Applied Microbiology and Biotechnology

, Volume 40, Issue 5, pp 699–709

Application of enzymatically synthesized short-chain-length hydroxy fatty acid coenzyme A thioesters for assay of polyhydroxyalkanoic acid synthases

  • Henry E. Valentin
  • Alexander Steinbüchel
Applied Microbial and Cell Physiology

Abstract

Various hydroxyacyl coenzyme A (CoA) thioesters were synthesized from the corresponding hydroxyalkanoic acid (such as e.g. [3-14C]d-(−)-hydroxybutyric acid, [1-14C]d-lactic acid, [1-14C]l-lactic acid, etc.) and from acetyl-CoA employing the propionate CoA transferase of Clostridium propionicum. Preparative isolation of the thioesters on hydrophobic matrices and analysis by HPLC are reported. These thioesters were subjected to a radiometric or a spectrometric assay of polyhydroxyalkanoic acid (PHA) synthase activity. The latter was based on the release of CoA from, for example, d-(−)-3-hydroxybutyryl-CoA, which was detected spectroscopically at 412 nm by reduction of 5,5′-dithiobis(2-nitrobenzoic acid) and provided a convenient assay of poly(3-hydroxybutyrate) synthase. When [1-14C]lactyl-CoA was used as substrate in a PHA synthase assay employing crude extracts obtained from various wild-type strains, [1-14C]lactyl-CoA was used as a substrate at a rate that was only less than 10−4 of the rate than with [3-14C]d-(−)-3-hydroxybutyryl-CoA or was negligible. One exception was a recombinant strain of Escherichia coli, which overexpressed the PHA synthase complex of Chromatium vinosum and which used [1-14C]d-lactyl-CoA as substrate at a relatively high rate.

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Copyright information

© Springer-Verlag 1994

Authors and Affiliations

  • Henry E. Valentin
    • 1
  • Alexander Steinbüchel
    • 1
  1. 1.Institut für Mikrobiologie der Georg-August-Universität GöttingenGöttingenGermany

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