Development of a Versatile Method to Construct Direct Electron Transfer-Type Enzyme Complexes Employing SpyCatcher/SpyTag System

The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method

Alteration of Electron Acceptor Preferences in the Oxidative Half-Reaction of Flavin-Dependent Oxidases and Dehydrogenases

In this review, recent progress in the engineering of the oxidative half-reaction of flavin-dependent oxidases and dehydrogenases is discussed, considering their current and future applications in bioelectrochemical studies, such as for the development of biosensors and biofuel cells. There have been two approaches in the studies of oxidative half-reaction: engineering of the oxidative half-reaction with oxygen, and engineering of the preference for artificial electron acceptors. The challenges for engineering oxidative half-reactions with oxygen are further categorized into the following approaches: (1) mutation to the putative residues that compose the cavity where oxygen may be located, (2) investigation of the vicinities where the reaction with oxygen may take place, and (3) investigation of possible oxygen access routes to the isoalloxazine ring. Among these approaches, introducing a mutation at the oxygen access route to the isoalloxazine ring represents the most versatile and effective strategy. Studies to engineer the preference of artificial electron acceptors are categorized into three different approaches: (1) engineering of the charge at the residues around the substrate entrance, (2) engineering of a cavity in the vicinity of flavin, and (3) decreasing the glycosylation degree of enzymes. Among these approaches, altering the charge in the vicinity where the electron acceptor may be accessed will be most relevant

Mediator Preference of Two Different FAD-Dependent Glucose Dehydrogenases Employed in Disposable Enzyme Glucose Sensors

Most commercially available electrochemical enzyme sensor strips for the measurement of blood glucose use an artificial electron mediator to transfer electrons from the active side of the enzyme to the electrode. One mediator recently gaining attention for commercial sensor strips is hexaammineruthenium(III) chloride. In this study, we investigate and compare the preference of enzyme electrodes with two different FAD-dependent glucose dehydrogenases (FADGDHs) for the mediators hexaammineruthenium(III) chloride, potassium ferricyanide (the most common mediator in commercial sensor strips), and methoxy phenazine methosulfate (mPMS). One FADGDH is a monomeric fungal enzyme, and the other a hetero-trimeric bacterial enzyme. With the latter, which contains a heme-subunit facilitating the electron transfer, similar response currents are obtained with hexaammineruthenium(III), ferricyanide, and mPMS (6.8 µA, 7.5 µA, and 6.4 µA, respectively, for 10 mM glucose). With the fungal FADGDH, similar response currents are obtained with the negatively charged ferricyanide and the uncharged mPMS (5.9 µA and 6.7 µA, respectively, for 10 mM glucose), however, no response current is obtained with hexaammineruthenium(III), which has a strong positive charge. These results show that access of even very small mediators with strong charges to a buried active center can be almost completely blocked by the protein

Mutagenesis Study of the Cytochrome c Subunit Responsible for the Direct Electron Transfer-Type Catalytic Activity of FAD-Dependent Glucose Dehydrogenase

The FAD-dependent glucose dehydrogenase from Burkholderia cepacia (FADGDH) is a hetero-oligomeric enzyme that is capable of direct electron transfer (DET) with an electrode. The cytochrome c (cyt c) subunit, which possesses three hemes (heme 1, heme 2, and heme 3, from the N-terminal sequence), is known to enable DET; however, details of the electron transfer pathway remain unknown. A mutagenesis investigation of the heme axial ligands was carried out to elucidate the electron transfer pathway to the electron mediators and/or the electrode. The sixth axial ligand for each of the three heme irons, Met109, Met263, and Met386 were substituted with His. The catalytic activities of the wild-type (WT) and mutant enzymes were compared by investigating their dye-mediated dehydrogenase activities and their DET abilities toward the electrode. The results suggested that (1) heme 1 with Met109 as an axial ligand is mainly responsible for the electron transfer with electron acceptors in the solution, but not for the DET with the electrode; (2) heme 2 with Met263 is responsible for the DET-type reaction with the electrode; and (3) heme 3 with Met386 seemed to be the electron acceptor from the catalytic subunit. From these results, two electron transfer pathways were proposed depending on the electron acceptors. Electrons are transferred from the catalytic subunit to heme 3, then to heme 2, to heme 1 and, finally, to electron acceptors in solution. However, if the enzyme complex is immobilized on the electrode and is used as electron acceptors, electrons are passed to the electrode from heme 2

Development of a Versatile Method to Construct Direct Electron Transfer-Type Enzyme Complexes Employing SpyCatcher/SpyTag System

The electrochemical enzyme sensors based on direct electron transfer (DET)-type oxidoreductase-based enzymes are ideal for continuous and in vivo monitoring. However, the number and types of DET-type oxidoreductases are limited. The aim of this research is the development of a versatile method to create a DET-type oxidoreductase complex based on the SpyCatcher/SpyTag technique by preparing SpyCatcher-fused heme c and SpyTag-fused non-DET-type oxidoreductases, and by the in vitro formation of DET-type oxidoreductase complexes. A heme c containing an electron transfer protein derived from Rhizobium radiobacter (CYTc) was selected to prepare SpyCatcher-fused heme c. Three non-DET-type oxidoreductases were selected as candidates for the SpyTag-fused enzyme: fungi-derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase (GDH), an engineered FAD-dependent d-amino acid oxidase (DAAOx), and an engineered FMN-dependent l-lactate oxidase (LOx). CYTc-SpyCatcher (CYTc-SC) and SpyTag-Enzymes (ST-GDH, ST-DAAOx, ST-LOx) were prepared as soluble molecules while maintaining their redox properties and catalytic activities, respectively. CYTc-SC/ST-Enzyme complexes were formed by mixing CYTc-SpyCatcher and SpyTag-Enzymes, and the complexes retained their original enzymatic activity. Remarkably, the heme domain served as an electron acceptor from complexed enzymes by intramolecular electron transfer; consequently, all constructed CYTc-SC/ST-Enzyme complexes showed DET ability to the electrode, demonstrating the versatility of this method

Employment of 1-Methoxy-5-Ethyl Phenazinium Ethyl Sulfate as a Stable Electron Mediator in Flavin Oxidoreductases-Based Sensors

In this paper, a novel electron mediator, 1-methoxy-5-ethyl phenazinium ethyl sulfate (mPES), was introduced as a versatile mediator for disposable enzyme sensor strips, employing representative flavin oxidoreductases, lactate oxidase (LOx), glucose dehydrogenase (GDH), and fructosyl peptide oxidase (FPOx). A disposable lactate enzyme sensor with oxygen insensitive Aerococcus viridans-derived engineered LOx (AvLOx), with A96L mutant as the enzyme, was constructed. The constructed lactate sensor exhibited a high sensitivity (0.73 ± 0.12 μA/mM) and wide linear range (0–50 mM lactate), showings that mPES functions as an effective mediator for AvLOx. Employing mPES as mediator allowed this amperometric lactate sensor to be operated at a relatively low potential of +0.2 V to 0 V vs. Ag/AgCl, thus avoiding interference from uric acid and acetaminophen. The lactate sensors were adequately stable for at least 48 days of storage at 25 °C. These results indicated that mPES can be replaced with 1-methoxy-5-methyl phenazinium methyl sulfate (mPMS), which we previously reported as the best mediator for AvLOx-based lactate sensors. Furthermore, this study revealed that mPES can be used as an effective electron mediator for the enzyme sensors employing representative flavin oxidoreductases, GDH-based glucose sensors, and FPOx-based hemoglobin A1c (HbA1c) sensors

An Fe-S cluster in the conserved Cys-rich region in the catalytic subunit of FAD-dependent dehydrogenase complexes

Several bacterial flavin adenine dinucleotide (FAD)-harboring dehydrogenase complexes comprise three distinct subunits: a catalytic subunit with FAD, a cytochrome c subunit containing three hemes, and a small subunit. Owing to the cytochrome c subunit, these dehydrogenase complexes have the potential to transfer electrons directly to an electrode. Despite various electrochemical applications and engineering studies of FAD-dependent dehydrogenase complexes, the intra/inter-molecular electron transfer pathway has not yet been revealed. In this study, we focused on the conserved Cys-rich region in the catalytic subunits using the catalytic subunit of FAD dependent glucose dehydrogenase complex (FADGDH) as a model, and site-directed mutagenesis and electron paramagnetic resonance (EPR) were performed. By co-expressing a hitch-hiker protein (γ-subunit) and a catalytic subunit (α-subunit), FADGDH γα complexes were prepared, and the properties of the catalytic subunit of both wild type and mutant FADGDHs were investigated. Substitution of the conserved Cys residues with Ser resulted in the loss of dye-mediated glucose dehydrogenase activity. ICP-AEM and EPR analyses of the wild-type FADGDH catalytic subunit revealed the presence of a 3Fe-4S-type iron-sulfur cluster, whereas none of the Ser-substituted mutants showed the EPR spectrum characteristic for this cluster. The results suggested that three Cys residues in the Cys-rich region constitute an iron-sulfur cluster that may play an important role in the electron transfer from FAD (intra-molecular) to the multi-heme cytochrome c subunit (inter-molecular) electron transfer pathway. These features appear to be conserved in the other three-subunit dehydrogenases having an FAD cofactor

Development of an Interdigitated Electrode-Based Disposable Enzyme Sensor Strip for Glycated Albumin Measurement.

Glycated albumin (GA) is an important glycemic control marker for diabetes mellitus. This study aimed to develop a highly sensitive disposable enzyme sensor strip for GA measurement by using an interdigitated electrode (IDE) as an electrode platform. The superior characteristics of IDE were demonstrated using one microelectrode of the IDE pair as the working electrode (WE) and the other as the counter electrode, and by measuring ferrocyanide/ferricyanide redox couple. The oxidation current was immediately reached at the steady state when the oxidation potential was applied to the WE. Then, an IDE enzyme sensor strip for GA measurement was prepared. The measurement of fructosyl lysine, the protease digestion product of GA, exhibited a high, steady current immediately after potential application, revealing the highly reproducible measurement. The sensitivity (2.8 nA µM-1) and the limit of detection (1.2 µM) obtained with IDE enzyme sensor strip were superior compared with our previously reported sensor using screen printed electrode. Two GA samples, 15 or 30% GA, corresponding to healthy and diabetic levels, respectively, were measured after protease digestion with high resolution. This study demonstrated that the application of an IDE will realize the development of highly sensitive disposable-type amperometric enzyme sensors with high reproducibility