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
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