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 [Tesi di dottorato] (Unpublished)

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Item Type: Tesi di dottorato
Language: English
Title: Pleurotus ostreatus hydrophobins: surface active proteins
Creators:
CreatorsEmail
Armenante, Annunziataarmenante@unina.it
Date: 24 November 2008
Number of Pages: 134
Institution: Università degli Studi di Napoli Federico II
Department: Chimica organica e biochimica
Doctoral School: Scienze biotecnologiche
PHD name: Scienze biotecnologiche
PHD cycle: 21
PHD Coordinator:
nameemail
Sannia, Giovannisannia@unina.it
Tutor:
nameemail
Giardina, Paolagiardina@unina.it
Date: 24 November 2008
Number of Pages: 134
Uncontrolled Keywords: hydrophobin,self-assembling, biofilm
MIUR S.S.D.: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Additional Information: Indirizzo del dottorato: Biotecnologie industriali
Date Deposited: 17 Nov 2009 09:59
Last Modified: 04 Dec 2014 12:01
URI: http://www.fedoa.unina.it/id/eprint/3411

Abstract

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.

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