Gravagnuolo, Alfredo Maria (2015) Hydrophobins from Pleurotus ostreatus in biotechnological industry. [Tesi di dottorato]

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Abstract

Biological interfacing of novel materials is a key step to improve their biocompatibility, biofunctionality and selectivity toward bio-technological applications such as nanomedicine, bioelectronics, and in bioanalytical chemistry. Self-assembling of proteins, "nanomachines" with a wide variety of functions, has been intensively studied in the past few decades as fundamental and green strategy to build hierarchical structures in both living systems and hybrid functional assemblies for bionanotechnological purposes. The Class I hydrophobin, called Vmh2, is a peculiar, surface active, and versatile fungal protein that is known to self-assemble into very chemically stable amphiphilic film, able to change wettability of surfaces and to strongly adsorb other proteins in their active form. Moreover the Vmh2 nanometer-scale layer is perfectly compatible with optical applications. However, the production and handling of this protein, which has been characterized in our laboratories, is very arduous due to its low solubility and natural propensity to self-assemble into amyloid-like stable nano-structures. As a first goal, productivity of protein extraction and purification has been sensibly increased to obtain adequate amount of protein for laboratory scale applications. Moreover the conditions for Vmh2 solubilization in aqueous media and for Vmh2 self-assembling have been explored characterizing the aggregated forms in solution and the stable film on Teflon support. A molecular model for Vmh2 self-assembly has also been proposed. The protein film has been exploited to easily coat the sample-loading steel plate used in MALDI-TOF mass spectrometry. The hybrid surface is able to stably and homogenously adsorb peptides and proteins whereas salts or denaturants can be washed out allowing fast and high-throughput on-plate desalting prior to MS analysis. The functions of the Vmh2 coating have been expanded immobilizing enzymes of interest in proteomics. Rapid and efficient multiple enzyme digestions have been performed to achieve high sequence coverage of model proteins and to analyze a whole proteome. Since Vmh2 can be de-polymerized in specific conditions, the functionalized supports can be reused for indefinite cycles. Moreover Vhm2 has been exploited to disperse quite high amount of highly hydrophobic graphene based materials, produced by ultrasonic wave exfoliation of low cost graphite. Notably, the non-covalent nature of the amphiphilic protein-carbon interactions preserves the band structure of sp2-carbon lattice. The bio-hybrid material is endowed with the self-assembling properties of Vmh2, controlled by environmental factors, and is a valuable material for biomedical applications. Finally, Vmh2 layer has been used as a facile method of glass surface coating and efficient substrate for the immobilization of proteins and nanomaterials, such as graphene oxide and home-made quantum dots. The functionalized slides have been tested in microarray technology. Notably, immobilized antibodies have proved functional and used in a bioassay. The time needed for fabrication of these new microarray slides is lower than that of the most efficient methods for chemical functionalization. In conclusion the unique properties of Vmh2 have provided new biotechnological solutions that could boost the applications of bio-hybrid materials in biomedical and bioanalytical fields.

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