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 (PhaZMsp) 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. PhaZMsp was stable at temperatures below 37 degrees 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. PhaZMsp was capable of hydrolyzing P(3HB), poly(3-hydroxypropionate), and poly(4-hydroxybutyrate).