<p>Hydrophobins are a class of small proteins (7-15kDa) found in filamentous fungi and are among the most surface active proteins known today. Because of this they have received attention for different applications, e.g. for the food industry as an alternative in emulsions. The goal of this project was to culture and extract hydrophobins from <em>Trichoderma reesei</em> and characterize it. This was done from a freeze-dried culture of <em>Trichoderma reesei</em>, which was cultured on PDA-plates and in liquid medium with glucose as carbon source. Extraction was made by breaking the cells, mechanically and by sonication, and then by shaking a seperating funnel to create foam from the surface-active proteins. The foam was washed and freeze-dried and the total protein concentration of the freeze-dried substance was determined with Bradford assay and the hydrophpbin was characterized with SDS-PAGE. The culturing of the fungi was successful. The amount of foam created was, however, less than expected. The Bradford assay gave a total protein concentration of 7.5% in the freeze-dried substance, but the SDS-PAGE didn't give any results. The reason for this probably depends on the culturing and the extraction of the hydrophobin. <em>T. reesei</em> hydrophobin HFBI, expressed in glucose containing media, is bound to the mycelium of the fungi and the breaking of the mycelium might not have been enough to release all the protein, which also would explain the small amounts of foam. One way to improve this could be to grow the fungi on lactose instead. This will result in that T. reesei produces HFBII instead, which is mainly released to the surrounding. The conclusion of the project is that the method for culturing and extraction needs to be improved to obtain hydrophobin from <em>T. reesei</em>.</p>

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Thesis (doctoral)--Åbo Akademi University, 2007. / Includes bibliographical references. Also available on the World Wide Web.

Thesis (doctoral)--University of Helsinki, 2007. / Includes bibliographical references. Also available on the World Wide Web.

Diss.Teknillinen korkeakoulu, 2006.

Hydrophobins are a class of small proteins (7-15kDa) found in filamentous fungi and are among the most surface active proteins known today. Because of this they have received attention for different applications, e.g. for the food industry as an alternative in emulsions. The goal of this project was to culture and extract hydrophobins from Trichoderma reesei and characterize it. This was done from a freeze-dried culture of Trichoderma reesei, which was cultured on PDA-plates and in liquid medium with glucose as carbon source. Extraction was made by breaking the cells, mechanically and by sonication, and then by shaking a seperating funnel to create foam from the surface-active proteins. The foam was washed and freeze-dried and the total protein concentration of the freeze-dried substance was determined with Bradford assay and the hydrophpbin was characterized with SDS-PAGE. The culturing of the fungi was successful. The amount of foam created was, however, less than expected. The Bradford assay gave a total protein concentration of 7.5% in the freeze-dried substance, but the SDS-PAGE didn't give any results. The reason for this probably depends on the culturing and the extraction of the hydrophobin. T. reesei hydrophobin HFBI, expressed in glucose containing media, is bound to the mycelium of the fungi and the breaking of the mycelium might not have been enough to release all the protein, which also would explain the small amounts of foam. One way to improve this could be to grow the fungi on lactose instead. This will result in that T. reesei produces HFBII instead, which is mainly released to the surrounding. The conclusion of the project is that the method for culturing and extraction needs to be improved to obtain hydrophobin from T. reesei.

Les hydrophobines sont des protéines fongiques caractérisées par leurs propriétés amphipathiques et un motif de quatre ponts disulfures. Leur forme soluble s’auto-assemble aux interfaces hydrophobe/hydrophile pour former une couche amphipatique. Ces protéines sont utilisées par les champignons pour franchir la barrière air/eau, former des hyphes aériennes ou recouvrir les spores les rendant hydrophobes, ce qui facilite leur dispersion dans l’air. L’hydrophobine RodA du pathogène opportuniste Aspergillus fumigatus forme une couche de fibres amyloïdes avec une morphologie en bâtonnets qui recouvre la surface des spores ce qui les rend inertes vis-à-vis du système immunitaire. Nous aspirons à décrire l’auto-association de RodA en bâtonnets, caractériser la structure des fibres et établir les potentiels liens entre structure et inertie immunologique. La protéine recombinante RodA exprimée chez E. coli peut être correctement repliée in vitro et s’auto-associe sous forme de fibres amyloïdes. Comme première étape, la structure et la dynamique de RodA ont été étudiées par RMN en solution. Par rapport aux autres hydrophobines, RodA présente de nouveaux éléments structuraux ainsi que d’autres conservés. Grâce à une étude de mutagénèse, des régions importantes dans la formation des fibres ont été identifiées, certaines impliquées dans le cœur des fibres et d’autres dans les interactions latérales des bâtonnets. Les relations entre la structure et les propriétés immunologiques ont également été établies. L’étude d’autres hydrophobines d’A. fumigatus, probablement impliquées dans la formation du biofilm ou importantes pour la conidiation et la survie des spores, a été initiée.dC), a été initiée. / Hydrophobins are fungal proteins characterised by their amphipatic properties and a pattern of four disulfide bridges. Their soluble form self-assembles at hydrophobic/hydrophilic interfaces to form an amphipatic layer. These proteins are used by fungi to breach the air/water barrier, to form aerial hyphae, or to cover spores rendering them hydrophobic, thus facilitating spore dispersal. The RodA hydrophobin of the opportunistic pathogen Aspergillus fumigatus forms an amyloid monolayer with a rodlet morphology that covers the surface of spores rendering them inert relative to the immune system. We aim at describing the self-association of RodA into rodlets, characterising the structure of the amyloid rodlets and shedding light on the possible relationships between structure and immunological inertness. Recombinant RodA expressed in Escherichia coli can be successfully refolded in vitro and it can auto-associate into amyloid rodlets. As a first step, we have studied the structure and dynamics of RodA by solution NMR and shown that the protein displays new as well as conserved structural features relative to other hydrophobins. A mutational analysis has highlighted important residues for rodlet formation that may be involved on the one hand in the spine of the amyloid fibres and on the other hand on the lateral association of the rodlets to form a monolayer. We have also established the relationship between structure and immunological inertness. We have initiated the study of other hydrophobins from A. fumigatus, that are most likely involved in biofilm formation or in conidiation and spore survival.

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