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

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.