THERMOSTABLE POLY(3-HYDROXYBUTYRATE) DEPOLYMERASE OF A THERMOPHILIC STRAIN OF LEPTOTHRIX SP. ISOLATED FROM A HOT-SPRING

A thermophilic bacterium that can grow on poly(3-hydroxybutyrate) (PHB ) was isolated from a hot spring. The Isolate, designated strain HS, w as a gram-negative aerobic rod and grew optimally at 45-50 degrees C. From its principal phenotypic characteristics, strain HS was revealed to be closely related to Leptothrix discophora. Sequence analysis of t he 16S rRNA gene also supported this result. PHB depolymerase was puri fied to electrophoretic homogeneity from cell-free culture broth by hy drophobic interaction chromatography using Toyopearl HW-55F in combina tion with ammonium sulfate precipitation. The molecular weight of the enzyme was calculated to be 4.6 x 10(4) by electrophoresis and 4.3 x 1 0(4) by gel filtration. The enzyme was stable up to 65 degrees C and t he optimum reaction temperature was 70 degrees C. A K-m of 0.018 g/l w as determined for PHB at 70 degrees C. An isoelectric point of pH 8.6 was obtained by isoelectric focusing. The enzyme activity was inhibite d by dithiothreitol but not by phenylmethylsulfonyl fluoride. The N-te rminal amino acid sequence was unlike that of any other bacterial PHB depolymerase. Secretion of the enzyme was accelerated by the addition to the culture medium of 3-hydroxybutyrate which is the product of the enzyme.