| 1. |
- Gonaus, Christoph, et al.
(författare)
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Analysis of Agaricus meleagris pyranose dehydrogenase N-glycosylation sites and performance of partially non-glycosylated enzymes
- 2017
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Ingår i: Enzyme and Microbial Technology. - : Elsevier BV. - 0141-0229. ; 99, s. 57-66
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Tidskriftsartikel (refereegranskat)abstract
- Pyranose Dehydrogenase 1 from the basidiomycete Agaricus meleagris (AmPDH1) is an oxidoreductase capable of oxidizing a broad variety of sugars. Due to this and its ability of dioxidation of substrates and no side production of hydrogen peroxide, it is studied for use in enzymatic bio-fuel cells. In-vitro deglycosylated AmPDH1 as well as knock-out mutants of the N-glycosylation sites N75 and N175, near the active site entrance, were previously shown to improve achievable current densities of graphite electrodes modified with AmPDH1 and an osmium redox polymer acting as a redox mediator, up to 10-fold. For a better understanding of the role of N-glycosylation of AmPDH1, a systematic set of N-glycosylation site mutants was investigated in this work, regarding expression efficiency, enzyme activity and stability. Furthermore, the site specific extend of N-glycosylation was compared between native and recombinant wild type AmPDH1. Knocking out the site N252 prevented the attachment of significantly extended N-glycan structures as detected on polyacrylamide gel electrophoresis, but did not significantly alter enzyme performance on modified electrodes. This suggests that not the molecule size but other factors like accessibility of the active site improved performance of deglycosylated AmPDH1/osmium redox polymer modified electrodes. A fourth N-glycosylation site of AmPDH1 could be confirmed by mass spectrometry at N319, which appeared to be conserved in related fungal pyranose dehydrogenases but not in other members of the glucose-methanol-choline oxidoreductase structural family. This site was shown to be the only one that is essential for functional recombinant expression of the enzyme.
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| 2. |
- Killyeni, Aniko, et al.
(författare)
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Effect of deglycosylation on the mediated electrocatalytic activity of recombinantly expressed Agaricus meleagris pyranose dehydrogenase wired by osmium redox polymer
- 2014
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Ingår i: Electrochimica Acta. - : Elsevier BV. - 0013-4686. ; 126, s. 61-67
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Tidskriftsartikel (refereegranskat)abstract
- The effect of deglycosylation of pyranose dehydrogenase (PDH) obtained from Agaricus meleagris (Am) and recombinantly expressed in Pichia pastoris on its electrocatalytic activity was investigated. Glycosylated (gAmPDH) and deglycosylated PDH (dgAmPDH) were immobilised on spectrographic graphite (G) simultaneously with an osmium redox polymer (Os-RP) using poly(ethylene glycol)(400) diglycidyl ether (PEGDGE) as cross-linking agent. The amperometric response to glucose, recorded at G/(Os-RP)-gAmPDH and G/(Os-RP)-dgAmPDH bioelectrodes, was optimised under flow injection conditions concerning the applied potential, enzyme loading, working pH and flow rate. The G/(Os-RP)dgAmPDH bioelectrode is characterised by better kinetic and electroanalytical parameters compared with the G/(Os-RP)-gAmPDH bioelectrode: (i) a higher value of the maximum catalytic current density, j(max) = (146.6 +/- 2.6) mu A cm(-2) vs. j(max) = (80.9 +/- 1.9) mu A cm(-2); (ii) a lower value of the apparent Michaelis-Menten constant, K-M(app) = (2.4 +/- 0.1) mu M vs. K-M(app) = (7.5 +/- 0.3) mM; (iii) a higher slope of the linear domain, (43.6 +/- 1.1) mu A cm(-2) mM(-1) vs. (9.74 +/- 0.16) mu A cm(-2) mM(-1). Additionally, the time dependent decay of the amperometric response to glucose shows a slightly better operational stability for the G/(Os-RP)-dgAmPDH bioelectrode than that for the G/(Os-RP)-gAmPDH. The enzyme deglycosylation induces significant changes in the order of substrate selectivity for gAmPDH and dgAmPDH. (C) 2013 Elsevier Ltd. All rights reserved.
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| 3. |
- Killyeni, Aniko, et al.
(författare)
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EFFECT OF ENZYME DEGLYCOSYLATION ON THE AMPEROMETRIC DETECTION OF GLUCOSE AT PDH-MODIFIED ELECTRODE
- 2012
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Ingår i: Studia Universitatis Babes-Bolyai, Chemia. - 2065-9520. ; 57:4, s. 87-99
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Tidskriftsartikel (refereegranskat)abstract
- The effect of deglycosylation of pyranose dehydrogenase (PDH), obtained from Agaricus meleagris and recombinantly expressed in Pichia pastoris, on the amperometric detection of glucose was investigated. Glycosylated (gPDH) and deglycosylated (dgPDH) PDH were immobilized on spectrographic graphite (G) simultaneously with an Os redox polymer (Os-RP). The amperometric response of G/Os-RP/gPDH and G/Os-RP/dgPDH to glucose was recorded using flow injection measurements and cyclic voltammetry. A significant increase in the maximum catalytic current density was observed for G/Os-RP/dgPDH [(148.7 +/- 0.14) mu A/cm(2)) compared with G/Os-RP/gPDH [(81.4 +/- 1.4) mu A/cm(2)]. Additionally, the deglycosylation of the enzyme resulted in a higher substrate-enzyme affinity (K-M (app) = 2.44 +/- 0.10 mM), compared with glycosylated PDH (K-M(app) = 7.52 +/- 0.34 mM).
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| 4. |
- Kittl, Roman, et al.
(författare)
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Molecular cloning of three pyranose dehydrogenase-encoding genes from Agaricus meleagris and analysis of their expression by real-time RT-PCR
- 2008
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Ingår i: Current Genetics. - : Springer Science and Business Media LLC. - 0172-8083 .- 1432-0983. ; 53:2, s. 117-127
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Tidskriftsartikel (refereegranskat)abstract
- Sugar oxidoreductases such as cellobiose dehydrogenase or pyranose oxidase are widespread enzymes among fungi, whose biological function is largely speculative. We investigated a similar gene family in the mushroom Agaricus meleagris and its expression under various conditions. Three genes (named pdh1, pdh2 and pdh3) putatively encoding pyranose dehydrogenases were isolated. All three genes displayed a conserved structure and organization, and the respective cDNAs contained ORFs translating into polypeptides of 602 or 600 amino acids. The N-terminal sections of all three genes encode putative signal peptides consistent with the enzymes extracellular secretion. We cultivated the fungus on different carbon sources and analyzed the mRNA levels of all three genes over a period of several weeks using real-time RT-PCR. The glyceraldehyde-3-phosphate dehydrogenase gene from A. meleagris was also isolated and served as reference gene. pdh2 and pdh3 are essentially transcribed constitutively, whereas pdh1 expression is upregulated upon exhaustion of the carbon source; pdh1 appears to be additionally regulated under conditions of oxygen limitation. These data are consistent with an assumed role in lignocellulose degradation.
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| 5. |
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| 6. |
- Kujawa, Magdalena, et al.
(författare)
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Properties of pyranose dehydrogenase purified from the litter-degrading fungus Agaricus xanthoderma
- 2007
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Ingår i: The FEBS Journal. - : Wiley. - 1742-464X .- 1742-4658. ; 274:3, s. 879-894
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Tidskriftsartikel (refereegranskat)abstract
- We purified an extracellular pyranose dehydrogenase (PDH) from the basidiomycete fungus Agaricus xanthoderma using ammonium sulfate fractionation and ion-exchange and hydrophobic interaction chromatography. The native enzyme is a monomeric glycoprotein (5% carbohydrate) containing a covalently bound FAD as its prosthetic group. The PDH polypeptide consists of 575 amino acids and has a molecular mass of 65 400 Da as determined by MALDI MS. On the basis of the primary structure of the mature protein, PDH is a member of the glucose-methanol-choline oxidoreductase family. We constructed a homology model of PDH using the 3D structure of glucose oxidase from Aspergillus niger as a template. This model suggests a novel type of bi-covalent flavinylation in PDH, 9-S-cysteinyl, 8-alpha-N3-histidyl FAD. The enzyme exhibits a broad sugar substrate tolerance, oxidizing structurally different aldopyranoses including monosaccharides and oligosaccharides as well as glycosides. Its preferred electron donor substrates are D-glucose, D-galactose, L-arabinose, and D-xylose. As shown by in situ NMR analysis, D-glucose and D-galactose are both oxidized at positions C2 and C3, yielding the corresponding didehydroaldoses (diketoaldoses) as the final reaction products. PDH shows no detectable activity with oxygen, and its reactivity towards electron acceptors is rather limited, reducing various substituted benzoquinones and complexed metal ions. The azino-bis-(3-ethylbenzthiazolin-6-sulfonic acid) cation radical and the ferricenium ion are the best electron acceptors, as judged by the catalytic efficiencies (k(cat)/K-m). The enzyme may play a role in lignocellulose degradation.
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| 7. |
- Kujawa, Magdalena, et al.
(författare)
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Structural basis for substrate binding and regioselective oxidation of monosaccharides at C3 by pyranose 2-oxidase
- 2006
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 281:46, s. 35104-35115
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Tidskriftsartikel (refereegranskat)abstract
- Pyranose2-oxidase(P2Ox) participates in fungal lignin degradation by producing the H2O2 needed for lignin-degrading peroxidases. The enzyme oxidizes cellulose- and hemicellulose-derived aldopyranoses at C2 preferentially, but also on C3, to the corresponding ketoaldoses. To investigate the structural determinants of catalysis, covalent flavinylation, substrate binding, and regios-electivity, wild-type and mutant P2Ox enzymes were produced and characterized biochemically and structurally. Removal of the histidyl-FAD linkage resulted in a catalytically competent enzyme containing tightly, but noncovalently bound FAD. This mutant (H167A) is characterized by a 5-fold lower k(cat), and a 35-mV lower redox potential, although no significant structural changes were seen in its crystal structure. In previous structures of P2Ox, the substrate loop (residues 452-457) covering the active site has been either disordered or in a conformation incompatible with carbohydrate binding. We present here the crystal structure of H167A in complex with a slow substrate, 2-fluoro-2-deoxy-D-glucose. Based on the details of 2-fluoro-2-deoxy-D-glucose binding in position for oxidation at C3, we also outline a probable binding mode for D-glucose positioned for regioselective oxidation at C2. The tentative determinant for discriminating between the two binding modes is the position of the O6 hydroxyl group, which in the C2-oxidation mode can make favorable interactions with Asp(452) in the substrate loop and, possibly, a nearby arginine residue (Arg(472)). We also substantiate our hypothesis with steady-state kinetics data for the alanine replacements of Asp(452) and Arg(472) as well as the double alanine 452/472 mutant.
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| 8. |
- O'Conghaile, Peter, et al.
(författare)
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Fully Enzymatic Membraneless Glucose|Oxygen Fuel Cell That Provides 0.275 mA cm-?2 in 5 mM Glucose, Operates in Human Physiological Solutions, and Powers Transmission of Sensing Data
- 2016
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 88:4, s. 2156-2163
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Tidskriftsartikel (refereegranskat)abstract
- Coimmobilization of pyranose dehydrogenase as an enzyme catalyst, osmium redox polymers [Os(4,4'-dimethoxy-2,2'-bipyridine)2(poly(vinylimidazole))10Cl]+ or [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl]+ as mediators, and carbon nanotube conductive scaffolds in films on graphite electrodes provides enzyme electrodes for glucose oxidn. The recombinant enzyme and a deglycosylated form, both expressed in Pichia pastoris, are investigated and compared as biocatalysts for glucose oxidn. using flow injection amperometry and voltammetry. In the presence of 5 mM glucose in phosphate-buffered saline (PBS) (50 mM phosphate buffer soln., pH 7.4, with 150 mM NaCl), higher glucose oxidn. current densities, 0.41 mA/cm2, are obtained from enzyme electrodes contg. the deglycosylated form of the enzyme. The optimized glucose-oxidizing anode, prepd. using deglycosylated enzyme coimmobilized with [Os(4,4'-dimethyl-2,2'-bipyridine)2(poly(vinylimidazole))10Cl]+ and carbon nanotubes, was coupled with an oxygen-reducing bilirubin oxidase on gold nanoparticle dispersed on gold electrode as a biocathode to provide a membraneless fully enzymic fuel cell. A max. power d. of 275 μW/cm2 is obtained in 5 mM glucose in PBS, the highest to date under these conditions, providing sufficient power to enable wireless transmission of a signal to a data logger. When tested in whole human blood and unstimulated human saliva max. power densities of 73 and 6 μW/cm2 are obtained for the same fuel cell configuration, resp.
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| 9. |
- Rafighi, Parvin, et al.
(författare)
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A novel membraneless β-glucan/O2 enzymatic fuel cell based on β-glucosidase (RmBgl3B)/pyranose dehydrogenase (AmPDH) co-immobilized onto buckypaper electrode
- 2022
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Ingår i: Bioelectrochemistry. - : Elsevier BV. - 1878-562X .- 1567-5394. ; 148
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Tidskriftsartikel (refereegranskat)abstract
- A novel membraneless β-glucan/O2 enzymatic fuel cell was developed by combining a bioanode based on buckypaper modified with co-immobilized Agaricus meleagris pyranose dehydrogenase (AmPDH) and Rhodothermus marinus β-glucosidase (RmBgl3B) (RmBgl3B-AmPDH/buckypaper) with a biocathode based on solid graphite modified with Myrothecium verrucaria bilirubin oxidase (MvBOx/graphite). AmPDH was connected electrochemically with the buckypaper using an osmium redox polymer in a mediated reaction, whereas MvBOx was connected with graphite in a direct electron transfer reaction.The fuel for the bioanode was produced by enzymatic hydrolysis of β-glucan by the exoglucanase RmBgl3B into d-glucose, which in turn was enzymatically oxidised by AmPDH to generate a current response. This design allows to obtain an efficient enzymatic fuel cell, where the chemical energy converted into electrical energy is higher than the chemical energy stored in complex carbohydrate based fuel.The maximum power density of the assembled β-glucan/O2 biofuel cell reached 26.3 ± 4.6 μWcm−2 at 0.36 V in phosphate buffer containing 0.5 % (w/v) β-glucan at 40 °C with excellent stability retaining 68.6 % of its initial performance after 5 days. The result confirms that β-glucan can be employed as fuel in an enzymatic biofuel cell.
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| 10. |
- Rafighi, Parvin, et al.
(författare)
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Substrate Preference Pattern of Agaricus meleagris Pyranose Dehydrogenase Evaluated through Bioelectrochemical Flow Injection Amperometry
- 2019
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Ingår i: ChemElectroChem. - : Wiley. - 2196-0216. ; 6:3, s. 801-809
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Tidskriftsartikel (refereegranskat)abstract
- Pyranose dehydrogenase (PDH) is a quinone-dependent extracellular flavoglycoprotein mainly produced by litter-decomposing fungi and contributes to the degradation of lignocellulose. PDH in terms of structure and catalytic features pertains to the glucose methanol-choline oxidoreductase family and oxidizes a wide substrate range of aldopyranoses including hexoses, pentoses, disaccharides and oligosaccharides with a high degree of regioselectivity. The purpose of this study was to rationalize the preference of PDH immobilized on an electrode with the structural features of various substrates and thus the kinetic constants were measured for various sugars. PDH was co-immobilized on the electrode with an osmium redox polymer. Response currents for different sugars were measured using flow injection amperometry at +0.3 V vs. Ag|AgCl, KCl (0.1 M). The Michaelis-Menten constants, the turnover numbers and the catalytic efficiency were calculated and revealed that type, orientation and configuration of the substituent play a major role on substrate preference. An OH-group at C-1 and C-6 are not essential and substrate specificities are little affected by the substitution at C-1. The presence and orientation of OH− at C-2 and C-3 are relevant for reactivity. Orientation of OH− at the C-4 position has little effect, and sugars with a substitution below the plane at C-5 are not suitable as substrate. Highest activity for oxidation of glucose, mannose and sucrose was detected at pH 8.5.
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