title: Immobilization of LccC Laccase from Aspergillus nidulans on Hard Surfaces via Fungal Hydrophobins.
abstract: 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.
  
  
  
  Although 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.
authors: Institute for Applied Biosciences, Department of Microbiology, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany oleksandra.fokina@kit.edu reinhard.fischer@kit.edu.
authors: Oleksandra
authors: O
authors: Fokina
authors: Institute for Applied Biosciences, Department of Microbiology, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
authors: Alex
authors: A
authors: Fenchel
authors: Institute for Applied Biosciences, Department of Microbiology, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany.
authors: Lex
authors: L
authors: Winandy
authors: Institute for Applied Biosciences, Department of Microbiology, Karlsruhe Institute of Technology (KIT), Karlsruhe, Germany oleksandra.fokina@kit.edu reinhard.fischer@kit.edu.
authors: Reinhard
authors: R
authors: Fischer
mesh_terms: D001233:Aspergillus nidulans / Q000201:enzymology* / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D004800:Enzymes, Immobilized / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005898:Glass / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011137:Polystyrenes / Q000737:chemistry; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties; D013501:Surface-Active Agents / Q000737:chemistry
chemical_list: D003470:Culture Media; C095254:DEWA protein, Asperigillus nidulans; D004800:Enzymes, Immobilized; D005656:Fungal Proteins; D011137:Polystyrenes; D011993:Recombinant Fusion Proteins; D013501:Surface-Active Agents; D042845:Laccase