In vitroActivities of Granule‐Bound Poly[(R)‐3‐Hydroxyalkanoate] Polymerase C1 ofPseudomonas oleovorans

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Abstract
A newly developed in vitro activity assay for medium‐chain‐length(mcl)‐poly(3‐hydroxyalkanoate) polymerases is described. Polymerase C1 of Pseudomonas oleovorans GPo1 attached to isolated granules was used as model enzyme. A direct correlation was found between (R)‐3‐hydroxyoctanoylCoA depletion and poly(3‐hydroxyalkanoate) synthesis due to polymerase C1 activity. Highest activities of 1.13 U/mg granule bound protein and highest specific activities of 2.3 U/mg polymerase C1 were determined towards (RS)‐3‐hydroxyoctanoylCoA. A first determination of a Km value for mcl poly(3‐hydroxyalkanoate) polymerases was performed leading to an estimated Km of 0.16 (± 0.1) mM for granule bound polymerase C1 with (RS)‐3‐hydroxyoctanoylCoA as substrate. Polymerase C1 showed no activity towards (RS)‐3‐hydroxybutyrylCoA and a specific activity of 0.28 U/mg polymerase C1 for (R)‐3‐hydroxyvalerylCoA. (R)‐3‐HydroxyoctanoylCoA and a mixture of (RS)‐3‐hydroxyoctanoylCoA were both depleted 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 polymerase C1 and poly(3‐hydroxyalkanoate) content of the granules, higher activities were measured when a higher substrate concentration or more enzyme was present in the in vitro activity assay.

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