Ralstonia eutropha H16の新規細胞内3-ビドロキシ酪酸オリゴマー分解酵素(2004年度神奈川大学総合理学研究所共同研究助成論文)

Ralstonia eutropha H16 mobilizes intracellularly accumulated poly(3-hydroxybutyrate) with intracellular poly(3-hydroxybutyrate) depolymerases. Different types of depolymerase, PhaZ1 and its homologs, account for the mobilization significantly but incompletely, and it is proposed that further unknown depolymerases or related enzymes complement the mobilization. A novel hydrolase was therefore investigated. The amino acid sequence of the intracellular 3HB-oligomer hydrolase of Acidovoraxsp. SA1 and the genome sequence of Ralstonia solanacearum, a close relative of R. eutropha, revealed a candidate for the hydrolase gene. The DNA sequence of its hydrolase was used for the design of a pair of primes. With chromosomal DNA of R. eutropha as a template, these primers were used to amplify about a 1.3-kbp fragment including the equivalent gene, phaZc. Then the phaZc gene was cloned by Southern hybridization using the amplified DNA fragment as a probe. The hydrolase (PhaZc) was purified from Escherichia coli overexpressing phaZc and characterized. Immunoblot analysis revealed that PhaZc exists mainly in the cytosolic fraction of R. eutropha. PhaZc degraded various 3-hydroxybutyrate-oligomers at a high rate and artificial amorphous poly (3-hydroxybutyrate) at a lower rate. A phaZ-rmLl mutation enhanced the deposition of PHB in the logarithmic phase in a nutrient-rich medium. PhaZc differs from the previously reported hydrolases in R. eutropha and is a novel type of intracellular 3HB-oligomer hydrolase, which participates in the mobilization of PHB along with other hydrolases.