Biological interfacing of graphene has become crucial to improve its biocompatibility,\ud dispersability, and selectivity. However, biofunctionalization of\ud graphene without yielding defects in its sp 2 -carbon lattice is a major challenge.\ud Here, a process is set out for biofunctionalized defect-free graphene\ud synthesis through the liquid phase ultrasonic exfoliation of raw graphitic\ud material assisted by the self-assembling fungal hydrophobin Vmh2. This\ud protein (extracted from the edible fungus Pleurotus ostreatus ) is endowed\ud with peculiar physicochemical properties, exceptional stability, and versatility.\ud The unique properties of Vmh2 and, above all, its superior hydrophobicity,\ud and stability allow us to obtain a highly concentrated (≈440–510 μg mL −1 ) and\ud stable exfoliated material ( ζ -potential, +40/+70 mV). In addition controlled\ud centrifugation enables the selection of biofunctionalized few-layer defect-free\ud micrographene fl akes, as assessed by Raman spectroscopy, atomic force\ud microscopy, scanning electron microscopy, and electrophoretic mobility.\ud This biofunctionalized product represents a high value added material for\ud the emerging applications of graphene in the biotechnological fi eld such as\ud sensing, nanomedicine, and bioelectronics technologies

Hydrophobins are small self-assembling proteins produced by fungi. A class I hydrophobin secreted by the basidiomycete fungus Pleurotus ostreatus was purified and identified. The pure protein is not water soluble, whereas complexes formed between the protein and glycans, produced in culture broth containing amylose, are soluble in water. Glycan structure matched to cyclic structures of alpha-(1-4) linked glucose containing from six to 16 monomers (cyclodextrins). Moreover, it was verified that not only pure cyclodextrins but also a linear oligosaccharide and even the simple glucose monomer are able to solubilize the hydrophobin in water. The aqueous solution of the protein-in the presence of the cyclic glucans-showed propensity to self-assembly, and conformational changes towards beta structure were observed on vortexing the solution. On the other hand, the pure protein dissolved in less polar solvent (60% ethanol) is not prone to self assembly, and no conformational change was observed. When the pure protein was deposited on a hydrophobic surface, it formed a very stable biofilm whose thickness was about 3 nm, whereas the biofilm was not detected on a hydrophilic surface. When the water-soluble protein-in the presence of the cyclic glucans-was used, thicker (up to 10-fold) biofilms were obtained on either hydrophilic or hydrophobic surfaces.

Fungal hydrophobins are amphipathic, highly surface-active, and self-assembling proteins. The class I hydrophobin Vmh2 from the basidiomycete fungus Pleurotus ostreatus seems to be the most hydrophobic hydrophobin characterized so far. Structural and functional properties of the protein as a function of the environmental conditions have been determined. At least three distinct phenomena can occur, being modulated by the environmental conditions: (1) when the pH increases or in the presence of Ca(2+) ions, an assembled state, β-sheet rich, is formed; (2) when the solvent polarity increases, the protein shows an increased tendency to reach hydrophobic/hydrophilic interfaces, with no detectable conformational change; and (3) when a reversible conformational change and reversible aggregation occur at high temperature. Modulation of the Vmh2 conformational/aggregation features by changing the environmental conditions can be very useful in view of the potential protein applications.

The spontaneous oxidative polymerization of 0.01-1% w/w 5,6-dihydroxyindole (DHI) in chicken egg white (CEW) in the absence of added solvents leads to a black, water-soluble, and processable artificial biomelanin (ABM) with robust and 1 order of magnitude stronger broadband light absorption compared to natural and synthetic eumelanin suspensions. Small angle neutron scattering (SANS) and transmission electron microscopy (TEM) analysis indicated the presence in the ABM matrix of isolated eumelanin nanoparticles (≤100 nm) differing in shape from pure DHI melanin nanoparticles (SANS evidence). Electron paramagnetic resonance (EPR) spectra showed a slightly asymmetric signal (g ∼ 2.0035) similar to that of solid DHI melanin but with a smaller amplitude (ΔB), suggesting hindered spin delocalization in biomatrix. Enhanced light absorption, altered nanoparticle morphology and decreased free radical delocalization in ABM would reflect CEW-induced inhibition of eumelanin aggregation during polymerization accompanied in part by covalent binding of growing polymer to the proteins (SDS-PAGE evidence). The technological potential of eumelanin nanosizing by biomimetic synthesis within a CEW biomatrix is demonstrated by the preparation of an ABM-based black flexible film with characteristics comparable to those of commercially available polymers typically used in electronics and biomedical applications.

The development of efficient and rapid methods for the identification with high sequence coverage of proteins is one of the most important goals of proteomic strategies today. The on-plate digestion of proteins is a very attractive approach, due to the possibility of coupling immobilized-enzymatic digestion with direct matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF)-mass spectrometry (MS) analysis. The crucial step in the development of on-plate immobilization is however the functionalization of the solid surface. Fungal self-assembling proteins, the hydrophobins, are able to efficiently functionalize surfaces. We have recently shown that such modified plates are able to absorb either peptides or proteins and are amenable to MALDI-TOF-MS analysis. In this paper, the hydrophobin-coated MALDI sample plates were exploited as a lab-on-plate for noncovalent immobilization of enzymes commonly used in protein identification/characterization, such as trypsin, V8 protease, PNGaseF, and alkaline phosphatase. Rapid and efficient on-plate reactions were performed to achieve high sequence coverage of model proteins, particularly when performing multiple enzyme digestions. The possibility of exploiting this direct on-plate MALDI-TOF/TOF analysis has been investigated on model proteins and, as proof of concept, on entire whey milk proteome.