IN-VITRO ACTIVITIES OF GRANULE-BOUND POLY[(R)-3-HYDROXYALKANOATE] POLYMERASE C1 OF PSEUDOMONAS-OLEOVORANS - DEVELOPMENT OF AN ACTIVITY TESTFOR MEDIUM-CHAIN-LENGTH-POLY(3-HYDROXYALKANOATE) POLYMERASES

A newly developed in vitro activity assay for medium-chain-length(mcl) -poly(3-hydroxyalkanoate) polymerases is described. Polymerase C1 of P seudomonas oleovorans GPo1 attached to isolated granules was used as m odel enzyme. A direct correlation was found between (R)-3-hydroxyoctan oylCoA depletion and poly(3-hydroxyalkanoate) synthesis due to polymer ase C1 activity. Highest activities of 1.13 U/mg granule bound protein and highest specific activities of 2.3 U/mg polymerase C1 were determ ined towards (RS)-3-hydroxyoctanoylCoA. A first determination of a K-m value for mel poly(3-hydroxyalkanoate) polymerases was pet-formed lea ding to an estimated K-m of 0.16 (+/-0.1) mM for granule bound polymer ase C1 with (R)-3-hydroxyoctanoylCoA as substrate. Polymerase C1 showe d no activity towards (RS)-3-hydroxybutyrylCoA and a specific activity of 0.28 U/mg polymerase C1 for (R)-3-hydroxyvalerylCoA. (R)-3-Hydroxy octanoylCoA and a mixture of (RS)-3-hydroxyoctanoylCoA were both deple ted for more than 75% by granule-bound polymerase C1, suggesting a non -rate-limiting epimerase activity attached to poly(3-hydroxyalkanoate) granules isolated from Pseudomonas putida GPp104[pGEc405]. Whereas no relationship was found between the activity of granule-bound polymera se C1 and poly(3-hydroxyalkanoate) content of the granules, higher act ivities were measured when a higher substrate concentration or more en zyme was present in the in vitro activity assay.