Cicatiello, Paola (2017) Marine fungi as source of protein biosurfactants. [Tesi di dottorato]

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Hydrophobins (HFBs) are small fungal surface-active proteins, which self-assemble spontaneously into amphipathic layers at air-water and water-environment interfaces, playing a key role in different fungal life cycles. Their properties have been demonstrated useful in biotechnological applications of several industrial fields. The knowhow about HFBs can be enforced by the discovery and characterization of new members of this family, thus also broadening the opportunities of their uses. Generally, environmental stressful conditions of fungal growth may induce the production of specific proteins, also endowed with peculiar features. In this view, the marine habitat can be fruitfully explored. Some selected marine fungi from Mycoteca Universitatis Taurinensis (MUT) were screened to identify new HFBs. Extraction methods were set up to isolate secreted or cell wall associated HFBs, allowing the identification of six new putative HFBs. Four of them formed very stable layers on silicon chips, whereas one of them was endowed with remarkable emulsification capacity. Two of the isolated HFBs, Pac2 and Pac3, were characterized in their soluble and aggregate forms. Using different techniques, i.e. circular dichroism, dynamic light scattering and fluorescence assay, Pac3 showed a higher propensity to form amyloid fibrils than Pac2. In addition, microscopy analysis allowed us to infer that the interaction of these proteins with specific surfaces can be crucial in the fibril formation and the assembly morphology. Furthermore, some different applications of the HFB Vmh2 from Pleurotus ostreatus, extensively studied in the research group where this PhD project has been carried out, were improved or developed. The Vmh2 layer was exploited to easily coat the sample-loading steel plate used in MALDI-TOF mass spectrometry to immobilize by adsorption a proteolytic enzyme in his active form, to achieve in-situ digestion of complex biological samples, i.e. the whole blood. This method allowed us to discriminate species-specific peptides in few minutes, encouraging its use in the forensic field. Moreover, a similar approach can be applied to analyse the distribution of biomolecules in biological tissues using the powerful tool of MALDI Imaging mass spectrometry (IMS). In particular, a rat brain tissue has been analysed identifying some putative metabolites and lipids. Recombinant Vmh2, fused to the GFP (Green Fluorescence Protein), was heterologously produced in the yeast Pichia pastoris. Since the two proteins were linked by the cutting site of the protease thrombin, this construct was used as the active biological element in the realization of a thrombin biosensor. Finally, the anti-biofilm activity of the Pac3 and Vmh2 layers against the nosocomial bacteria Staphilococcus epidermidis was analysed on different surfaces generally used in medical field. Pac3 showed better performances than Vmh2, reducing tenfold the biofilm thickness and preserving the cell vitality. On the whole this work contributes to strengthen the knowledge of this family of proteins, broadening the practical perspectives.

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