The Oxalacetate Decarboxylase of Azotobacter Vinelandii

In 1941 Krampitz and Werkman (1) discovered an enzyme in Micrococcus lysodeikticus which catalyzed the decarboxylation of oxalacetate.By means of an exchange reaction they were able to demonstrate the incorporation of isotopic carbon dioxide into the p-carboxyl group of oxalacetic acid in the presence of this enzyme (2).Similar results were obtained by Evans, Vennesland, and Slotin (3) with an enzyme system from pigeon liver.Further studies on the C+Cl condensation have been reviewed by Wood (4) and will, therefore, not be discussed here.The apparent importance of oxalacetate decarboxylase as one of the gateway enzymes for the fixation of carbon dioxide into compounds of the Krebs isocitric acid cycle made it of interest to attempt to purify this enzyme and to study some of its properties.The biotin content of the enzyme fractions at various stages of purity was determined in view of the recent implication of biotin in the metabolism of carbon dioxide (5-11).Lee, Burris, and Wilson (12) demonstrated that cell-free preparations of Azotobacter vinelandii have oxalacetate decarboxylase activity.Since this organism is easily grown on a large scale (13), cell-free extracts of this bacterium were used as the source material for this study. EXPERIMENTAL PuriJication of EnzymeAzotobacter vinelandii was grown on the nitrogen-free medium of Lee and Burris (13) in 259 liter quantities with vigorous agitation and aeration at 30" in 100 gallon iron tanks.After 24 hours the cells were harvested with a Sharples centrifuge.Usually 400 to 600 gm. of cell paste were obtained.The bacterial centrifugate was suspended with vigorous agitation in 6 liters of acetone at 5".The suspension was filtered with suction.The filter cake, still wet with acetone, was resuspended in 6 liters of cold acetone with vigorous agitation.After titration with suction the residue was placed in a vacuum desiccator containing calcium chloride and shaved