Purification and properties of D-3-hydroxybutyrate dehydrogenase from Paracoccus denitrificans

D-3-Hydroxybutyrate dehydrogenase from Paracoccus denitrificans has been purified to near homogeneity. The enzyme was prepared using DEAE-cellulose chromatography, affinity chromatography on immobilized Cibacron blue (Matrex Gel Blue A) and gel permeation chromatography. The pure enzyme was obtained by chromatofocusing as the final isolation step. The purification procedure yielded the enzyme with a specific activity of about 100 units/mg protein. The enzyme is specific for D-3-hydroxybutyrate and NAD and it exhibits anomalous kinetics (hysteresis) at low enzyme and coenzyme concentrations. It is relatively stable in the presence of EDTA at pH 7–8 higer salt concentrations. D-3-Hydroxybutyrate dehydrogenase is a tetramer with a molecular weight of 130 000 ± 10 000, its isoelectric point equals 5.10 ± 0.05. The enzyme is applicable to the determination of acetoacetate and D-3-hydroxybutyrate concentrations.