Purification and properties of beta-hydroxybutyrate dehydrogenase from Mycobacterium phlei ATCC354.

beta-Hydroxybutyrate dehydrogenase (EC 1.1.1.30) was purified 145-fold from Mycobacterium phlei ATCC354 by ammonium sulphate fractionation and DEAE-cellulose chromatography. The pH optima for oxidation and reduction reactions were 8.4 and 6.8 respectively. The purified enzyme was specific for NAD, NADH, acetoacetate and D(-)-beta-hydroxybutyrate. Km values for DL-beta-hydroxybutyrate and NAD were 7.4 mM and 0.66 mM respectively. The enzyme was inactivated by mercurial thiol inhibitors and by heat, but could be protected by NADH, Ca2+ and partially by Mn2+. The enzyme did not require metal ions and was insensitive to EDTA, glutathione, dithiothreitol, beta-mercaptoethanol and cysteine.