Sorrentino, Ilaria (2018) FUNGAL SELF-ASSEMBLING PROTEIN LAYERS: NEW BIOTECH-TOOLS FOR BIO/NON-BIO HYBRID DEVICES. [Tesi di dottorato]

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Tipologia del documento: Tesi di dottorato
Lingua: English
Titolo: FUNGAL SELF-ASSEMBLING PROTEIN LAYERS: NEW BIOTECH-TOOLS FOR BIO/NON-BIO HYBRID DEVICES
Autori:
AutoreEmail
Sorrentino, Ilariailaria.sorrentino@unina.it
Data: 16 Dicembre 2018
Numero di pagine: 141
Istituzione: Università degli Studi di Napoli Federico II
Dipartimento: Scienze Chimiche
Dottorato: Biotecnologie
Ciclo di dottorato: 31
Coordinatore del Corso di dottorato:
nomeemail
Sannia, Giovannisannia@unina.it
Tutor:
nomeemail
Giardina, Paola[non definito]
Data: 16 Dicembre 2018
Numero di pagine: 141
Parole chiave: self-assembling protein, chimera proteins, biosensing platforms
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Depositato il: 16 Gen 2019 10:08
Ultima modifica: 16 Giu 2020 09:20
URI: http://www.fedoa.unina.it/id/eprint/12704

Abstract

During recent years the demand of valuable scaffolds to biofunctionalize nanomaterials has been rapidly increasing. Self-assembling proteins, such as amyloid fibrils, are promising candidates for the functionalization of nanomaterials due to their chemical and mechanical stability. These fibrillar structures are typically associated to neurodegenerative diseases. However, it has been recently observed that many organisms, such as fungi and some bacteria, take advantage of the ability of polypeptides to form amyloids. Hydrophobins represent an example of functional amyloid fibrils produced at different growth stages by filamentous fungi. They are a large family of very active surface proteins and can be grouped into two distinct classes based on the stability of the amphipathic layers that they form. In particular, layers formed by class I share many structural properties with amyloid fibrils, solubilized only after harsh acid treatments Immobilization of active proteins on the hydrophobin layer has been proven as a simple approach for the functionalization of different surfaces useful in biotechnological applications. For most of these applications, a critical aspect concerns protein orientation with respect to the surface. To overcome this problem, design and recombinant production of fused engineered proteins combining the adhesive moiety of hydrophobins to target proteins can be a valuable choice. This PhD project has been focused on recombinant production in different hosts (Pichia pastoris and Escherichia coli) of chimera proteins built by selected target proteins (laccase, antimicrobial peptides and antibody) fused to an adhesive self-assembling class I hydrophobin, and on their exploitation in some application fields. The selected class I hydrophobin was Vmh2, extracted from the mycelium of the fungus Pleurotus ostreatus, one of the most hydrophobic hydrophobin able to spontaneously form stable and homogeneous layers on hydrophilic or hydrophobic surfaces, changing their wettability. As a first goal, the hydrophobin Vmh2 was fused to the laccase POXA1b from P. ostreatus, a high redox potential oxidase endowed with a remarkable stability at high temperature and at alkaline pH. The resulting fusion enzyme was secreted into the culture medium by the heterologous yeast P. pastoris and directly used for coating different surfaces, i.e. graphene and polystyrene, without additional purification steps. The immobilized enzyme was exploited to develop innovative optical and electrochemical biosensing platform for the detection of phenolic compounds. Subsequently, two chimeric proteins were recombinantly expressed in E. coli: in the first case, Vmh2 was fused to the antimicrobial peptide LL37, a small cationic amphiphilic peptide belonging to the family of cathelicidins which has an effective activity against a wide range of bacteria and fungi. The LL37-Vmh2 chimeric protein was successfully produced and its adhesive and antibiofilm capabilities, against Staphylococcus epidermidis, tested by coating polystyrene surfaces. The chimera HN1-Vmh2, the fusion between the hydrophobin and the anti-mesothelin ScFv antibody, was produced in the soluble fraction of E. coli whilst its purification and exploiation needs to be optimized. In conclusion, chimeric proteins obtained by the genetic fusion of the hydrophobin Vmh2 to target proteins represent a proof of concept of versatile and straightforward solutions of surface functionalizations that could be explored in several fields. Moreover, during the six months stay at the CNRS of Grenoble, the work was focused on carbon nanotubes (CNT) functionalization using the native laccase POXC from P. ostreatus. POXC is a promising biocathode for enzymatic biofuel cells (EBFCs), since it shows efficient ORR (Oxygen Reduction Reaction) performances and it is capable to operate in a conventional H2/air proton-exchange membrane fuel cell.

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