| Research Abstract |
Several bacteria which degrade bacterial polyester (PHB) and also perform denitrification were isolated. We attempted the isolation of extracellular PHB depolymerases and cloning of their genes, but failed. When small fish (gold fish) were kept in the presence of PHB, a considerable reduction of nigrogen compounds in water was observed. These results indicate that the important role of the bacteria with ability of both PHB-degradation and denitrification in environment. On the other hand, we coned the gene of an intracellular PHB depolymerase (phaZl) from Ralstonia eutropha H16 and charaterization its product (PhaZ1). The amino acid sequence of PhaZ1 was novel. The molecular mass of PhaZ1 was 47 Kda. PhaZ1, which localized solely in PHB inclusions, degraded artificial amorphous PHB granules, but not crystalline PHB granules. We further cloned several intracellular 3-hydroxybutyrate-oligomer hydrolases. One of them, an intracellular 3HB-oligomer hydrolase (PhaZ2) from R. eutropha, showed both 3HB-oligomer hydrolase and PHB depolymerase activities, PhaZ2 localizedd both in cytosol and PHB inclusions, The amounts of PhaZ1 and PhaZ2 increased with the content of PHB in cells. The null mutant lacking both PhaZ1and PhaZ2 revealed the increase PHB deposition in cells. These results indicate that PhaZ1 and PhazZ2 cooperate in intracellular degradation of PHB
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