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. 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...

Comparative analysis of surface coating properties of five hydrophobins from Aspergillus nidulans and Trichoderma reseei

“…Due to the anchor they are less suitable for application in soluble form. The deletion of the anchor, as shown for the DewB protein, results in almost complete loss of surface binding properties for this protein 14 . The function and surface binding properties of other hydrophobins from A .…”

“…Various applications, that have been investigated for hydrophobins, include coating and modification of solid surfaces, emulsion and foam stabilization, increasing enzyme activity or antifouling 14 – 18 . However, the preparation of hydrophobins from wild type strains is complex and results mostly in milligram amounts of purified protein, due to the low natural production levels or strong binding of the proteins to the fungal cell wall 13 , 19 .…”

“…DewA contributes to the spore hydrophobicity and has been so far established as the first-choice hydrophobin from A . nidulans for biosynthetic surface modification 14 , 15 , 25 , 26 . Hydrophobins RodA and DewB both possess a glycosylphosphatidylinositol (GPI) anchor for immobilization on the spore surface and both contribute to the hydrophobicity of the conidiospore surface of A .…”

“…Several of them (DewA, DewB, RodA and DewC) are induced in the presence of lignocellulose, with RodA and DewC directly contributing to A . nidulans growth on lignocellulose 14 , 27 . It can be assumed that they all fulfil specific functions and that their biochemical or biophysical properties vary.…”

Applications of Functional Amyloids from Fungi: Surface Modification by Class I Hydrophobins

“…Platforms for protein immobilization were developed by using HGFI from Grifola frondosa native [ 31 ] and recombinant source [ 32 ]. On the other hand, the HFBs DewA and DewB from A. nidulans were fused to the laccase LccC from the same fungal source, developing an efficient system for laccase immobilization on polystyrene multiwell plates [ 33 ].…”