Immobilisation of active enzymes on novel GFP protein particles : a thesis submitted in complete fulfilment of the requirements of the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand
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Date
2013
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Massey University
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Abstract
Inclusion bodies were previously thought to be aggregations of inactive, mis-folded
proteins. However, there is a growing body of evidence that suggests otherwise. In 2011
Jahns et al demonstrated a self-assembling GFP protein particle (GFP particle) that not
only exhibited fluorescence, but was also able to display functional antibody and ligand
binding sites. These functional GFP particles exhibited reasonable activity, and in many
cases outperformed commercially available particles. The GFP particles consisted of an
aggregation of fusion proteins. These fusion proteins in turn consisted of an Nterminally
extended enhanced GFP protein which was fused at its C-terminus to an
inactive polyester synthase (PhaC(C319A)) from Ralstonia eutropha, and a
functionality, e.g. antibody/ ligand binding site. In this study, GFP particles were
investigated to ascertain whether they could serve as a support for the immobilization
and display of active enzymes; and provide a technology that is potentially more
efficient and cost-effective than other enzyme immobilization methods. Furthermore,
their inherent fluorescence would provide an additional advantage. The enzymes used
for functionality tests were: a thermostable α-amylase from Bacillus licheniformis that
lacked its signal sequence (Bla(-ss)); N-acetyl-D-neuraminic acid aldolase (NanA) from
Escherichia coli; and organophosphohydrolase (OpdA) from Agrobacterium
radiobacter. These enzymes were chosen for their differing quaternary structuremonomer,
tetramer, and dimer, respectively- and were fused to the C-termini of GFP
fusion proteins. The results of this investigation showed that it is possible to generate
fluorescent GFP particles inside recombinant E. coli BL21(DE3) cells which are also
able to display active enzyme. These enzyme-bearing GFP particles exhibited
considerable stability across a range of temperature, pH, and storage conditions, and
could also be reused. The activity of the particles was also compared to a similar
technology- functionalized PHA beads; however, the PHA beads consistently exhibited
stronger enzyme activity under all conditions tested. GFP protein particles represent a
novel method for the immobilization and display of enzymes. Their ability to
immobilise and display active enzymes of different quaternary structure under a range
of conditions makes GFP particles particularly attractive to industrial biocatalysis
processes. Potential applications include diagnostic assays, food production,
pharmaceutical production, and bioremediation.
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Keywords
Inclusion bodies, Immobilised enzymes, GFP, Green fluorescent protein, Fusion proteins, Enzymes, Enzyme immobilisation, Industrial biocatalysis