Studies on Acetoacetate Decarboxylase by the Polyacrylamide Gel Disc Electrophoresis : Activity Staining Method of Acetoacetate Decarboxylase

The activity staining method for acetoacetate decarboxylase (EC4. 1. 1. 4) was developed by the use of acrylamide gel disc electrophoresis. The gel was immersed in a buffer solution of a pH indicator dye of pH 5.0. It was then incubated in the substrate solution. As the pH changes on the enzyme band due to the decomposition of acetoacetate to acetone and carbon dioxide, an alkaline colored band appeared in the acid colored background of the gel. pH indicator dyes tested were BTB, CR, PR, NP, MR and PP. Among them, BTB was most sensitive and MR and PP were ineffective. The activity staining method described here was applied to the purification steps of the acetoacetate decarbozylase and was proven to be useful. Culture method of Clostridium acetobutylicum and purification of the enzyme from it were also discussed.