Direct Electron Transfer-type Glucose Research Articles

Microgravity environment grown crystal structure information based engineering of direct electron transfer type glucose dehydrogenase

The heterotrimeric flavin adenine dinucleotide dependent glucose dehydrogenase is a promising enzyme for direct electron transfer (DET) principle-based glucose sensors within continuous glucose monitoring systems. We elucidate the structure of the subunit interface of this enzyme by preparing heterotrimer complex protein crystals grown under a space microgravity environment. Based on the proposed structure, we introduce inter-subunit disulfide bonds between the small and electron transfer subunits (5 pairs), as well as the catalytic and the electron transfer subunits (9 pairs). Without compromising the enzyme’s catalytic efficiency, a mutant enzyme harboring Pro205Cys in the catalytic subunit, Asp383Cys and Tyr349Cys in the electron transfer subunit, and Lys155Cys in the small subunit, is determined to be the most stable of the variants. The developed engineered enzyme demonstrate a higher catalytic activity and DET ability than the wild type. This mutant retains its full activity below 70 °C as well as after incubation at 75 °C for 15 min – much higher temperatures than the current gold standard enzyme, glucose oxidase, is capable of withstanding.

Strategic design and improvement of the internal electron transfer of heme b domain-fused glucose dehydrogenase for use in direct electron transfer-type glucose sensors

A fusion enzyme composed of an Aspergillus flavus-derived flavin adenine dinucleotide glucose dehydrogenase (AfGDH) and an electron transfer domain of Phanerochaete chrysosporium-derived cellobiose dehydrogenase (Pcyb) was previously reported to show the direct electron transfer (DET) ability to an electrode. However, its slow intramolecular electron transfer (IET) rate from the FAD to the heme, limited the sensor signals. In this study, fusion FADGDH (Pcyb-AfGDH) enzymes were strategically redesigned by performing docking simulation, following surface-electrostatic potential estimation in the predicted area. Based on these predictions, we selected the amino acid substitution on Glu324, or on Asn408 to Lys to increase the positive charge at the rim of the interdomain region. Pcyb-AfGDH mutants were recombinantly produced using Pichia pastoris as the host microorganism, and their IET was evaluated. Spectroscopic observations showed that the Glu324Lys (E324K) and Asn408Lys (N408K) Pcyb-AfGDH mutants showed approximately 1.70- and 9.0-fold faster IET than that of wildtype Pcyb-AfGDH, respectively. Electrochemical evaluation revealed that the mutant Pcyb-AfGDH-immobilized electrodes showed higher DET current values than that of the wildtype Pcyb-AfGDH-immobilized electrodes at pH 6.5, which was approximately 9-fold higher in the E324K mutant and 15-fold higher in the N408K mutant, than in the wildtype. Glucose enzyme sensors employing N408K mutant was able to measure glucose concentration under physiological condition using artificial interstitial fluid at pH 7.4, whereas the one with wildtype Pcyb-AfGDH was not. These results indicated that the sensor employed the redesigned mutant Pcyb-AfGDH can be used for future continuous glucose monitoring system based on direct electron transfer principle. (247 words).

Construction of Super-Stabilized Direct Electron Transfer Type Glucose Dehydrogenase for Long Term Continuous Glucose Sensing System

Introduction: The development of super-stabilized flavin adenine dinucleotide glucose dehydrogenase (FADGDH) was achieved based on the rational biomolecular engineering.The FADGDH from Burkholderia cepacia (BcGDH) is the ideal enzyme for continuous glucose monitoring (CGM) sensor, because of its inherent DET ability (Yamazaki 2008, Lee 2018). BcGDH is composed of three subunits, which are the catalytic subunit harboring FAD and iron-sulfur (3Fe-4S) cluster, the electron transfer subunit harboring three heme c, and the small subunit which functions as a “hitchhiker” protein. We recently reported the BcGDH catalytic subunit complexed with small subunit (Yoshida et al., 2019, PDB 6a2u), thereby elucidated the intra-molecular electron transfer pathway, and inherent disulfide bond between catalytic subunit and small subunit.BcGDH shows two distinct temperature profiles due to variations in its quaternary structure (Sode 1996, Yamazaki 1999). BcGDH composed of three subunits showed an optimum reaction temperature of approximately 45 ̊C. By increasing temperature, enzyme activity gradually decreases, due to the dissociation of electron transfer subunit. However, at temperature above 60 ̊C, enzyme activity increases again with the increase the temperature and shows second peak at around 70 ̊C, which is derived from the complex composed of catalytic subunit and small subunit. This phenomenon suggested that the dissociation of the electron transfer subunit dominates the stability of BcGDH three subunit complex, which is the essential structure to show DET ability. Namely, to stabilize BcGDH enzyme complex, the stabilization of quaternary structure is the most rational strategy for this enzyme.We recently succeeded in the elucidation of crystal structure of BcGDH complex together with the electron transfer subunit. The availability of quaternary structure of this enzyme enabled us to design inter-subunit disulfide bond in BcGDH. In this study, the designing and characterization of super-stabilized BcGDH is presented. We also characterize the DET ability of the engineered BcGDH, which is the mandatory characteristic of this enzyme for the development of the future innovative biosensing systems. Methods: Amino acid substitution to cysteine residues were carried out at the residues existing on the surface of each subunit, to form inter-subunit disulfide bonds. Recombinantly prepared BcGDH mutant complexes were purified by affinity chromatography. The catalytic activity and thermal stability were investigated. Then DET-type sensors were constructed by immobilization on the gold electrode with self-assembled monolayer (SAM) layer, and investigated their stability by continuous operation. Results and Discussion: Based on the 3D structure of BcGDH complex, disulfide bonds between electron transfer subunit and either catalytic subunit or small subunit were designed. By the screening of the cystaine residue substituted mutants, some mutants showed remarkable high thermal stability as BcGDH complex without the decrease of the catalytic activity. Furthermore, multiple inter-subunit disulfide bonds made the enzyme as super-stabilized which showed high catalytic activity even by the incubation at 70 ̊C. Thus developed engineered enzyme revealed to show DET ability with electrode, indistinguishable to the wild type one. This super-stabilized enzyme will contribute the improvement of the operational stability of enzyme sensors. References Lee et al., Bioelectrochemistry. 121 (2018) 1–6Sode et al., Enzyme Microb. Technol. 19 (1996) 82–85Yamazaki et al., Appl. Biochem. Biotechnol. 77 (1999) 325–336Yamazaki et al., Anal. Lett. 41 (2008) 2363-2373.Yoshida et al., Acta Crystallogr. Sect. D Struct. Biol. 75 (2019) 841–851

Third generation impedimetric sensor employing direct electron transfer type glucose dehydrogenase

Faradaic electrochemical impedance spectroscopy (faradaic EIS) is an attractive measurement principle for biosensors. However, there have been no reports on sensors employing direct electron transfer (DET)-type redox enzymes based on faradaic EIS principle. In this study, we have attempted to construct the 3rd-generation faradaic enzyme EIS sensor, which used DET-type flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase (GDH) complex, to elucidate its characteristic properties as well as to investigate its potential application as the future immunosensor platform. The gold disk electrodes (GDEs) with DET-type FADGDH prepared using self-assembled monolayer (SAM) showed the glucose concentration dependent impedance change, which was confirmed by the change in the charge transfer resistance (Rct). The Δ(1/Rct) values were also affected by DC bias potential and the length of SAM. Based on the Nyquist plot and Bode plot simulations, glucose sensing by imaginary impedance monitoring under fixed frequency (5 mHz) was carried out, revealing the higher sensitivity at low glucose concentration with wider linear range (0.02–0.2 mM). Considering this high sensitivity toward glucose, the 3rd-generation faradaic enzyme EIS sensor would provide alternative platform for future impedimetric immunosensing system, which does not use redox probe.

Minimally Invasive Microneedle Array Electrodes Employing Direct Electron Transfer Type Glucose Dehydrogenase for the Development of Continuous Glucose Monitoring Sensors

Closed loop systems hinge on the accuracy and precision of the continuous glucose monitoring sensors. Most of the commercially available continuous glucose monitoring sensors is implanted subcutaneously for a period of 7-14 days. The subsequent biofouling effects have implications on the performance of the sensors over time especially at low glucose concentrations. In addition, the commercially available sensors are sensitive to the presence of interfering species such as acetaminophen in the skin compartment. We report here on the marriage of minimally invasive, continuous glucose sensors and a direct electron transfer type glucose dehydrogenase enzymatic system. Whilst the microneedles here are designed to sit in the dermal interstitial fluid over a 24-48 hour period to minimize the biofouling effect, the direct electron transfer enzyme allows operation of the electrochemical sensor at lower potentials to minimize the effect of interference. The microneedle structure design also enables the use of compensation electrodes for background subtraction to further nullify the effects of interference.

Continuous operation of an ultra-low-power microcontroller using glucose as the sole energy source

An ultimate goal for those engaged in research to develop implantable medical devices is to develop mechatronic implantable artificial organs such as artificial pancreas. Such devices would comprise at least a sensor module, an actuator module, and a controller module. For the development of optimal mechatronic implantable artificial organs, these modules should be self-powered and autonomously operated. In this study, we aimed to develop a microcontroller using the BioCapacitor principle. A direct electron transfer type glucose dehydrogenase was immobilized onto mesoporous carbon, and then deposited on the surface of a miniaturized Au electrode (7mm2) to prepare a miniaturized enzyme anode. The enzyme fuel cell was connected with a 100 μF capacitor and a power boost converter as a charge pump. The voltage of the enzyme fuel cell was increased in a stepwise manner by the charge pump from 330mV to 3.1V, and the generated electricity was charged into a 100μF capacitor. The charge pump circuit was connected to an ultra-low-power microcontroller. Thus prepared BioCapacitor based circuit was able to operate an ultra-low-power microcontroller continuously, by running a program for 17h that turned on an LED every 60s. Our success in operating a microcontroller using glucose as the sole energy source indicated the probability of realizing implantable self-powered autonomously operated artificial organs, such as artificial pancreas.

The development of an autonomous self-powered bio-sensing actuator

The inherent low power supplies from biofuel cells limits their application as power sources in implantable devices. To overcome these limitations, we have previously developed a novel device designated as a “BioCapacitor” (Hanashi et al., 2009). The BioCapacitor generates sufficient stable power to operate biosensing devices and signal transducers. In this paper, we report an autonomous, self-powered, sensing actuator that employs the principle of BioCapacitor as the core technology. The device is composed of a BioCapacitor, which uses a direct electron transfer-type glucose enzyme fuel cell, connected to a stepper motor as the actuator. In the presence of glucose, the BioCapacitor was able to generate enough electricity to operate the actuator. The torque and rotational speed of the stepper motor was dependent on the capacitance of the capacitor used in the BioCapacitor, and on the glucose concentration. A higher glucose concentration provided the stepper motor with higher torque and speed, resulting from the charge/discharge frequency of the capacitor as a function of the enzyme reaction at the anode. The actuator was operated entirely by the energy derived from glucose oxidation, and its performance was regulated by the glucose concentration. We also present the performance of a liquid-pumping system that employs the autonomous, self-powered, sensing actuator, which demonstrates its potential for application as a drug delivery/pumping system.

A novel wireless glucose sensor employing direct electron transfer principle based enzyme fuel cell

In this paper we present a novel wireless glucose biosensing system employing direct electron transfer principle based enzyme fuel cell. Using the glucose dehydrogenase complex, which is composed of a catalytic subunit containing FAD, the cytochrome c subunit that harbors heme c as the electron transfer subunit, and chaperone-like subunit, a direct electron transfer-type glucose enzyme fuel cell was constructed. The enzyme glucose fuel cell generated electric power, and the open-circuit voltage showed glucose concentration dependence, which suggests potential applications for this glucose-sensing system. We constructed a miniaturized “all-in-one” glucose enzyme fuel cell, which represents a compartmentless fuel that is based on the direct electron transfer principle. This involved the combination of a wireless transmitter system and a simple and miniaturized continuous glucose monitoring system, which operated continuously for about 3 days with stable response. This is the first demonstration of an enzyme-based direct electron transfer-type enzyme fuel cell and fuel cell-type glucose sensor which can be utilized as a subcutaneously implantable system for continuous glucose monitoring.

PQQ glucose dehydrogenase with novel electron transfer ability

PQQ glucose dehydrogenase from Acinetobacter calcoaceticus (GDH-B) is one of the most industrially attractive enzymes, as a sensor constituent for glucose sensing, because of its high catalytic activity and insensitivity to oxygen. We attempted to engineer GDH-B to enable electron transfer to the electrode in the absence of artificial electron mediator by mimicking the domain structure of the quinohemoprotein ethanol dehydrogenase (QH-EDH) from Comamonas testosteroni, which is composed of a PQQ-containing catalytic domain and a cytochrome c domain. We genetically fused the cytochrome c domain of QH-EDH to the C-terminal of GDH-B. The constructed fusion protein showed not only intra-molecular electron transfer, between PQQ and heme of the cytochrome c domain, but also electron transfer from heme to the electrode, thereby allowing the construction of a direct electron transfer-type glucose sensor.