Back to Search
Start Over
Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme.
Analysis of the Thiocapsa pfennigii polyhydroxyalkanoate synthase: subcloning, molecular characterization and generation of hybrid synthases with the corresponding Chromatium vinosum enzyme.
- Source :
-
Applied microbiology and biotechnology [Appl Microbiol Biotechnol] 2000 Aug; Vol. 54 (2), pp. 186-94. - Publication Year :
- 2000
-
Abstract
- The PHA synthase structural gene of Thiocapsa pfennigii was identified and subcloned on a 2.8-kbp BamHI restriction fragment, which was cloned recently from a genomic 15.6-kbp EcoRI restriction fragment. Nucleotide sequence analysis of this fragment revealed three open reading frames (ORFs), representing coding regions. Two ORFs encoded for the PhaE (Mr 40,950) and PhaC (Mr 40,190) subunits of the PHA synthase from T. pfennigii and exhibited high homology with the corresponding proteins of the Chromatium vinosum (52.8% and 85.2% amino acid identity) and the Thiocystis violacea (52.5% and 82.4%) PHA synthases, respectively. This confirmed that the T. pfennigii PHA synthase was composed of two different subunits. Also, with respect to the molecular organization of phaE and phaC, this region of the T. pfennigii genome resembled very much the corresponding regions of C. vinosum and of Thiocystis violacea. A recombinant strain of Pseudomonas putida, which overexpressed phaE and phaC from T. pfennigii, was used to isolate the PHA synthase by a two-step procedure including chromatography on Procion Blue H-ERD and hydroxyapatite. The isolated PHA synthase consisted of two proteins exhibiting the molecular weights predicted for PhaE and PhaC. Hybrid PHA synthases composed of PhaE from T. pfennigii and PhaC from C. vinosum and vice versa were constructed and functionally expressed in a PHA-negative mutant of P. putida; and the resulting PHAs were analyzed.
- Subjects :
- Acyltransferases chemistry
Acyltransferases isolation & purification
Amino Acid Sequence
Base Sequence
Chromatium genetics
Cloning, Molecular
Codon
Escherichia coli genetics
Fatty Acids metabolism
Genes, Bacterial
Molecular Sequence Data
Molecular Weight
Mutagenesis, Site-Directed
Protein Biosynthesis
Protein Subunits
Recombinant Proteins metabolism
Sequence Alignment
Substrate Specificity
Thiocapsa genetics
Acyltransferases genetics
Acyltransferases metabolism
Chromatium enzymology
Thiocapsa enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 0175-7598
- Volume :
- 54
- Issue :
- 2
- Database :
- MEDLINE
- Journal :
- Applied microbiology and biotechnology
- Publication Type :
- Academic Journal
- Accession number :
- 10968631
- Full Text :
- https://doi.org/10.1007/s002530000375