Solid-support immobilization of a “swing” fusion protein for enhanced glucose oxidase catalytic activity

Takatsuji, Yoshiyuki; Yamasaki, Ryota; Iwanaga, Akira; Lienemann, Michael; Linder, Markus B.; Haruyama, Tetsuya

doi:10.1016/j.colsurfb.2013.07.051

Cited by 27 publications

(29 citation statements)

References 46 publications

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“…The immobilization with the fusion protein alone would likely cause a reduction in the laccase activity due to the steric hindrance between single LccC molecules. Previous studies with the class II hydrophobin HFBI fused to a glucose oxidase revealed that a molar ratio between 1:1 and 1:19 of GOx-HFBI to HFBI allowed the highest enzyme activity on surfaces (18). Therefore, we tested different ratios between DewA and the DewA-LccC fusion protein (Fig.…”

Section: ϫ4mentioning

confidence: 99%

“…Since the immobilization was performed with crude culture supernatant containing native exoproteins from A. nidulans, including DewA, it can be assumed that the natural production increase of hydrophobins in the presence of straw (Fig. 2b) was sufficient for effective coating with laccase-fused DewA and did not require additional supplements, as was the case for heterologically produced and purified GOx-HFBI (18).…”

Section: ϫ4mentioning

confidence: 99%

“…A DewA-enzyme fusion presents a tempting alternative to conventional methods of highly stable surface functionalization with an enzyme. So far, a functional enzyme fusion was achieved to class II hydrophobins to enhance the activity of cutinases in solution and to create self-organized membranes with glucose oxidase on a solid surface (18)(19)(20). Extracellular fungal laccases present a suitable target for fusion with hydrophobins for immobilization due to their monomeric structure, high stability, and great biotechnological potential (21,22).…”

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Immobilization of LccC Laccase from Aspergillus nidulans on Hard Surfaces via Fungal Hydrophobins

Fokina

Fenchel

Winandy

et al. 2016

Appl Environ Microbiol

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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...

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Section: ϫ4mentioning

confidence: 99%

Section: ϫ4mentioning

confidence: 99%

mentioning

confidence: 99%

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Immobilization of LccC Laccase from Aspergillus nidulans on Hard Surfaces via Fungal Hydrophobins

Fokina

Fenchel

Winandy

et al. 2016

Appl Environ Microbiol

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“…This "swingable molecular layer" makes it possible to fuse functional dissociation in a solidmolecular interface or a molecular-liquid interface. Regarding the swingable molecular layer, we have reported on the immobilized enzyme 2 and the affinity sensor using photoexcited current. 3 Charge transfer efficiency i.e., electron transfer, is dependent on the distance through which the electron must travel.…”

Section: Introductionmentioning

confidence: 99%

Smooth Electron Transfer from a Photoexcited Dye to Semiconductor Electrode Through a Swingable Molecular Interface

et al. 2016

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The transfer of photoexcited electrons from a fluorescent dye molecule to a semiconductor electrode is regulated by the interfacial design between the electrode and the dye. In the case of larger functional molecules, the regulation becomes observable because of molecular steric hindrance on the solid electrode surface. We have studied the effectiveness of a "swingable" functional molecule layer on a solid substrate for immobilized catalysts and electrochemical reactions. The swingable molecular interface offers the possibility of a similar outcome in photoexcited current generation. The swingable design of the molecular layer may allow smooth current flow from the dye to the semiconductor electrode, because the dye molecules come in close proximity to the semiconductor surface through the swinging action of the linker molecules. In the present study, FTO (fluorinedoped tin oxide) electrodes were prepared with two types of dye-tagged molecular layers, differing only in the type of linker between the dye and the FTO surface: one with a flexible peptide linker capable of swinging back and forth and around, and the other with a rigid peptide linker. The two dye-sensitized FTO electrodes were assayed using photoexcited current measurements. As per the experimental results, the FTO electrode modified with the swingable linker molecule produced higher photoexcited current than that of the FTO modified with the rigid molecule linker.

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“…It has been shown that in solution, HFBI forms selforganized membranes at the air/water interface. [9][10][11][12] In this study, we investigated the structural and electrochemical properties of self-organized HFBI membranes on two types of electrode materials. Two requirements must be fulfilled for the selection of substrates.…”

Section: Introductionmentioning

confidence: 99%

Structural and Electrochemical Properties of Self-organized HFBI Membranes on Different Types of Substrates

et al. 2015

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The HFBI protein, a type of hydrophobin, from Trichoderma reesei self-organizes in an orderly manner at either air/ water or water/solid interfaces. The structural and electrochemical properties of self-organized membranes on different types of solid substrates (electrode materials) were investigated. Two types of substrates, highly oriented pyrolytic graphite (HOPG) and single crystal Au(111), were used for this study. Atomic force microscopy (AFM) showed that the self-organized HFBI membranes (prepared at the air/water interface) exhibited different structures on the two types of electrodes. In terms of the electrochemical results, both HFBI/HOPG and HFBI/Au(111) electrodes performed electrochemical reactions. Self-organized HFBI membranes did not provide insulation from electron transfer. Our findings that HFBI can be tagged with functional proteins, such as enzymes, by genetic fusion and utilized as a molecular carrier to fabricate molecular interfaces on an electrode.

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Solid-support immobilization of a “swing” fusion protein for enhanced glucose oxidase catalytic activity

Cited by 27 publications

References 46 publications

Immobilization of LccC Laccase from Aspergillus nidulans on Hard Surfaces via Fungal Hydrophobins

Immobilization of LccC Laccase from Aspergillus nidulans on Hard Surfaces via Fungal Hydrophobins

Smooth Electron Transfer from a Photoexcited Dye to Semiconductor Electrode Through a Swingable Molecular Interface

Structural and Electrochemical Properties of Self-organized HFBI Membranes on Different Types of Substrates

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