Isolation and purification of the D(–)β-hydroxybutyric dehydrogenase of Azotobacter vinelandii

It has been possible to isolate and purify the D(–)β-hydroxybutyric dehydrogenase from cell-free extracts of Azotobacter vinelandii. Standardized resting cell suspensions were disrupted by sonic oscillation, and purification of the enzyme was achieved by use of protamine sulfate, ammonium sulfate, and hydroxyapatite or by direct use of a Svensson-Porath preparatory column electrophoretic unit. The D(–)β-hydroxybutyric dehydrogenase was found to be a classical, soluble, NAD+-dependent dehydrogenase and neither bound nor associated with any intracellular membranous structure. The highest specific activity achieved was 17 μmoles NAD+ reduced per minute per milligram protein at 37°. Two unusual features were noted with this dehydrogenase: (a) loss of activity occurred when the enzyme was dialyzed in the absence of metal ion; and (b) marked stability was observed when the enzyme was exposed to alkaline pH even at 40 °C for hours.