenzonitrile as sole carbon source. Int. J. Biochem. 17: 677–683.Google Scholar
  23. Hughes J, Armitage YC, Symes KC & Brooke AP (1997) Processes for the production of dry acrylamide polymer particles having reduced residual acrylamide monomer contamination using amidase enzyme. WO patent application no. 97/00317.Google Scholar
  24. Ingvorsen K, Yde B, Godtfredsen SE & Tsuchiya RT (1988) Microbial hydrolysis of organic nitriles and amides. CIBA Found. Symp. 140: 16–31.Google Scholar
  25. Kato A & Kenji H (1976) Treating waste water containing nitriles and cyanides. US patent no. 3,940,332.Google Scholar
  26. Kobayashi M, Nagasawa T & Yamada H (1988) Regiospecific hydrolysis of dinitrile compounds by nitrilase from Rhodococcus rhodochrous J1. Appl. Microbiol. Biotechnol. 29: 231–233.Google Scholar
  27. Kobayashi M, Nagasawa T, Yanaka N & Yamada H (1989) Nitrilase-catalysed production of p-aminobenzoic acid from p-aminobenzonitrile Rhodococcus rhodochrous J1. Biotechnol. Lett. 11: 27–30.Google Scholar
  28. Kobayashi M, Yanaka N, Nagasawa T & Yamada H (1990) Purification and characterisation of a novel nitrilase of Rhodococcus rhodochrous K22 that acts on aliphatic nitriles. J. Bacteriol. 172: 4807–4815.Google Scholar
  29. Kobayashi M, Nagasawa T & Yamada H (1992a) Enzymatic synthesis of acrylamide: A success story not yet over. TIBTECH 10: 402–408.Google Scholar
  30. Kobayashi M, Komeda H, Yanaka N, Nagasawa T & Yamada H (1992b) Nitrilase from Rhodococcus rhodochrous J1. J. Biol. Chem. 29: 20746–20751.Google Scholar
  31. Kobayashi M, Yanaka N, Nagasawa T & Yamada H (1992c) 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. Biochem. 31: 9000–9007.Google Scholar
  32. Kobayashi M, Komeda H, Nagasawa T, Yamada H & Shimizu S (1993) Occurrence of amidases in the industrial microbe Rhodococcus rhodochrous J1. Biosci. Biotech. Biochem. 57: 1949–1950.Google Scholar
  33. Lande SS, Bosch SJ & Howard PH (1979) Degradation and leaching of acrylamide in soil. J. Environ. Qual. 8: 133–137.Google Scholar
  34. Lee S-T, Lee S-B & Park Y-H (1991) Characterisation of a pyridine-degrading branched Gram-positive bacterium isolated from the anoxic zone of an oil shale column. Appl. Microbiol. Biotechnol. 35: 824–829.Google Scholar
  35. Linardi VR, Dias JCT & Rosa CA (1996) Utilisation of acetonitrile and other aliphatic nitriles by a Candida famata strain. FEMS Microbiol. Lett. 144: 67–71.Google Scholar
  36. Linton EA & Knowles CJ (1986) Utilisation of aliphatic amides and nitriles by Nocardia rhodochrous LL100–21. J. Gen. Microbiol. 132: 1493–1501.Google Scholar
  37. Maestracci M, Bui K, Thiery A, Arnaud A & Glazy P (1988) The amidases from a Brevibacterium strain: Study and applications. Adv. Biochem. Eng. Biotechnol. 36: 69–115.Google Scholar
  38. Mimura A, Kawano T & Yamaga K (1969) Application of microorganisms to petrochemical industry. J. Ferment. Technol. 47: 631–638.Google Scholar
  39. Nagasawa T & Yamada H (1989) Microbial transformations of nitriles. TIBTECH 7: 153–158.Google Scholar
  40. Nagasawa T & Yamada H (1990a) Application of nitrile converting enzymes for the production of useful compounds. Pure Appl. Chem. 62: 1441–1444.Google Scholar
  41. Nagasawa T & Yamada H (1990b) Large-scale bioconversion of nitriles into useful amides and acids. In: Abramowicz DA (Ed) Biocatalysis (pp 277–318). van Nostrand Reinhold, New York.Google Scholar
  42. Nagasawa T, Mathew CD, Mauger J & Yamada H (1988) Nitrile hydratase-catalysed production of nicotinamide from 3-cyanopyridine in Rhodococcus rhodochrous J1. Appl. Environ. Microbiol. 54: 1766–1769.Google Scholar
  43. Nagasawa T, Nakamura T & Yamada H (1990a) Production of acrylic acid and methacrylic acid using Rhodococcus rhodochrous J1 nitrilase. Appl. Microbiol. Biotechnol. 34: 322–324Google Scholar
  44. Nagasawa T, Nakamura T & Yamada H (1990b) & #x220A;-Caprolactam, a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase. Arch. Microbiol. 155: 13–17.Google Scholar
  45. Nagasawa T, Takeuchi K, Nardi-Dei V, Mihara Y & Yamada H (1991a) Optimum culture conditions for the production of cobalt-containing nitrile hydratase by Rhodococcus rhodochrous J1. Appl. Microbiol. Biotechnol. 34: 783–788.Google Scholar
  46. Nagasawa T, Takeuchi K, & Yamada H (1991b) Characterisation of a new cobalt-containing nitrile hydratase purified from ureainduced cells of Rhodococcus rhodochrous J1. Eur. J. Biochem. 196: 581–589Google Scholar
  47. 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.Google Scholar
  48. Narayanasamy K, Shukla S & Parekh LJ (1990) Utilisation of acrylonitrile by bacteria isolated from petrochemical wastewaters. Indian J. Exp. Biol. 28: 968–971.Google Scholar
  49. Nawaz MS, Franklin W, Campbell WL, Heinze TM & Cerniglia CE (1991) Metabolism of acrylonitrile by Klebsiella pneumoniae. Arch. Microbiol. 156: 231–238.Google Scholar
  50. Nawaz MS, Franklin W & Cerniglia CE (1993) Degradation of acrylamide by immobilised cells of a Pseudomonas sp. and Xanthomonas maltophilia. Can. J. Microbiol. 39: 207–212.Google Scholar
  51. Nawaz MS, Kahn AA, Seng JE, Leakey JE, Siitonem PH & Cerniglia CE (1994) Purification and characterisation of an amidase from an acrylamide-degrading Rhodococcus sp. Appl. Environ. Microbiol. 60: 3343–3348.Google Scholar
  52. Ramakrishna C & Desai JD (1992) Induction of iron and cobalt dependent acrylonitrile hydratases in Arthrobacter sp. IPCB-3. Biotechnol. Lett. 14: 827–830.Google Scholar
  53. Robinson WG & Hook RH (1964) Ricine nitrilase. J. Biol. Chem. 239: 4257–4267.Google Scholar
  54. Shanker R, Ramakrishna C & Seth PK (1990) Microbial degredation of acrylamide monomer. Arch. Microbiol. 154: 192–198.Google Scholar
  55. Stevenson DE, Feng R, Dumas F, Groelau D, Mihoc A & Storer AC (1992) Mechanistic and structural studies on Rhodococcus ATCC 39484 nitrilase. Biotechnol. Appl. Biochem. 15: 283–302.Google Scholar
  56. Symes KC & Hughes J (1997) Production of ammonium acrylate. WO patent application no. 96/03081.Google Scholar
  57. 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.Google Scholar
  58. Watanabe I, Satoh Y & Enomoto K (1987) Screening, isolation and taxonomical properties of microorganisms having acrylonitrile-hydrating activity. Agric. Biol. Chem. 51: 3193–3199.Google Scholar
  59. Wyatt JM & Linton EA (1988) The industrial potential of microbial nitrile biochemistry. CIBA Found. Symp. 140: 32–48.Google Scholar
  60. Webster NA (1996) The development of a biocatalytic process for the conversion of acrylonitrile to ammonium acrylate. PhD Thesis, University of Huddersfield, UK.Google Scholar
  61. Yamada H, Asano Y, Hino T & Tani Y (1979) Microbial utilisation of acrylonitrile. J. Ferment. Technol. 57: 8–14.Google Scholar
  62. Yamada H & Kobayashi M (1996) Nitrile hydratase and its application to industrial production of acrylamide. Biosci. Biotech. Biochem. 60: 1391–1400.Google Scholar
  63. Yamada H, Asano Y & Tani Y (1980) Microbial utilisation of glutaronitrile. J. Ferment. Technol. 58: 495–500.Google Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Jonathan Hughes
    • 1
  • Yvonne C. Armitage
    • 1
  • Kenneth C. Symes
    • 1
  1. 1.Allied Colloids Ltd.Low Moor BradfordUK

Personalised recommendations