The activator of the Rhodospirillum rubrum PHB depolymerase is a polypeptide that is extremely resistant to high temperature (121 degrees C) and other physical or chemical stresses.

Hydrolysis of native (amorphous) polyhydroxybutyrate (nPHB) granules isolated from different sources by soluble PHB depolymerase of Rhodospirillum rubrum in vitro requires the presence of a heat-stable compound (activator). The activator was purified and was resistant against various physical and chemical stresses such as heat (up to 130 degrees C), pH 1-12, dryness, oxidation by H2O2, reducing and denaturing compounds (2-mercaptoethanol, 5 M guanidinium-HCl) and many solvents including phenol/chloroform. The activator coding gene was identified by N-terminal sequencing of the purified protein, and the deduced protein showed significant homology to magnetosome-associated protein (Mms16) of magnetotactic bacteria. Analysis of the activation process in vitro showed that the activator acts on nPHB granules but not on the depolymerase. The effect of the activator could be mimicked by pretreatment of nPHB granules with trypsin or other proteases but protease activity of the purified activator was not detected. Evidence is shown that different mechanisms were responsible for activation of nPHB by trypsin and activator, respectively. PHB granule-associated protein (PhaP) of Ralstonia eutropha nPHB granules were cleaved by trypsin but no cleavage occurred after activator treatment. Hydrolysis of artificial protein-free PHB granules coated with negatively charged detergents (sodium dodecyl sulfate (SDS), cholate but not cetyltrimethyl-ammonium bromide (CTAB)) did not require activation and confirmed that surface layer proteins of nPHB granules are the targets of the activator rather than lipids. All experimental data are in agreement with the assumption that trypsin and the activator enable the PHB depolymerase to find and to bind to the polymer surface: trypsin by removing a portion of proteins from the polymer surface, the activator by modifying the surface structure in a not yet understood manner presumably by interaction with phasins of the proteinous surface layer of nPHB.