Polyhydroxyalkanoate (PHA) is a linear, optically active polyester which is a widespread intracellular storage compound among prokaryotic organisms. Due to their biodegradable and thermoplastic properties, PHAs have attracted considerable attention for many years. Pseudomonads generally synthesize medium chain length PHAs (mcl-PHAs) formed of monomers of 6-14 carbons. To date, more than 100 different monomers have been found in PHA in which the side chains contain a wide variety of functional groups [1].

PHA polymerases are the key enzymes of the polymerisation reaction which use CoenzymeA activated monomers as substrates. Due to the broad substrate specificity of mcl-PHA polymerases, there is an increasing interest for the in vitro production of novel polymers. Pseudomonas oleovorans GPo1 contains two different PHA polymerases encoded by phaC1 and phaC2 [2]. These PHA polymerases are attached to PHA granules and were partially purified by isolation of the PHA granules. Protein analysis of these isolated PHA granules revealed that besides the PHA polymerase, several other proteins were associated to the PHA granules. Activity measurements of the granule-bound polymerases revealed an activity of 5.8 U/mg. However, when the PHA polymerases were purified to homogeneity (via release from the granule by incubation with mild detergents), activity was reduced at least 10-fold.

Focus is now set on the purified PHA polymerase which will be examined for re-activation. Furthermore, kinetic studies will be done in order to get mechanistic insight of the priming-initiation, elongation and termination reactions. These data, together with substrate specificity studies will increase our understanding of the PHA polymerisation process.

[1] Steinbüchel, A. and Valentin, H. E. FEMS Microbiol. Lett. 128, 219-228, 1995.

[2] Huisman, G. W., Wonink, E., Meima R., Kazemier, B., Terpstra, P and Witholt, B. J. Biol. Chem. 266, 2191-2198, 1991.