Partial purification and resolution of acetoacetic decarboxylase.

Commerical interest in the acetone-butanol fermentation has prompted considerable investigation into methods of solvent production. The bulk of the work on the subject, however, has been concerned chiefly with methods of increasing solvent yields, whereas relatively few studies have dealt with the intermediary metabolism involved. Johnson, Peterson, and Fred (1933) first demonstrated clearly the enzymatic decarboxylation of acetoacetic acid to form acetone. Davies (1942, 1943), employing the method of manometric measurement, obtained a cell-free preparation of the decarboxylase and purified it in several steps including adsorption on alumina C-y, elution, and fractional precipitation by ammonium sulfate and by ethyl alcohol. He showed further that this purified enzyme preparation when diluted sufficiently would partially dissociate into the component apoenzyme and coenzyme. The presence in diaphorase of a factor that acted as the coenzyme in stimulating the decarboxylation of acetoacetic acid by this partially resolved enzyme, the stability of this factor to boiling and to acid hydrolysis, and its inactivation by alkaline hydrolysis, led Davies to postulate that the coenzyme for acetoacetic decarboxylation might be riboflavin phosphate. Prior to our efforts to characterize this coenzyme, a different method of purification and a more effective method of resolution of the acetoacetic decarboxylase was found and is described here.