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  • In vitro synthesis of poly(3-hydroxydecanoate): Purification and enzymatic characterization of type II polyhydroxyalkanoate synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa

    Author(s)
    Qi, Q
    Steinbuchel, A
    Rehm, BHA
    Griffith University Author(s)
    Rehm, Bernd
    Year published
    2000
    Metadata
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    Abstract
    For the first time, the purification has been achieved of the type II polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa applying N-terminal His6-tag fusions and metal chelate affinity chromatography. In vivo His6-tagged PHA synthase activity was confirmed by functional expression of the corresponding genes in Escherichia coli, and PHA synthase activity could also be measured in vitro with the enzymes. The specific enzyme activity of PHA synthases PhaC1 and PhaC2 was 0.039 U mg−1 and 0.035 U mg−1 protein, respectively. Kinetic studies showed a lag phase for both PHA synthases using (R,S)-3-hyd ...
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    For the first time, the purification has been achieved of the type II polyhydroxyalkanoate (PHA) synthases PhaC1 and PhaC2 from Pseudomonas aeruginosa applying N-terminal His6-tag fusions and metal chelate affinity chromatography. In vivo His6-tagged PHA synthase activity was confirmed by functional expression of the corresponding genes in Escherichia coli, and PHA synthase activity could also be measured in vitro with the enzymes. The specific enzyme activity of PHA synthases PhaC1 and PhaC2 was 0.039 U mg−1 and 0.035 U mg−1 protein, respectively. Kinetic studies showed a lag phase for both PHA synthases using (R,S)-3-hydroxydecanoyl-CoA as substrate. Specific enzyme activity was increased to 0.055 U mg−1 when the phasin GA24 from Ralstonia eutropha was added to the assay. CoA inhibited PHA synthase activity, and a Ki of 85 μM was determined. A two-enzyme system was established, employing commercially available acyl-CoA synthetase and PHA synthase, which allowed the in vitro de novo PHA granule formation and the in vitro synthesis of poly(3-hydroxydecanoate) exhibiting a weight average molar mass of 9.8 × 104 g mol−1, and which occurred independently of pre-existing PHA granules.
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    Journal Title
    Applied Microbiology and Biotechnology
    Volume
    54
    Issue
    1
    DOI
    https://doi.org/10.1007/s002530000357
    Subject
    Microbiology not elsewhere classified
    Publication URI
    http://hdl.handle.net/10072/373508
    Collection
    • Journal articles

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