Applied Microbiology and Biotechnology

, Volume 40, Issue 5, pp 699–709 | Cite as

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
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Revised:
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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.

Keywords

Hydroxy Fatty Acid Hydroxybutyric Acid Preparative Isolation Hydroxyalkanoic Acid Spectrometric Assay 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
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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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