International Journal of
Molecular Sciences Article
Bimolecular Rate Constants for FAD-Dependent Glucose Dehydrogenase from Aspergillus terreus and Organic Electron Acceptors Nozomu Tsuruoka, Takuya Sadakane, Rika Hayashi and Seiya Tsujimura * Division of Materials Science, Faculty of Pure and Applied Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan;
[email protected] (N.T.);
[email protected] (T.S.);
[email protected] (R.H.) * Correspondence:
[email protected]; Tel.: +81-29-853-5358; Fax: +81-29-753-4490 Academic Editor: Deepak Pant Received: 13 December 2016; Accepted: 8 March 2017; Published: 10 March 2017
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. Keywords: flavin adenine dinucleotide; glucose dehydrogenase; phenothiazine; quinone; redox mediator
1. Introduction 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 [1–3]. In fact, FAD-GDHs 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]. Although some bacterial FAD-GDHs have the ability to transfer electrons directly from the enzyme to the surface of electrode [5,6], FAD-GDHs from Aspergillus species require redox mediators to shuttle electrons from the FAD site to the electrode [2,3]. For example, hexacyanoferrate ([Fe(CN)6 ]3− ; ferricyanide) has been used as a redox mediator in commercially available FAD-GDH-based blood glucose sensor strips because of its high solubility in water, low cost, and high stability [2,4]. However, the reactivity between FAD-GDH and ferricyanide is quite low, where the bimolecular rate constant (k2 = kcat /KM ) has been documented to be as low as 1 × 103 M−1 ·s−1 in phosphate buffer (pH 7.0) at room temperature [2]. This low kinetic constant could be attributed to the low affinity between the active site of FAD-GDH and ferricyanide (i.e., an immeasurably high Michaelis–Menten constant). For the further development and performance
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improvement of bioelectrochemical devices, selection of a suitable mediator is critical. A mediator that has a low redox potential, but exhibits high electron transfer activity, is desired for high-power biofuel cells and for highly sensitive biosensors which is not affected by interfering substances such as uric acid or acid [7–9]. Int. ascorbic J. Mol. Sci. 2017, 18, 604 2 of 9 Int. Mol. Sci. 2017, 604 22 of Int.J.J.J.Mol. Mol.Sci. Sci.2017, 2017, 18, 18,604 604 of999 Int. of Osmium (Os) 18, complexes, a group of metal complex-type redox mediators used 2with GOxs, appear (i.e., an immeasurably high Michaelis–Menten constant). For the further development and (i.e., an immeasurably high Michaelis–Menten constant). For the further development and to react much more efficiently FAD-GDH constant). [3,10,11]. Osmium complex-type mediators are useful (i.e., an immeasurably immeasurably high with Michaelis–Menten constant). For For the further further development and (i.e., an high Michaelis–Menten the development and performance improvement of bioelectrochemical devices, selection of a suitable mediator is critical. performance improvement of bioelectrochemical devices, selection of suitable mediator is critical. performance improvement of bioelectrochemical bioelectrochemical devices, selection of aaa suitable suitable mediator isself-exchange critical. performance improvement of devices, selection of mediator is critical. because, in addition to maintaining high kinetic constants through their efficient electron A mediator that has a low redox potential, but exhibits high electron transfer activity, is desired for A mediator that has low redox potential, but exhibits high electron transfer activity, is desired for A mediator mediator that that has has aaa low low redox redox potential, potential, but but exhibits exhibits high high electron electron transfer transfer activity, activity, is is desired desired for for A transfer capabilities, they make it possible to biosensors control the potential at which the reaction proceeds high-power biofuel cells and for highly sensitive which is not affected by interfering high-power biofuel cells and for highly sensitive biosensors which is not affected by interfering high-power biofuel biofuel cells cells and and for for highly highly sensitive sensitive biosensors biosensors which which is is not not affected affected by by interfering interfering high-power such as uric acid or ascorbic acid [7–9]. solelysubstances by replacing the ligand [7,12–16]. However, Os complexes are expensive and non-sustainable, substances such as uric acid or ascorbic acid [7–9]. substances such such as as uric uric acid acid or or ascorbic ascorbic acid acid [7–9]. [7–9]. substances (Os) complexes, aa group of metal complex-type redox mediators used with GOxs, Osmium (Os) complexes, group of metal complex-type redox mediators used with GOxs, renderingOsmium them impractical for use in low-cost and disposable FAD-GDH-based electrodes. Thus, there Osmium (Os) (Os) complexes, complexes, aa group group of of metal metal complex-type complex-type redox redox mediators mediators used used with with GOxs, GOxs, Osmium appear to react much more efficiently with FAD-GDH [3,10,11]. Osmium complex-type mediators appear to react much more efficiently with FAD-GDH [3,10,11]. Osmium complex-type mediators appear to react much more efficiently with FAD-GDH [3,10,11]. Osmium complex-type mediators appear react much more efficiently with FAD-GDH [3,10,11]. complex-type mediators is a need to toselect or identify other organic compounds or Osmium metalthrough complexes based on earth abundant are useful because, in addition to maintaining high kinetic constants their efficient selfare useful because, in addition to maintaining high kinetic constants through their efficient selfare useful useful because, because, in in addition addition to to maintaining maintaining high high kinetic kinetic constants constants through through their their efficient efficient selfselfare metals that can efficiently mediate redox reactions with FAD-GDH while also being economically exchange electron transfer capabilities, they make it possible to control the potential at which the exchange electron transfer capabilities, they make it possible to control the potential at which the exchange electron electron transfer transfer capabilities, capabilities, they they make make it it possible possible to to control control the the potential potential at at which which the the exchange proceeds solely by replacing the ligand [7,12–16]. However, Os complexes are expensive and viablereaction for low-cost bioelectrical devices. Recently, naphthoquinone and the naphthoquinone-based reaction proceeds solely by replacing the ligand [7,12–16]. However, Os complexes are expensive and reactionproceeds proceedssolely solelyby byreplacing replacingthe theligand ligand[7,12–16]. [7,12–16].However, However,Os Oscomplexes complexesare areexpensive expensiveand and reaction non-sustainable, rendering them impractical for use in low-cost and disposable FAD-GDH-based non-sustainable, rendering them impractical for use in low-cost and disposable FAD-GDH-based redoxnon-sustainable, polymer hydrogel were to for an FAD-GDH-based electrode to improve its biofuel cell non-sustainable, rendering themapplied impractical for use use in low-cost low-cost and and disposable disposable FAD-GDH-based rendering them impractical in FAD-GDH-based electrodes. Thus, there is a need to select or identify other organic compounds or metal complexes electrodes. Thus, there is need to select or identify other organic compounds or metal complexes electrodes.[17]. Thus,Naphthoquinone/phenanthrenequinone there is is aaa need need to to select select or or identify identify other other organic organic compounds compounds or metal metal complexes electrodes. Thus, there or performance derivatives havecomplexes alsowhile been investigated based on earth abundant metals that can efficiently mediate redox reactions with FAD-GDH based on earth abundant metals that can efficiently mediate redox reactions with FAD-GDH while based on on earth earth abundant abundant metals metals that that can can efficiently efficiently mediate mediate redox redox reactions reactions with with FAD-GDH FAD-GDH while while based for their application in blood glucose sensors [18,19]. TheRecently, choice naphthoquinone of mediator isand very important as it also being economically viable for low-cost bioelectrical devices. the also being economically viable for low-cost bioelectrical devices. Recently, naphthoquinone and the also being being economically economically viable viable for for low-cost low-cost bioelectrical bioelectrical devices. devices. Recently, Recently, naphthoquinone naphthoquinone and and the the also naphthoquinone-based redox polymer hydrogel were applied to an FAD-GDH-based electrode to determines the potential at which the reaction proceeds, as well as the reaction rate (current density). naphthoquinone-based redox polymer hydrogel were applied to an FAD-GDH-based electrode to naphthoquinone-based redox redox polymer polymer hydrogel hydrogel were were applied applied to to an an FAD-GDH-based FAD-GDH-based electrode electrode to to naphthoquinone-based improve its biofuel cell performance [17]. Naphthoquinone/phenanthrenequinone derivatives have improve its biofuel cell performance [17]. Naphthoquinone/phenanthrenequinone derivatives have However, it is not easy to obtain information on the optimum properties of potential mediators for improve its its biofuel biofuel cell cell performance performance [17]. [17]. Naphthoquinone/phenanthrenequinone Naphthoquinone/phenanthrenequinone derivatives derivatives have have improve also been investigated for their application in blood glucose sensors [18,19]. The choice of mediator also been investigated for their application in blood glucose sensors [18,19]. The choice of mediator also been investigated for their application in blood glucose sensors [18,19]. The choice of mediator also been investigated for their application in blood glucose sensorsproceeds, [18,19]. choice of mediator FAD-GDHs. Even with structural and chemical information about The the active center pocket obtained is very important as itit determines the potential at which the reaction as well as the reaction is very important as determines the potential at which the reaction proceeds, as well as the reaction isvery veryimportant importantas asit itdetermines determinesthe the potential potentialat atwhich whichthe thereaction reactionproceeds, proceeds,as aswell wellas asthe thereaction reaction is by crystal structure analysis of the enzyme, it would be difficult to predict the suitable mediator to rate (current density). However, it is not easy to obtain information on the optimum properties of rate (current density). However, it is not easy to obtain information on the optimum properties of rate (current (current density). density). However, However, it it is is not not easy easy to to obtain obtain information information on on the the optimum optimum properties properties of of rate potential mediators for FAD-GDHs. Even with structural and chemical information about the active use. Although there isfor anFAD-GDHs. increasing number of reports the discovery recombinant production potential mediators for FAD-GDHs. Even with structural and chemical information about the active potential mediators mediators for FAD-GDHs. Even with structural structural andon chemical informationand about the active active potential Even with and chemical information about the center pocket obtained by crystal structure analysis of the enzyme, itit would be difficult to predict the center pocket obtained by crystal structure analysis of the enzyme, would be difficult to predict the of new FAD-GDHs fromby fungi [20–26], isenzyme, not realistic from the viewpoint of time and cost center pocketobtained obtained byvarious crystalstructure structure analysisof ofitthe the enzyme, itwould would bedifficult difficult topredict predictthe the center pocket crystal analysis it be to suitable mediator to use. Although there is an increasing number of reports on the discovery and suitable mediator to use. Although there is an increasing number of reports on the discovery and suitable mediator to use. use. Although there is an an increasing increasing number of of reports reports on the theTherefore, discovery and and suitable to Although is number on discovery to clarify themediator mediator structure forthere all these newly discovered enzymes. in this study, we recombinant production of new FAD-GDHs from various fungi [20–26], it is not realistic from the recombinant production of new FAD-GDHs from various fungi [20–26], it is not realistic from the recombinant production production of of new new FAD-GDHs FAD-GDHs from from various various fungi fungi [20–26], [20–26], itit is is not not realistic realistic from from the the recombinant propose a strategy for determining the optimum mediator for FAD-GDHs by screening a set of organic viewpoint of time and cost to clarify the mediator structure for all these newly discovered enzymes. viewpoint of time and cost to clarify the mediator structure for all these newly discovered enzymes. viewpoint of of time time and and cost cost to to clarify clarify the the mediator mediator structure structure for for all all these these newly newly discovered discovered enzymes. enzymes. viewpoint in this study, we propose aa strategy for determining the optimum mediator for FAD-GDHs redoxTherefore, mediators via their bimolecular rate constants for the enzyme reaction. For this purpose, a set of Therefore, in this study, we propose strategy for determining the optimum mediator for FAD-GDHs Therefore,in inthis thisstudy, study,we wepropose proposeaastrategy strategyfor fordetermining determiningthe theoptimum optimummediator mediatorfor forFAD-GDHs FAD-GDHs Therefore, by screening aa set of organic redox mediators via their bimolecular rate constants for the enzyme by screening set of organic redox mediators via their bimolecular rate constants for the enzyme quinones and phenothiazines (Table 1) were used, since their structure and redox potentials can be by screening screening aa set set of of organic organic redox redox mediators mediators via via their their bimolecular bimolecular rate rate constants constants for for the the enzyme enzyme by reaction. For this purpose, aa set of quinones and phenothiazines (Table 1) were used, since their reaction. For this purpose, set of quinones and phenothiazines (Table 1) were used, since their reaction. For this purpose, a set of quinones and phenothiazines (Table 1) were used, since their reaction. purpose, a set ofbequinones and phenothiazines (Table 1) were used, since their easilystructure tuned For by this changing the substituents. The findingsthe ofsubstituents. this study may be useful as the starting and redox potentials can easily tuned by changing The findings of this structure and redox potentials can be easily tuned by changing the substituents. The findings of this structure and and redox redox potentials potentials can can be be easily easily tuned tuned by by changing changing the the substituents. substituents. The The findings findings of of this this structure pointstudy for the retrosynthetic analysis of new mediators. may be useful as the starting point for the retrosynthetic analysis of new mediators. study may be useful as the starting point for the retrosynthetic analysis of new mediators. study may may be be useful useful as as the the starting starting point point for for the the retrosynthetic retrosynthetic analysis analysis of of new new mediators. mediators. study
Table Redox mediators and the enzyme oxidation rates measured at pH 7 and 25 °C. Table 1. Redox mediators and the enzyme oxidation rates measured at pH and 25 °C. Table 1. 1. Redox and enzyme oxidation rates measured at pH 7 and 25 ◦ C. Table 1. Redox Redoxmediators mediators and and thethe enzyme oxidation rates measured measured at pH pH 777 and and 25 25°C. °C. Table 1. mediators the enzyme oxidation rates at Compound Compound Compound Compound Compound
E (V vs. E (V vs. (Vvs. vs. EE(V Ag/AgCl
Ag/AgCl EAg/AgCl (V vs. Ag/AgCl (sat. (sat. Ag/AgCl (sat.(sat. (sat. KCl)) KCl)) KCl)) KCl)) KCl))
2-Methyl-1,42-Methyl-1,42-Methyl-1,42-Methyl-1,4-
naphthoquinone naphthoquinone 2-Methyl-1,4naphthoquinone naphthoquinone (MeNQ) (MeNQ) naphthoquinone (MeNQ) (MeNQ)
DM × D M× D M6×× D M 10
Structure Structure Structure Structure Structure
log (k2 for log (k for log (k222for for log (k FAD-
log (k2 for log (k2 for
6 log(k (k2−1 2for for −1) log 10 FADlog (k2GOx/M for DM 106 6 6× −1·s 10 FAD2·s −1) −1·s−1) 10 FAD·s−1) GOx/M (cm −11 −1 −1·s−1) −1 ·s− GOx/M 2 ·−1 −1 GDH/M ·s·s)−1−1)) GOx/M (cm GDH/M FAD-GDH/M (cm222·s·s −1·s−1 −1 (cm ·s−1−1s))) ) GDH/M GDH/M (cm ·s −1))
−0.20 −0.20
7.0 7.0 7.0 7.0 7.0
5.7 ± 0.0 2.8 5.7 0.0 2.8 5.7±±±0.0 0.0 5.7 ± 0.0 2.8 2.8 5.7
−0.20 −0.20 −0.20
log (k2 for GOx/M−1 ·s−1 )
2.8
(MeNQ)
Toluidine blue Toluidine Toluidine blue blue (TB) (TB)
−0.19 −0.19 −0.19 −0.19
−0.19
2.4 2.4 2.4 2.4 2.4
6.2 ± 0.1 4.3 ± 0.1 6.2 0.1 4.3 0.1 6.2±±±0.1 0.1 6.2 ± 0.1 4.3±±±0.1 0.1 6.2 4.3
4.3 ± 0.1
AzureA AzureA (AA) (AA) (AA)
−0.19 −0.19 −0.19 −0.19
3.6 3.6 3.6 3.6 3.6
6.2 ± 0.0 4.1 ± 0.1 6.2 0.0 4.1 0.1 6.2±±±0.0 0.0 6.2 ± 0.0 4.1±±±0.1 0.1 6.2 4.1
4.1 ± 0.1
−0.18 −0.18 −0.18
6.06.0 6.0 6.0 6.0
4.2 ± 0.1 4.2 ± 0.1 8.2 ± 0.1 8.2 ± 0.1 4.2 4.2 ±±0.1 8.2 0.1 3.6 *0.1 3 of 9 8.2±±±0.1 0.1 8.2 3.6 3.6*** 3.6
4.2 ± 0.1 3.6 *
4.3 6.1 4.3 4.3 4.3 4.3
3.0 ± 0.2 6.1 ± 0.2 6.1 ± 0.2 3.5 ± 0.0 7.1 6.1 0.2 3.5 ± *0.0 3 of 9 3.5 6.1±±±0.2 0.2 3.5 0.0 6.1 3.5 ±±0.0
3.5 ± 0.0
2.6 6.16.1
3.0 7.0 ± 0.0 5.1 ± 0.2 0.0 7.1 ± 0.2 7.1 ± 0.2 3.5 *
3.0 ± 0.2 3.5 *
4.3 2.6
6.5 ± 0.1 7.0 ± 0.0
4.5 ± 0.2 5.1 ± 0.0
6.1 4.3
8.1 ± 0.1 0.2 6.5
5.5 ± 0.2 4.5 ± 0.2 5.0 *
3.0 6.1
6.6 ±± 0.2 0.1 8.1
4.0 ±± 0.2 0.1 5.5 2.9 ** 5.0
Toluidine blue (TB) Toluidine blue (TB) (TB)
AzureA AzureA AzureA (AA) (AA)
9,10-Phenanthrenequinone 9,10-Phenanthrenequinone 9,10-Phenanthrenequinone 9,10-Phenanthrenequinone 9,10-Phenanthrenequinone Int. J. Mol. Sci.(PQ) 2017, 18, 604 (PQ) (PQ) (PQ) (PQ)
Methylene blue 1,4-Naphthoquinone (14NQ) (MB) (MB) (MB)
Methylene blue Methylene blue (MB) Methylene blue Methylene (MB) Int. J. Mol. Sci. 2017,blue 18, 604
−0.19
−0.18 −0.18 O O
O O OO
O OO
−0.17 −0.15 −0.17 −0.17
−0.17 −0.17 O O
Thionine
1,4-Naphthoquinone 1,4-Naphthoquinone (TH) (14NQ) (14NQ)
−0.14 −0.15 −0.15 O
Methylene green Thionine (MG) (TH)
−0.06 −0.14 O
1,2-Naphthoquinone Methylene green (12NQ) (MG) 1,2-Naphthoquinone-41,2-Naphthoquinone sulfonate (12NQ) (NQS)
O
−0.05 −0.06 O
0.01 −0.05
O
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of 99 333 of of 99 3 of
Table 1. Cont. O OO O O
1,4-Naphthoquinone 1,4-Naphthoquinone 1,4-Naphthoquinone 1,4-Naphthoquinone (14NQ) (14NQ) (14NQ) Compound (14NQ)
E (V vs. −0.15 −0.15 −0.15 −0.15 Ag/AgCl (sat. KCl))
6.1
6.1× 106 D6.1 M 6.1 (cm2 ·s−1 )
Structure O OO O O
3.0 ±± 0.2 0.2 3.0 3.0 ±± 0.2 0.2 7.1 ±± 0.2 0.2 3.0 7.1 7.1 ±± 0.2 0.2 log (k2 for 3.5 ** 7.1 3.5 3.5 * 3.5 − 1 − FAD-GDH/M ·s * 1 )
log (k2 for GOx/M−1 ·s−1 )
Thionine Thionine Thionine Thionine (TH)
−0.14 −0.14 −0.14
−0.14 −0.14
2.6 2.6 2.62.6 2.6
7.0 ±± 0.0 0.0 5.1 ±± 0.0 0.0 7.0 5.1 7.0 ±± 0.0 0.0 7.0 ± 0.0 5.1 ±± 0.0 0.0 7.0 5.1
5.1 ± 0.0
Methylene green green Methylene Methylene green (MG) (MG)
−0.06 −0.06 −−0.06 0.06 −0.06
4.3 4.3 4.34.3 4.3
6.5 ±± 0.1 0.1 4.5 ±± 0.2 0.2 6.5 4.5 6.5 ±± 0.1 0.1 6.5 ± 0.1 4.5 ±± 0.2 0.2 6.5 4.5
4.5 ± 0.2
−0.05
6.16.1 6.1 6.1 6.1
5.5 ±± 0.2 0.2 5.5 5.5 ±± 0.2 0.2 8.1 ±± 0.2 0.2 8.1 ± 0.2 5.5 8.1 8.1 ±± 0.2 0.2 5.0 ** 8.1 5.0 5.0 ** 5.0
5.5 ± 0.2 5.0 *
0.01 0.01 0.01 0.01
3.03.0 3.0 3.0 3.0
4.0 ±± 0.1 0.1 4.0 4.0 ±± 0.1 0.1 6.6 ±± 0.1 0.1 6.6 ± 0.1 4.0 6.6 6.6 ±± 0.1 0.1 2.9 ** 6.6 2.9 2.9 2.9 **
4.0 ± 0.1 2.9 *
8.48.4 8.4 8.4 8.4
5.4 ±± 0.2 0.2 5.4 5.4 ±± 0.2 0.2 7.9 ±± 0.2 0.2 7.9 ± 0.2 5.4 7.9 7.9 ±± 0.2 0.2 5.2 ** 7.9 5.2 5.2 5.2 **
5.4 ± 0.2 5.2 *
(TH) (TH) Thionine (TH) (TH)
Methylene green Methylene green (MG) (MG) (MG)
1,2-Naphthoquinone 1,2-Naphthoquinone 1,2-Naphthoquinone 1,2-Naphthoquinone 1,2-Naphthoquinone (12NQ) (12NQ) (12NQ) (12NQ) (12NQ)
O O O O O
O O O O O
−0.05 −−0.05 0.05 −0.05
1,2-Naphthoquinone-41,2-Naphthoquinone-4-
1,2-Naphthoquinone-41,2-Naphthoquinone1,2-Naphthoquinone-4sulfonate sulfonate sulfonate 4-sulfonate (NQS) sulfonate (NQS) (NQS) (NQS) (NQS) (NQS)
1,4-Benzoquinone 1,4-Benzoquinone (BQ) (BQ) (BQ) (BQ)
1,4-Benzoquinone 1,4-Benzoquinone (BQ) 1,4-Benzoquinone (BQ)
0.01
0.09 0.09 0.09 0.09
O O O O O
0.09
O O O O O
From J. Kuly, N. Cenas, Biochim. Biophys. Acta 1983, 744, 57–63 [27]. GOx glucose oxidase; FAD*** From J. N. Biochim. Biophys. Acta 1983, 744, 57–63 glucose oxidase; FAD* From J. Kuly, N. Cenas, Biochim. Biophys. [27].GOx GOx==== = glucose oxidase; FAD-GDH = flavin From J. Kuly, Kuly, N. Cenas, Cenas, Biochim. Biophys.Acta Acta1983, 1983,744, 744, 57–63 57–63 [27]. [27]. GOx glucose oxidase; FAD* From J. Kuly, N. Cenas, Biochim. Biophys. Acta 1983, 744, 57–63 [27]. GOx glucose oxidase; FADM = diffusion coefficient GDH = flavin adenine dinucleotide-dependent glucose dehydrogenasese; D Mcoefficient adenine dinucleotide-dependent glucose dehydrogenasese; D = diffusion of the mediator; E = formal M = diffusion coefficient GDH = flavin adenine dinucleotide-dependent glucose dehydrogenasese; D M M = diffusion coefficient GDH == flavin flavin adenine adenine dinucleotide-dependent dinucleotide-dependent glucose glucose dehydrogenasese; dehydrogenasese; D DM = diffusion coefficient GDH ==the oxidation rate constant of the mediator. of the mediator; E formal potential of the mediator; potential the mediator; k2 =potential oxidation constantkkkof mediator. 2222 = oxidation rate of E of the oxidation rate constant constant of of the the mediator. mediator. of the theofmediator; mediator; E === formal formal potential of rate the mediator; mediator; of the mediator; E = formal potential of the mediator; k2 = oxidation rate constant of the mediator.
2. and Discussion 2. Results Results and Discussion 2. Results Discussion 2. Results andand Discussion In this study, In this this study, study, an an electrochemical electrochemical method method was was applied applied to to evaluate evaluate the the bimolecular bimolecular rate rate constants constants In an electrochemical method was applied to evaluate the bimolecular rate constants In this study, an electrochemical method was applied to evaluate the bimolecular of the selected electron acceptors for FAD-GDH and GOx. Since the difference in the of the the selected selected electron electron acceptors acceptors for for FAD-GDH FAD-GDH and and GOx. GOx. Since Since the the difference difference in in the the extinction extinction rate constants of extinction oxidized reduced is color was of thecoefficients selectedof acceptors for FAD-GDH and aaaGOx. Sincemeasurement the difference in the extinction coefficients ofelectron oxidized and and reduced quinones quinones is quite quite small, small, color change change measurement was not not coefficients of oxidized and reduced quinones is quite small, color change measurement was not adequate for the kinetic analyses. Furthermore, the solubility of some quinones is so low that it is adequate for the kinetic analyses. Furthermore, the solubility of some quinones is so low that it is coefficients reduced quinones is quite of small, color change was not adequateof foroxidized the kineticand analyses. Furthermore, the solubility some aquinones is so lowmeasurement that it is cat value). We first difficult to increase their concentration to observe the maximum turnover rate (k cat difficult to to increase increase their their concentration concentration to to observe observe the the maximum maximum turnover turnover rate rate (k (kcat cat value). value). We We first first difficult adequate for the kinetic analyses. Furthermore, the solubility of some quinones is so low that it is confirmed confirmed the the electrochemical electrochemical behavior behavior of of the the electron electron acceptors acceptors by by cyclic cyclic voltammetry. voltammetry. Figure Figure 111 confirmed the electrochemical behavior of the electron acceptors by cyclic voltammetry. Figure shows the cyclic voltammograms (CVs) of 0.1 mM toluidine blue (TB) and 0.1 mM 1,4difficult to increase their concentration to observe the maximum turnover rate (k value). We first shows the the cyclic cyclic voltammograms voltammograms (CVs) (CVs) of of 0.1 0.1 mM mM toluidine toluidine blue blue (TB) (TB) and and 0.1 0.1 mM mM cat 1,4shows 1,4naphthoquinone (14NQ), which were recorded in the presence or absence of FAD-GDH in phosphate confirmed the electrochemical behavior of the electron acceptors by cyclic voltammetry. Figure 1 naphthoquinone (14NQ), which were recorded in the presence or absence of FAD-GDH in phosphate naphthoquinone (14NQ), which were recorded in the presence or absence of FAD-GDH in phosphate buffer (pH glucose. these CVs, the potentials of buffer (pH 7) 7) containing containing glucose. From From these CVs,mM the formal formal potentials (i.e., mid-point potential of shows the cyclic voltammograms (CVs) of 0.1 toluidine blue (i.e., (TB)mid-point and 0.1 potential mM 1,4-naphthoquinone buffer (pH 7) containing glucose. From these CVs, the formal potentials (i.e., mid-point potential of oxidation oxidation and and reduction reduction peak peak potentials) potentials) of of TB TB and and 14NQ 14NQ were were evaluated evaluated to to be be −0.19 −0.19 and and −0.15 −0.15 V, V, oxidation and reduction peak potentials) of TB and 14NQ were evaluated to be −0.19 −0.15 V, (14NQ), which were recorded in the presence or absence of FAD-GDH in and phosphate buffer (pH 7) respectively, vs. Ag/AgCl (sat. KCl). In this same way, the formal potentials of all the other chemicals respectively, vs. Ag/AgCl (sat. KCl). In this same way, the formal potentials of all the other chemicals respectively, vs. Ag/AgCl (sat. KCl). In this same way, the formal potentials of all the other chemicals containing glucose. From these CVs, the formal potentials (i.e., mid-point potential of oxidation and were evaluated and are listed in Table 1. In the presence of FAD-GDH in the glucose-containing were evaluated evaluated and and are are listed listed in in Table Table 1. 1. In In the the presence presence of of FAD-GDH FAD-GDH in in the the glucose-containing glucose-containing were phosphate buffer, clear glucose oxidation currents were observed (Figure 1). Typical sigmoidal reduction peak potentials) of TB and 14NQ were evaluated to be − 0.19 and − 0.15 V, respectively, vs. phosphate buffer, clear glucose oxidation currents were observed (Figure 1). Typical sigmoidal phosphate buffer, clear glucose oxidation currents were observed (Figure 1). Typical sigmoidal curves were observed for both TB and 14NQ, but the steady-state catalytic current density and the curves were observed for both TB and 14NQ, but the steady-state catalytic current density and the Ag/AgCl In for thisboth same way,14NQ, the formal potentialscatalytic of all the other chemicals curves(sat. wereKCl). observed TB and but the steady-state current density and thewere evaluated potential potential at at which which the the catalytic catalytic current current reached reached the the steady steady state state differed. differed. The The catalytic catalytic current current density density potential at which the catalytic current reached the steady state differed. The catalytic current density and are listed in Table 1. In the presence of FAD-GDH in the glucose-containing phosphate buffer, depended depended on on the the oxidation oxidation kinetics kinetics of of the the enzyme, enzyme, as as determined determined by by the the electron electron acceptors acceptors (details (details depended on the oxidation kinetics of the enzyme, as determined by the electron acceptors (details in the following section). clear discussed glucose oxidation currents were observed (Figure 1). Typical sigmoidal curves were observed discussed in in the the following following section). section). discussed Based on the CVs of the phenothiazines and quinones examined in the presence of the enzyme for both TB and but steady-state current in density and of the Based on 14NQ, the CVs CVs of of thethe phenothiazines andcatalytic quinones examined examined in the presence presence of thepotential enzyme at which the Based on the the phenothiazines and quinones the the enzyme and we were to applied potential for constant potential and glucose, glucose, wereached were able ablethe to determine determine the applied potential for the the constantcurrent potential amperometry amperometry catalytic current steady the state differed. The catalytic density depended on the and glucose, we were able to determine the applied potential for the constant potential amperometry (chronoamperometry) (chronoamperometry) for for measuring measuring the the steady-state steady-state catalytic catalytic glucose glucose oxidation oxidation current. current. Figure Figure 2A 2A (chronoamperometry) for measuring the steady-state catalytic glucose oxidation current. Figure 2A oxidation kinetics of the enzyme, as determined by thedensity electron acceptors (details discussed in the shows the TB-mediated FAD-GDH-catalyzed reaction current (j) at 0.05 V vs. Ag/AgCl (sat. shows the TB-mediated FAD-GDH-catalyzed reaction current density (j) at 0.05 V vs. Ag/AgCl (sat. shows the TB-mediated FAD-GDH-catalyzed reaction current density (j) at 0.05 V vs. Ag/AgCl (sat. following section). KCl) as a function of the electrolysis time for various TB concentrations. The steady-state catalytic KCl) as as aa function function of of the the electrolysis electrolysis time time for for various various TB TB concentrations. concentrations. The The steady-state steady-state catalytic catalytic KCl) current with concentration, evident By Based on theincreased CVs of linearly the phenothiazines and quinonesas examined in the2B. presence current density density increased linearly with increasing increasing TB TB concentration, as evident in in Figure Figure 2B. By using using of the enzyme current density increased linearly with increasing TB concentration, as evident in Figure 2B. By using these chronoamperometry readings of the dependence of the steady-state jj on the mediator these chronoamperometry readings of the dependence of the steady-state on the mediator and glucose, we were able toreadings determine thedependence applied potential for the constant these chronoamperometry of the of the steady-state j on the potential mediator amperometry concentration, concentration, the the bimolecular bimolecular kinetic kinetic constants constants for for FAD-GDH FAD-GDH oxidation oxidation by by the the phenothiazines phenothiazines and and concentration, the bimolecular kinetic constants for FAD-GDH oxidation by the phenothiazines and (chronoamperometry) for measuring the steady-state catalytic glucose oxidation current. Figure 2A cat/KM quinones quinones (k (k2222 === kkkcat cat/K /KM M)) ) could could be be evaluated evaluated from from the the following following equation equation [28]: [28]: quinones (k cat M could be evaluated from the following equation [28]:
shows the TB-mediated FAD-GDH-catalyzed reaction current density (j) at 0.05 V vs. Ag/AgCl (sat. KCl) as a function of the electrolysis time for various TB concentrations. The steady-state catalytic current density increased linearly with increasing TB concentration, as evident in Figure 2B. 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]: s j = 2F
DM kcat [E] [M] KM
(1)
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[E] DMMkkcat D cat[E] [M] [M] jj == 22FF KMM K
(1) (1)
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where F, F, D DMM,, [E], [E], and and [M] [M] are are the the Faraday Faraday constant, constant, diffusion diffusion coefficient coefficient of of the the mediator, mediator, where where F, D , [E], and [M] are the Faraday constant, diffusion coefficient of the mediator, concentration M concentration of the enzyme, and concentration of the mediator, respectively. Figure 3 shows the concentration of the enzyme, and concentration of the mediator, respectively. Figure 3 shows the ofdependence the enzyme, and concentration of the mediator, respectively. Figure 3 shows the dependence of the dependence of of the the logarithmic logarithmic bimolecular bimolecular rate rate constants constants (k (k22 == kkcat cat/K /KMM)) on on the the formal formal potential potential of of the the logarithmic bimolecular rate constants (k = k /K ) on the formal potential of the phenothiazines cat 2 M phenothiazines (blue triangles) and quinones (red circles) for FAD-GDH (closed symbols) and for phenothiazines (blue triangles) and quinones (red circles) for FAD-GDH (closed symbols) and for (blue triangles) and quinones (red circles) for FAD-GDH (closed symbols) and for GOx (open symbols). GOx (open (open symbols). symbols). GOx
Figure 1. Cyclic voltammograms of (A) 0.1 mM toluidine blue and(B) 0.1 mM 1,4-naphthoquinone in Figure Cyclic voltammograms (A) 0.1 mM toluidine blue and(B) 0.1 mM 1,4-naphthoquinone Figure 1.1. Cyclic voltammograms ofof (A) 0.1 mM toluidine blue and(B) 0.1 mM 1,4-naphthoquinone inin the presence (red) or absence (black) of FAD-GDH (1.0 μM) in a phosphate phosphate buffer (pH 7.0) containing the presence (red) absence (black) FAD-GDH (1.0 μM) buffer (pH 7.0) containing the presence (red) oror absence (black) ofof FAD-GDH (1.0 µM) inin a aphosphate buffer (pH 7.0) containing −1. −1 0.1 M glucose. Scan rate of 10·mV·s − 1 . 0.1 M glucose. Scan rate of 10·mV·s 0.1 M glucose. Scan rate of 10·mV·s .
Figure 2. (A) Dependence of the current density on the constant potential electrolysis time for various Figure (A) Dependence the current density the constant potential electrolysis time for various Figure 2.2. (A) Dependence ofof the current density onon the constant potential electrolysis time for various toluidine blue (TB) concentrations; curves (a) 5.0 μM, (b) 8.4 μM, (c) 12 μM, (d) 16 μM, and (e) 24 μM toluidine blue (TB) concentrations; curves 5.0 μM, (b) 8.4 μM, μM, (d) μM, and μM toluidine blue (TB) concentrations; curves (a)(a) 5.0 µM, (b) 8.4 µM, (c)(c) 1212 µM, (d) 1616 µM, and (e)(e) 2424 µM TB; (B) Dependence of the steady-state catalytic current on the TB concentration. The error bars were TB; (B) Dependence the steady-state catalytic current the concentration.The Theerror errorbars barswere were TB; (B) Dependence ofof the steady-state catalytic current onon the TBTB concentration. evaluated by Student’s t-distribution at confidence 90% confidence confidence level.lines Dashed linesthe represent the evaluated by by a Student’s t-distribution at a 90% level. Dashed represent regression evaluated aa Student’s t-distribution at aa 90% level. Dashed lines represent the regression linethe considering the weight of the the error. error. line considering weight ofthe theweight error. of regression line considering
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Figure Dependence the logarithmic bimolecular rate constantsfor for FAD-GDH (closed symbols) Figure 3. 3. Dependence ofof the logarithmic bimolecular rate constants FAD-GDH (closed symbols) and glucoseoxidase oxidase(open (opensymbols) symbols)on onthe theformal formal potential potential of the and glucose the mediators mediatorsat atpH pH7.0: 7.0:MeNQ MeNQ(1), (1),TB (3), PQPQ (4),(4), MBMB (5),(5), 14NQ (6),(6), THTH (7), (7), MGMG (8), 12NQ (9), 12NQS (10), (10), and BQ TB(2), (2),AA AA (3), 14NQ (8), 12NQ (9), 12NQS and(11). BQ (See (11).Table (See 1 for the abbreviation definitions.) The error bars were evaluated by a Student’s t-distribution at a 90% Table 1 for the abbreviation definitions.) The error bars were evaluated by a Student’s t-distribution at confidence level. Phenothiazines are depicted as blue triangles, and quinones are depicted as red a 90% confidence level. Phenothiazines are depicted as blue triangles, and are depicted as red circles. The glucose oxidase from the Penicillium vitale–quinone system shownasasgreen greensquares squares[27]. [27]. circles. The glucose oxidase from the Penicillium vitale–quinone system is is shown Black solid, black dashed, and green dashed lines represent regression for FAD-GDH Black solid, black dashed, and green dashed lines represent the the regression lineslines for FAD-GDH fromfrom A. terreus and GOx fromfrom A. sp.A.and fromfrom P. vitale [27]. [27]. A. terreus and GOx sp. GOx and GOx P. vitale
For GOx the Penicillium vitale–quinone systemsquares (green insquares in the Figure 3), the For thethe GOx fromfrom the Penicillium vitale–quinone system (green Figure 3), regression regression line for the klogarithmic k2 values as a function of the formal potential of the mediators was line for the logarithmic 2 values as a function of the formal potential of the mediators was analyzed analyzed on the basis of the Marcus (i.e., terms of the self-exchange rate of constants of the on the basis of the Marcus theory (i.e.,theory in terms ofin the self-exchange rate constants the enzyme −1 −1 − 1 − 1 enzyme and mediators); ·s )+=8.4 4.9 × + 8.4 × E(modified med (modified from original paper reference and mediators); log k2 (M log·sk2 (M ) = 4.9 Emed from thethe original paper onon reference electrodes) [27,29]. In the −0.2 to 0.1 V range, the logarithm of each k 2 value for FAD-GDH and those electrodes) [27,29]. In the −0.2 to 0.1 V range, the logarithm of each k2 value for FAD-GDH and those for the GOx from the Aspergillus species also increased linearly as a function of the formal potential for the GOx from the Aspergillus species also increased linearly as a function of the formal potential the phenothiazinesand andquinones. quinones.The Theregression regressionlines linesfor forFAD-GDH FAD-GDH(solid (solidline) line)and andGOx GOx(black (black ofof the phenothiazines dashed line), as shown in Figure 3, can be represented roughly by the following equations, based dashed line), as shown in Figure 3, can be represented roughly by the following equations, based onon the GOx (from vitale)-mediator system [27]: the GOx (from P. P. vitale)-mediator system [27]: log (k2 for FAD-GDH (M−1·s−1)) = 8.1 + 8.4 × Emed (2) log (k2 for FAD-GDH (M−1 ·s−1 )) = 8.1 + 8.4 × Emed (2) log (k2 for GOx from Aspergillus sp. (M−1·s−1)) = 5.5 + 8.4 × Emed (3) log (k2 for GOx from Aspergillus sp. (M−1 ·s−1 )) = 5.5 + 8.4 × Emed (3) The results suggest that electron transfer from the reduced FAD active site—found deep within theThe enzyme—is strongly governed the outer-sphere electron transfer mechanism. deep Interestingly, results suggest that electron by transfer from the reduced FAD active site—found within theenzyme—is kinetic parameter not depend onouter-sphere whether the electron mediatortransfer was a quinone or a phenothiazine, the stronglydid governed by the mechanism. Interestingly, but instead dependeddid mainly on the formal potential the molecules. Overall, or weaobserved that the the kinetic parameter not depend on whether the of mediator was a quinone phenothiazine, bimolecular rate constant of on each mediator was higher for FAD-GDH for GOx by approximately but instead depended mainly the formal potential of the molecules. than Overall, we observed that the 2.5 orders rate of magnitude. It is possible difference is related structural differences bimolecular constant of each mediatorthat was this higher for FAD-GDH thanto forthe GOx by approximately two enzymes. GOx that has this a homodimeric structure with a narrow active site pocket, 2.5between orders ofthese magnitude. It is possible difference is related to the structural differences between resulting in a minimum distance of 13 Å between protein surface andsite thepocket, FAD site [30,31]. these two enzymes. GOx has a homodimeric structurethe with a narrow active resulting crystal structure of FAD-GDH Aspergillus flavus [32],In this enzyme inIna contrast, minimumaccording distance to of the 13 Å between the protein surface from and the FAD site [30,31]. contrast, has a broader substrate-binding pocket than that of GOx, flavus which[32], allows easier access of redox according to the crystal structure of FAD-GDH from Aspergillus this enzyme has a broader molecules to thepocket FAD site. rate constant ofaccess pyrroloquinoline quinone-dependent substrate-binding thanThe that bimolecular of GOx, which allows easier of redox molecules to the FAD glucose dehydrogenase (PQQ-GDH) for a variety of quinones showed similar potential-dependent
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site. The bimolecular rate constant of pyrroloquinoline quinone-dependent glucose dehydrogenase (PQQ-GDH) for a variety of quinones showed similar potential-dependent behavior, although PQQ-GDH exhibits potential-dependent activation-controlled as well as potential-independent diffusion-controlled regions [33]. The k2 value of PQQ-GDH in the potential-dependent region was 2 orders of magnitude higher than the maximum k2 value of FAD-GDH, which suggests that the difference could be due to the structure of the active site pocket. The long-range electron transfer rate also depends on the electron transfer distance, which would be affected by the accessibility of the molecule to the FAD site, which is the closest transfer distance [30,31]. 9,10-Phenanthrenequinone (PQ) showed higher k2 values than expected from the regression line, which could be due to some attractive forces between the active site and the molecule, including π–π interactions (π stacking), π–cation interactions, or hydrophobic interactions, as well as the ortho-quinone structure, which is favorable for electron transfer [17]. On the contrary, 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 therefore potentially disturb the access to FAD. However, the rate constant for 1,4-benzoquinone (BQ), whose molecular structure is the smallest among the molecules examined, was lower than expected from the calibration line. This might be due to the inverted region of the Marcus theory or to the weak interactions (or a lack of favorable interactions) between BQ and the active site of the enzymes. Intriguingly, among all the molecules used in this study, only BQ lacked an aromatic ring. The higher catalytic currents seen with FAD-GDH, in spite of the low formal potential, was likely due to the decreased steric hindrance in terms of access to the FAD active site compared with that for GOx. With regard to the formal potential, higher k2 values were found for ortho-quinones (including PQ and 1,2-naphthoquinone) than for para-quinones in the reactions with FAD-GDH and GOx. Thus, 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). 3. Materials and Methods 3.1. Materials FAD-GDH from Aspergillus terreus was kindly donated by Ikeda Tohka Industries Co., Ltd. (Fukuyama, Japan) [2]. The GOx from Aspergillus species was purchased from Toyobo (product code GLO-201; Osaka, Japan). The FAD-GDH and GOx concentrations were determined spectrophotometrically using a molar extinction coefficient of free FAD of 11.3 × 103 M−1 ·cm−1 at 465 nm and 13.1 × 103 M−1 ·cm−1 at 450 nm, respectively [2]. The following phenothiazines were used in this study: thionine acetate (Wako Pure Chemical Industries, Osaka, Japan), azureA chloride (Sigma-Aldrich, St. Louis, MO, USA), basic blue17 (toluidine blue; Tokyo Chemical Industry Co., Tokyo, Japan), methylene blue hydrate (Tokyo Chemical Industry Co.), and basic green 5 (methylene green; Tokyo Chemical Industry Co.). The following quinones were used in this study and were purchased from Wako Pure Chemical Industries: 1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonate, 1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone, and 9,10-phenanthrenequinone. All materials were used without further purification. Table 1 illustrates the structures of the phenothiazines and quinones in their oxidized forms. All had a redox potential in the range of −0.20–0.09 V at pH 7.0 relative to an Ag/AgCl (sat. KCl) reference electrode. 3.2. Homogeneous Redox Reactions of FAD-GDH with the Electron Acceptors A gold (Au) electrode, 3 mm in diameter, was polished with an alumina slurry and rinsed with distilled water. Electrochemical measurements were performed using an electrochemical analyzer
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(BAS CV 50 W; BAS Inc., West Lafayette, IN, USA), with a platinum wire and an Ag/AgCl (sat. KCl) electrode as the counter and reference electrodes, respectively. Cyclic voltammetry of the redox molecules (0.1 mM) was carried out in phosphate buffer (0.1 M, pH 7) containing glucose (0.1 M), in the presence or absence of FAD-GDH (1.0 µM), at a scan rate of 10 mV·s−1 . Chronoamperometry for the kinetic analysis was carried out at the potential where the steady-state current was observed, in phosphate buffer (0.1 M) containing FAD-GDH (1.0 µM) or GOx (5.0 µM), and glucose (0.1 M). For the kinetic analysis, the diffusion coefficient of each mediator (0.1 mM) was evaluated from the dependence of its plateau current value on the electrode rotation rate (1000, 2000, 3000, 4000, and 5000 rpm), using the Levich equation (Table 1). All measurements were carried out at room temperature (25 ± 1 ◦ C). Prior to each experiment, the solutions were bubbled with Argon (Ar) for 10 min. Glucose stock solutions were stored overnight in order to achieve mutarotative equilibrium. 4. Conclusions The bimolecular rate constants for FAD-GDH oxidation by phenothiazines and quinones at pH 7.0 varied between 106 and 108 M−1 ·s−1 depending on the formal potential of the redox mediator. The overall results suggest that the kinetics determined by the formal potential of the mediator, and the electron transfer distance between the redox active site of the molecules and the FAD site of the glucose dehydrogenase, greatly affect the electron transfer rate. More specifically, ortho-type redox active sites with π-electrons are favorable, whereas bulky functional groups, especially hydrophilic (charged) functional groups, close to the redox site are unfavorable. There is a strong need for more cost-effective mediators for the bioelectrochemical application of FAD-GDH that can allow high biocatalytic activity at low catalyst-mediator overpotential. The choice of a mediator is very important, as it determines the potential at which the reaction proceeds as well as the reaction rate. Further kinetic analysis and spectroscopic analysis, in combination with structural information about the active site, will no doubt provide a clearer understanding of the factors governing the mediator reactions. In the meantime, our kinetic analysis strategy using a set of quinones and phenothiazines provides an important guideline for the identification, potential design, and selection of more efficient redox mediators for FAD-GDH. The approach presented here can also be used to fully exhibit the performances of other new or mutant FAD-GDHs with minimized kinetic and steric hindrances. Acknowledgments: This work was partially supported by a grant from JSPS KAKENHI (15K14684) and JST A-STEP (AS272S004a). Author Contributions: Seiya Tsujimura conceived and designed the experiments and wrote the paper; Nozomu Tsuruoka, Takuya Sadakane, and Rika Hayashi performed the experiments and analyzed the data. Conflicts of Interest: The authors declare no conflicts of interest.
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