Purification and some properties of a novel ethanol dehydrogenase with high activity against dulcitol 1-phosphate from Salmonella typhimurium IFO 12529

A novel ethanol dehydrogenase with high activity against dulcitol 1-phosphate (D1P-EDH) was purified from Salmonella typhimurium IFO 12529 grown in a medium containing dulcitol as a carbon source. D1P-EDH was purified from a crude extract of S. typhimurium cells by (NH₄)₂SO₄precipitation and column chromatographies on Blue-Cellulofine, Sephacryl S-300, and Zorbax GF-250. D1P-EDH was purified 277-fold with an activity yield of 21.3%. The purified preparation gave a single band on an electrophoregram. The activity staining of the electrophoregram of the (NH₄)₂SO₄precipitate indicated that there was no isozyme of D1P-EDH in the extract. The molecular weight of D1P-EDH was estimated to be 158, 000 by gel filtration and 40, 000 by SDS-polyacrylamide gel electrophoresis. D1P-EDH showed its maximal activity in a pH range from 9.0 to 9.5. D1P-EDH was stable in a pH range from 6.0 to 10.0 and was also stable at 30°C for 120 min. The purified preparation oxidized fructose 6-phosphate and galactose 6-phosphate to the same extent as D1P and oxidized much more ethanol than D1P. D1P-EDH activity was strongly inhibited by p-chloromercuribenzoic acid and NaN₃though it was activated by Al^{3 +}, Ba^{2 +}, Ca^{2 +}, and Fe^{2 +}.