Characterization of methylated azopyridine as a potential electron transfer mediator for electroenzymatic systems

Article abstract of DOI:10.1016/j.procbio.2017.01.006

N,N'-dimethyl-4,4'-azopyridinium methyl sulfate (MAZP) was characterized as an electron transfer mediator for oxidation reactions catalyzed by NAD⁺- and pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases. The bimolecular rate constant of NADH reactivity with MAZP was defined as (2.2?±?0.1)?×?10⁵?M^?1?s^?1, whereas the bimolecular rate constant of reactivity of the reduced form of PQQ-dependent alcohol dehydrogenase with MAZP was determined to be (4.7?±?0.1)?×?10⁴?M^?1?s^?1. The use of MAZP for the regeneration of the cofactors was investigated by applying the electrochemical oxidation of the mediator. The total turnover numbers of mediator MAZP and cofactor NADH for ethanol oxidation catalyzed by NAD⁺-dependent alcohol dehydrogenase depended on the concentration of the substrate and the duration of the electrolysis, and the yield of the reaction was limited by the enzyme inactivation and the electrochemical process. The PQQ-dependent alcohol dehydrogenase was more stable, and the turnover number of the enzyme reached a value of 2.3?×?10³. In addition, oxidation of 1,2-propanediol catalyzed by the PQQ-dependent alcohol dehydrogenase proceeded enantioselectively to yield L-lactic acid.

Full text of DOI:10.1016/j.procbio.2017.01.006

Accepted Manuscript
Title: CHARACTERIZATION OF METHYLATED
AZOPYRIDINE AS A POTENTIAL ELECTRON
TRANSFER MEDIATOR FOR ELECTROENZYMATIC
SYSTEMS
Author: <ce:author id="aut0005"
author-id="S1359511316304755-
146e21d92e6a18c0aad6c38573b986c4"> Lidija
Tetianec<ce:author id="aut0010"
author-id="S1359511316304755-
46a7d1f4d6627eed4c9a183b07add97f"> Ana
Chaleckaja<ce:author id="aut0015"
author-id="S1359511316304755-
24aefa232c47040f75c8485e38f6093f"> Juozas
Kulys<ce:author id="aut0020"
author-id="S1359511316304755-
924bfc27b1035a88c3c871624b35f2b4"> Regina
Janciene<ce:author id="aut0025"
author-id="S1359511316304755-
493c2e38f6c7c559da3bc97c3777b6d4"> Liucija
Marcinkeviciene<ce:author id="aut0030"
author-id="S1359511316304755-
3875e68633d92f88f3516a4856af5a5c"> Rita
Meskiene<ce:author id="aut0035"
author-id="S1359511316304755-
fa3f3d54c5f75c236515f226d1b75c54"> Jonita
Stankeviciute<ce:author id="aut0040"
author-id="S1359511316304755-
e6bec7c07bc9bb8eef5f560ef42d0389"> Rolandas
Meskys
PII:
S1359-5113(16)30475-5
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.procbio.2017.01.006
PRBI 10904
To appear in:
Process Biochemistry
Received date:
Revised date:
Accepted date:
26-9-2016
3-1-2017
4-1-2017

Please cite this article as: Tetianec Lidija, Chaleckaja Ana, Kulys Juozas, Janciene
Regina, Marcinkeviciene Liucija, Meskiene Rita, Stankeviciute Jonita, Meskys
Rolandas.CHARACTERIZATION OF METHYLATED AZOPYRIDINE AS A
POTENTIAL ELECTRON TRANSFER MEDIATOR FOR ELECTROENZYMATIC
SYSTEMS.Process Biochemistry http://dx.doi.org/10.1016/j.procbio.2017.01.006
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CHARACTERIZATION OF METHYLATED AZOPYRIDINE AS
A
POTENTIAL
ELECTRON TRANSFER MEDIATOR FOR ELECTROENZYMATIC SYSTEMS
Lidija Tetianeca,b, Ana Chaleckajaa, Juozas Kulysa,b, Regina Jancienea, Liucija Marcinkevicienea, Rita
Meskienea, Jonita Stankeviciutea, Rolandas Meskysa
aInstitute of Biochemistry, Life Science Center, Vilnius University, Sauletekio al. 7, LT-10257, Vilnius, Lithuania
bDepartment of Chemistry and Bioengineering, Faculty of Fundamental Sciences, Vilnius Gediminas Technical
University, Sauletekio al. 11, LT-10223, Vilnius, Lithuania
Corresponding author: Lidija Tetianec, e-mail lidija.tetianec@bchi.vu.lt, Phone: +370-5-223 4391.
1

Graphical abstract
Highlights
 MAZP has moderate potential and high reactivity with NADH;
 NAD+- and PQQ-dependent ADH were used in an electroenzymatic system;
 High TTN of PQQ-dependent ADH was achieved;
 PQQ-dependent ADH-catalyzed reaction is enantioselective.
2

Abstract
N,N'-dimethyl-4,4'-azopyridinium methyl sulfate (MAZP) was characterized as an electron
transfer mediator for oxidation reactions catalyzed by NAD+- and pyrroloquinoline quinone
(PQQ)-dependent alcohol dehydrogenases. The bimolecular rate constant of NADH reactivity
with MAZP was defined as (2.2±0.1)×105 M-1s-1, whereas the bimolecular rate constant of
reactivity of the reduced form of PQQ-dependent alcohol dehydrogenase with MAZP was
determined to be (4.7±0.1)×104 M-1s-1. The use of MAZP for the regeneration of the cofactors
was investigated by applying the electrochemical oxidation of the mediator. The total turnover
numbers of mediator MAZP and cofactor NADH for ethanol oxidation catalyzed by NAD+-
dependent alcohol dehydrogenase depended on the concentration of the substrate and the
duration of the electrolysis, and the yield of the reaction was limited by the enzyme inactivation
and the electrochemical process. The PQQ-dependent alcohol dehydrogenase was more stable,
and the turnover number of the enzyme reached a value of 2.3×103. In addition, oxidation of 1,2-
propanediol catalyzed by the PQQ-dependent alcohol dehydrogenase proceeded
enantioselectively to yield L-lactic acid.
Keywords
Methylated azopyridine; quinoprotein; alcohol dehydrogenase; NADH; cofactor regeneration;
enantioselective conversion.
3

Introduction
The application of dehydrogenases in the conversion of organic compounds is very attractive for
biocatalysis because of the appropriate catalytic activity and substrate specificity of the enzymes
[1-4]. However, for the successful use of dehydrogenases in bioconversion systems, effective
schemes for in situ regeneration of the expensive cofactors must be created [5-7].
The procedures of the regeneration of cofactors can be divided into either mediator-dependent or
mediator-independent classes. With mediator-independent regeneration, the cofactor is oxidized
using auxiliary enzymes and substrates, or direct electron transport occurs between the electrode
and the cofactor. Hence, the enzymatic oxidation of NAD(P)H can be achieved by the
application of NADH oxidase [8] or supplementary dehydrogenases and appropriate co-
substrates such as pyruvate for lactate dehydrogenase, 2-ketoglutarate for glutamate
dehydrogenase, or acetaldehyde/acetone for alcohol dehydrogenase (ADH) [5,7]. NADH oxidase
uses oxygen as a terminal electron acceptor, but the main disadvantage of this approach is the
limited stability of such cofactor-regenerating enzymes under reaction conditions. The
equilibrium of the reaction in the dehydrogenase-coupled schemes is favorable, but their
application is limited either by high costs of the co-substrate or by low reactivity. The substrate-
coupled approach entails the use of an additional sacrificial substrate and the same production
enzyme [9,10]. The main problem of such an approach is a huge molar surplus of the
supplementary substrate. Furthermore, because of the formation of the co-product, additional
stages of the purification of the desired product are needed. The direct electrochemical oxidation
of NAD(P)H requires a relatively high voltage, which causes fouling of the electrode by strong
absorption of NAD(P)+. Moreover, enzymatically inactive forms of the cofactor are formed [11].
Direct electron transfer has been observed for several multi-cofactor dehydrogenases [6,12,13],
4

but the current of the electrolysis is too low for the application of these dehydrogenases to the
bioconversion of substrates.
Common mediator-dependent schemes of regeneration of cofactors consist of mediator-reducing
and mediator-oxidizing steps. The reduction of the oxidized form of the mediator occurs during
reactions with the reduced form of the cofactor, which may either be freely diffusing or attached
to an apoenzyme. Then an enzyme can be used to re-oxidize the mediator or the mediator can be
oxidized on an electrode (Fig. 1). The enzymatic oxidation of the mediator can be catalyzed by
laccases, which are active toward a broad spectrum of organic compounds and use oxygen as an
electron acceptor [7,14-16]. However, the applicability of these enzymes is limited because of
poor overlap between pH profiles of dehydrogenases and laccases as most laccases show a
maximum activity at acidic pH. Nevertheless, some laccase-mediator systems are active in an
alkaline reaction medium [17]. Electrochemical regeneration of the oxidized mediator form is a
convenient method considering the absence of by-products in an enzymatic reactor [18]. During
an electrochemical reaction, the anodic and cathodic compartments are separated by an ion-
selective membrane or in other ways, which ensures the charge transfer between the
compartments. The working electrode, on which the mediator is oxidized, is an anode, and the
enzymatic reaction of substrate oxidation occurs in this electrode chamber. The counter electrode
is the cathode, on which the secondary electrochemical reaction (usually the reduction of the
proton resulting in the production of molecular hydrogen) occurs [19]. The separated
compartments of the working electrode and the counter electrode facilitate the procedure of
purification of the desired product. Moreover, the reduction of the regeneration scheme to only
one biocatalyst increases the preparative value of the biocatalytic synthesis.
5

A mediated electrochemical oxidation of NAD(P)H usually requires a lower working potential
than the mediator-independent process. The mediators used for this purpose have been
summarized in a number of reviews [11,18,20]. A one-electron transducer, namely 2,2'-azino-
bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), has been used as an efficient mediator in the
biocatalytic system for the regeneration of nicotinamide cofactors [21,22]. Electrochemically
oxidized mediators are also suitable in reactors with glucose oxidase [19] and quinoproteins [6].
ADHs are enzymes that contain various cofactors including NAD(P)+, pyrroloquinoline quinone
(PQQ), and heme c, and because of their substrate specificity, the enzymes are very promising
for the industrial enantioselective oxidation of various alcohols [2,23,24].
ADHs containing PQQ as the prosthetic group are currently of special interest in the field of
biosensors and catalytic systems [25-32]. Quinohemoproteins catalyze the oxidation of a great
variety of primary or secondary aliphatic and cyclic alcohols. These enzymes are active toward
both mono- and polyhydroxylic compounds. In contrast to NAD(P)+-dependent ADHs,
quinoprotein ADHs do not require external addition of expensive cofactors. Many natural and
artificial electron acceptors including cytochromes, conducting polymers, benzoquinones,
ferrocene derivatives, phenazines, metal complexes, and various nanostructures have been used
to transfer electrons from the reduced PQQ-dependent enzymes to the electrode [2,4,6,31-38].
Recently, N-methylated azopyridine has been characterized and tested as an electron transfer
mediator for the electrocatalytic oxidation of NADH [39]. This mediator demonstrates reversible
electrochemistry and a moderate redox potential. These properties encouraged us to investigate
the possibility of using N-methylated azopyridine as an electron transfer mediator for the
electrochemical regeneration of cofactors.
6

Materials and Methods
Materials
PQQ-dependent ADH (ADH IIG) was purified from Pseudomonas putida HK-5 as described in
[37]. P. putida HK5 strain [40] was kindly gifted by Prof. O. Adachi and Dr. H. Toyama. The
concentration of the enzyme in solution was determined using the molecular mass of the
monomer enzyme 72 kDa [40], and the concentration of the protein was estimated by the Lowry
method [41] with bovine serum albumin as a standard.
ADH from Saccharomyces cerevisiae was obtained from Sigma–Aldrich (Germany). The
enzyme was used as received. The activity of the enzyme was determined by manufacturer and
was equal to 369 U/mg. The enzyme solution was prepared using deionized water, and the molar
concentration was calculated assuming the molecular mass of the enzyme to be 141 kDa.
The gene of lactate oxidase (LO) from Aerococcus viridans IFO 12219 (gene accession number
AEU12219, [42]) was synthesized and purchased from Genscript (USA) and then cloned into
pET28a vector. Escherichia coli cells harboring the recombinant plasmid were cultivated with
aeration in flasks with 100 ml of brain heart infusion broth (Oxoid) supplemented with
kanamycin (40 µg/ml) at 30 °C up to A600 0.8. Then the flasks were cooled down to 20 °C, 0.1
mM IPTG was added, and cultivation was continued at 20 °C for 17 h. The cells were collected
by centrifugation (3000 ×g); suspended in 50 mM sodium phosphate buffer containing 1 mM
PMSF, 1 mM EDTA, and 10% glycerol; and disrupted by ultrasonic disintegration. The pellets
were separated by centrifugation (10000 ×g), and LO was salted out from the cell-free
supernatant with NH4(SO4)2 (35–65%). Precipitated proteins were collected and dialyzed against
5 mM sodium phosphate buffer supplemented with 1 mM EDTA and 2% sucrose (NaP buffer).
The dialyzed enzyme was applied to the DEAE 650 M column (2.5 × 12 cm) (Tosoh, Japan)
7

equilibrated with the NaP buffer. LO was eluted by a linear gradient of 0–1 M NaCl in the same
buffer. Fractions with enzyme activity were collected, concentrated by ultrafiltration using 30
kDa cut-off membrane (Millipore, USA), and dialyzed against NaP buffer. The purified enzyme
had a specific activity of 18.6 U/mg of protein.
NADH, dimethyl sulphate, azopyridine, and L-glyceric acid were from Sigma-Aldrich
(Germany). Ethyl alcohol, 1,2-propanediol, (2S,3S)-(+)-2,3-butanediol, (2R,3R)-(-)-2,3-
butanediol, meso-2,3-butanediol, acetoin, and N-ethyl-3-(3-dimethylamino-propy1)carbodiimide
hydrochloride were from Fluka. Glycerol was from Carl Roth GmbH + Co. KG (Germany). DL-
glyceraldehyde was from Reachim (Russia). Solutions of the alcohols, ketone, and aldehyde
were prepared in deionized water. All buffer reagents and other chemicals were of analytical
grade.
N,N'-dimethyl-4,4'-azopyridinium methyl sulfate (MAZP) was synthesized as described
previously [39]: 20 ml of dimethyl sulfate was added to 2 g (11 mmol) of azopyridine and the
o
suspension was stirred at 70 C for 12 h to form MAZP. The product was filtered and washed
o
thrice with dry diethyl ether. Then, the precipitate was dried in vacuum at 75 C for 6 h. Yield:
3.3 g (69.7%) of MAZP, orange crystals, melting point 197–200 oC.
MAZP spectra and titration with NADH
MAZP spectral investigations were performed using a computerized “Nicolet evolution 300”
spectrophotometer. The molar extinction coefficients of MAZP and the stoichiometry of the
reaction of MAZP with NADH were estimated from the spectra of MAZP and its change after
titration with NADH. The change of concentration of MAZP was calculated from the change of
its absorption at 480 nm.
8

Kinetic measurements and calculations
Stopped-flow measurements of MAZP reactivity with NADH were performed with a stopped-
flow spectrometer (Otsuka Electronics RA-401, Japan). The absorbance kinetics were monitored
at 340 nm in 50 mM phosphate buffer solution, pH 7.2, under anaerobic conditions. The
anaerobic conditions were achieved by bubbling the solutions with argon for 15 min before the
measurements. Five sets of measurements with various initial concentrations of MAZP and
NADH were performed, and each set consisted of 16 iterations.
The NADH and MAZP reactivity constant was determined by fitting the kinetic data to the
integral of the equation of the rate dependence of a bimolecular irreversible reaction A+B→C+D
on the initial concentrations of the reactants:
(A exp(k t  B0  k t  A  ln(A ) ln(B0 )) B0 )
0
0
0
A  A 
(1)
t
0
(1 exp(k t  B0  k t  A  ln(A ) ln(B0 ))
0
0
where At is the concentration of reagent A at time moment t, A0 and B0 are the initial
concentrations of reagent A and B, respectively, t is time, and k is the bimolecular constant of
reactivity of reagents A and B. For the analysis, the value of molar extinction coefficient of
NADH (6.22 mM−1cm−1 at 340 nm) [43] and the experimentally determined values of molar
extinction coefficient of MAZP and its reduced form (Table 1) were used.
The reactivity of MAZP with ADH IIG was investigated spectrophotometrically in 50 mM
phosphate buffer solution at 25 °C, pH 7.1, by monitoring the absorbance of the MAZP at 480
nm. Spectra and kinetic assays were performed using a computerized “Nicolet evolution 300”
spectrophotometer.
9

The kinetic curves of reduction of MAZP by ADH IIG were analyzed, and the constants of the
reaction were calculated by applying the ping-pong enzyme reaction scheme, which included
reversible inhibition of enzyme with excess of substrate:
k1
k2
Eox  S ES Ered  P
(2)
k1
k3
Ered  Mox Eox  Mred
(3)
(4)
Kin
ES  S Ein
where Eox and Ered are the oxidized and reduced forms of the ADH IIG, respectively, ES is the
enzyme-substrate complex, Ein is the inactive enzyme form, S and P are substrate and its
oxidized product, respectively, Mox and Mred are the oxidized and reduced forms of the mediator
MAZP, respectively, enzyme catalytic constant kcat=k2, Michaelis constant for substrate KM=(k-
1+k2)/k1, bimolecular enzyme and substrate reactivity constant kred=k1k2/(k-1+k2), bimolecular
enzyme and mediator reactivity constant kox=k3, and the enzyme inactivation constant is Kin.
Fitting was performed using the software rModeler by Ubique calculus Ltd.. The software for the
fitting of experimental data and calculation of parameters uses the algorithm described
previously [44].
Electrochemical measurements
The cell of electrochemical measurements consisted of two chambers, namely anodic and
cathodic chambers, which were interconnected by a bridge of electroconductive agarose gel.
Rolled platinum wires (diameter 0.3 mm, length of each unfolded wire approximately 30 mm)
were used as working (anode) and auxiliary (cathode) electrodes. The reference electrode was a
10

saturated calomel electrode (SCE) placed in the chamber of working electrode. The kinetic
curves of the anodic current were registered maintaining a fixed potential of the working
electrode (300 mV vs SCE). MAZP (mediator in oxidized form), NADH (in the case of ADH),
substrates, and enzymes (ADH or ADH IIG) were added to the working electrode chamber,
which was filled with buffer solution. Charge (Q, C) passed during the electrolysis time (tel, s)
was calculated by integrating kinetic curves of current according time, whereas Q normalized by
Faraday constant (F= 9.648534×104 C mol-1) gives the number of moles of electrons transferred
during the electrolysis (N, mol). Assuming the stoichiometry of redox conversions of the
mediator and substrate, N divided by 2 (N/2, mol) was used to calculate the yield of the
electrolysis (Y%) and the total turnover numbers of mediator, cofactor, and enzyme (TTNM,
TTNNADH, and TTNE, respectively).
The estimation of products of alcohol conversion
The 2,4-dinitrophenylhydrazone derivative of acetaldehyde (the product of ADH-catalyzed
ethanol oxidation) was detected by liquid chromatography-mass spectrometry (LC-MS) as
described previously [17]. The products of glycerol oxidation were analyzed using a
derivatization method as described previously [45]. The ortho-nitrophenylhydrazone derivatives
of glyceric acid and glyceraldehyde were detected using a high-performance liquid
chromatography system (CMB-20A controller, two LC-2020AD pumps, SIL-30AC autosampler,
and CTO-20AC column oven, Shimadzu, Japan) equipped with a photodiode array detector
(SPD-M20A Prominence diode array detector; Shimadzu, Japan) and a mass spectrometer
(LCMS-2020, Shimadzu, Japan) equipped with an electrospray ionization source.
Chromatographic separation was performed using YMC-Pack Pro C18 column, 4 mm x 150 mm
11

(YMC, Japan) at 40 ºC and a mobile phase that consisted of 0.1% formic acid (solvent A) and
acetonitrile (solvent B) delivered in gradient elution mode (5-90%) at a flow rate of 0.4 mL min-
1. UV absorbance peaks were detected at 360 nm. Mass scans were measured from m/z 1000 at
350 ºC interface temperature, 250 ºC DL temperature, ±4.500 V interface voltage, and neutral
DL/Qarray using N2 as a nebulizing and drying gas. Mass spectrometry data were acquired in
both positive and negative ionization modes and analyzed using LabSolutions LCMS software.
Calibration curves were obtained using freshly prepared standard solutions of L-glyceric acid
and DL-glyceraldehyde in 5 mM phosphate buffer solution, pH 7.0.
The L-enantiomer of lactic acid as a product of the reaction was detected using the highly
specific FMN-dependent LO, which, in the presence of oxygen, catalyzed the oxidation of L-
lactate to pyruvic acid and hydrogen peroxide [46,47]. A homemade hydrogen peroxide
electrode was used to determine the concentration of hydrogen peroxide produced in the reaction
after LO was added. The electrode consisted of a platinum anode and a silver/silver chloride
(Ag/AgCl) cathode, which were insulated from the reaction mixture by the dialysis membrane
(MWCO 12,000–14,000, Spectra/Por). The hydrogen peroxide oxidation at the platinum anode
was measured at an electrode potential of 0.6 V vs. Ag/AgCl 0.1 M KCl. The electrode was
calibrated using freshly prepared hydrogen peroxide solution made from a 30% stock solution
(Carl Roth GmbH + Co. KG, Germany). The concentration of hydrogen peroxide in the solutions
for calibration was determined spectrophotometrically at 240 nm using an extinction coefficient
(ε240) of 39.4 M-1cm-1 [48].
Results and Discussion
The reaction of MAZP with NADH
12

The reactivity of MAZP and NADH is one of the factors defining the efficiency of the mediator
in the regeneration of the cofactor. The data of titration of MAZP with NADH (Fig. 2) suggested
that the stoichiometric ratio of NADH to MAZP in the reaction was 1:1. This ratio corresponds
to the data of the electrochemical reaction of MAZP, which has been reported to be a two-
electron and two-proton transfer process [39]. The titration revealed that the reaction of NADH
with MAZP was irreversible, and no equilibrium was established. This allowed us to estimate the
values of the molar extinction coefficients of oxidized and reduced forms of the mediator (Table
1).
A stopped-flow technique was applied to determine the rate of the reaction. The change in
absorbance at 340 nm was monitored for 1000 ms. The rate of the reaction depended on the
initial concentrations of MAZP and NADH (Fig. 3). The calculated bimolecular constant of the
cross-reaction between MAZP and NADH was equal to (2.2±0.1)×105 M-1s-1 at pH 7.2. It should
be noted that the obtained value is much higher than the value (1.31×103 M-1s-1) established from
the analysis of the electrocatalytic oxidation of NADH at the MAZP-modified electrode surface
[39]. The difference in the values can be explained by the contribution of diffusion limitations,
which is considerable for the reaction between the surface-immobilized MAZP and the free
NADH.
The obtained values of NADH reactivity with MAZP were higher than the reactivity of NADH
toward various compounds: quinones (values in the range of 0.05 M-1s-1 to 3×103 M-1s-1 [49]),
ABTS cation radical (5.6−6.45×103 M-1s-1 [22]), tetramethoxy azobismethylene quinone
(1.0×104 M-1s-1 [17]), or Meldola’s blue (2.0×104 M-1s-1 [15]). Thus, the high reactivity of the
mediator with the cofactor defined MAZP as a relevant compound for NADH oxidation.
13

The apparent parameters of MAZP reactivity with ADH IIG
The reactivity of ADH IIG with MAZP was investigated by measuring the absorbance of MAZP
at 480 nm. Glycerol was used as a substrate to reduce the enzyme. The kinetic curves were
registered at different initial MAZP (in the interval from 0.18 mM to 2.3 mM) and glycerol (in
the interval from 1.2 mM to 19.6 mM) concentrations (Fig. 4).
The ping-pong enzyme reaction scheme with enzyme inhibition by substrate (Eqs. 2−4) was used
to analyze the kinetic curves and calculate the constants of ADH IIG catalyzed oxidation of
glycerol in the presence of MAZP. The ping-pong scheme of enzyme action and inhibition of the
quinoproteins by their substrates were demonstrated in previous kinetic studies [50,51]. Visual
comparison of simulated and experimental data (Fig. 4) showed that the model fitted the
experimental data well. The calculated model constants are listed in Table 2. The value of the
bimolecular constant of ADH IIG reactivity with MAZP is comparable to the values of reactivity
constants of ADH IIG with hexacyanidoferrate(III) and heterocycle-pentacyanoferrate(III)
complexes (bimolecular constant kox = 3.1 × 104−8.7 × 105 M−1s−1) [37]. The electrochemistry of
hexacyanidoferrate(III) was often complicated by the formation of Prussian blue [52]; however,
MAZP electrochemistry was characterized as reversible, and no polymerization reaction was
detected [39]. Thus, MAZP with its proper electrochemistry and moderate reactivity with ADH
IIG can be a very promising electrochemical mediator for the enzyme.
The study of kinetics of MAZP reduction varying the initial concentrations of the reducing
substrate glycerol (Fig. 4, curves 1, 2, and 3) allowed us to determine the values of the apparent
constants of reductive half reaction of ADH IIG with glycerol, i.e., kred, KM, and Kin (Table 2).
NAD+ regeneration in the presence of MAZP
14

The use of MAZP for regeneration of NAD+ was investigated through the electrochemical
oxidation of the mediator. The kinetic curves of the anodic current and of the calculated number
of moles of electrons transferred during electrolysis time are shown in Fig. 5A and Fig. 5B,
respectively. When MAZP was added to the chamber of the working electrode, the registered
current did not exceed the value of 1 µA. After NADH was added to the cell, the reaction
between NADH and the mediator began:
MAZP  NADH  MAZP  NAD
(5)
red
The reduced form of MAZP (MAZPred) and NAD+ were generated in this reaction. MAZPred was
oxidized on the surface of the working electrode, resulting in an increase in the current (Fig. 5A).
The number of moles of electrons passed during the electrolysis corresponded to the added
amount of NADH, considering that the oxidation of NADH and MAZP involved two electrons
(Fig. 5B).
Ethanol and NAD+-dependent ADH were added to the cell when almost all the NADH was
oxidized. After the addition of the substrate and the enzyme, the reaction of the oxidation of
ethanol proceeded according to the reaction scheme:
ADH
NAD C2H5OH  NADH C2H4O
(6)
During reaction (6), NADH was generated and reacted with the electrochemical mediator MAZP
(reaction 5), forming MAZPred. MAZPred was further re-oxidated on the electrode, resulting in
the current, and N/2 respectively increased (Fig. 5). This increase proved that the NAD+ formed
15

in the reaction (5) was enzymatically active. During prolonged electrolysis, the current
decreased. When one more portion of ADH was added to the cell, a second rise of the current
was registered. This second increase indicated that the activity of the enzyme in the
electrochemical cell dropped during the reaction. The results of four experiments with different
concentrations of the reagents are summarized in Table 3. The maximum product yield was
obtained using the lowest initial concentrations of the substrate, but only modest TTN of the
mediator and the cofactor were observed in this case (Table 3, #1). At higher concentrations of
the substrate and in the case of prolonged electrolysis, larger TTNM and TTNNADH were obtained,
but the yield of the product decreased. The decrease in the conversion yield was partially related
to an inactivation of the enzyme, but limitations of the electrochemical process also occurred.
Thus, concentrations of the reagents used in experiment #4 were larger than those in experiment
#3; however, the number of moles of the transferred electrons was the same in both the cases.
The formation of acetaldehyde, the product of the oxidation of the ethanol, was followed by
monitoring its 2,4-dinitrophenylhydrazone derivative by LC-MS. When the initial amount of
ethanol (6.6×10-7 mol) was added to the electrolysis cell, after the 3 h of electrolysis, the
estimated amount of acetaldehyde (2.5×10-7 mol) corresponded to a yield of 38% (Table 3, #3).
The yield of the product was less than the yield of the electrolysis calculated from the number of
moles of the electrons. The difference may be explained by the leakage of acetaldehyde, which is
a volatile and reactive compound.
Previously, TTNs of 1860 for the mediator and of 93 for the cofactor were obtained using ABTS
as the mediator in a three-dimensional flow electrochemical cell, with the working electrode
having a large surface area [21]. In our case, the TTN of the cofactor was 11-fold lower;
however, the lower oxidation potential of MAZP and the much lower price can be beneficial
16

compared with ABTS. To improve the MAZP-dependent system, further optimization of the
electrochemical cell and working electrode is needed.
Regeneration of oxidized ADH IIG mediated by electrochemically oxidized MAZP
The possibility to apply the electrochemical mediator MAZP for the regeneration of other
cofactors such as PQQ and heme was studied using ADH IIG as a model enzyme. The enzyme
was selected because of its substrate specificity and possible applications in enantioselective
synthesis [2]. Moreover, ADH IIG was expected to be a more stable enzyme and result in higher
turnover numbers than NAD+-dependent ADH. In the case of ADH IIG, the TTN of the enzyme
(TTNE) showed the number of turnovers of both attached cofactors as a whole.
The kinetic curves of the anodic current and the corresponding curves of calculated number of
moles of the electrons transferred during electrolysis are shown in Fig. 6A and Fig. 6B,
respectively. The increase in the current was registered when ADH IIG, substrate, and MAZP
were added to the cell (Fig. 6A). The increase in number of moles of the electrons was
simultaneously registered (Fig. 6B). The oxidation of the substrate catalyzed by ADH IIG
proceeded according to the ping-pong scheme (Eqs. 2 and 3, inhibition reaction Eqs. 4 was
omitted as the used concentrations of the substrate were lower than the inhibition constant). The
product of MAZP reaction with ADH IIG was MAZPred, which was re-oxidized on the surface of
the working electrode, resulting in the increase in the current (Fig. 6A). The increase in the
current depended on the added amount of the mediator (Fig. 6A). During electrolysis, the current
decreased and the increase in N/2 reached the saturation point. A decline in current was related
to the consumption of the substrate but not to the deficiency of ADH IIG or MAZP according to
the number of electrons transferred during electrolysis (Fig. 6B). The number of moles of the
17

electrons and the values of the yield of the electrolysis depended on the chosen substrate and the
concentration of the mediator (Fig. 6B, Table 4). The presented Y values equaled to 100% and
87% for glyceraldehyde and (2S,3S)-(+)-2,3-butanediol, respectively. Hence, the oxidation of
these substrates to corresponding acid or ketone occurred by a single stage of two-electron
transfer. Only when mediator and ADH IIG were added to the chamber of the working electrode,
the background amount of the transferred electrons was equal to 1.8×10-8 mol. When (2R,3R)-(-
)-2,3-butanediol, meso-2,3-butanediol, or acetoin was used as the substrate, the number of
electrons transferred during the electrolysis was at the background level. This is in agreement
with previously published results that ADH IIG is not active toward these compounds [2]. In
contrary, the obtained yields of electrolysis in the case of 1,2-propanediol and glycerol were
much higher than 100%, and even reached 200%. This indicated that the oxidation of the
substrates proceeded not only to the corresponding aldehydes but also further to the
corresponding acids or dioxo compounds. The LC-MS analysis of the oxidation products showed
that the major product of the glycerol conversion was glyceric acid (yield 53%), although some
amount of glyceraldehyde (yield 15%) was also detected (Table 4). The number of moles of
electrons and the yield of conversion were very similar to the values of N/2 and Y determined
from the chronoamperometric curves (Table 4). ADH IIG used in this work catalyzed the
oxidation of glyceraldehyde, and the yield of the electrolysis in this case was 100% (Table 4).
1,2-propanediol was oxidized to lactaldehyde, and the latter was further oxidized to lactic acid.
The formation of L-lactate in the reaction mixture was validated in our work by using L-LO as
an auxiliary enzyme. These results were in agreement with the fact that quinohemoprotein ADHs
could oxidize aldehydes to the corresponding carboxylic acids [31,50].
18

As was expected, the measured values of the TTNE were much higher than the value of
TTNNADH, mainly because of the higher stability of ADH IIG. The maximal TTNE of up to
~2300 was achieved during the longer period of electrolysis (Table 4.). In comparison with the
NAD+-dependent dehydrogenase and ABTS mediator system [21], the TTNM value obtained
with our system was low. To analyze the factors influencing the TTNs of the mediator and the
enzyme, we invoked the data of ADH IIG-catalyzed glycerol oxidation at two different
concentrations of the mediator (Fig. 6). We acquired N/2 from the segments of
chronoamperometric curves during the limited period of time, i.e., 10 min after enzyme addition.
We assumed that at this period of electrolysis, no inactivation of the enzyme occurred and the
concentration of the substrate was high enough to not limit the electrolysis current. N/2 for lower
mediator concentration (Fig. 6 curve 1) was 7.3×10-8 mol of pairs of electrons per 10 min, and
N/2 for two-fold higher concentration of mediator (Fig. 6 curve 2) was 1.2×10-7 mol of pairs of
electrons per 10 min, i.e., although we increased the concentration of the mediator two-fold, the
N/2 increased only 1.6-fold. The same trend was observed for the growth of the current level
(Fig. 6A), which was achieved soon after the addition of the enzyme, with maximal current
differences of 1.6-fold for the two concentrations of the mediator. We suggested that the limit of
electrochemical process originates in this manner. To overcome this limit, the electrochemical
cell and working electrode must be optimized, i.e., optimization of the diffusion of the mediator
to and from the surface of the electrode and the increase in the surface area of the working
electrode.
Various compounds used as electron transfer mediators for quinoproteins and quinoprotein-
containing bacterial cells and the main applications of these electroenzymatic systems in
biosensors and biofuel cells have been summarized previously [6,31,38]. In many cases, high
19

sensitivity of biosensors and high current densities of the bioanodes were achieved when
mediators with higher redox potential were used. Considering the MAZP redox potential, its
reactivity with ADH IIG, and the measured high values of the current (Fig. 6), this mediator can
be also adapted for application in biosensors and biofuel cells.
Enantioselective conversion of 1,2-propanediol to L-lactate
To investigate whether ADH IIG-catalyzed oxidation of substrates is enantioselective, the
product of the oxidation of the 1,2-propandiol, i.e., lactic acid, was synthesized in a larger
electrochemical cell (bulk of 4 ml). The amounts of 1,2-propanediol, MAZP, and ADH IIG were
1.0×10-6, 9.8×10-8, and 7.2×10-10 mol, respectively. After 5 h of electrolysis, 3.6×10-7 moles of
L-lactate were detected using L-LO. The primary substrate 1,2-propanediol is a racemic mixture
of (S)- and (R)- enantiomers. L-lactic acid, the amount of which was determined in this work,
originates from (S)-1,2-propanediol. In theory, 5×10-7 moles of L-lactate can be produced using
1×10-6 moles of racemic 1,2-propanediol. Thus, the obtained L-lactate yield is 72% of the
theoretical maximum. The calculated number of moles of electrons transferred during the
electrolysis N/2=7.8×10-7 mol, which shows that 92% of the electrons were received by the
oxidation of S-enantiomer to L-lactic acid and the synthesis is enantioselective.
Conclusions
The electrochemical method of regeneration of the cofactors of ADH by using MAZP as a
mediator was efficient. The mediator showed high reactivity with NADH [bimolecular rate
constant (2.2±0.1)×105 M-1s-1] compared to other compounds that were applied as mediators for
NADH oxidation [15,17,22,49]. Enzymatically active NAD+ was formed in the reaction of
20

NADH and MAZP. In the presence of ADH and ethanol, the calculated TTNs of the mediator
and cofactor were 4–17 and 1.9–8.5, respectively. The main reasons of the low TTNs were the
enzyme inactivation and the limitations of the electrochemical process. The positive effect of the
mediator on the biocatalytic system was the change in the equilibrium of the ethanol oxidation
reaction. As is well known, equilibrium of the reaction catalyzed by NAD+-dependent ADH is
shifted far to acetaldehyde reduction [53]. In the presented electroenzymatic system, a
conversion of ethanol to acetaldehyde proceeded efficiently and reached up to 91%.
Unlike ADH, quinohemoprotein ADH IIG has tightly bound cofactors: PQQ and heme c.
Although the bimolecular rate constant of MAZP with ADH IIG is lower than that of MAZP
with NADH, higher values of TTNs in the electroenzymatic system were reached: ~35 for the
mediator and ~2300 for the enzyme. Moreover, ADH IIG enantioselectively catalyzed the
oxidation of 1,2-propanediol to lactic acid; therefore, the enzyme preferred the S-enantiomer of
the substrate, and the major product of the electroenzymatic conversion was L-lactic acid.
Acknowledgments
The work was supported by European Social Foundation and the Government of the Republic of
Lithuania, project No. VP1-3.1-ŠMM-08-K-01-001. Dr. J. Stankeviciute and Dr. L. Tetianec are
grateful to the Dr. Bronislovas Lubys Support and Charity Foundation for the financial support.
The authors thank Prof. O. Adachi and Dr. H. Toyama for the HK5 strain. The authors are
grateful to Dr. Ravinder N.M. Sehgal for reading the manuscript.
21

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