Oxidation of n-alkanes: Isolation of alkane hydroxylase from Pseudomonas putida

  • Neville M. Fish
  • Stuart Harbron
  • David J. Allenby
  • Malcolm D. Lilly
Applied Microbiology


The disruption of Pseudomonas putida cells capable of n-alkane assimilation was investigated by enzymic lysis and mechanical disruption in a high pressure-homogeniser, with a view to the isolation of alkane hydroxylase activity. Examination of the conditions for enzymic lysis showed that disruption with lysozyme/EDTA could be replaced effectively with lysozyme alone in phosphate buffer, pH 8.0 (I=0.05). This allowed inclusion of DNase during the lysis procedure for high bacterial concentrations and gave improved cell disruption. Mechanical disruption resulted in the solubilisation of alkane hydroxylase activity. In contrast enzymic lysis allowed the isolation of an insoluble fraction containing alkane hydroxylase activity, and although some solubilisation of the enzyme system did occur much of the activity was retained in the insoluble fraction. This fraction also contained a high level of n-alkane or diethoxymethane inducible, NAD-independent alcohol dehydrogenase activity.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Allenby DJ (1982) The enzymic oxidation of n-alkanes. Ph.D. Thesis, LondonGoogle Scholar
  2. Asbell MA, Eagon RG (1966) Role of multivalent cations in the organization, structure and assembly of the cell wall of Pseudomonas aeruginosa. J Bacteriol 92: 380–387Google Scholar
  3. Baptist JN, Gholson RK, Coon MJ (1963) Hydrocarbon oxidation by a bacterial enzyme system. I Products of octane oxidation. Biochim Biophys Acta 69: 40–47Google Scholar
  4. Benson S, Fennewald M, Shapiro J, Huettner C (1977) Fractionation of inducible alkane hydroxylase activity in Pseudomonas putida and characterization of hydroxylase-negative plasmid mutations. J Bacteriol 132: 614–621Google Scholar
  5. Benson S, Shapiro J (1976) Plasmid-determined alcohol dehydrogenase activity in alkane-utilizing strains of Pseudomonas putida. J Bacteriol 126: 794–798Google Scholar
  6. Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911–917Google Scholar
  7. Day DF, Marceau-Day ML, Ingram JM (1978) Protein-lipopolysaccharide interactions. 1 The reaction of lysozyme with Pseudomonas aeruginosa LPS. Can J Microbiol 24: 196–199Google Scholar
  8. Eagon RG, Simmons GP, Carson KJ (1965) Evidence for the presence of ash and divalent metals in the cell wall of Pseudomonas aeruginosa. Can J Microbiol 11: 1041–1042Google Scholar
  9. Eyk J van, Bartels TJ (1970) Paraffin oxidation in Pseudomonas aeruginosa. II Gross fractionation of the enzyme system into soluble and particulate components. J Bacteriol 104: 1065–1073Google Scholar
  10. Fish NM, Allenby DJ, Lilly MD (1982) Oxidation of n-alkanes: Growth of Pseudomonas putida. Eur J Appl Microbiol Biotechnol 14: 259–262Google Scholar
  11. Gornall AG, Bardawill CJ, David MM (1949) Determination of serum proteins by means of the biuret reaction. J Biol Chem 177: 751–766Google Scholar
  12. Gürtler H (1980) Procedure for determination of DNA (in bacterial fermentation broths). Personal communicationGoogle Scholar
  13. Hetherington PJ, Follows M, Dunnill P, Lilly MD (1971) Release of protein from baker's yeast by disruption in an industrial homogeniser. Trans Inst Chem Eng 49: 142–148Google Scholar
  14. Lode ET, Coon MJ (1973) Role of rubredoxin in fatty acid and hydrocarbon hydroxylation reactions. In: Lovenberg (ed) Iron-sulphur proteins, vol I, Biological properties. Academic Press, New York London, pp 173–191Google Scholar
  15. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265–275Google Scholar
  16. Matsushita K, Adachi O, Shinagawa E, Ameyama M (1978) Isolation and characterization of outer and inner membranes from Pseudomonas aeruginosa and effect of EDTA on membranes. J Biochem 83: 171–181Google Scholar
  17. McKenna EJ, Coon MJ (1970) Enzymatic ω-oxidation. IV Purification and properties of the ω-hydroxylase of Pseudomonas oleovorans. J Biol Chem 245: 3882–3889Google Scholar
  18. Repaske R (1956) Lysis of Gram-negative bacteria by lysozyme. Biochim Biophys Acta 22: 189–191Google Scholar
  19. Rogers SW, Gilleland HE Jr, Eagon RG (1969) Characterization of a protein-lipopolysaccharide complex released from cell walls of Pseudomonas aeruginosa by ethylenediaminetetra-acetic acid. Can J Microbiol 15: 743–748Google Scholar
  20. Ruettinger RT, Griffith GR, Coon MJ (1977) Characterization of the ω-hydroxylase of Pseudomonas olevorans as a non-heme iron protein. Arch Biochem Biophys 183: 528–537Google Scholar
  21. Tassin J-P, Celier C, Vandecastelle J-P (1973) Purification and properties of a membrane-bound alcohol dehydrogenase involved in oxidation of long-chain hydrocarbons by Pseudomonas aeruginosa. Biochim Biophys Acta 315: 220–232Google Scholar
  22. Tauchert H, Grunow M, Aurich H (1978) Regulation und einige Eigenschaften einer partikularen, akzeptorabhängigen Alkoholdehydrogenase aus Pseudomonas putida beim Wachstum auf n-Alkane. Z Allg Mikrobiol 18: 675–680Google Scholar
  23. Ueda T, Lode ET, Coon MJ (1972) Enzymatic ω-oxidation. VI Isolation of homogenous NADH: rubredoxin reductase. J Biol Chem 247: 2109–2116Google Scholar
  24. Van der Linden AC, Huybregtse R (1969) Occurence of inducible and NAD (P)-independant primary alcohol dehydrogenases in an alkane-oxidizing Pseudomonas. Antonie van Leeuwenhoek 35: 344–360Google Scholar

Copyright information

© Springer-Verlag 1983

Authors and Affiliations

  • Neville M. Fish
    • 1
  • Stuart Harbron
    • 1
  • David J. Allenby
    • 1
  • Malcolm D. Lilly
    • 1
  1. 1.Department of Chemical and Biochemical EngineeringUniversity College londonLondonEngland

Personalised recommendations