Purification and Some Properties of a Novel Ethanol Dehydrogenase with High Activity against Dulcitol 1-Phosphate from Salmonella Typhimurium IFO 12529.

Abstract

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 columnchromatographies on Blue-Cellulofine, Sephacryl S-300, and Zorbax GF-250. D1P-EDHwas 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-EDHin the extract. The molecular weight of D1P-EDHwas estimated to be 158,000 by gel filtration and 40,000 by SDS-polyacrylamide gel electrophoresis.D1P-EDHshowed its maximal activity in a pH range from 9.0 to 9.5. D1P-EDHwas stable in a pH range from 6.0 to 10.0 and was also stable at 30°C for 120min. 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-EDHactivity was strongly inhibited by /7-chloromercuribenzoic acid and NaN3though it was activated by Al3+, Ba2+, Ca2 +, andFe2+.