Identification of a Marine Benthic P(3HB)-Degrading Bacterium Isolate and Characterization of Its P(3HB) Depolymerase

A poly[(R)-3-hydroxybutyrate] (P(3HB))-degrading marine bacterium (strain NK-1, JCM10458) was isolated from the Pacific Ocean deep-sea floor (1165 m in depth) in Japan. The organism was a motile and Gram negative, aerobic, and rod-shaped bacterium, and its DNA had a guanine-plus-cytosine content of 57.7 mol %. On the basis of several phenotypic characters and a phylogenetic analysis of the gene coding for 16S rRNA, this strain was identified as Marinobacter sp. The strain required sodium salt for growth in the medium and secreted a P(3HB) depolymerase into the supernatant when it was cultivated on (S)-3-hydroxybutyric acid or P(3HB) as the sole carbon source. The P(3HB) depolymerase (PhaZ M sp) was purified to homogeneity from the culture supernatant of Marinobacter sp. by hydrophobic and ion exchange column chromatography and showed a molecular mass of 70 kDa. PhaZ M sp was stable at temperatures below 37 °C and at pH values of 7.5−10.0. The N-terminal amino acid sequences of both the purified enzyme and the truncated one shared high homologies to the N-terminal and internal sequences of Pseudomonas stutzeri depolymerase, respectively. High-performance liquid chromatography analysis revealed that the enzymatic products of P(3HB) yielded monomer, dimer, and trimer of 3-hydroxybutyric acid. PhaZ M sp was capable of hydrolyzing P(3HB), poly(3-hydroxypropionate), and poly(4-hydroxybutyrate).