Abstract

To produce a novel amyloid mash material decorated with PET degrading enzymes, we will first produce chimera protein monomers using the pET/BL21 protein expression system. The chimera proteins will consist of the amyloid forming part of the yeast protein Sup35 (called Sup35NM) linked to PETase, MHETase or BHETase. These three enzymes will provide Sup35NM amyloid fibrils with enzymatic activities that catalyse a series of reactions that breaks down PET into ethylene glycol and terephthalic acid. Secondly, we will make constructs containing the three catalytic chimera proteins for expression and extracellular export into the media as monomers, where they can assemble in situ into large fibril networks, using the Curli-dependant amyloid generator (C-DAG) system. We will also assemble the chimera proteins into amyloid fibril networks decorated with PET degrading enzymes in vitro, which we will confirm by kinetics and AFM imaging experiments. Finally, we will assess the structure and the PET degrading activity of the fibril mesh material formed in vitro and in situ. Thus, this projeect will produce a novel biomaterial which is a robust yet malleable, non-toxic substance that could act as microplastics capturing and degrading filters.