PARTIAL-PURIFICATION AND CHARACTERIZATION OF BETA-HYDROXYBUTYRIC ACID DEHYDROGENASE OF A METHYLOTROPHIC BACTERIUM, PSEUDOMONAS-135

Abstract

An intracellular enzyme, D(-)-beta-hydroxybutyric acid dehydrogenase involved in an intracellular poly-D(-)-beta-hydroxybutyric acid degradation was isolated from a facultative methylotrophic bacterium, Pseudomonas 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to 11.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation Of D(-)-beta-hydroxybutyric acid (Dbeta-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5-6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at -20-degrees-C. The K(m) values for oxidation and reduction reactions were determined as 1.84 mm for Dbeta-HB, 0.244 mm for NAD+, 0. 319 mm for acetoacetate and 0.032 mm for NADH, respectively. It was also found that D-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mM for D-lactate, 0.196 mM for NADH and 1.82 mM for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive.