Molecular Carrier-Gox Fusion Protein for Electro-Catalytic Reaction

Abstract

Smart functions of biomolecules (e.g. enzyme, antibody, and DNA) have been employed for various practical applications, such as immobilized catalyst, biosensor, bio-battery, clinical assay. Because that biomolecules have an excellent force of molecular recognition and molecular selectivity, and they considered useful. As the existence examples, there is a glucose sensor (enzyme based sensor) for diabetes to check the glucose concentration in their blood. Antibody had been employed for affinity sensors that are used in clinical assay. In this study, we try to fabricate a glucose oxidase (GOx) modified electrode using GOx – molecular carrier fusion molecule for electro-catalytic reaction. Smart fabrication of enzyme modified electrode could be performed by using the molecular carrier fusion enzyme. The molecular carrier can adsorb solid electrode surface and form single molecular membrane through self-organization. Using the molecular-carrier system, enzyme modified electrode can be prepared by very simple easy process. However, in order to perform efficient electro catalytic reaction (electron transfer between modified molecules and an electrode), careful optimization of enzyme modification on an electrode is required. We have paid attention and employed hydrophobin as molecular carrier. The hydrophobin is derived from filamentous bacterium and is divided into two classes by the hydropathy patterns of their amino acid sequences and the difference of their solubility in solvents. In this research, we focused on the class II hydrophobin, HFBI especially. HFBI is an amphiphilic protein and the size of it is very small, about 2nm cubic molecule. It has four disulfide bond, and the existence of them makes HFBI strong. Most important characteristic, one face of the cubic is dominantly hydrophobic and is adsorption to solid material surface. Another important function of HFBI is self-organization. It forms monomolecular film on liquid/air interface and liquid/solid interface. These characteristic that molecular-carrier is needed. We already reported that the HFBI can form coated membrane on solid substrate surface [1]. In this study, we prepared genetically tagged HFBI - glucose oxidase (GOx) molecule. The fusion protein immobilize GOx on solid surface with non-rigidity (swing-ability of immobilized GOx). The swing motion lets GOx have efficient catalytic activity [2]. 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 previous 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 [3]. In this study, catalytic electrochemical reaction was performed by HFBI-GOx fusion protein adsorbed electrode. Catalytic electrochemical manner is compared with condition of HFBI-GOx on an electrode which is observed by AFM. HFBI makes random structure on GC substrate and it is far from the one on HOPG substrate. HFBI-coated HOPG electrode performs electron transfer between the electrode and solution phase through the dense HFBI molecular layer. This is because the HFBI self-organized membrane has a honeycomb-like structure, with penetrating holes [4]. The difference due to the substrate makes possibility the proper use depend on the type of devices in the future. Hereafter, it is necessary to examine the electrolytic property of the electrode covered with HFBI-GOx fusion protein by overlaying the number of electrochemical measurement. Reference Hitoshi Asakawa, Shinya Tahara, Momoka Nakamichi, Kenji Takehara, Shinya Ikeno, Markus Linder, and Tetsuya Haruyama, The amphiphilic protein HFBII as a genetically taggable molecular carrier for the formation of a self-organized functional protein layer on a solid surface, Langmuir, 25(16), 8841-8844 (2009) Yoshiyuki Takatsuji, Ryota Yamasaki, Atsushi Iwanaga, Michael Lienemann, Markus B. Linder, Tetsuya Haruyama, Solid-support immobilization of a “swing” fusion protein for enhanced glucose oxidase catalytic activity, Colloids and Surfaces B, 112, 186-191 (2013) Ryota Yamasaki, Hitoshi Asakawa, Takeshi Fukuma, and Tetsuya Haruyama, Structual and Electrochemical Properties of Self-organized HFBI Membranes on Different Types of Substrates, Electrochemistry, 83(11), 969-973 (2015) Ryota Yamasaki, Yoshiyuki Takatsuji, Michael Lienemann, Hitoshi Asakawa, Takeshi Fukuma, Markus Linder, Tetsuya Haruyama, Electrochemical properties of honeycomb-like structured HFBI self-organized membranes on HOPG electrodes, Colloids and Surfaces B, 123, 803-808 (2014)