PURIFICATION AND PARTIAL CHARACTERIZATION OF A D(-)-LACTATE DEHYDROGENASE FROM DESULFOVIBRIO-DESULFURICANS (ATCC 7757)

A membrane-bound d(-)-lactate dehydrogenase (LDH), an important enzyme in carbon and energy metabolism in sulfate-reducing bacteria of the genus Desulfovibrio, was solubilized from the membrane fraction of Desulfovibrio desulfuricans (ATCC 7757). The enzyme was purified 84-fold to a final specific activity of 525 nmol DCPIP-reduced/min/mg protein by ammonium sulfate precipitation, chloroform extraction, gel filtration with Sephadex G-150, and hydrophobic column chromatography with N-octylamine Sepharose 4B. The enzyme eluted off a Sephacryl S-300 column as a single peak with a molecular weight of 400 000±40 000 Da. Denaturing gel electrophoresis showed it to be composed of 5 protein bands. The oxidized and dithionite reduced spectra of LDH resembles the spectra of c-type cytochromes found in Desulfovibrio species. The addition of lactate to LDH resulted in a partially reduced spectrum. The flavin/cytochrome c/non-heme iron content per 400 000 Da LDH molecular weight was found to be 1:1.6:4.5. The LDH activity was specific for d(-)-lactate and had a Km for d(-)-lactate of 4.3×10-4 M. The pH optimum was between 6.5 and 8.5. © 1990 Society for Industrial Microbiology.