Archives of Microbiology

, Volume 152, Issue 3, pp 244–250

Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum

  • B. Meinecke
  • J. Bertram
  • G. Gottschalk
Original Papers


The pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum was purified to homogeneity and partially characterized. A 9.2-fold purification was achieved in a three step purification procedure: ammonium sulfate fractionation, chromatography on Phenyl Sepharose and on Procion Blue H-EGN12. The pure enzyme exhibited a specfic activity of 25 U/mg of protein. Homogeneity of the pyruvate-ferredoxin oxidoreductase was confirmed by native polyacrylamide gel electrophoresis and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. The molecular weight was determined to be 123,000/monomer. The subunit composition of the native enzyme could not be determined because of the instability of the pure enzyme. The pyruvate-ferredoxin oxidoreductase is sensitive to oxygen and dilution during purification. The dilution inactivation could be partially overcome by the addition of 300 μM coenzyme A or 50% ethyleneglycol. A thiamine pyrophosphate content of 0.39 mol per mol of enzyme monomer was found, the iron and sulfur content was 4.23 and 0.91, respectively. The pH-optimum was at pH 7.5 and the temperature optimum was at 60°C. Kinetic constants were measured in the forward reaction. The apparent Km for pyruvate and coenzyme A were 322 μM and 3.7 μM, respectively. With 2-ketobutyrate the pyruvate-ferredoxin oxidoreductase showed 12.5% of the activity compared to pyruvate. No activity was found with 2-ketoglutarate. Ferredoxin from Clostridium pasteurianum could be used as physiological electron acceptor.

Key words

Clostridium acetobutylicum Acetone-butanol fermentation Lactate co-metabolism Pyruvateferredoxin oxidoreductase 

Non-standard abbreviations


nicotinamide adenine dinucleotide (reduced)


nicotinamide adenine dinucleotide phosphate (reduced)




phenazine methosulfate


nitro blue tetrazolium chloride


dimethyl sulfoxide




3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl-tetrazolium bromide


triphenyltetrazolium chloride


flavin adenine dinucleotide


flavin mononucleotide


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andersch W, Bahl H, Gottschalk G (1983) Level of enzymes involved in acetate, butyrate, acetone and butanol formation by Clostridium acetobutylicum. Eur J Appl Microbiol Biotechnol 18:327–332Google Scholar
  2. Atkinson T, Hammond PM, Hartwell RD, Hughes P, Scawen MD, Sherwood RF, Small DAP, Bruton CJ, Harrey MJ, Lowe CR (1981) Triazine-dye ligand chromatography. Biochem Soc Trans 9:290–293Google Scholar
  3. Bahl H, Andersch W, Gottschalk G (1982) Continuous production of acetone and butanol by C. acetobutylicum in a two-stage phosphate limited chemostat. Eur J Appl Microbiol Biotechnol 15:201–205Google Scholar
  4. Bahl H, Gottwald M, Kuhn A, Rale V, Andersch W, Gottschalk G (1986) Nutritional factors affecting the ratio of solvents produced by Clostridium acetobutylicum. Appl Environ Microbiol 52:169–172Google Scholar
  5. Bergmeyer HU (1974) Methoden der enzymatischen Analyse. Verlag Chemie, WeinheimGoogle Scholar
  6. Bryant MP (1972) Commentary on the HUNGATE-technique for culture of anaerobic bacteria. Am J Clin Nutr 25:1324–1328Google Scholar
  7. Bush RS, Sauer FD (1976) Enzymes of 2-oxo-acid degradation and biosynthesis in cell-free extracts of mixed rumen microorganism. Biochem 157:325–331Google Scholar
  8. Drake HL, Shou-Ih H, Wood HG (1981) Purification of five components from Clostridium thermoaceticum which catalyze synthesis of acetate from pyruvate and methyltetrahydrofolate. J Biol Chem 256:11137–11144Google Scholar
  9. Dürre P, Kuhn A, Gottwald M, Gottschalk G (1987) Enzymatic investigations on butanol dehydrogenase and butyraldehyde dehydrogenase in extracts of Clostridium acetobutylicum. Appl Microbiol Biotechnol 26:268–272Google Scholar
  10. Friedemann TE, Haugen GE (1943) Pyruvic acid II. The determination of keto acids in blood and urine. J Biol Chem 147:415–442Google Scholar
  11. Gehring U, Arnon DI (1972) Purification and properties of aketoglutarate synthase from a photosynthetic becterium. J Biol Chem 247:6963–6969Google Scholar
  12. Hartmanis MGN, Gatenbeck S (1984) Intermediary metabolism in Clostridium acetobutylicum: levels of enzymes in the formation of acetate and butyrate. Appl Environ Microbiol 47:1277–1283Google Scholar
  13. Hungate RE (1969) A role tube method for cultivation of strict anaerobes. In: Norris JR, Ribbons DW (eds) Methods in microbiology, vol 3b. Academic Press, London, pp 117–132Google Scholar
  14. Keech DB, Wallace JC (1985) Pyruvate carboxylase. CRC Press, Boca Raton, FloridaGoogle Scholar
  15. Kerscher L, Oesterhelt D (1981a) Purification and properties of two 2-oxo-acid: ferredoxin oxidoreductases from Halobacterium halobium. Eur J Biochem 116:587–594Google Scholar
  16. Kerscher L, Oesterhelt D (1981b) The catalytic mechanism of 2-oxo-acid: ferredoxin oxidoreductases from Halobacterium halobium. Eur J Biochem 116:595–600Google Scholar
  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  18. Levin LM, Wei R (1966) Microassay of thiamine and its phosphate esters after separation by paper chromatography. Anal Biochem 16:29–35Google Scholar
  19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folinphenol reagent. J Biol Chem 193:265–275Google Scholar
  20. Marczak R, Ballongue J, Petitdemange H, Gay H (1984) Regulation of the biosynthesis of NADH-rubredoxin oxidoreductase in Clostridium acetobutylicum. Curr Microbiol 10:165–168Google Scholar
  21. Ogata M, Yagi T (1986) Pyruvate dehydrogenase and the lactate degradation in Desulfovibrio vulgaris Miyazaki F1. J Biochem 10:311–318Google Scholar
  22. O'Brien RW, Morris JG (1971) Oxygen and the growth and metabolism of Clostridium acetobutylicum. J Gen Microbiol 68:307–318Google Scholar
  23. Palosaari NR, Rogers P (1988) Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum. J Bacteriol 170:2971–2976Google Scholar
  24. Petitdemange H, Cherrier C, Raval G, Gay R (1976) Regulation of the NADH and NADPH-ferredoxin oxidoreductases in clostridia of the butyric group. Biochim Biophys Acta 421:334–347Google Scholar
  25. Petidemange H, Desbor J, Maugras M (1969) Etude de la formation du n-butanol chez Clostridium acetobutylicum. Bull Soc Chem Biol 51:157–165Google Scholar
  26. Petitdemange H, Marczak R, Blusson H, Gay R (1979) Isolation and properties of reduced adenine dinucleotide rubredoxin oxidoreductase of Clostridium acetobutylicum. Biochim Biophys Res Commun 91:1258–1265Google Scholar
  27. Raeburn S, Rabinowitz JC (1971a) Pyruvate:ferredoxin oxidoreductase I. The pyruvate CO2 exchange reaction. Arch Biochem Biophys 146:9–20Google Scholar
  28. Richards EG, Coll JA (1965) Disc electrophoresis of ribonucleic acid in polyacrylamide gels. Anal Biochem 12:452–471Google Scholar
  29. Sauer FD, Bush RS, Stevenson IL (1976) The separation of pyruvate:ferredoxin oxidoreductase from Clostridium pasteurianum into two enzymes catalyzing different reactions. Biochim Biophys Acta 445:518–520Google Scholar
  30. Suhara K, Takemon S, Katagiri M, Wada K, Kobayashi H, Matsubara H (1975) Estimation of labile sulfide in iron-sulfurproteins. Anal Biochem 68:632–636Google Scholar
  31. Trinder P (1956) The improved determination of iron in serum. J Clin Path 9:170Google Scholar
  32. Uyeda K, Rabinowitz JC (1971a) Pyruvate:ferredoxin oxidoreductase III. Purification and properties of the enzyme. J Biol Chem 246:3111–3119Google Scholar
  33. Uyeda K, Rabinowitz JC (1971b) Pyruvate:ferredoxin oxidoreductase IV. Studies on the reaction mechanism. J Biol Chem 246:3120–3125Google Scholar
  34. Wahl RC, Orme-Johnson WH (1987) Clostridial pyruvate oxidoreductase and the pyruvate-oxidizing enzyme specific to nitrogen fixation in Klebsiella pneumoniae are similar enzymes. J Biol Chem 262:10489–10496Google Scholar
  35. Weber K, Osborn M (1969) The reliability of molecular weight determination by dodecylsulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412Google Scholar
  36. Zehnder AJB, Wuhrmann K (1976) Titan-(III)-citrate as a nontoxic oxidation-reduction buffering system for the culture of obligate anaerobes. Science 194:1165–1166Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • B. Meinecke
    • 1
  • J. Bertram
    • 1
  • G. Gottschalk
    • 1
  1. 1.Institut für MikrobiologieUniversität GöttingenGöttingenFederal Republic of Germany
  2. 2.Fa. Seratec Gesellschaft für Biotechnologie mbHGöttingenFRG

Personalised recommendations