Purification and Properties of d(−)‐3‐Hydroxybutyrate‐Dimer Hydrolase from Zoogloea ramigera I‐16‐M

Publisher Website
Google Scholar
Abstract
D(-)-3-Hydroxybutyrate dimer hydrolase from Z. ramigera I-16-M was purified 7000-fold to electrophoretic homogeneity. The MW of the purified enzyme was 28,000 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 for D(-)-3-[D(-)-3-hydroxybutyryloxy]butyric acid (DD-dimer) but also hydrolyzed D(-)-3-[L(+)-3-hydroxybutyryloxy]butyric acid (LD-dimer) at reduced rates. The enzyme did not attack L(+)-3-[L(+)-3-hydroxybutyryloxy]butyric acid (LL-dimer) at all. The purified hydrolase hydrolyzed several oligomeric esters of D(-)-3-hydroxybutyric acid (DDD-trimer, DDDD-tetramer and DDDDD-pentamer) faster than DD-dimer. Time course experiments with these oligomers and analysis of hydrolytic products of DDDD-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.