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D. Jendrossek, R. Handrick, and S. Reinhardt

Institut für Mikrobiologie der Universität Stuttart, Germany

PHB depolymerases can be distinguished by their substrate specificity for native (amorphous) PHB (nPHB; nPHB depolymerases) or for denatured (partially crystalline) PHB (dPHB; dPHB depolymerases. Many extracellular dPHA depolymerases (EC 3.1.1.75 and 3.1.1.76) have been characterized on the molecular level during the last decade but little is known about (intracellular) nPHA depolymerases. In was only until recently that up to 5 genes coding for putative intracellular nPHB depolymerases were found in Wautersia eutropha H16 (formerly Ralstonia eutropha). Intracellular nPHB depolymerases are not related to extracellular dPHB depolymerases with respect to amino acid sequence but share significant amino acid similarities between each other.

In this contribution we investigated nPHB depolymerases of W. eutropha and of Rhodospirillum rubrum. Both bacteria do not have any detectable extracellular PHB depolymerase activity. Fusions of selected depolymerase genes with variant genes of the green fluorescent protein (EGFP) were constructed and expression and localization of the depolymerases were followed fluorescence-microscopically. PhaZ1-EGFP was expressed in wild type H16 and in a phaZ1 deletion mutant during PHB accumulation phase and during starvation, i.e. PHB mobilization phase. Fluorescence microscopical analysis revealed that PhaZ1-EGFP colocalized with PHB granules. Our results confirmed earlier observations that PHB accumulation and PHB mobilization can occur simultaneously.

An unexpected and surprising result was obtained for putative intracellular PHB depolymerase of R. rubrum: PHB depolymerase PhaZ1 was located in the periplasm. Moreover, its amino acid sequence was unrelated to intracellular PHB depolymerases of W. eutropha but was significantly related to extracellular dPHB depolymerases. A second gene, phaZ2, was found in the R. rubrum genome that apparently encoded the true intracellular PHB depolymerase.

論文來(lái)源：International Symposium on Biological Polyesters ,Auguest 22-27, 2004