Pleurotus ostreatus hydrophobins: surface active proteins.
[Tesi di dottorato]
Tesi di dottorato
||Pleurotus ostreatus hydrophobins: surface active proteins
||24 November 2008
|Number of Pages:
||Università degli Studi di Napoli Federico II
||Chimica organica e biochimica
||24 November 2008
|Number of Pages:
||Area 05 - Scienze biologiche > BIO/10 - Biochimica
||Indirizzo del dottorato: Biotecnologie industriali
||17 Nov 2009 09:59
||30 Oct 2014 08:26
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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