Towards a better understanding of the polyhydroxyalkanoate synthase from Ralstonia eutropha : protein engineering and molecular biometrics : a thesis presented to Massey University in partial fulfilment of the requirement for the degree of Doctor of Philosophy in Microbiology
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Date
2009
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Massey University
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Abstract
Polyhydroxyalkanoates (PHAs) are polyesters composed of (R)-3-hydroxy-fatty acids.
A variety of gram-positive as well as gram-negative bacteria and some archaea are able
to produce these biopolymers as energy and carbon storage materials. In times of
unbalanced growth, when carbon is available in excess but other nutrients are limited,
PHA inclusions are formed. These granules are water-insoluble, stored intracellularly
and can be maintained outside the cell as beads. The key enzyme for the formation of
PHA inclusions is the PHA synthase PhaC, which catalyses the polymerization of (R)-
3-hydroxyacyl-CoA to PHA with the concomitant release of CoA.
The PHA synthase from Ralstonia eutropha (currently Cupriavidus necator), which is
covalently bound to the PHA granule surface, tolerates fusions to its N terminus
without loss of activity. In this study it was investigated if it would also tolerate
translational fusions to its C terminus. A specially designed linker was employed,
aiming at maintaining the hydrophobic surroundings of the R. eutropha synthase C
terminus to allow proper folding and activity. Two reporter proteins were tested as
fusion partners, the maltose binding protein MalE and the green fluorescent protein
GFP. As GFP is a hydrophobic protein itself, no additional linker between the PHA
synthase and the reporter protein was necessary to produce PHA granules displaying
the functional fusion protein on the surface. Principally, the PHA synthase PhaC
tolerates translational fusions to its C terminus but the nature of the fusion partner
influences the functionality.
Recently, PHA granules have often been acknowledged as bio-beads. A one-step
production allows the formation of functionalised beads without the need for further
cross-linking to impart desired surface properties. PHA beads displaying a gold- or
silica-binding peptide at the N terminus of PhaC were constructed and tested for their
applicability. Additionally, these beads were able to bind IgG due to the ZZ domain of
the IgG binding protein A, which was employed as a linker sequence. These
functionalised beads can be used as molecular tools in bioimaging and biomedicine,
combining organic core with inorganic-binding shell structures.
In a different biomimetic approach, the display of ten lysine residues at the granule
surface was achieved using the phasin protein PhaP as the anchoring matrix. Extensive
work was performed in an attempt to also employ the synthase protein, but was
unsuccessful. These positively charged bio-beads can be used for dispersion or crosslinking
experiments as well as silica binding.
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Keywords
PHA granules, Bio-beads, Polyesters, Biopolymers, Ralstonia eutropha, PHA synthase, Molecular biomimetics