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

Content Provider	Springer Nature Link
Author	Qi, Q. Steinbüchel, A. Rehm, B. H. A.
Copyright Year	2000
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-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 K i 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.
Starting Page	37
Ending Page	43
Page Count	7
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	54
Issue Number	1
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2000-07-14
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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