Production of Protein Nanofibers and their Application in the Development of Innovative Materials


Protein nanofibers, also known as amyloid fibrils, are gaining much attention due to their peculiar morphology, mechanical strength and functionalities. These nanofibers are characterized as fibrillar assemblies of monomeric proteins or peptides that underwent unfolding-refolding transition into stable β-sheet structures and are emerging as building nanoblocks for the development of innovative functional materials for application in distinct fields, for instance, in biosensors, bioactive membranes and tissue engineering scaffolds. However, one of the main limitations pointed out for the exploitation of protein nanofibers is their high production time since fibrillation is a time-consuming process that can take hours, days, and even weeks. The use of alternative solvents, such as ionic liquids (ILs), as fibrillation agents has been recently reported with considerable reduction in the fibrillation time. This fact encouraged us to study the fibrillation of a model protein, hen egg white lysozyme (HEWL), in the presence of several ILs based on imidazolium and cholinium cations combined with different anions derived from organic acids. All ILs used were shown to fibrillate HEWL within a few hours with conversion ratios over than 80% and typically worm-like nanofibers were obtained. In another study, a deep eutectic solvent (DES) based on cholinium chloride and acetic acid (1:1) was studied as a possible promoter of HEWL fibrillation, and a considerably reduction of the fibrillation time from 8-15 h to just 2-3 h was also observed. Temperature has a key role in the acceleration of the fibrillation and both temperature and pH significantly influence the nanofibers dimensions, in terms of length and width. In what concerns the nanofibers aspect-ratio, several DES combining cholinium chloride and mono-, di- and tri-carboxylic acids were studied. It was observed that carboxylic acid plays an important role on the length of the nanofibers produced with aspect-ratios always higher than those obtained by fibrillation with cholinium chloride alone. The potential of the obtained protein nanofibers as reinforcing elements was evaluated by preparing pullulan-based nanocomposite films containing lysozyme nanofibers with different aspect-ratios, resulting in highly homogenous and transparent films with improved mechanical performance, particularly for the nanofibers with higher aspect-ratios. Furthermore, the incorporation of lysozyme nanofibers in the pullulan films imparted them also with bioactive functionalities, namely antioxidant capacity and antibacterial activity against Staphylococcus aureus. The results showed that the antioxidant and antibacterial effectiveness increased with the content of nanofibers, supporting the use these films as, for example, eco-friendly edible films for active packaging. Lysozyme nanofibers were also blended with nanocellulose fibers to produce a sustainable sorbent film to be used in the removal of mercury (II) from natural waters. Homogenous and translucent films were obtained by vacuum filtration and the incorporation of these nanofibers in a nanocellulose film promoted a considerable mechanical reinforcement. In terms of the capacity to remove mercury (II) from natural water, the presence of lysozyme nanofibers demonstrated to increase expressively the mercury (II) removal with efficiencies of 82% (pH 7) < 89% (pH 9) < 93% (pH 11), using realistic concentrations of mercury (II) under the limit established in the European Union regulations (50 μg L-1). In sum, it was demonstrated in this thesis that the use of ionic liquids and deep eutectic solvents can accelerate the formation of long and thin lysozyme nanofibers that can be explored as nanosized reinforcing elements for the development of bionanocomposites with applications ranging from food packaging to water purification systems and nanotechnology.


Nuno H. C. S. Silva

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Isabel M. Marrucho, Carmen S. R. Freire

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