Adenine Dinucleotide-dependent Glucose Dehydrogenase 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.

Electrochemical modification at multiwalled carbon nanotube electrodes with Azure A for FAD- glucose dehydrogenase wiring: structural optimization to enhance catalytic activity and stability

Electrochemical grafting is a suitable technology for fabricating electrode surfaces with new chemical functionalities whilst maintaining the bulk properties of the electrode, and electrochemical amine oxidation and diazonium salt reduction are two widely used techniques to achieve this end. Herein, we report the electrochemical reductive grafting of Azure A onto multiwalled carbon nanotube (MWCNT) electrodes for the efficient wiring of flavin adenine dinucleotide (FAD) dependent glucose dehydrogenase. The diazonium salt of Azure A is formed in situ and subsequently grafted onto the electrode surface through electrochemical reduction. The formal potential of the resultant Azure-A-modified electrode shifted to −0.05 V vs. Ag/AgCl upon radical coupling to the MWCNT electrode. Electron transfer from FAD buried in the protein shell to the electrode via Azure A was then observed in the presence of glucose in the buffer solution. This study focused on the important effect of CNT mass loading on Azure-A loading as well as bioelectrocatalytic activity and storage stability. The three-dimensional porous structure of the MWCNT electrode was determined to be favorable for the immobilization of flavin adenine dinucleotide dependent glucose dehydrogenase and efficient electron transfer via the Azure-A functionalities. The optimized 300 µg CNT-loaded modified electrode on glassy carbon (3 mm diameter) retains its initial activity for 3 d and 25% of its initial activity after 10 d. Furthermore, we show that grafted Azure A is stably immobilized on the MWCNTs for 1 month; therefore, the limiting stability factor is enzyme leaching and/or deactivation.

Photoelectrochemically-assisted biofuel cell constructed by redox complex and g-C3N4 coated MWCNT bioanode

Herein, we report a membraneless glucose and air photoelectrochemical biofuel cell (PBFC) with a visible light assisted photobioanode. Flavin adenine dinucleotide dependent glucose dehydrogenase (FADGDH) was immobilized on the combined photobioanode for the visible light assisted glucose oxidation (GCE|MWCNT|g-C3N4|Ru-complex|FADGDH) with a quinone mediated electron transfer. Bilirubine oxidase (BOx) immobilized on MWCNT coated GCE (GCE|BOx) was used as the cathode with direct electron transfer (DET). An improvement of biocatalytic oxidation current was observed by 6.2% due in part to the light-driven electron-transfer. The large oxidation currents are probably owing to the good contacting of the immobilized enzymes with the electrode material and the utilization of light assisted process. Under the visible light, the photobioanode shows an anodic photocurrent of 1.95 μA cm2 at attractively low potentials viz. −0.4 vs Ag/AgCl. The lower-lying conduction band of g-C3N4 as compared to Ru-complexes decreases the rate of hole and electron recombination and enhances the charge transportation. The bioanode shows maximum current density for glucose oxidation up to 6.78 μA cm−2 at 0.2 V vs Ag/AgCl at pH:7. The performance of three promising Ru-complexes differing in chemical and redox properties were compared as electron mediators for FADGDH. Upon illumination, the PBFC delivered a maximum power density of 28.5 ± 0.10 μW cm−2 at a cell voltage of +0.4 V with an open circuit voltage of 0.64 V.

First Eurasian conference on nanotechnology, Baku, Azerbaijan photoelectrochemically-assisted bioanode constructed by Ru-complex and g-C3N4 coated MWCNT electrode

Abstract In this study, a novel photobioanode was constructed using a Ruthenium complex (Ru-complex) sensitized graphitic carbon nitride (g-C3N4) nanomaterial deposited on multi-walled carbon nanotubes (MWCNT). g-C3N4 coated MWCNT bearing Ru-complex acts as a platform for the immobilization of flavin adenine dinucleotide dependent glucose dehydrogenase (FADGDH). The formed photosensitive bioanode showed an enhancement in glucose oxidation current under visible light irradiation. The electrocatalytic current increase for in glucose oxidation are possibly due to the good electrical contacting of the immobilized FADGDH on the electrode surface and utilization of light energy. The spectroscopic properties of g-C3N4 and Ru-complex in dimethylformamide (DMF) were determined by UV–visible absorption spectra demonstrating the characteristic absorption peaks in the visible light region. Upon visible light illumination, the enzymatic photoanode revealed a prompt rise in photocurrent (ca. 1.44 μA cm−2) compared to the photoelectrode whithout enzyme (0.65 µA cm−2). Light irradiation of the bioanode improved the overall performance of the electrode as glucose oxidation was reinforced by photoelectrochemical (PEC) process.

Гостра токсичність та гіпоглікемічна активність 7-хлоро-4-тіозаміщених хіноліну

The search for effective and safe pharmacological drugs for the treatment of diabetes is remaining highly valuable issue due to the chronic course of the disease and the need to use antipyretic drugs during the rest of life. Currently, various hypoglycemic agents are presented in the pharmaceutical market and the safety of their application is in priority. The activity of 4-thio-7-chloroquinoline derivatives as potential hypoglycemic drugs was studied here. The hypoglycemic activity was determined using a One Touch Ultra Plus Flex meter. The approach to glucose determination by this method is based on the reaction of glucose contained in the blood sample with flavin adenine dinucleotide dependent glucose dehydrogenase in the test strip, with the formation of a small electric current. The strength of this current calculates the level of glucose in the blood serum. Sodium 3-(7-chloroquinolin-4-ylthio)propionate demonstrates a high hypoglycemic activity after its intragastric administration. This compound significantly reduces glucose levels in rats up to 18.1% when compared with baseline and other compounds. 2-(7-Chloroquinoline-4-ylthio)propane and 2-acetamido-3-(7-chloroquinolin-4-ylthio)propanoic acid had little or no effect on serum glucose. The administration of compound 3 3-(7-chloroquinolin-4-ylthio)propionic acid in experimental animals increases the serum glucose on 19,6% comparing to the basic level. Acute toxicity of 4-thio7-chloro-substituted quinoline was determined with the Prozorovsky express method. The substances were administered once intraperitoneally as a thin aqueous suspension in saline solution (stabilizer - Twin 80) or as a solution of not more than 1 ml. The relationship between the structure of the test compounds and their toxicity is analyzed. It has been established that the mean lethal dose (LD50) of these compounds ranges from 200-800 mg/kg and is classified as low toxic that corresponds to the fourth class of toxicity.

Structural Based Investigation of Artificial Electron Acceptor Interaction of Fungus Derived Flavin Adenine Dinucleotide Dependent Glucose Dehydrogenase

[Introduction]Self-monitoring of blood glucose (SMBG) is inevitable for the glycemic control of the diabetic patients, and plays an indispensable role in the current diabetes management. The current most popular principle for the sensor for SMBG is based on the so-called “second generation” type that are the use of the dye mediated dehydrogenase reactions. The principle employs the artificial electron acceptor and monitoring the concentration/quantity of thus reacted (reduced) artificial electron acceptor on the electrode. Fungi-derived flavin adenine dinucleotide dependent glucose dehydrogenases (FADGDHs) have recently been paid significant attention, focusing its narrow specificity, especially as the enzyme not active toward maltose. However, interaction between FADGDHs and artificial electron acceptor has not yet been investigated. Recently, we have reported on the first 3D structures of FADGDH, using Aspergillus flavus derived FADGDH (AfGDH). Here we report the investigation of interaction between mediator and enzyme by elucidating the 3D structure of AfGDH complex with potassium ferricyanide. Mutagenesis studies were also carried out to investigate the role of residues elucidated to interact with ferricyanide. [Methods]The structural gene of AfGDH was inserted into an expression vector and the enzyme was recombinantly expressed using E. coli BL21 (DE3). Soluble AfGDH was harvested and purified by hydrophobic interaction chromatography and anion exchange chromatography. Purified protein was concentrated, and crystallized by sitting-drop vapor-diffusion method. In order to obtain a ternary complex structure with FAD and ferricyanide, a single crystal was soaked in the potassium ferricyanide solution. X-ray diffraction data were collected and structure was determined. Based on the 3D structure, several mutants were constructed by site-directed mutagenesis and evaluated GDH activity, which was determined using either phenazine methosulphate/2,6-dichlorophenol indophenol (PMS/DCIP) or potassium ferricyanide as the electron acceptors. [Results & Discussion] A ternary complex structure of AfGDH with FAD and ferricyanide complex was obtained. Two potassium ferricyanide molecules were observed in the protein structure. One potassium ferricyanide interacted with one lysine residue, whereas the other molecules interacted with two lysine residues. In order to elucidate the role of these lysine residues, mutant AfGDHs were constructed by substituting lysine residues with alanine. The structural gene of AfGDH mutants were inserted into an expression vector and the enzyme was recombinantly prepared using E. coli BL21 (DE3). All mutants were expressed at soluble fraction. These mutants were subjected for anion exchange chromatography for purification. Interestingly, the mutant enzymes were eluted at different salt concentration than those eluted in the purification of wild type enzyme, suggesting that surface net charge was significantly changed by these mutations. Some of the mutant AfGDH showed unique property in the specificity toward electron acceptors. The GDH activities were compared by using either PMS/DCIP or ferricyanide as the electron acceptor, for both wild type and mutant enzymes. Although the alanine substitutions to these lysine residues did not significantly influenced PMS/DCIP based dye mediated GDH activity, but some mutation affected the ratio of dye mediated GDH activities of those using PMS/DCIP and of using ferricyanide. These results, together with the results of the change in the elution properties during anion exchange chromatography, suggested the lysine residues, we focused, play important role in the affinity toward ferricyanide during oxidative half reaction. (1) K. Mori, et al., Biotechnol Lett. 33(11) 2255-63(2011) (2) G. Sakai, et al., Biotechnol Lett. 37(5) 1091-99(2015) (3) H. Yoshida, et al. Sci. Rep. 5:13498. doi:10.1038/srep13498 (2015)

Identification of Substrate Binding Site of Fungus Derived Flavin Adenine Dinucleotide Dependent Glucose Dehydrogenase

【Introduction】 The fungi derived flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenases (GDHs) are flavoproteins that catalyze the oxidation of first hydroxyl group of glucose and other sugar molecules, using FAD as the primary electron acceptor (systematic name; D-glucose:acceptor 1-oxidoreductase). Recently, fungi-derived FADGDHs have received attention as the enzymes for blood glucose monitoring in diabetes patients, especially for the use in the self-monitoring of blood glucose (SMBG), because of their oxygen insensitivity and relatively narrow substrate specificity, in particular their lack of activity toward maltose. However, unlike glucose oxidase, a gold standard enzyme for blood glucose monitoring, especially for the strictly narrow specificity toward glucose, fungi derived FADGDHs react with xylose as the substrate. We have previously reported on the characterization of Aspergillus flavus derived FADGDH (AfGDH) from genome database 1, and also the stabilization of AfGDH by introducing a disulfide bond 2. Recently we have reported on the first 3D structure of fungus derived FADGDH3. In order to construct an engineered enzyme meeting with the current increasing demands in the accuracy of sensors for SMBG, further advance in our understanding of the mechanism of substrate recognition of this enzyme is necessary.In this study, we report the site directed mutagenesis studies toward the residues expected to influence the binding of substrate, based on the structural analyses of AfGDH crystal structure (PDB ID：4ynu). 【Methods】The residues responsible for the substrate binding and/or recognition of AfGDH and Aspergillus niger derived GOx (AnGOx), were predicted based on the reported crystal structure (PDB ID:4ynu, 1cf3). According to this prediction, several mutant AfGDHs were designed and constructed by the sited directed mutagenesis. AfGDH wild type (WT) and mutants were prepared using recombinant Escherichia coli. The enzyme activities and substrate specificities were determined by measuring dye mediated dehydrogenase activity, spectrometrically, using phenazine methosulphate as the electron mediator and 2,6-dichlorophenolindophenol(DCIP) as the final electron acceptor, and by monitoring decrease in the absorbance of oxidized DCIP at 600nm. 【Results and Discussion】Several residues existing within the distance which may interact with glucono-δ-lactone by their side chain were observed from the ternary complex structure of AfGDH (4ynu). Assuming that this side chain of each residue may recognize and interact with glucose, AfGDH structure was compared with AnGOx. As results, residues, possibly recognize 1st, 2nd, 3rd or 4th hydroxyl group of glucose are conserved within AfGDH and AnGOx. Especially, two residues recognize 1st hydroxyl group of glucose are completely conserved. Mutational analyses resulted in the complete loss of the catalytic activity, thus suggesting that these residues are the catalytic residues. The residue interacting 4th hydroxyl group is conserved, and has been recognized that plays an important role in discrimination of monosaccharide from disaccharides, such as maltose. Interestingly, the introduction of mutation on this residue did not result in the significant change in the substrate specificity, especially toward maltose. The results suggested that the predicted residue may have a limited role in the determination of substrate specificity, and other residues may have more crucial roles. Significant difference in the substrate interaction was observed at the position where may recognize 6th hydroxyl group, which would be the reason of the AfGDH substrate specificity reacting with xylose. Further mutational analyses will be reported.(1) K. Mori, et al., Biotechnol Lett. 33(11) 2255-63(2011) (2) G. Sakai, et al., Biotechnol Lett. 37(5) 1091-99(2015) (3) H. Yoshida, et al., Sci. Rep. 5:13498 doi: 10.1038/srep13498(2015)

High Power Glucose/O2 Biofuel Cell Constructed from MgO-Temlated Carbon Modified Carbon Cloth

Enzymatic biofuel cells (EBFCs) based on enzyme electrocatalysts are devices to directly convert chemical energy of glucose and alcohol to electronical energy. EBFCs gather attention as a power source of wearable electronic sensor devices because they have high biocompatibility. In this paper, we report a glucose/O2 EBFC using flexible carbon cloth electrodes modified with mesoporous carbon particle, which shows high power density as high as 2 mW cm-2. Moreover, the capacity of the BFCs can be controlled by the mole number of glucose added. Porous carbon electrode was fabricated by modifying the MgO-templated mesoporous carbon (MgOC) on carbon cloth in order to enlarge surface area and to immobilize enzyme and redox mediator on electrode surface. MgOC is a kind of pore size-controlled porous carbon; mesopore size can be controlled by alternating the MgO template size. For the anode catalyst, flavin adenine dinucleotide dependent glucose dehydrogenase (FAD-GDH) from Aspergillus terreus was used. Naphthoquinones (NQ) were used as anode mediator. FAD-GDH and NQ were modified on the anode 0.19 and 1.5 mg cm-2, respectively. For the oxygen-reducing cathode, bilirubin oxidase (BOD) was used as enzyme, and 2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid ammonium salt) (ABTS) as mediator. BOD and ABTS were modified on the cathode 1.25 and 1.25 mg cm-2, respectively. 1.0 M phosphate buffer (pH = 7.0) was used as an electrolyte. A cellophane sheet was sandwiched between the anode and cathode as a separator. Figure 1 shows the cell power (solid lines) and cell voltage (broken lines) as a function of current density of the EBFCs. The maximum output power density of BFCs using 1,2-NQ and 1,4-NQ for anode mediators were 1.69 and 2.06 mW cm-2, receptively, because the redox potential of 1,4-NQ is more negative than that of 1,2-NQ. Figure 2 shows the cell capacity of the BFCs as a function of glucose concentration added to the cell. The capacity of the cell increased linear with an increase of the mole number of glucose. Figure 1

Electron-Transfer Studies with a New Flavin Adenine Dinucleotide Dependent Glucose Dehydrogenase and Osmium Polymers of Different Redox Potentials

A new extracellular flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase from Glomerella cingulata (GcGDH) was electrochemically studied as a recognition element in glucose biosensors. The redox enzyme was recombinantly produced in Pichia pastoris and homogeneously purified, and its glucose-oxidizing properties on spectrographic graphite electrodes were investigated. Six different Os polymers, the redox potentials of which ranged in a broad potential window between +15 and +489 mV versus the normal hydrogen electrode (NHE), were used to immobilize and "wire" GcGDH to the spectrographic graphite electrode's surface. The GcGDH/Os polymer modified electrodes were evaluated by chronoamperometry using flow injection analysis. The current response was investigated using a stepwisely increased applied potential. It was observed that the ratio of GcGDH/Os polymer and the overall loading of the enzyme electrode significantly affect the performance of the enzyme electrode for glucose oxidation. The best-suited Os polymer [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(PVI)Cl](+) had a potential of +309 mV versus NHE, and the optimum GcGDH/Os polymer ratio was 1:2 yielding a maximum current density of 493 μA·cm(-2) at a 30 mM glucose concentration.