Esposito, Alessandra (2025) Amyloid fibrils for the construction of innovative, safe and sustainable functional materials. [Tesi di dottorato]

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Abstract

Catalytic amyloids are an emerging class of functional nanomaterials that combine the selectivity and efficiency of enzymatic catalysis with the robustness and recyclability of heterogeneous systems, all within peptide-based nanostructures. This PhD thesis aims at constructing amyloid-based catalytic nanostructures by immobilizing FeMC6*a, a miniaturized artificial metalloenzyme, within such peptide frameworks. In detail, FeMC6*a is a synthetic mini-enzyme belonging to the Mimochrome family, featuring a metallo-porphyrin core embedded within two short, designed peptides. The compact structure of MC6*a can host different metal ions, allowing it to switch among different kinds of reactivities with exceptional efficiency, often surpassing natural metalloenzymes. In particular, the iron-based analogue (FeMC6*a) demonstrated outstanding catalytic performances in peroxidase-like reactions. When covalently bound onto gold nanomaterials, FeMC6*a forms functional nanoconjugates useful in biocatalytic applications, where stability and reusability are critical. The immobilization of this mini-enzyme on peptide-based supports aims at improving its robustness and promoting catalyst recycling effectively addressing key challenges in sustainable catalysis. In this Ph.D. thesis, two innovative strategies are developed to maximize catalytic FeMC6*a potential when immobilized within peptide-based nanostructures, which are detailed in two Parts. Part A explores the functionalization of amyloid fibrils with FeMC6*a to construct efficient and reusable catalytic nanomaterials, while Part B investigates the encapsulation of FeMC6*a within amyloid-derived microparticles for integration into microfluidic flow systems. These approaches are designed to incorporate FeMC6*a into robust peptide-based frameworks, unlocking its full potential for continuous and cyclic catalytic processes. The first Part focuses on the development of a peptide-based catalytic nanomaterial by functionalizing amyloid fibrils with FeMC6*a. In particular, by using the amyloidogenic peptide TTR(105-115) and its azide-modified derivative, TTRLysN3, the enzyme was anchored onto fibrils using click chemistry. Two functionalization strategies were evaluated: post-assembly attachment of the enzyme to the formed fibrils and pre-assembly conjugation of the enzyme to the peptide monomers. The pre-assembly method proved to be the best choice, yielding fibrils with enhanced stability and catalytic activity. These functionalized fibrils, tested for peroxidase activity using ABTS as a substrate, showed improvement over the free enzyme, highlighting the beneficial effect of immobilization on fibrils. Additionally, the fibrils were integrated into a flow catalytic system using PVDF membranes, enabling continuous substrate conversion over 40 reaction cycles while retaining activity. This system demonstrates the dual advantages of durability and recyclability, paving the way for practical applications in flow biocatalysis. The results also underscore the potential for customization, as the azide groups on the fibril surface can facilitate the integration of various functional molecules. These findings highlight the promise of amyloid fibrils as versatile scaffolds for catalytic applications in fields such as environmental science and material engineering.The second Part extends the investigation into microfluidic systems, focusing on the encapsulation of FeMC6*a within amyloid-derived microparticles for flow applications. The work was conducted at the Center of Misfolding Diseases, University of Cambridge, under the supervision of Professor Tuomas Knowles. A composite nanomaterial combining TTR peptides with the rapid self-assembling silk fibroin was developed. This hybrid material formed stable fibril-based microparticles capable of enzyme immobilization. Using droplet microfluidics, monodisperse FeMC6*a-loaded microparticles were fabricated with high reproducibility. The immobilized enzyme retained its catalytic activity across multiple reaction cycles under flow conditions, demonstrating excellent recyclability. The fibrillar network within the microparticles provided a protective microenvironment, shielding the enzyme from deactivation and ensuring structural integrity throughout repeated use. This system showcases the potential of microfluidic technology for precise enzyme immobilization and reuse, aligning with sustainability and cost-efficiency goals. Future work will optimize the fibrillar composition and device design to enable the platform applicability to biotechnological challenges, establishing it as a versatile tool for biocatalysis. Overall, this thesis explores innovative strategies for using the artificial metalloenzyme FeMC6*a within peptide-based systems to address key challenges in sustainable catalysis. By immobilizing this mini-enzyme onto peptide-based supports, significant improvements in catalytic efficiency and reusability were achieved. The results obtained demonstrate the applicability of FeMC6*a in flow catalysis, paving the way for broader applications and offering promising solutions for sustainable and high-performance catalytic systems.

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