Longobardi, Sara (2011) Hydrophobins from Pleurotus ostreatus: self-assembling proteins for nanobiotechnological applications. [Tesi di dottorato] (Unpublished)

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Hydrophobins are a large family of small proteins (about 100 aminoacids), produced by filamentous fungi at different developmental stages, self-assembling at hydrophobic/hydrophilic interfaces into amphipathic biofilm. A hydrophobin secreted by the basidiomycete fungus Pleurotus ostreatus, identified as Vmh2, has been purified both from cultural broth and mycelia. Vmh2 extracted from cultural broth (5-10 mg L-1) was found complexed with glucans, identified as cyclodextrins. After separation from glucidic fraction, the protein was not soluble in water but only in less polar solvents. The Vmh2 extraction from P. ostreatus mycelia has been optimized, obtaining a higher amount of protein (about 100 mg L-1). The recombinant expression of Vmh2 in Escherichia coli and Pichia pastoris has been also performed, with a yield of about 50 and 30 mg L-1, respectively. The behavior of Vmh2 has been analyzed in different conditions, as solvents, pH, temperature, presence of salts or glucans. When the protein is dissolved in low polar solvents (i.e. 60% ethanol) its structure is characterized by a high content of -helix, and it is stable in solution. When the pH increases or in the presence of Ca2+ ions, a conformational change occurs and a self-assembled -sheet rich state is formed, rapidly followed by precipitation. When the solvent polarity increases the protein shows an increased tendency to reach hydrophobic/hydrophilic interfaces, with no detectable conformational change. On the other hand a reversible conformational change and reversible aggregation occurs at high temperature. The interaction with glucans (such as glucose) enables the protein to be water soluble. In this condition Vmh2 adopts a  -structure stable in solution, whilst the self-assembled -sheet rich state occurs after agitation of the solution. Vmh2 dissolved in ethanol, forms very stable nanometric biofilm by deposition on solid surfaces, such as silicon, changing their wettability (from hydrophobic to hydrophilic). Vmh2 biofilm has been formed also in presence of glucose; in this case the hydrophilicity of the surface increased. Locally, on the top of Vmh2 biofilm, the presence of some structures rodlet-like has been observed by atomic force microscopy (AFM). The rodlet formation at the water/air interface has been studied in more details preparing Vmh2 biofilm by Langmuir techniques. These rodlets appear to correspond to a hydrophobic bilayer, where conformational changes lead to more rigid structures. The ability of Vmh2 biofilm to immobilize biological macromolecules has been also verified. Acting as a bioactive substrate to bind other proteins to an inert surface, the biofilm can be used for the fabrication of a new class of hybrid devices, such as biosensors, or for proteomic applications. Vmh2 biofilm has been also used to modify the wettability of clothing fabrics. In particular, after Vmh2 coating, nylon fabrics became more hydrophobic, hence more waterproof. On the other hand wool (hydrophobic) becomes strongly hydrophilic, improving the wearing comfort and facilitating dyeing process. Moreover, the emulsification capability of Vmh2 solution has been analysed using three different oils: olive, peanut and almond oil. The stabilization of the emulsion has been evaluated: the emulsions obtained in the presence of Vmh2 are stable at least for two days, and even longer in the cases of the peanut and almond oils. This work has allowed an improvement of the Vmh2 availability, both as recombinant or extractive protein, to clarify the behaviour of the protein and to verify its applicability in several fields.

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