Characterization and partial purification of β-hydroxybutyrate dehydrogenase from sporulating cells of Bacillus cereus T
A soluble NAD+-dependent β-hydroxybutyrate (βHB) dehydrogenase was shown to appear 3 to 4 h after the onset of sporulation of Bacillus cereus T. The enzyme was stable in Tris-chloride buffer when frozen, but required 0.05 to 0.1 M of MgCl2 or other divalent cation such as Mn2+, Ba2+, or Ca2+ for stability at 4C. In the presence of phosphate buffer or EDTA, the enzyme lost all activity within 2 min. βHB dehydrogenase was partially purified and shown to have a molecular weight of about 93 000, pH optimum of 8.0 in 0.1 M Tris-chloride buffer, Michaelis constants, Km, of 2.3 × 10−3 M for β-hydroxybutyrate and 9.5 × 10−4 M for NAD+, and was inhibited 40% by 1 × 10−3 M p-hydroxymercuribenzoate. The enzyme from B. cereus T was compared in these respects with βHB dehydrogenases isolated from several non-sporeforming bacteria.