u( -)-3-Hydroxybutyrate-dimer hydrolase from Zoogloea ramigera I-16-M was purified 7000-fold to electrophoretic homogeneity. The molecular weight of the purified enzyme was 28000 as determined by Sephadex G-100 gel filtration, and 30 000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric point was at pH 5.7. The pH optimum for the enzyme reaction was 8.0. The dimer hydrolase was stereospecific forIn addition, the purified hydrolase hydrolyzed several oligomeric esters Of D( -)-3-hydroxybutyric acid (DDD-trimer, DmD-tetramer and DDDDD-pentamer) faster than DD-dimer. Time course experiments with these oligomers and analysis of hydrolytic products of DmD-tetramer methyl ester with the hydrolase indicated that the enzyme attacked these substrates from the free hydroxyl terminus, releasing monomer units one at a time.The polymeric ester of D( -)-3-hydroxybutyric acid, poly(3-hydroxybutyrate), is accumulated by diverse species of bacteria as a principal intracellular reserve of organic carbon, and is digested when the external carbon supply becomes limited [I -31. However, owing to the extreme complexity of the initial stage of degradation, the enzymatic mechanism of poly(3-hydroxybutyrate) digestion in bacterial cells has not been fully clarified yet demonstrated that poly(3-hydroxybutyrate) granules isolated from Bacillus megaterium KM, which did not undergo self-digestion, were hydrolyzed by a depolymerizing enzyme system present in the soluble fraction of poly( 3-hydroxybutyrate)depleted Rhodospirillum rubrum cells, yielding D( -)-3-hydroxybutyrate as a main product and its dimeric ester (DD-dimer) as a minor product [4]. The latter product was further hydrolyzed to the monomer by a hydrolase (hydroxybutyrate-dimer hydrolase) partially purified from R. rubrum. However, this enzyme hydrolyzed the presumed trimeric ester of D( -)-3-hydroxybutyrate (DDD-trimer) faster than DD-dimer [7]. In contrast, a similar dimer hydrolase partially purified from Pseudomonas lemoignei did not attack DD-trimer to an appreciable extent [S].During studies on the pathway of poly(3-hydroxybutyrate) synthesis in Zoogloea ramigera I-16-M isolated from activated