1,721,101 research outputs found
Hydrophobins from Pleurotus ostreatus: self-assembling proteins for nanobiotechnological applications
No full textHydrophobins are a large family of small proteins (about 100 aminoacids), produced by filamentous fungi at different developmental stages, self-assembling at hydrophobic/hydrophilic interfaces into amphipathic biofilm.
A hydrophobin secreted by the basidiomycete fungus Pleurotus ostreatus, identified as Vmh2, has been purified both from cultural broth and mycelia. Vmh2 extracted from cultural broth (5-10 mg L-1) was found complexed with glucans, identified as cyclodextrins. After separation from glucidic fraction, the protein was not soluble in water but only in less polar solvents. The Vmh2 extraction from P. ostreatus mycelia has been optimized,
obtaining a higher amount of protein (about 100 mg L-1). The recombinant expression of Vmh2 in Escherichia coli and Pichia pastoris has been also performed, with a yield of about 50 and 30 mg L-1, respectively. The behavior of Vmh2 has been analyzed in different conditions, as solvents, pH, temperature, presence of salts or glucans. When the protein is dissolved in low polar
solvents (i.e. 60% ethanol) its structure is characterized by a high content of -helix, and it is stable in solution. When the pH increases or in the presence of Ca2+ ions, a conformational change occurs and a self-assembled -sheet rich state is formed, rapidly followed by precipitation. When the solvent polarity increases the protein shows an increased tendency to reach hydrophobic/hydrophilic interfaces, with no detectable
conformational change. On the other hand a reversible conformational change and reversible aggregation occurs at high temperature. The interaction with glucans (such as
glucose) enables the protein to be water soluble. In this condition Vmh2 adopts a - structure stable in solution, whilst the self-assembled -sheet rich state occurs after agitation of the solution.
Vmh2 dissolved in ethanol, forms very stable nanometric biofilm by deposition on solid surfaces, such as silicon, changing their wettability (from hydrophobic to hydrophilic).
Vmh2 biofilm has been formed also in presence of glucose; in this case the hydrophilicity of the surface increased.
Locally, on the top of Vmh2 biofilm, the presence of some structures rodlet-like has been observed by atomic force microscopy (AFM). The rodlet formation at the water/air interface
has been studied in more details preparing Vmh2 biofilm by Langmuir techniques. These rodlets appear to correspond to a hydrophobic bilayer, where conformational changes lead
to more rigid structures. The ability of Vmh2 biofilm to immobilize biological macromolecules has been also verified.
Acting as a bioactive substrate to bind other proteins to an inert surface, the biofilm can be used for the fabrication of a new class of hybrid devices, such as biosensors, or for proteomic applications. Vmh2 biofilm has been also used to modify the wettability of clothing fabrics. In particular, after Vmh2 coating, nylon fabrics became more hydrophobic, hence more waterproof. On the other hand wool (hydrophobic) becomes strongly hydrophilic, improving the wearing comfort and facilitating dyeing process. Moreover, the emulsification capability of Vmh2 solution has been analysed using three different oils: olive, peanut and almond oil. The stabilization of the emulsion has been
evaluated: the emulsions obtained in the presence of Vmh2 are stable at least for two days, and even longer in the cases of the peanut and almond oils.
This work has allowed an improvement of the Vmh2 availability, both as recombinant or extractive protein, to clarify the behaviour of the protein and to verify its applicability in several fields
Pleurotus ostreatus hydrophobins: surface active proteins
No full textHydrophobins 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
Development of a biosensing platform based on a laccase-hydrophobin chimera
No full textA simple and stable immobilization of a laccase from Pleurotus ostreatus was obtained through genetic fusion with a self-assembling and adhesive class I hydrophobin. The chimera protein was expressed in Pichia pastoris and secreted into the culture medium. The crude culture supernatant was directly used for coatings of polystyrene multi-well plates without additional treatments, a procedure that resulted in a less time-consuming and chemicals reduction. Furthermore, the gene fusion yielded a positive effect with respect to the wild-type recombinant enzyme in terms of both immobilization and stability. The multi-well plate with the immobilized chimera was used to develop an optical biosensor to monitor two phenolic compounds: L-DOPA ((S)-2-amino-3-(3,4-dihydroxyphenyl) propanoic acid) and caffeic acid (3-(3,4-dihydroxyphenyl)-2-propenoic acid); the estimation of which is a matter of interest in the pharmaceutics and food field. The method was based on the use of the analytes as competing inhibitors of the laccase-mediated ABTS oxidation. The main advantages of the developed biosensor are the ease of preparation, the use of small sample volumes, and the simultaneous analysis of multiple samples on a single platform
Transcriptional analysis of Pleurotus ostreatus laccase genes
No full textFungal laccases (p-diphenol:oxygen oxidoreductase; EC 1.10.3.2) are multi-copper-containing oxidases that catalyse the oxidation of a great variety of phenolic compounds and aromatic amines through simultaneous reduction of molecular oxygen to water. Fungi generally produce several laccase isoenzymes encoded by complex multi-gene families. The Pleurotus ostreatus genome encodes 11 putative laccase coding genes, and only six different laccase isoenzymes have been isolated and characterised so far.
Laccase expression was found to be regulated by culture conditions and developmental stages even if the redundancy of these genes still raises the question about their respective unctions in vivo. In this context, laccase transcript profiling analysis has been used to unravel the physiological role played by the different isoforms produced by P. ostreatus. Even if reported results depict a complex picture of the transcriptional responses exhibited by the analysed laccase genes, they were allowed to speculate on the isoform role in vivo. Among the produced laccases, LACC10 (POXC) seems to play a major role during vegetative growth, since its transcription is downregulated when the fungus starts the fructification process. Furthermore, a new tessera has been added to the puzzling mosaic of the heterodimeric laccase LACC2 (POXA3). LACC2 small subunit seems to play an additional physiological role during fructification, beside that of LACC2 complex activation/stabilisation
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