Fungal hydrophobins are small amphiphilic proteins that self-assemble into monolayers on hydrophobic:hydrophilic interfaces and can be used for surface coatings. Because e.g. Aspergillus nidulans contains six different hydrophobins, it is likely that they have different properties and are used for different “applications” in the fungus. We established a method for recombinant production of different class hydrophobins in Escherichia coli. We produced DewA, DewC, DewD, DewE from A. nidulans and HFBI from Trichoderma reesei and compared surface coating properties of these hydrophobins. All tested proteins formed coatings on glass, strongly increasing the hydrophobicity of the surface, and showed emulsion-stabilizing properties. But whereas the typical class I hydrophobin DewA formed the most stable coating on glass, the intermediate class hydrophobins DewE and DewD were more effective in stabilization of oil:water emulsions. This work gives insights into correlations between structural characteristics of hydrophobins and their behaviour as surface binding agents. It could help with the clarification of their biological functions and lead to novel biotechnological applications.

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Fungal hydrophobins are small amphiphilic proteins that can be used for coatings on hydrophilic and hydrophobic surfaces. Through the formation of monolayers, they change the hydrophobicity of a given surface. Especially, the class I hydrophobins are interesting for biotechnology, because their layers are stable at high temperatures and can only be removed with strong solvents. These proteins self-assemble into monolayers under physiological conditions and undergo conformational changes that stabilize the layer structure. Several studies have demonstrated how the fusion of hydrophobins with short peptides allows the specific modification of the properties of a given surface or have increased the protein production levels through controlled localization of hydrophobin molecules inside the cell. Here, we fused the Aspergillus nidulans laccase LccC to the class I hydrophobins DewA and DewB and used the fusion proteins to functionalize surfaces with immobilized enzymes. In contrast to previous studies with enzymes fused to class II hydrophobins, the DewA-LccC fusion protein is secreted into the culture medium. The crude culture supernatant was directly used for coatings of glass and polystyrene without additional purification steps. The highest laccase surface activity was achieved after protein immobilization on modified hydrophilic polystyrene at pH 7. This study presents an easy-to-use alternative to classical enzyme immobilization techniques and can be applied not only for laccases but also for other biotechnologically relevant enzymes. IMPORTANCEAlthough fusion with small peptides to modify hydrophobin properties has already been performed in several studies, fusion with an enzyme presents a more challenging task. Both protein partners need to remain in active form so that the hydrophobins can interact with one another and form layers, and so the enzyme (e.g., laccase) will remain active at the same time. Also, because of the amphiphilic nature of hydrophobins, their production and purification remain challenging so far and often include steps that would irreversibly disrupt most enzymes. In our study, we present the first functional fusion proteins of class I hydrophobins from A. nidulans with a laccase. The resulting fusion enzyme is directly secreted into the culture medium by the fungus and can be used for the functionalization of hard surfaces. Immobilization of enzymes is of increasing importance in biotechnology. It provides various advantages compared to the application of free enzymes in solution, like increased stability, easy recovery, and reuse of the enzymes (1, 2). In some cases, binding to certain surfaces even improved enzyme activity (3). Several methods of immobilization are distinguished and are based on chemical and physical interactions between the enzyme and the surface, each having its own advantages and disadvantages (1, 2). Basic parameters, like maintenance of high enzyme activity, prevention of enzyme leaching, and contamination of the product, are relevant for choosing the ri...

The conservation of architectural heritage is a big challenge in times with increasing air pollution with aggressive gases. A second major threat to buildings is the combination of water and air contaminants which may be used by microorganisms for their metabolism. Hence, myriads of different bacteria and fungi populate stone surfaces and penetrate into the fine pores and cracks. Whereas epoxid-based paintings (or other paintings) may protect the coated surfaces from water and aggressive gases, these chemicals seal the stone surface and prevent also the evaporation of vapor from the inside of the buildings. Here, we tested a natural, fungal protein-based coating method. Fungi use small, amphiphilic proteins to turn their surfaces hydrophobic. We found that Aspergillus nidulans hydrophobin DewA and Trichoderma reesei HFBI confer hydrophobicity to stones but keep their pores open. The effect resembles “Gore-tex” fabric material.