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- 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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- 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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- Shao, Minling, et al.
(författare)
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Optimization of a Membraneless Glucose/Oxygen Enzymatic Fuel Cell Based on a Bioanode with High Coulombic Efficiency and Current Density
- 2013
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Ingår i: ChemPhysChem. - : Wiley. - 1439-7641 .- 1439-4235. ; 14:10, s. 2260-2269
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Tidskriftsartikel (refereegranskat)abstract
- After initial testing and optimization of anode biocatalysts, a membraneless glucose/oxygen enzymatic biofuel cell possessing high coulombic efficiency and power output was fabricated and characterized. Agaricus meleagris (AmPDH) and flavodehydrogenase domains of various cellobiose dehydrogenases (DHCDH) were tested during the pre-screening. Myrothecium verrucaria adsorbed on graphite. Optimization showed that the current density for the mixed enzyme electrode could be further improved by using a genetically engineered variant of the non-glycosylated flavodehydrogenase domain of cellobiose dehydrogenase from Corynascus thermophilus expressed in E. coli (ngDH(CtCDHC310Y)) with a high glucose-turnover rate in combination with an Os-complex-modified redox polymer with a high concentration of Os complexes as well as a low-density graphite electrode. AmPDH/ngDH(CtCDHC310Y) anode showed not only a similar maximum voltage as with the biofuel cell based only on the ngDH(CtCDHC310Y) anode (0.55 V) but also a substantially improved maximum power output (20 Wcm(-2)) at 300 mV cell voltage in air-saturated physiological buffer. Most importantly, the estimated half-life of the mixed biofuel cell can reach up to 12 h, which is apparently longer than that of a biofuel cell in which the bioanode is based on only one single enzyme.
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4. |
- Yakovleva, Maria, et al.
(författare)
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Further Insights into the Catalytical Properties of Deglycosylated Pyranose Dehydrogenase from Agaricus meleagris Recombinantly Expressed in Pichia pastoris
- 2013
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 1520-6882 .- 0003-2700. ; 85:20, s. 9852-9858
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Tidskriftsartikel (refereegranskat)abstract
- The present study focuses on fragmented deglycosylated pyranose dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris. Fragmented deglycosylated PDH is formed from the deglycosylated enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when stored in a buffer solution at 4 degrees C. The remaining larger fragment has a molecular weight of similar to 46 kDa and exhibits higher volumetric activity for glucose oxidation compared with the deglycosylated and glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic voltammetry were used to assess and compare the catalytic activity of the three investigated forms of PDH, "wired" to graphite electrodes with two different osmium redox polymers: [Os(4,4'-dimethyl-2,2'-bipyridine)(2)(poly(vinylimidazole))(10)Cl](+) [Os(dmbpy)PVI] and [Os(4,4'-dimethoxy-2,2'-bipyridine)(2)(poly-(vinylimidazole))(10)Cl](+) [Os(dmobpy)PVI]. When "wired" with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with J(max). 13- and 6-fold higher than that observed for gPDH- and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily attractive for further biotechnological applications. An easier access of the substrate to the active site and improved communication between the enzyme and mediator matrix are suggested as the two main reasons for the excellent performance of the fdgPDH when compared with that of gPDH and dgPDH. Three of the four glycosites in PDH: N-75, N-175, and N-252 were assigned using mass spectrometry in conjunction with endoglycosidase treatment and tryptic digestion. Determination of the asparagine residues carrying carbohydrate moieties in PDH can serve as a solid background for production of recombinant enzyme lacking glycosylation.
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5. |
- Yakovleva, Maria, et al.
(författare)
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Recombinant pyranose dehydrogenase-A versatile enzyme possessing both mediated and direct electron transfer
- 2012
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Ingår i: Electrochemistry Communications. - : Elsevier BV. - 1388-2481. ; 24, s. 120-122
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Tidskriftsartikel (refereegranskat)abstract
- The catalytical properties of glycosylated pyranose dehydrogenase (gPDH) and deglycosylated PDH (dgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris were studied. Both gPDH and dgPDH were "wired" to an osmium redox polymer on graphite electrodes mounted in a flow-injection system. The current from oxidation of glucose by immobilised gPDH and dgPDH was compared using flow injection amperometry and cyclic voltammetry. An increase in the current density was observed for dgPDH (190 mu A cm(-2)) compared with that for gPDH (90 mu A cm(-2)) due to the improved electron transfer between the active site and the electrode. Additionally, the ability of dgPDH for direct electron transfer (DET) was discovered, which is rather unique among FAD-containing enzymes. The ability to oxidise a variety of sugars at a rather low potential makes dgPDH attractive for construction of biofuel cells with high power output. (C) 2012 Elsevier B.V. All rights reserved.
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