Pleurotus ostreatus hydrophobins: surface active proteins

Armenante, Annunziata (2008) Pleurotus ostreatus hydrophobins: surface active proteins. [Tesi di dottorato] (Inedito)

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Hydrophobins are a large family of small cysteine rich proteins (about 100 amino acids) that appear to be ubiquitous in the Fungi kingdom. The ability of hydrophobins to modify surface properties by interfacial self-assembly and their high surface activity provide a potential for several applications. A hydrophobin secreted by the basidiomycete fungus Pleurotus ostreatus has been purified, and identified as vmh2-1 (TrEMBL entry Q8WZI2_PLEOS). The hydrophobin production has been optimized using different conditions, the highest production (~60 mg/l) has been obtained when P. ostreatus mycelium was grown in minimum medium under static conditions. The pure protein is insoluble in water, whereas complexes formed between the hydrophobin and glycans, present in culture broth containing amylose (PDY), are water soluble. The structure of these glycans, analyzed by GC-MS, MALDI-MS and NMR, matches to cyclic structures of α 1-4 linked glucose containing from 6 to 16 monomers (cyclodextrins). In the presence of these glycans, the hydrophilicity of the hydrophobin increases, nevertheless the protein is prone to self aggregation. On the other hand when the pure hydrophobin is dissolved in 60% ethanol, its self assembly is prevented. Recombinant P. ostreatus hydrophobin has been expressed in a host microorganism, Escherichia coli. The recombinant protein has been obtained fused to GST, separated by an aminoacidic sequence recognized by TEV protease. Purification of the recombinant protein has been achieved using the self-assembling properties of the native hydrophobin. The set up procedure has allowed us to obtain about 12 mg/litre of the pure, correctly structured (by CD analysis) recombinant hydrophobin. The pure protein from P. ostreatus, deposited on a silicon hydrophobic surface, forms a very stable biofilm, whereas the biofilm has not been detected on a oxidized silicon hydrophilic surface. When the water-soluble cyclodextrin-hydrophobin complex was used, thick biofilms have been obtained on both surfaces. The hydrophobin biofilm is resistant to hot 2% SDS and it is able to protect silicon surface from basic dissolution, a procedure used in micromachining process. The pure hydrophobin self-assembles also on other surfaces, like porous silicon and oxidized porous silicon, changing the wettability of these surfaces (from hydrophobic to hydrophilic and vice versa) but leaving unaltered the sensing ability of the surface. The features of the Languimir Blodgett (LB) film formed by the P. ostreatus hydrophobin have been investigated. When the LB film is transferred onto a silicon substrate, AFM observations revealed the coexistence of a LB monolayer and rodlets. The observed rodlets have a hydrophilic character and are formed by hydrophobin bilayers embedded in the LB monolayer. We have also demonstrated that the hydrophobin biofilm is suitable for peptides and proteins immobilization. The monolayer acts as a bioactive substrate to bind other proteins. These results can be the starting point in the manufacture of a new generation of hybrid devices for proteomics applications.

Tipologia di documento:Tesi di dottorato
Parole chiave:hydrophobin,self-assembling, biofilm
Settori scientifico-disciplinari MIUR:Area 05 Scienze biologiche > BIO/10 BIOCHIMICA
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Stato del full text:Accessibile
Data:24 Novembre 2008
Numero di pagine:134
Istituzione:Università Federico II
Dipartimento o Struttura:Dipartimento di Chimica organica e Biochimica
Tipo di tesi:Dottorato
Stato dell'Eprint:Inedito
Scuola di dottorato:Scienze Biotecnologiche
Denominazione del dottorato:Scienze Biotecnologiche Ind. Industriale
Ciclo di dottorato:XXI
Numero di sistema:3411
Depositato il:17 Novembre 2009 10:59
Ultima modifica:17 Novembre 2009 10:59

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