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1.
J Chem Ecol ; 45(11-12): 972-981, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31713110

RESUMO

Microplitis croceipes is a solitary parasitoid that specializes on noctuid larvae of Helicoverpa zea and Heliothis virescens. Both the parasitoid and its hosts are naturally distributed across a large part of North America. When parasitoids deposit their eggs into hosts, venom and polydnaviruses (PDVs) are also injected into the caterpillars, which can suppress host immune responses, thus allowing parasitoid larvae to develop. In addition, PDVs can regulate host oral cues, such as glucose oxidase (GOX). The purpose of this study was to determine if parasitized caterpillars differentially induce plant defenses compared to non-parasitized caterpillars using two different caterpillar host/plant systems. Heliothis virescens caterpillars parasitized by M. croceipes had significantly lower salivary GOX activity than non-parasitized caterpillars, resulting in lower levels of tomato defense responses, which benefited parasitoid performance by increasing the growth rate of parasitized caterpillars. In tobacco plants, parasitized Helicoverpa zea caterpillars had lower GOX activity but induced higher plant defense responses. The higher tobacco defense responses negatively affected parasitoid performance by reducing the growth rate of parasitized caterpillars, causing longer developmental periods, and reduced cocoon mass and survival of parasitoids. These studies demonstrate a species-specific effect in different plant-insect systems. Based on these results, plant perception of insect herbivores can be affected by parasitoids and lead to positive or negative consequences to higher trophic levels depending upon the particular host-plant system.


Assuntos
Lycopersicon esculentum/parasitologia , Mariposas/fisiologia , Tabaco/parasitologia , Vespas/fisiologia , Animais , Feminino , Glucose Desidrogenase/metabolismo , Glucose Oxidase/metabolismo , Interações Hospedeiro-Parasita , Larva/metabolismo , Lycopersicon esculentum/metabolismo , Oviposição/fisiologia , Parasitos , Doenças das Plantas/parasitologia , Extratos Vegetais/química , Extratos Vegetais/metabolismo , Folhas de Planta/química , Folhas de Planta/metabolismo , Especificidade da Espécie , Tabaco/metabolismo
2.
Acta Crystallogr D Struct Biol ; 75(Pt 9): 841-851, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31478907

RESUMO

The bacterial flavin adenine dinucleotide (FAD)-dependent glucose dehydrogenase complex derived from Burkholderia cepacia (BcGDH) is a representative molecule of direct electron transfer-type FAD-dependent dehydrogenase complexes. In this study, the X-ray structure of BcGDHγα, the catalytic subunit (α-subunit) of BcGDH complexed with a hitchhiker protein (γ-subunit), was determined. The most prominent feature of this enzyme is the presence of the 3Fe-4S cluster, which is located at the surface of the catalytic subunit and functions in intramolecular and intermolecular electron transfer from FAD to the electron-transfer subunit. The structure of the complex revealed that these two molecules are connected through disulfide bonds and hydrophobic interactions, and that the formation of disulfide bonds is required to stabilize the catalytic subunit. The structure of the complex revealed the putative position of the electron-transfer subunit. A comparison of the structures of BcGDHγα and membrane-bound fumarate reductases suggested that the whole BcGDH complex, which also includes the membrane-bound ß-subunit containing three heme c moieties, may form a similar overall structure to fumarate reductases, thus accomplishing effective electron transfer.


Assuntos
Burkholderia cepacia/enzimologia , Glucose Desidrogenase/química , Domínio Catalítico , Cristalografia por Raios X/métodos , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/química , Modelos Moleculares , Proteínas Recombinantes/química
3.
Chemphyschem ; 20(16): 2082-2092, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31233266

RESUMO

The studied enzyme-based biocatalytic system mimics NXOR Boolean logic gate, which is a logical operator that corresponds to equality in Boolean algebra. It gives the functional value true (1) if both functional arguments (input signals) have the same logical value (0,0 or 1,1), and false (0) if they are different (0,1 or 1,0). The output signal producing reaction is catalyzed by pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH), which is inhibited at acidic and basic pH values. Two other reactions catalyzed by esterase and urease produce acetic acid and ammonium hydroxide, respectively, shifting solution pH from the optimum pH for PQQ-GDH to acidic and basic values (1,0 and 0,1 input combinations, respectively), thus switching the enzyme activity off (output 0). When the input signals are not applied (0,0 combination) or both applied compensating each other (1,1 combination) the optimum pH is preserved, thus keeping PQQ-GDH running at the high rate (output 1). The biocatalytic cascade mimicking the NXOR gate was characterized optically and electrochemically. In the electrochemical experiments the PQQ-GDH enzyme communicated electronically with a conducting electrode support, thus resulting in the electrocatalytic current when signal combinations 0,0 and 1,1 were applied. The logic gate operation, when it was realized electrochemically, was also extended to the biomolecular release controlled by the gate. The release system included two electrodes, one performing the NXOR gate and another one activated for the release upon electrochemically stimulated alginate hydrogel dissolution. The studied system represents a general approach to the biocatalytic realization of the NXOR logic gate, which can be included in different catalytic cascades mimicking operation of concatenated gates in sophisticated logic circuitries.


Assuntos
Computadores Moleculares , Esterases/química , Glucose Desidrogenase/química , Lógica , Urease/química , Acetatos/química , Alginatos/química , Animais , Canavalia/enzimologia , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Eletrodos , Fluoresceína-5-Isotiocianato/química , Corantes Fluorescentes/química , Concentração de Íons de Hidrogênio , Ferro/química , Nanotubos de Carbono/química , Suínos , Ureia/química
4.
Bioelectrochemistry ; 128: 94-99, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-30959399

RESUMO

We report on a hybrid bioelectrochemical system that integrates an energy converting part, viz. a glucose/oxygen enzymatic fuel cell, with a charge-storing component, in which the redox features of the immobilized redox protein cytochrome c (cyt c) were utilized. Bilirubin oxidase and pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) were employed as the biocatalysts for dioxygen reduction and glucose oxidation, respectively. A bi-protein PQQ-GDH/cyt c signal chain was created that facilitates electron transfer between the enzyme and the electrode surface. The assembled supercapacitor/biofuel cell hybrid biodevice displays a 15 times higher power density tested in the pulse mode compared to the performance achieved from the continuously operating regime (4.5 and 0.3 µW cm-2, respectively) with an 80% residual activity after 50 charge/discharge pulses. This can be considered as a notable step forward in the field of glucose/oxygen membrane-free, biocompatible hybrid power sources.


Assuntos
Fontes de Energia Bioelétrica , Citocromos c/metabolismo , Enzimas Imobilizadas/metabolismo , Glucose Desidrogenase/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismo , Técnicas Eletroquímicas/instrumentação , Eletrodos , Transporte de Elétrons , Glucose/metabolismo , Oxirredução
5.
Biosci Biotechnol Biochem ; 83(6): 1171-1179, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30777491

RESUMO

Acetic acid bacteria are used in the commercial production of lactobionic acid (LacA). However, the lactose-oxidizing enzyme of these bacteria remains unidentified. Lactose-oxidizing activity has been detected in bacterial membrane fractions and is strongly inhibited by d-glucose, suggesting that the enzyme was a membrane-bound quinoprotein glucose dehydrogenase, but these dehydrogenases have been reported to be incapable of oxidizing lactose. Thus, we generated m-GDH-overexpressing and -deficient strains of Komagataeibacter medellinensis NBRC3288 and investigated their lactose-oxidizing activities. Whereas the overexpressing variants produced ~2-5-fold higher amounts of LacA than the wild-type strains, the deficient variant produced no LacA or d-gluconic acid. Our results indicate that the lactose-oxidizing enzyme from acetic acid bacteria is membrane-bound quinoprotein glucose dehydrogenase. Abbreviations: LacA: lactobionic acid; AAB: acetic acid bacterium; m-GDH: membrane-bound quinoprotein glucose dehydrogenase; DCIP: 2,6-dichlorophenolindophenol; HPAEC-PAD: high-performance anion-exchange chromatography with pulsed amperometric detection.


Assuntos
Acetobacteraceae/enzimologia , Dissacarídeos/metabolismo , Glucose Desidrogenase/metabolismo , Membrana Celular/enzimologia , Glucose/metabolismo , Lactose/metabolismo , Oxirredução , Especificidade por Substrato
6.
Biochemistry ; 58(10): 1388-1399, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30742415

RESUMO

A combination of bioinformatics, steady-state kinetics, and NMR spectroscopy has revealed the catalytic functions of YcjQ, YcjS, and YcjR from the ycj gene cluster in Escherichia coli K-12. YcjS was determined to be a 3-keto-d-glucoside dehydrogenase with a kcat = 22 s-1 and kcat/ Km = 2.3 × 104 M-1 s-1 for the reduction of methyl α-3-keto-d-glucopyranoside at pH 7.0 with NADH. YcjS also exhibited catalytic activity for the NAD+-dependent oxidation of d-glucose, methyl ß-d-glucopyranoside, and 1,5-anhydro-d-glucitol. YcjQ was determined to be a 3-keto-d-guloside dehydrogenase with kcat = 18 s-1 and kcat/ Km = 2.0 × 103 M-1 s-1 for the reduction of methyl α-3-keto-gulopyranoside. This is the first reported dehydrogenase for the oxidation of d-gulose. YcjQ also exhibited catalytic activity with d-gulose and methyl ß-d-gulopyranoside. The 3-keto products from both dehydrogenases were found to be extremely labile under alkaline conditions. The function of YcjR was demonstrated to be a C4 epimerase that interconverts 3-keto-d-gulopyranosides to 3-keto-d-glucopyranosides. These three enzymes, YcjQ, YcjR, and YcjS, thus constitute a previously unrecognized metabolic pathway for the transformation of d-gulosides to d-glucosides via the intermediate formation of 3-keto-d-guloside and 3-keto-d-glucoside.


Assuntos
Proteínas de Escherichia coli/metabolismo , Glucose Desidrogenase/genética , Glucosídeos/metabolismo , Catálise , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Glucose/química , Glucose Desidrogenase/metabolismo , Glucosídeos/genética , Cinética , Família Multigênica , Oxirredução , Oxirredutases/metabolismo , Especificidade por Substrato
7.
Protein Expr Purif ; 156: 58-65, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30629972

RESUMO

The novel isolated Rhizobium sp. S10 was identified as d-glucoside 3-dehydrogenase (G3DH) producing microbe. Therefore, the gene encoding for G3DH from Rhizobium sp. S10 was cloned and overexpressed in Escherichia coli strain JM109 as a soluble enzyme complex. The recombinant G3DH (rG3DH) was purified with relatively high specific activity of 38.54 U/mg compared to the previously characterized and cloned G3DHs. The purified rG3DH showed the highest activity at pH 7.0, 40 °C toward cellobiose. It can also oxidize a broad range of mono-disaccharides including saccharide derivatives. The glycosides oxidizing activity combined with chemical reaction, could produce d-gulose from lactitol via 3-ketolactitol.


Assuntos
Escherichia coli , Glucose Desidrogenase , Hexoses/biossíntese , Proteínas Recombinantes , Rhizobium/enzimologia , Clonagem Molecular , Glucose Desidrogenase/biossíntese , Glucose Desidrogenase/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
8.
Biosens Bioelectron ; 123: 114-123, 2019 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-30057265

RESUMO

Fungi-derived flavin adenine dinucleotide glucose dehydrogenases (FADGDHs) are currently the most popular and advanced enzymes for self-monitoring of blood glucose sensors; however, the achievement of direct electron transfer (DET) with FADGDHs is difficult. In this study, a designer FADGDH was constructed by fusing Aspergillus flavus derived FADGDH (AfGDH) and a Phanerochaete chrisosporium CDH (PcCDH)-derived heme b-binding cytochrome domain to develop a novel FADGDH that is capable of direct electron transfer with an electrode. A structural prediction suggested that the heme in the CDH may exist in proximity to the FAD of AfGDH if the heme b-binding cytochrome domain is fused to the AfGDH N-terminal region. Spectroscopic observations of recombinantly produced designer FADGDH confirmed the intramolecular electron transfer between FAD and the heme. A decrease in pH and the presence of a divalent cation improved the intramolecular electron transfer. An enzyme electrode with the immobilized designer FADGDH showed an increase in current immediately after the addition of glucose in a glucose concentration-dependent manner, whereas those with wild-type AfGDH did not show an increase in current. Therefore, the designer FADGDH was confirmed to be a novel GDH that possesses electrode DET ability. The difference in the surface electrostatic potentials of AfGDH and the catalytic domain of PcCDH might be why their intramolecular electron transfer ability is inferior to that of CDH. These relevant and consistent findings provide us with a novel strategic approach for the improvement of the DET properties of designer FADGDH. (241 words).


Assuntos
Aspergillus flavus/enzimologia , Técnicas Biossensoriais , Glicemia/isolamento & purificação , Glucose Desidrogenase/química , Aspergillus flavus/química , Domínio Catalítico , Eletrodos , Transporte de Elétrons , Flavina-Adenina Dinucleotídeo/química , Heme/química
9.
Artigo em Inglês | MEDLINE | ID: mdl-30577417

RESUMO

Quinoprotein glucose dehydrogenase (GDH) is the most important enzyme of inorganic phosphorus-dissolving metabolism, catalyzing the oxidation of glucose to gluconic acid. The insoluble phosphate in the sediment is converted into soluble phosphate, facilitating mass reproduction of algae. Therefore, studying the diversity of gcd genes which encode GDH is beneficial to reveal the microbial group that has a significant influence on the eutrophication of water. Taking the eutrophic Sancha Lake sediments as the research object, we acquired samples from six sites in the spring and autumn. A total of 219,778 high-quality sequences were obtained by DNA extraction of microbial groups in sediments, PCR amplification of the gcd gene, and high-throughput sequencing. Six phyla, nine classes, 15 orders, 29 families, 46 genera, and 610 operational taxonomic units (OTUs) were determined, suggesting the high genetic diversity of gcd. Gcd genes came mainly from the genera of Rhizobium (1.63⁻77.99%), Ensifer (0.13⁻56.95%), Shinella (0.32⁻25.49%), and Sinorhizobium (0.16⁻11.88%) in the phylum of Proteobacteria (25.10⁻98.85%). The abundance of these dominant gcd-harboring bacteria was higher in the spring than in autumn, suggesting that they have an important effect on the eutrophication of the Sancha Lake. The alpha and beta diversity of gcd genes presented spatial and temporal differences due to different sampling site types and sampling seasons. Pearson correlation analysis and canonical correlation analysis (CCA) showed that the diversity and abundance of gcd genes were significantly correlated with environmental factors such as dissolved oxygen (DO), phosphorus hydrochloride (HCl⁻P), and dissolved total phosphorus (DTP). OTU composition was significantly correlated with DO, total organic carbon (TOC), and DTP. GDH encoded by gcd genes transformed insoluble phosphate into dissolved phosphate, resulting in the eutrophication of Sancha Lake. The results suggest that gcd genes encoding GDH may play an important role in lake eutrophication.


Assuntos
Sedimentos Geológicos/microbiologia , Glucose Desidrogenase/genética , Lagos/microbiologia , Fósforo/metabolismo , Proteobactérias/genética , China , Eutrofização/fisiologia , Variação Genética , Estações do Ano
10.
Anal Chem ; 90(24): 14500-14506, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30427170

RESUMO

Antibody-enzyme complexes (AECs) are ideal sensing elements, especially when oxidoreductases are used as the enzymes in the complex, with the potential to carry out rapid electrochemical measurements. However, conventional methods for the fabrication of AECs, including direct fusion and chemical conjugation, are associated with issues regarding the generation of insoluble aggregates and production of homogeneous AECs. Here, we developed a convenient and universal method for the fabrication of homogeneous AECs using the SpyCatcher/SpyTag system. We used an anti-epidermal growth factor receptor (EGFR) variable domain of a heavy chain antibody (VHH) and a glucose dehydrogenase (GDH) derived from Aspergillus flavus ( AfGDH) as the model antibody and enzyme, respectively. Both SpyTag-fused VHH and SpyCatcher-fused AfGDH were successfully prepared using an Escherichia coli expression system, whereas anti-EGFR AECs were produced by simply mixing the two fusion proteins. A bivalent AEC, AfGDH with two VHH at both terminals, was also prepared and exhibited an increased affinity. A soluble EGFR was successfully detected in a dose-dependent manner using immobilized anti-EGFR immunoglobulin G (IgG) and bivalent AEC. We also confirmed the universality of this AEC fabricating method by applying it to another VHH. This method results in the convenient and universal preparation of sensing elements with the potential for electrochemical measurement.


Assuntos
Complexo Antígeno-Anticorpo/metabolismo , Glucose Desidrogenase/metabolismo , Cadeias Pesadas de Imunoglobulinas/metabolismo , Complexo Antígeno-Anticorpo/genética , Aspergillus/enzimologia , Técnicas Biossensoriais , Receptores ErbB/análise , Receptores ErbB/imunologia , Escherichia coli/metabolismo , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glucose Desidrogenase/genética , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Ressonância de Plasmônio de Superfície
11.
Appl Microbiol Biotechnol ; 102(22): 9531-9540, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30218379

RESUMO

Pyrroloquinoline quinone (PQQ)-dependent dehydrogenases (quinoproteins) of acetic acid bacteria (AAB), such as the membrane-bound alcohol dehydrogenase (ADH) and the membrane-bound glucose dehydrogenase, contain PQQ as the prosthetic group. Most of them are located on the periplasmic surface of the cytoplasmic membrane, and function as primary dehydrogenases in cognate substance-oxidizing respiratory chains. Here, we have provided an overview on the function and molecular architecture of AAB quinoproteins, which can be categorized into six groups according to the primary amino acid sequences. Based on the genomic data, we discuss the types of quinoproteins found in AAB genome and how they are distributed. Our analyses indicate that a significant number of uncharacterized orphan quinoproteins are present in AAB. By reviewing recent experimental developments, we discuss how to characterize the as-yet-unknown enzymes. Moreover, our bioinformatics studies also provide insights on how quinoproteins have developed into intricate enzymes. ADH comprises at least two subunits: the quinoprotein dehydrogenase subunit encoded by adhA and the cytochrome subunit encoded by adhB, and the genes are located in a polycistronic transcriptional unit. Findings on stand-alone derivatives of adhA encourage us to speculate on a possible route for ADH development in the evolutional history of AAB. A combination of bioinformatics studies on big genome sequencing data and wet studies assisted with genetic engineering would unravel biochemical functions and physiological role of uncharacterized quinoproteins in AAB, or even in unculturable metagenome.


Assuntos
Ácido Acético/metabolismo , Bactérias/enzimologia , Genoma Bacteriano , Oxirredutases/metabolismo , Cofator PQQ/metabolismo , Álcool Desidrogenase/metabolismo , Oxirredutases do Álcool/metabolismo , Bactérias/genética , Proteínas de Bactérias/metabolismo , Biologia Computacional , Transporte de Elétrons , Engenharia Genética , Glucose Desidrogenase/metabolismo
12.
Enzyme Microb Technol ; 116: 57-63, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29887017

RESUMO

A new approach to deposition of electroactive ZnO thin films have been carried out, by one-pot chemical bath deposition with Al dopant and incorporation of neutral red as organic mediator. The morphological, structural and functional characterization of the neutral red incorporated, Al-doped ZnO (NR-AZO) film was carried out using electron microscopy, FTIR, XRD and EIS respectively. The incorporated neutral red was found to induce strain in the crystal of AZO proportional to the concentration used in depositing solution which further affected the charge transfer resistance of the films in solution. One mM neutral red was found to be the optimum concentration for both conductivity and response to NADH/NADPH. The response of the films was further validated by immobilizing NAD+ dependent alcohol dehydrogenase (ADH) and NADP+ dependent glucose dehydrogenase (GDH) independently. The ADH/NR-AZO showed a sensitivity of 3.2 µA cm-2 mM-1 with a LoD of 1.7 µM of ethanol in the range 5.6 µM-7 mM, whereas GDH/NR-AZO showed a sensitivity of 4.33 µA cm-2 mM-1 with a LoD of 27 µM of glucose in the range 90 µM-4 mM. This method serves as a simple alternative to immobilize the organic redox dyes into the inorganic thin films in a single step making it electroactive towards specific biomolecules.


Assuntos
Álcool Desidrogenase/química , Glucose Desidrogenase/química , NADP/química , NAD/química , Vermelho Neutro/química , Óxido de Zinco/química , Alumínio/química , Biocatálise , Glucose/química
13.
Biosens Bioelectron ; 112: 8-17, 2018 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-29684749

RESUMO

In this study, polythiophene copolymers have been used as modifier for electrode surfaces in order to allow the immobilization of active pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH) and to simultaneously improve the direct electrical connection of the enzyme with the electrode. Polymer films are electrosynthesized in aqueous solution without the need of surfactants onto carbon nanotubes modified gold electrodes from mixtures of 3-thiopheneacetic acid (ThCH2CO2H) and 3-methoxythiophene (ThOCH3) using a potentiostatic pulse method. Polythiophene deposition significantly improves the bioelectrocatalysis of PQQ-GDH: the process starts at - 200 mV vs. Ag/AgCl and allows well-defined glucose detection at 0 V vs. Ag/AgCl with high current density. Several parameters of the electro-polymerization method have been evaluated to maximize the anodic current output after enzyme coupling. The polymer deposited by this new procedure has been morphologically and chemically characterized by different methods (SEM, EDX, FT-IR, UV-Vis, XPS and Raman spectroscopy). The bioelectrocatalytic response towards increasing glucose concentrations exhibits a dynamic range extending from 1 µM to 2 mM. The low applied potential allows to avoid interferences from easily oxidizable substances such as uric acid and ascorbic acid. Short and long-term stability has been evaluated. Finally, the PQQ-GDH electrode has been coupled to a bilirubin oxidase (BOD)- and carbon nanotube-based cathode in order to test its performance as anode of a biofuel cell. The promising results suggest a further investigation of this kind of polymers and, in particular, the study of the interaction with other enzymes in order to employ them in building up biosensors and biofuel cells.


Assuntos
Técnicas Biossensoriais , Enzimas Imobilizadas/química , Glucose Desidrogenase/química , Glucose/isolamento & purificação , Glucose/química , Humanos , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/química , Cofator PQQ/química , Polímeros/química , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Tiofenos/química
14.
Bioelectrochemistry ; 122: 115-122, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29625423

RESUMO

Glucoside 3­dehydrogenase (G3DH) is a flavin adenine dinucleotide (FAD)-containing oxidoreductase that catalyzes the oxidation of the hydroxy group on the C-3 position of pyranose and shows broad substrate specificity by oxidizing many saccharides. Due to unique site specificity and wide substrate specificity, G3DHs can be used for synthesis of sugar derivatives, anodic catalysis in biofuel cells, multi-sugar analysis using enzyme electrode, and for enzymatic detection of 1,5­anhydro­d­glucitol, a clinical marker for diabetes. However, few studies have focused on the fundamental biochemical properties of G3DH, including its electron transfer pathway. In this study, we isolated the G3DH gene from Rhizobium radiobacter, a homologue of marine bacterial G3DH, and reported that the isolated gene fragment contains the genes encoding the G3DH catalytic subunit (subunit I), G3DH hitch-hiker subunit (subunit II), and cytochrome c-like molecule (CYTc). Furthermore, we report the recombinant expression of G3DH from R. radiobacter in Escherichia coli, the characterization of recombinant G3DH and the investigation of the molecular electron pathway of G3DH. We first prepared the G3DH subunit I-II complex using a co-expression vector for both subunits. The G3DH subunit I-II complex showed dye-mediated G3DH activity toward methyl­α­d­glucoside (MαG). Electron paramagnetic resonance (EPR) and inductively coupled plasma optical emission spectroscopy (ICP-OES) analyses revealed that subunit I contains an iron-sulfur cluster. We, then, prepared recombinant CYTc and revealed that it is capable of accepting electrons from the catalytic subunit of G3DH by absorption spectrum analysis. These results suggested that R. radiobacter G3DH possesses an iron­sulfur cluster that may play an important role in the electron transfer from FAD to cytochrome c like molecule, which is an external electron acceptor of G3DH. Furthermore, we demonstrated that CYTc mediate the electron transfer from G3DH to electrode without the artificial electron mediator.


Assuntos
Agrobacterium tumefaciens/enzimologia , Glucose Desidrogenase/metabolismo , Proteínas com Ferro-Enxofre/metabolismo , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/genética , Sequência de Aminoácidos , Domínio Catalítico , Transporte de Elétrons , Genes Bacterianos , Glucose Desidrogenase/química , Glucose Desidrogenase/genética , Proteínas com Ferro-Enxofre/química , Proteínas com Ferro-Enxofre/genética , Família Multigênica , Alinhamento de Sequência
15.
Bioelectrochemistry ; 121: 185-190, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29471242

RESUMO

Enzyme based electrochemical biosensors are divided into three generations according to their type of electron transfer from the cofactors of the enzymes to the electrodes. Although the 3rd generation sensors using direct electron transfer (DET) type enzymes are ideal, the number of enzyme types which possess DET ability is limited. In this study, we report of a glucose sensor using mediator-modified glucose dehydrogenase (GDH), that was fabricated by a new quick-and-easy method using the pre-functionalized amine reactive phenazine ethosulfate (arPES). Thus mediator-modified GDH obtained the ability to transfer electrons to bulky electron acceptors as well as electrodes. The concentration of glucose was successfully measured using electrodes with immobilized PES-modified GDH, without addition of external electron mediators. Therefore, continuous monitoring systems can be developed based on this "2.5th generation" electron transfer principle utilizing quasi-DET. Furthermore, we successfully modified two other diagnostically relevant enzymes, glucoside 3-dehydrogenase and lactate oxidase, with PES. Therefore, various kinds of diagnostic enzymes can achieve quasi-DET ability simply by modification with arPES, suggesting that continuous monitoring systems based on the 2.5th generation principle can be developed for various target molecules.


Assuntos
Técnicas Biossensoriais/métodos , Botrytis/enzimologia , Enzimas Imobilizadas/química , Glucose 1-Desidrogenase/química , Glucose/análise , Aerococcus/enzimologia , Agrobacterium tumefaciens/enzimologia , Glicemia/análise , Transporte de Elétrons , Glucose Desidrogenase/química , Humanos , Oxigenases de Função Mista/química , Fenazinas/química , Proteínas Recombinantes/química
16.
J Phys Chem B ; 121(51): 11465-11471, 2017 12 28.
Artigo em Inglês | MEDLINE | ID: mdl-29185751

RESUMO

An artificial Ca2+-regulated PQQ glucose dehydrogenase (PQQ-GDH) enzyme was electrically connected to conducting electrodes and semiconductor interfaces. Direct electron transfer from the enzyme to the conducting electrode support was stimulated by the addition of Ca2+ cations resulting in reversible enzyme activation. A signal-switchable biofuel cell and biomolecular release have been realized using the Ca2+-activated enzyme immobilized on conducting electrodes. Interfacing the signal-switchable enzyme with a semiconductor chip allowed electronic read out of the enzyme ON-OFF states. The developed approach based on the signal-regulated PQQ-GDH enables numerous bioelectrochemical/bioelectronic applications of the developed systems in signal-activated biosensors and biofuel cells, as well as in biomolecular computing/logic systems.


Assuntos
Cálcio/metabolismo , Técnicas Eletroquímicas , Glucose Desidrogenase/química , Glucose Desidrogenase/metabolismo , Eletrodos , Semicondutores
18.
Sensors (Basel) ; 17(11)2017 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-29144384

RESUMO

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.


Assuntos
Glucose/análise , Técnicas Biossensoriais , Flavina-Adenina Dinucleotídeo , Glucose Desidrogenase
19.
Enzyme Microb Technol ; 106: 114-118, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28859805

RESUMO

Enterobacter aerogenes was metabolically engineered for acetoin production. To remove the pathway enzymes that catalyzed the formation of by-products, the three genes encoding a lactate dehydrogenase (ldhA) and two 2,3-butanediol dehydrogenases (budC, and dhaD), respectively, were deleted from the genome. The acetoin production was higher under highly aerobic conditions. However, an extracellular glucose oxidative pathway in E. aerogenes was activated under the aerobic conditions, resulting in the accumulation of 2-ketogluconate. To decrease the accumulation of this by-product, the gene encoding a glucose dehydrogenase (gcd) was also deleted. The resulting strain did not produce 2-ketogluconate but produced significant amounts of acetoin, with concentration reaching 71.7g/L with 2.87g/L/h productivity in fed-batch fermentation. This result demonstrated the importance of blocking the glucose oxidative pathway under highly aerobic conditions for acetoin production using E. aerogenes.


Assuntos
Acetoína/metabolismo , Enterobacter aerogenes/metabolismo , Engenharia Metabólica/métodos , Aerobiose , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Reatores Biológicos/microbiologia , Enterobacter aerogenes/genética , Fermentação , Deleção de Genes , Genes Bacterianos , Gluconatos/metabolismo , Glucose Desidrogenase/genética , Glucose Desidrogenase/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , L-Lactato Desidrogenase/genética , L-Lactato Desidrogenase/metabolismo , Lactato Desidrogenase 5 , Redes e Vias Metabólicas/genética
20.
J Biol Chem ; 292(33): 13823-13832, 2017 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-28667014

RESUMO

Glycolysis and the pentose phosphate pathway both play a central role in the degradation of glucose in all domains of life. Another metabolic route that can facilitate glucose breakdown is the gluconate shunt. In this shunt glucose dehydrogenase and gluconate kinase catalyze the two-step conversion of glucose into the pentose phosphate pathway intermediate 6-phosphogluconate. Despite the presence of these enzymes in many organisms, their only established role is in the production of 6-phosphogluconate for the Entner-Doudoroff pathway. In this report we performed metabolic profiling on a strain of Schizosaccharomyces pombe lacking the zinc-responsive transcriptional repressor Loz1 with the goal of identifying metabolic pathways that were altered by cellular zinc status. This profiling revealed that loz1Δ cells accumulate higher levels of gluconate. We show that the altered gluconate levels in loz1Δ cells result from increased expression of gcd1 By analyzing the activity of recombinant Gcd1 in vitro and by measuring gluconate levels in strains lacking enzymes of the gluconate shunt we demonstrate that Gcd1 encodes a novel NADP+-dependent glucose dehydrogenase that acts in a pathway with the Idn1 gluconate kinase. We also find that cells lacking gcd1 and zwf1, which encode the first enzyme in the pentose phosphate pathway, have a more severe growth phenotype than cells lacking zwf1 We propose that in S. pombe Gcd1 and Idn1 act together to shunt glucose into the pentose phosphate pathway, creating an alternative route for directing glucose into the pentose phosphate pathway that bypasses hexokinase and the rate-limiting enzyme glucose-6-phosphate dehydrogenase.


Assuntos
Glucose Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/metabolismo , Via de Pentose Fosfato , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/enzimologia , Fatores de Transcrição/metabolismo , Metabolismo Energético , Deleção de Genes , Gluconatos/metabolismo , Glucose Desidrogenase/genética , Glucosefosfato Desidrogenase/genética , Metabolômica/métodos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Fatores de Transcrição/genética
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