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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dc.contributor.authorJahns, Anika Carolin
dc.date.accessioned2009-10-21T03:02:12Z
dc.date.availableNO_RESTRICTIONen_US
dc.date.available2009-10-21T03:02:12Z
dc.date.issued2009
dc.description.abstractPolyhydroxyalkanoates (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.en_US
dc.identifier.urihttp://hdl.handle.net/10179/1081
dc.language.isoenen_US
dc.publisherMassey Universityen_US
dc.rightsThe Authoren_US
dc.subjectPHA granulesen_US
dc.subjectBio-beadsen_US
dc.subjectPolyestersen_US
dc.subjectBiopolymersen_US
dc.subjectRalstonia eutrophaen_US
dc.subjectPHA synthaseen_US
dc.subjectMolecular biomimeticsen_US
dc.subject.otherFields of Research::290000 Engineering and Technology::290100 Industrial Biotechnology and Food Sciences::290105 Other industrial biotechnologyen_US
dc.titleTowards 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 Microbiologyen_US
dc.typeThesisen_US
massey.contributor.authorJahns, Anika Carolin
thesis.degree.disciplineMicrobiologyen_US
thesis.degree.grantorMassey Universityen_US
thesis.degree.levelDoctoralen_US
thesis.degree.levelDoctoralen
thesis.degree.nameDoctor of Philosophy (Ph.D.)en_US
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