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)₂SO₄ precipitation 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)₂SO₄ precipitate 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-poIyacrylamide 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 DIP and oxidized much more ethanol than DIP. D1P-EDH activity was strongly inhibited by p-chloromercuribenzoic acid and NaN₃ though it was activated by Al³⁺, Ba²⁺, Ca²⁺, and Fe²⁺.