Abstract
The flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) from Aspergillus species require suitable redox mediators to transfer electrons from the enzyme to the electrode surface for the application of bioelectrical devices. Although several mediators for FAD-GDH are already in use, they are still far from optimum in view of potential, kinetics, sustainability, and cost-effectiveness. Herein, we investigated the efficiency of various phenothiazines and quinones in the electrochemical oxidation of FAD-GDH from Aspergillus terreus. At pH 7.0, the logarithm of the bimolecular oxidation rate constants appeared to depend on the redox potentials of all the mediators tested. Notably, the rate constant of each molecule for FAD-GDH was approximately 2.5 orders of magnitude higher than that for glucose oxidase from Aspergillus sp. The results suggest that the electron transfer kinetics is mainly determined by the formal potential of the mediator, the driving force of electron transfer, and the electron transfer distance between the redox active site of the mediator and the FAD, affected by the steric or chemical interactions. Higher k2 values were found for ortho-quinones than for para-quinones in the reactions with FAD-GDH and glucose oxidase, which was likely due to less steric hindrance in the active site in the case of the ortho-quinones.
Highlights
Flavin adenine dinucleotide-dependent glucose dehydrogenases (FAD-GDHs) have recently garnered considerable attention as bioelectrocatalysts for glucose biosensors and biofuel cells because of their unique characteristics, including oxygen insensitivity, high biocatalytic activity, and substrate specificity [1,2,3]
flavin adenine dinucleotide-dependent glucose dehydrogenase (FAD-GDH) isolated from several Aspergillus species have already been employed as catalysts in disposable blood glucose sensor strips, indicating that they may be promising alternatives to glucose oxidases (GOxs) [4]
The rate constants for the bimolecular reaction of 2-methyl-1,4-naphthoquinone (MeNQ), methylene green (MG), and 1,2-naphthoquinone-4-sulfonate (NQS) with FAD-GDH and GOx were lower than expected from the potential-dependent calibration line
Summary
Flavin adenine dinucleotide-dependent glucose dehydrogenases (FAD-GDHs) have recently garnered considerable attention as bioelectrocatalysts for glucose biosensors and biofuel cells because of their unique characteristics, including oxygen insensitivity, high biocatalytic activity, and substrate specificity [1,2,3]. Ag/AcccuuugrrrCvvveeelsss(wswwaeeetrrr.eeeKooobbbCssselee)rrr.vvveIeenddd ffftooohrrribsbbooostttahhhmTTTBBBe aaawnnnddday111,444tNNNhQQQe,,,fbbbouuurtttmttthhhaeeelssspttteeeoaaatdddeyyyn---sssttttiaaaattteleescccoaaatttfaaalallyyyltttliiicccthcccuuuerrrrorreeetnnnhtttedddreeennncshssiiitettyyymaaaninncdddatttlhhhseeewere evaluated aclnedarapddpddpddgroeiooeieieslssptppttcucceeeeueeluunnncninnssststtodsddissiitaeaaeeseeeeldlldddedddaaaiiitttoooonnnwiwwnnnxnttthhhhhhittthdhehieeiiTccceeeafhaffhhooootoobtlttllixhlxxhhlloloooiiieeeedddwnwwcaccaa1iaiiaatttncnnit.iittoaoougaaggnlInnllyrsyyssnertkeetktkiciiecciciitcctnttnnnhiiiccceoeeooutuuetttnnnsiriirrc)cc))rprr.s..wsseeernnnoooeetfttffsrrrrttteeeheehhaaaeneecocchcehheebneenneesdzddzzeoyyytttrmhmmfhhveeeFeeees,s,s,AtdttaaaeeesDssaaa(ddddddF-yeyyeeGitttgsesseeDtttrurraaammmtttHreeeieiinnndddeie1eiiinfdffdd)fffe.eebbbtrrrTyheyyeedyddettt.h..hhpTgTTeeeihhhlceeeueeeallleeecccclcccaaaotstttttrrrasiaaoooglellnynnyym-tttcaiaaiiccccoccoccccccnieueeuudppptrrrartartrtoeooeeilnrnnnrrscsstttiud(ndd((dddeeregeeennnvtttsassaapeiiiiiitttslhllyyysssowseprheaotbesbeurvffeedr, for both TBBBaaaBssseeeadddnooodnnn t1tthhh4eeeNCCCVQVVsss,ooobfffuttthhhteeetphpphhheeeensnnotooettthhhaiiidaaazzzyiiinnn-seeestssaaaatnnnedddcqqqauuutiiiannnloooynnnteeeisssceeecxxxuaaammmrriiinenneeendddt iiidnnnettthhhneeespipptrrryeeessseaeennnncccdeee oootfffhtttehhheeepeeeonnntzzzeyyynmmmteeeial at which the catalyaaantnnidddcgggcllluuuucccrooorssseeeen,,, wwwt eeerwewwaeeecrrreeehaaaebbbdllleeetttthoooedddeeesttteteererrmmmadiiinnnyeee tttshhhteeeaaaatpppepppdllliiieeeidfddfpepporooettteeednnn.tttiiiaaaTlll fffhoooerrr ttthchheaeetcccaooolnnnyssstttaaaincnntttcpppuoootrtteeernnnetttniiiaaatlll aaadmmmepppneeesrrrioootmmmyeeedtttrrryeyypended on the ofoxlildoawK(sK(sK(sctchchhChiiCChhooonorlrrllwww)o))oongnsnanssaaosskoosstttheahhaaaiaamecmmneeftffTepuTTppuuioBtenBBeennir-rrcn--cccomoommttts)imiimmoo.oeeenodnneddeetittfiiroaroroaayyytfttftfe)eeh))tttdddhfhhffeoooeeeFFFrrreAAAeeemmmnlllDDDeeezeeeccc---aaatyGttGGrsrrssomuooDuuDDlllrrryHyyHHieiinsnnss-,--iiiggcgsccssaaaatttttttstthihhiiaaammmeeellldyyyeeessszezztttefeeeffeetooodaddeaarrrdddrrrrvymvvyyeeea-aaa--aasssrcrricctttitiinttaoaaooiiiotoottueuueeennnsdsscccccTcaTTaaubuutBttBBaraarryrlrrllcccyeyyeeooottntntnhininintcttcccccedddgegegeeneelnnlleunuunntttlrcrrsccsseaaaoiooiitctttttsssyiyyiiteoeeoor(n((nnojooojj)s))ssxxxn...aaaiiidTddtTTttaahaa0hh00tctt.e..eei0ii00cooo55s5ssnenntttVeVVpeecccaaautuuvdvvddorrrssysyyrrr.r..-e--eeAssAAssnnntttagtaaggtt(...td/tt//eFAeeFAFAeiiicggccggggtaaaCuCCauutttralriraalleleel(ll((ysyysss222ataattAidAAiitttccc...iscussed in the Bcccuuuarrrsrrreeennndttt dddoeeennnnssstiiihtttyyyeiiinnnCcccrVrreeeasaasssoeeedddf ltlliiihnnneeeaaaprrrlllyhyy wewwniiitttohhhtiiihnnnciccrarreeezaaaisssniiinnnegggsTTTaBBBncccdooonnnqccceueennnitttnrrraaaotttniiioooennns,,, aaaesssxeeeavvvmiiidddeeeinnnnttteiiindnn FFFiiiinggguuutrrrheeee222BBBp...rBBBeyyysueuusnssiiinnncgegg of the enzyme and gttthlhhueeesscseeeoscccehhh,rrrooownnnoeooaaawmmmpeppereererrooomammbeeeltttrerryyytorrreeedaaadddeiiitnnnegggrsssmoooifffnettthhhteeehedddeeeappppeeenpnndddlieeeennndccceeepooooffftettthhnheeetiassstttleeeafaaodddryyy---tssshtttaaaettteeecojjj nooosnnntatttnhhhetee pmmmoeeetdddeiiinaaatttoooiarrr l amperometry (schhorwocqcqcqnsooouuuonnniiitnanncccheoeeoomennnnnntteteeprrrTsssaaaeBt(tt((irkiikkooo-2o222nmnn===m,,,kkktettcchcechhaaadattetttee///KriKKbbbayMMMMiiitmmm))))ecccfodoooooolllueuueerFclcclldudduumAlllbbbaaaDeereerrake-kkeesGvivviinnnuaaaeDeelllrutuuttiiiiHacaancctttcecgcee-odooddcnnntafffhsssrrrttttoooeaaaammmnnnlsyttttstssttzhehhfffeeaoeeoodrdrrfffoooFFFylllrAlAAll-oeoosDDDwwwat---aciiiGGGnnnttgDiggDeDoHeHHeecnqqqauouuootcxaxxaaauitiittdliddiioryooaaanrnntttteiiii[o[[oocn222nnn888tg]b]]bb:l::dyyyuettcthhhnoeeesspppiethhhyoeeennnx(ooojit)dtthhhaiaiiaaattzzziioii0nnnn.eee0sssc5aaaunnnVdrddrevnst By using these chronoamperometry readings of the dependence of the steady-state j on the mediator concentration, the bimolecular kinetic constants for FAD-GDH oxidation by the phenothiazines and quinones (k2 = kcat/KM) could be evaluated from the following equation [28]:. The rate constants for the bimolecular reaction of 2-methyl-1,4-naphthoquinone (MeNQ), methylene green (MG), and 1,2-naphthoquinone-4-sulfonate (NQS) with FAD-GDH and GOx were lower than expected from the potential-dependent calibration line This could be due to steric hindrance, since the functional groups of these molecules (a methyl group in MeNQ, a nitro group in MG, and a sulfo group in NQS) are close to the redox active site and could potentially disturb the access to FAD. The mechanism for effective electron transfer could be explained by steric hindrance (i.e., the electron transfer distance between the FAD site and the redox site of the quinone molecules)
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