Engineering novel amyloid-based biofilm for capture and degradation of micro-plastics

PET-based microplastics represent a significant environmental pollutant, with deleterious effects recorded across the human body once ingested and a near-complete penetration in the environment, including within drinking water. The aim of this project, therefore, is to engineer an amyloid-based meshwork decorated with enzymes, known as PETases, capable of breaking down PET materials into products that can be more easily metabolised by the body. The resulting meshwork could act as a water filter. To this end, Sup35NM was chosen as the amyloid protein with HotPETase, LCC ICCG and PHL7 as the chosen PETases. The N- and M-regions of Sup35 are responsible for fibril formation- facilitating the production of chimeric proteins capable of forming the necessary amyloid-based meshwork- with the enzymes of interest added onto the C-terminal end of Sup35NM. Two constructs were successfully produced and isolated: Sup35NM-HotPETase and Sup35NM-LCC ICCG. The corresponding fusion protein of Sup35NM-LCC ICCG, along with the wildtype Sup35NM protein, were recombinantly produced and their assembly characterised by ThT f luorescence kinetics and atomic force microscopy imaging. The results showed protein mixtures containing low levels of Sup35NM-LCC ICCG were able to form fibrils, suggesting incorporation of the fusion protein into the wildtype fibril structure is feasible and has a quantifiable impact on amyloid suprastructure; this therefore warrants further exploration. Ultimately, the generation and morphological characterization of Sup35NM-PETase proteins represents a significant step towards amyloid-based biomaterials capable of environmental plastic degradation.