Intracellular depolymerase functionality and location in Pseudomonas oleovorans inclusions containing polyhydroxyoctanoate

Microbial poly-3-hydroxyoctanoate inclusion bodies produced by Pseudomonas oleovorans when grown on n-octanoic acid, are complex macromolecular structures consisting of polyester, organized paracrystalline lattice arrays and lipids. While it is known that the polymer in the granules maintains its native, amorphous state while it is surrounded by the components of this complex, the precise functions of the various components during polymer production and utilization have yet to be established. By utilizing electron microscopy, SDS-PAGE, and gel filtration chromatography along with in vitro assays for depolymerase activity, the present study demonstrates that a protein species with molecular weight of similar to 32 kDa is the depolymerase protein of the polymer inclusion. When exogenous carbon was exhausted, cell viability required utilization of the stored polyester. Under these conditions, the concentration of the depolymerase increased while the concentrations of the polymerase decreased. Thus, the association of the depolymerase with the granules was shown to be under metabolic regulation relative to the polymerase. The results from the present studies show that careful manipulation of the substrate concentration can selectively, and differentially, alter the level of inclusion associated proteins as well as the quantity and quality of the polyester which is accumulated.