Biobased and Biological Polysaccharide-Amyloid Composites
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2025
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Doctoral Thesis
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Abstract
Amyloids are insoluble, fibrillar protein aggregates that are rich in β-sheet structures. In both their pathological and functional roles, amyloids are often found closely associated with another type of biopolymer, namely polysaccharides. This is the case, for example in the extracellular matrix of microorganisms. Individually, both amyloids and polysaccharides have been extensively studied to generate a palette of functional materials. In this thesis, I explore the natural synergy between amyloids and polysaccharides and the potential of ex-ploiting these synergies for the generation of new functional composites.
For this purpose, this thesis covers three types of amyloid-polysaccharide composites, namely, engineered composites, natural composites, and living composites. The polysaccha-rides discussed in this thesis are cellulose (specifically, cellulose nanofibers or 'CNFs'), the most abundant biopolymer in the world and schizophyllan (SPG), an exopolysaccharide and cell wall constituent produced by the fungus Schizophyllum commune. Additionally, the pro-tein hen egg white lysozyme (HEWL) was used as model amyloid-forming protein along with the microbial amyloid proteins, fungal hydrophobins. Both proteins are functional, in that the former is an antimicrobial protein, while the latter is a surface active, amphipathic protein.
First, TEMPO-oxidized CNFs were explored as a potential template to induce and accelerate amyloid formation in proteins, specifically in HEWL. The negatively charged surface of the CNFs combined with their high surface-to-volume ratio were expected to attract the oppo-sitely charged protein monomers and cause them to aggregate. First, the four disulfide bonds in the native HEWL were cleaved with the help of the reducing agent TCEP (~pH 2.5). Then the pH was adjusted as desired (3.5 or 4.5) and these denatured HEWL solutions were ex-posed to aerogels composed of TEMPO-CNFs. Protein aggregation on the surface of the aer-ogels as well as in the bulk was monitored.
At the lowest pH value, the aerogels did not accelerate bulk aggregation as evidenced by DLS measurements, however, the FTIR spectra of the aerogels indicated β-sheet rich struc-tures on their surface. Thus, amyloid formation of HEWL appears to be triggered on the sur-face of the aerogels. Upon increasing the pH, aggregation at both the nano- (pH 3.5) and mi-cro-scale (pH 4.5) is clearly visible in the bulk of the solution. Presumably, amyloid struc-tures detach from the surface of the aerogels and enucleate further aggregation and amyloid formation in the bulk. Polysaccharides can thus be used to generate amyloids, and thus amy-loid-polysaccharide composites in situ for a host of functional applications.
In nature, as in the above engineered composites, polysaccharides are known to induce amy-loid formation to form composite structures. One such composite can be found in the cell wall and extracellular matrix of S. commune. Here, the β-glucan schizophyllan and fungal hydrophobins are found naturally associated with each other. While each component has been separately purified and explored for various materials applications, here their proper-ties as a composite were explored. This schizophyllan-hydrophobin nanocomposite (SHNC) can, for instance, tune the rheological properties of cellulose nanocrystal (CNC) suspen-sions. Depending on their concentration, the SHNC may disrupt or reinforce CNC networks, lowering or raising their viscosity as needed.
If the SHNC is sonicated, the resultant reduction in SPG fiber size is accompanied by a loss of the attached hydrophobins. Both the SHNC and the sonicated short fibers seem to disturb the chiral nematic arrangement of CNCs. However, shorter fibers do seem to allow the for-mation of structural colors, through the cholesteric pitch of the CNCs is clearly affected. Such hybrid materials combine the mechanical robustness of cellulosic structures with the functional nature of the SHNC components.
Similar to purified hydrophobins, the SHNC can also be used to stabilize emulsions. Unlike previous emulsions, however, the SHNC emulsions involve minimal downstream processing and chemical treatments. Further, the highly viscous nature of the SPG acts as a natural rhe-ology modifier, preventing creaming and reducing oil droplet coalescence which prolonged the shelf life of the emulsions. Thus, the inherent synergies of this naturally secreted micro-bial nanocomposite served as a platform for creating new biobased materials.
While such natural composites may be extracted and utilized ex situ for the creation of new materials, nature has, in a sense already created an optimized polysaccharide-amyloid com-posite structure through millennia of evolution. The fungal hypha is in essence one such composite replete with polysaccharides and coated with hydrophobin proteins. This provides a blend of a structurally sound fiber-like material infused with dynamic properties through the advantage of life.
These fungal 'living fibers' were dispersed, through a gentle milling process in their own ex-tracellular matrix, composed primarily of the natural nanocomposite SHNC. This living fi-ber dispersion (LFD) could be converted into self-standing films similar to other fibrillar materials. Unlike other fibers, however, LFD films could naturally crosslink and strengthen themselves upon exposure to humidity. Similarly, through the formation of aerial hyphae, the films could modify their water contact angle from hydrophobic (~80°) to superhydro-phobic (~150°). LFD films also had the unique combination of possessing surface hydro-phobicity while being extremely responsive to humidity. As a result, films could act as prox-imity or humidity sensors, outperforming current cellulose-based sensors in both speed and curvature.
Next, like the SHNC, the LFD could be used to generate stable oil in water or water in oil emulsions. Unlike the SHNC, however, the living fibers could continuously secrete hydro-phobins into the emulsions. To study the impact of these secreted hydrophobins, unstable LFD emulsions were prepared, and their rate of phase separation was monitored. After al-lowing the separated emulsion to stand for two weeks, they were re-emulsified. Notably, these emulsions had reduced rates as well as degrees of phase separation indicating a unique emulsion with increasing stability over time. The LFD is thus a combination of each of the three classes of composites, inducing hydrophobin assembly through the polysaccharide schizophyllan, providing a natural composite of the two for material applications, and lastly enhancing the structural and functional properties of said materials through life.
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08821 - Fischer, Peter (Tit.-Prof.)