PMID: 9428695Jan 15, 1998Paper

In vitro activities of granule-bound poly[(R)-3-hydroxyalkanoate]polymerase C1 of Pseudomonas oleovorans--development of an activity test for medium-chain-length-poly(3-hydroxyalkanoate) polymerases

European Journal of Biochemistry
M N KraakB Witholt

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 (R)-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-bou...Continue Reading

References

Dec 1, 1980·Proceedings of the National Academy of Sciences of the United States of America·G DittaD R Helinski
Jul 3, 1995·Proceedings of the National Academy of Sciences of the United States of America·T U Gerngross, D P Martin
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Citations

Jul 23, 1999·International Journal of Biological Macromolecules·K SudeshY Doi
Mar 14, 2001·Journal of Biotechnology·B Kessler, B Witholt
Jun 11, 1999·Current Opinion in Biotechnology·B Witholt, B Kessler
Jun 17, 2009·Analytical Biochemistry·Der-Shyan SheuWen-Ming Chen
Mar 6, 1999·Microbiology and Molecular Biology Reviews : MMBR·L L Madison, G W Huisman

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