INTRACELLULAR DEPOLYMERASE FUNCTIONALITY AND LOCATION IN PSEUDOMONAS-OLEOVORANS INCLUSIONS CONTAINING POLYHYDROXYOCTANOATE

Microbial poly-3-hydroxyoctanoate inclusion bodies produced by Pseudom onas oleovorans when grown on n-octanoic acid, are complex macromolecu lar structures consisting of polyester, organized paracrystalline latt ice arrays and lipids. While it is known that the polymer in the granu les maintains its native, amorphous state while it is surrounded by th e components of this complex, the precise functions of the various com ponents during polymer production and utilization have yet to be estab lished. 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 w eight of similar to 32 kDa is the depolymerase protein of the polymer inclusion. When exogenous carbon was exhausted, cell viability require d utilization of the stored polyester. Under these conditions, the con centration of the depolymerase increased while the concentrations of t he polymerase decreased. Thus, the association of the depolymerase wit h 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 diff erentially, alter the level of inclusion associated proteins as well a s the quantity and quality of the polyester which is accumulated.