Polyhydroxyalkanoate granules : surface protein topology and rational design of functionalised biobeads : a thesis presented to Massey University in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Microbiology at Massey University, Manawatu, New Zealand

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This thesis examined aspects of the polyhydroxyalkanoate (PHA) biobead system for immobilisation of proteins. Three separate studies have expanded the scope of this platform technology into different applications. New flexible regions along the length of the PhaC protein were discovered and functionalised with IgG binding domains. The bioremediation and fine-chemical synthesis aspects of the PHA biobeads were developed with active enzymes of interest immobilised to the bead surface. Additionally, functional dual fusion of enzymes to both the N- and C-terminus of PhaC was demonstrated for the first time. The enhanced scope of the PHA biobeads will lead to further applications in fields such as protein purification, vaccines, and diagnostics. The first study assessed the ability of the PHA synthase (PhaC) based immobilisation system to tolerate dual enzyme fusions allowing the recapitulation of a biosynthetic pathway. N-acetyl neuraminic acid aldolase and N-acetyl glucosamine 2-epimerase allow for the synthesis of the medically relevant fine-chemical N-acetyl neuraminic acid (Neu5Ac). Ultimately, biobeads establishing the entire Neu5Ac synthesis pathway were able to convert up to 22% of the initial N-acetyl glucosamine into Neu5Ac which compares favourably with the theoretical maximum from chemi-enzymatic synthesis of 33%. Despite intense research interest, the structure of PhaC has not yet been solved. Structural information of the exposed regions of granule-associated PhaC was gathered by the application of biotinylation labels. Six amino acid sites were found to be surface exposed and four were able to tolerate FLAG-tag insertion. Three of these sites were chosen to functionalise with the IgG binding domain. These beads were able to mediate the binding and elution of IgG, with a maximum capacity of 16 mg IgG/g wet PHA beads. The enhanced carbonic anhydrase from Desulfovibrio vulgaris str. "Miyazaki F" (DvCA) was fused the N-terminus of PhaC and immobilised on the surface of PHA beads. The DvCA beads had a specific activity of 114 U/mg enzyme. PHA-immobilised DvCA retained 54% of its initial activity after incubation at 90 °C for 1 h and 77% of its initial activity after incubation at pH 12 for 30 min. This stability indicates its usefulness in the challenging industrial environments where it may be deployed.
Chapter 6 excluded for copyright reasons. Available as: Hooks, D. O. & Rehm, B. H. A. (2015). Surface display of highly-stable Desulfovibrio vulgaris carbonic anhydrase on polyester beads for carbon dioxide capture. Biotechnology Letters, 37(7), p. 1415-1421. doi:10.1007/s10529-015-1803-7
Microbial polymers, Microbial biotechnology, Biobeads, Research Subject Categories::TECHNOLOGY::Bioengineering::Biochemical process engineering