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

Abstract

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

NAD(H)

nicotinamide adenine dinucleotide (reduced)

NADP(H)

nicotinamide adenine dinucleotide phosphate (reduced)

DTE

dithioerythritol

PMS

phenazine methosulfate

NBT

nitro blue tetrazolium chloride

DMSO

dimethyl sulfoxide

DCPIP

dichlorophenolindophenol

MTT

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

TTC

triphenyltetrazolium chloride

FAD

flavin adenine dinucleotide

FMN

flavin mononucleotide

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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

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