PURIFICATION AND PROPERTIES OF ENZYMES INVOLVED IN THE PROPIONIC ACID FERMENTATION

Abstract

Chromatographic procedures are described for the separation and purification of phosphotransacetylase, acetyl-kinase, malic-dehydrogenase and coenzyme-A (CoA)-transferase. Purity of the enzymes was judged by homogeneity in an ultracentrifuge and by specific activity. Phosphotransacetylase was obtained 85% pure with a specific activity of 27.1. The preparation of acetyl-kinase was a homogeneous protein with a specific activity of 531. The malic-dehydrogenase likewise was homogeneous with a specific activity of 938. The CoA-transferase, which was about 56% pure with a specific activity of 42.6, is the purest preparation of this enzyme yet described. The pH optimum was 6.5 to 7.8, and the Km for succinyl-CoA in the transfer of CoA to acetate was found to be 1.3 x 10-4 [image]; for acetate, in the same transfer, the Km was 7.0 x 10-3 [image]; for succinyl-CoA to propionate in was 6.8 x 10-5 [image], and for propionate, in the same reaction, 6.2 x 10-4 [image]. Methods are described for the enzymatic production of methyl-nalonyl-CoA, malonyl-CoA, propionyl-CoA, acetyl-CoA and succinyl-CoA. The role of these enzymes in the propionic-acid fermentation as well as the possible mechanism responsible for the high yields of adenosine-triphosphate from glucose are considered.