A spectroscopic characterization of a phenolic natural mediator in the laccase biocatalytic reaction

Article abstract of DOI:10.1016/j.molcatb.2013.08.013

Multi-frequency ESR combined with NALDI-TOF MS has been used for the characterization of 3,5-dimethoxy-4-hydroxyacetophenone radical intermediate and by-products formed during the Coriolopsis gallica laccase catalytic reaction. A stable radical species is formed and an intense and well-structured ESR spectrum was detected and fully characterized at S-, X- and W-bands. The presence of by-products generated as the result of by-reactions has been investigated and analyzed through NALDI-TOF MS, performing the experiments versus time. The superior radical stability of such phenoxy radical, due to steric hindrance in ortho to the phenol group and the great delocalization of the unpaired electron on the acetyl substituent, makes acetosyringone particularly interesting for biotechnological applications. This represents a good example for the development of new stable laccase mediator molecules.

Full text of DOI:10.1016/j.molcatb.2013.08.013

Journal of Molecular Catalysis B: Enzymatic 97 (2013) 203–208
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
A spectroscopic characterization of a phenolic natural mediator in the
laccase biocatalytic reaction
Andrea Martoranaa, Lorenzo Soraceb, Harry Boerc, Rafael Vazquez-Duhaltd,
Riccardo Basosia,∗, Maria Camilla Barattoa,∗∗
a Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, 53100 Siena, Italy
b Department of Chemistry and INSTM RU, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy
c VTT Technical Research Centre of Finland, P.O. BOX 1000, FI-02044 Espoo, Finland
d Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Apartado Postal 150-3, Cuernavaca, Moorelos 62210, Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 March 2013
Received in revised form 21 August 2013
Accepted 22 August 2013
Available online 4 September 2013
Multi-frequency ESR combined with NALDI-TOF MS has been used for the characterization of 3,5-
dimethoxy-4-hydroxyacetophenone radical intermediate and by-products formed during the Coriolopsis
gallica laccase catalytic reaction. A stable radical species is formed and an intense and well-structured
ESR spectrum was detected and fully characterized at S-, X- and W-bands. The presence of by-products
generated as the result of by-reactions has been investigated and analyzed through NALDI-TOF MS, per-
forming the experiments versus time. The superior radical stability of such phenoxy radical, due to steric
hindrance in ortho to the phenol group and the great delocalization of the unpaired electron on the
acetyl substituent, makes acetosyringone particularly interesting for biotechnological applications. This
represents a good example for the development of new stable laccase mediator molecules.
© 2013 Elsevier B.V. All rights reserved.
Keywords:
Acetosyringone
Radical intermediate
Laccase
Multifrequency ESR
NALDI-TOF MS
1. Introduction
and O2 and an accurate determination of the magnetic parameters,
through a multifrequency (S-, X-, W-band) ESR approach, has been
Several studies have been carried out using 3,5-dimethoxy-
4-hydroxyacetophenone, also called acetosyringone, as natural
laccase mediator. Acetosyringone and some other natural lignin-
derived phenols, such as syringaldehyde, are promising eco-
friendly mediators for dye degradation in terms of both efficiency
and velocity of oxidation [1,2].
Among the dimethoxy phenols, acetosyringone resulted the
first compound showing a considerable radical stability and could
represent a good example for the production of new mediator
molecules. The large amount of free radical produced has allowed
the identification and full characterization of the radical interme-
diate generated during the catalytic reaction mediated by laccase.
In the past, evidence of such a stable radical was observed at the
X-band ESR (Electron Spin Resonance) [3,4], but it was produced
by horseradish peroxidase and ligninase in the presence of H2O2.
In this work, for the first time, the radical was generated by laccase
achieved.
Laccase (p-diphenol:oxygen oxidoreductase E.C.1.10.3.2)
belongs to copper-containing oxidases that catalyze the oxi-
dation of a wide range of substrates using molecular oxygen
as co-substrate. During its catalytic process, laccase removes a
single electron from the substrate generating free radicals, with
the concomitant reduction of oxygen to water [5]. Due to steric
hindrance factors and to its redox potential between 0.5 and 0.8 V,
laccase is unable to oxidize non-phenolic lignin units which have a
high-redox potential (>1.5 V) and it can only oxidize phenolic units
at the substrate surface. Furthermore it is known that fatty acids
present in lignocellulosic pulps are responsible for “pitch prob-
lems” which can be overcome using laccase, modifying liphophilic
extractives [6–9]. To overcome the accessibility problem and to
extend the ability of non-phenolic lignin unit oxidation, laccase is
often used in the presence of an oxidizing small molecule, called
mediator, which acts as a sort of electron shuttle between the
enzyme and the lignin [10–14] (see Scheme 1). Once oxidized,
the mediator (Medox), represented by the phenolic compound,
diffuses away from the enzymatic pocket and in turn oxidizes
other molecules, extending the range of substrates susceptible
to the enzymatic action. In this way, laccases are able to oxidize
∗
Corresponding author. Tel.: +39 0577 234240; fax: +39 0577 234239.
Corresponding author. Tel.: +39 0577 234247; fax: +39 0577 234239.
E-mail addresses: riccardo.basosi@unisi.it (R. Basosi),
∗∗
mariacamilla.baratto@unisi.it (M.C. Baratto).
1381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molcatb.2013.08.013

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A. Martorana et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 203–208
T1
Cu2+
T3
Cu2+
OH
HO
O
T2
H O
O
O
Cu2+
Cu2+
.
+
O
T3
HO
H2O
H+
O
O
Cu2+
Cu2+
H+
OH
Sub(ox)
Sub(red)
Cu2+
Cu2+
H2O
O2
O
O
OOH
Cu2+
O
Cu2+
H2O
O
Cu2+
Cu2+
Scheme 1. The role of the mediator, represented by the phenolic compound, during the substrate oxidation by laccase.
compounds with a redox potential higher than 0.8 V and the direct
interaction between the enzyme and the target substrate is no
longer necessary.
intermediate generated during the catalytic oxidation. Nano-
Assisted Laser Desorption/IonizationTime-of-Flight spectrometry
(NALDI-TOF) was also applied to investigate the possible by-
products formed in the reaction.
An ideal redox mediator (i) must be a good laccase sub-
strate, (ii) must have a stable oxidized form and (iii) its redox
conversion must be cyclic [15]. For industrial applications, lac-
case mediators should be environmentally friendly and available
at low cost [1,9,16–20]. Several compounds have been used
as laccase mediators. Generally they are products of chemical
synthesis such as 2,2ꢀ-azino-bis(3-etilenbenzotiazolin-6-sulphonic
acid) (ABTS), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), vio-
luric acid (VIOL) and 1-hydroxybenzotriazole (HBT) [21–23]. Their
most relevant disadvantage is their high cost, toxicity and forma-
tion of many by-products. In this regard, the use of new and more
effective mediators among natural phenolic compounds that are
cheaper, non-toxic and more eco-friendly, has recently aroused
great interest. In particular, lignin-derived phenols have been
reported as efficient mediators and among them, dimethoxyphenol
compounds were described as the fastest and most efficient lac-
case natural mediators. However, until now, no information about
radical structures of natural mediators generated during catalytic
reactions was present in the literature.
Laccase activity involving phenols is enhanced by the presence
of electron-donating substituents on the benzene ring that decrease
its electrochemical potential. Moreover, two features are important
for improving the stability of phenoxy radicals: (i) steric hindrance
of the OH group on the phenol, leading to a great density of the
unpaired electron on the oxygen atom in the radical form and (ii) a
substituent group able to delocalize the unpaired electron [24,25].
It is known that laccase can act through two different path-
ways: an electron transfer mechanism (ET route) or a H-abstraction
mechanism (HAT route) and in both pathways a radical species is
formed as final product. In Scheme 2, the two different pathways
of 3,5-dimethoxy-4-hydroxyacetophenone (also named acetosy-
ringone) oxidation and the delocalization of the unpaired electron
are shown. Until now, the only structural characterization of medi-
ator radicals was performed on ABTS and VIOL [26] and on a
precursor radical such as 4-methyamino benzoic acid [27].
In the present work we report a multifrequency ESR study on
the acetosyringone oxidation, catalyzed by laccase from C. gallica
[28] combined with two-dimensional X-band ESR measurements
in order to obtain a more complete description of the radical
2. Materials and methods
Laccase from C. gallica was produced and purified as described
previously [29]. The enzyme preparation used in this study showed
80% purity estimated by SDS-PAGE electrophoresis and an activity
of 11200 U/mL (313 U/mg of protein) determined with syringal-
dazine as substrate [30].
3,5-dimethoxy-4-hydroxyacetophenone was purchased from
Sigma Aldrich and used without further purification.
The catalytic constants of acetosyringone transformation by
laccase from C. gallica were estimated by measuring the transfor-
mation rate in 1 mL reaction mixture containing 0.22 nM of purified
laccase and acetosyringone ranging from 0.01 to 3.0 mM in 50 mM
succinate buffer (pH 4.5) containing 20% ACN. The reaction progress
was monitored by an HPLC system (Perkin-Elmer 200 series)
equipped with a reverse phase C18 column (250 mm × 4 mm)
Nucleosil 5um (Macherey-Nagel) and coupled to a diode array
detector (Perkin-Elmer 235C). The decrease in the concentration
of substrates was determined by measuring the decrease in peak
area at 280 nm. All experiments were done in triplicate.
UV–vis spectra were recorded on a Hewlett Packard 8453 spec-
trometer using a quartz cuvette of 0.5 mL.
Continuous wave (CW) S-band ESR spectra were obtained
using an S-band bridge (v= 3.8 GHz) SB-1111 Jagmar (Poland). Low
temperatures were controlled with a Bruker ER 4111VT variable
temperature unit. CW X-band (9.87 GHz) measurements were per-
formed on a Bruker E500 Elexsys Series using the Bruker ER 4122
SHQE cavity and an Oxford helium continuous flow cryostat (ESR
900). W-band (94.17 GHz) experiments were recorded on a Bruker
Elexsys E600 spectrometer operating in continuous wave equipped
with a 6T split-coils superconducting magnet (Oxford Instrument)
using a continuous helium flow cryostat (Oxford Instrument).
The enzymatic reaction was carried out in 200 ␮L of aqueous
solution containing 15 ␮M of purified laccase from C. gallica and
30 mM of phenolic mediator in 0.1 M acetate buffer pH 4.5, con-
taining ACN 20% (v/v). After the addition of laccase to the reaction

A. Martorana et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 203–208
205
O
.
+
ET route
O
O
- H+
Medox
O
OH
O
Medrid
HAT route
O
O
O
O
Med
O
Med
OH
O
OH
O
O
O
O
O
.
.
O
O
O
O
O
O
O
O
O
O
O
Scheme 2. Mechanism of oxidation of acetosyringone mediated by laccase (top) and delocalization of unpaired electron (bottom).
mixture, the samples were quickly placed into 1 mm ID quartz cap-
illary tubes and then placed inside standard suprasil ESR tubes.
For the TF-ESR measurements the spectra were recorded for
1.30 min per spectrum using 5 s of delay time until the intensity
was close to zero.
Nano Assisted Laser/Desorption Ionization Time of Flight
(NALDI-TOF) mass spectra were acquired using an Autoflex II mass
spectrometer (Bruker Daltonics, Germany) using a 337 nm nitro-
gen laser as the ionization source at a repetition rate of 50 Hz. The
acceleration voltage was 20 kV in the reflector mode. For all spec-
tra, 150 shots in the positive mode were accumulated. A NALDITM
target (Bruker Daltonics, Germany) was used, directly spotting
1 ␮L of the sample solution diluted ten times with a solution of
water/acetonitrile 1/1 (10 mg/mL) on the target and then the mea-
surement was performed.
and 400 nm with an isosbestic point at 444 nm showing that two
species are linearly related. The signal at 400 nm decreases quickly
to zero, suggesting that this band is relative to a transient species.
At the same time, the band at 528 nm increases with time to
reach a plateau, which indicates the formation of the new prod-
uct. In the inset the time traces for the reaction has been reported
and the kinetic constants for the acetosyringone oxidation are:
kcat = 50 min−1 and KM = 0.74 mM.
Experiments performed under the same conditions with the
same molar ratios using laccase from Trametes versicolor have pro-
vided the same results, but with slower oxidation kinetics.
To obtain a full characterization of the radical intermediate
formed during the catalytic reaction, the system has been fully
investigated through multifrequency ESR spectroscopy (3.8 GHz, S-
band; 9.8 GHz, X-band; 94.17 GHz, W-band), as shown in Fig. 2. All
the instrumental conditions are reported in the caption. ESR spec-
tra simulations were performed by Easyspin software package [31]
(ESI: ESR spectra simulation).
3. Results and discussion
In Fig. 2A, the S-band ESR spectrum of the radical at room tem-
perature is shown. The spectrum is characterized by a giso = 2.0045
and a linewidth of 1.5 mT and it was simulated considering two dif-
ferent sets of protons as main hyperfine coupling: two equivalent
aromatic hydrogens with A = 0.17 mT and six equivalent hydrogens
with A = 0.14 mT belonging to the methoxy groups. The X-band ESR
spectrum at room temperature reported in Fig. 2B shows a very
structured spectrum centred at giso = 2.0045. The set of coupling
constants used for the two frequencies were the same. However,
due to the improved resolution of the X-band spectrum, it was
necessary to include in this case a superhyperfine coupling of
A = 0.029 mT, relative to the three equivalent hydrogens of the
acetyl group. This unambiguously indicated that all the hydrogen
atoms present in the molecule are involved in the delocalization
of the unpaired electron, providing a good stabilization of the rad-
ical intermediate. The well-structured pattern exhibited at S and
X-band spectra of acetosyringone radical in liquid solution col-
lapsed to an anisotropic, unresolved spectrum when the sample
was frozen at 120 K or 70 K (ESI: ESR measurements and Figure S1).
In order to assign the radical species unequivocally and to deter-
mine its g principal values, a W-band ESR spectrum was recorded
on a frozen solution (Fig. 2C). At this frequency it is indeed often
possible to resolve the g-tensor (gx, gy, gz) anisotropies, providing
a molecular fingerprint for the organic radical assignment [32]. In
our case, due to the unresolved hyperfine splitting and the conse-
quent linewidth, the gx and gy components are partially overlapped,
but complete simulation of the spectrum allowed us to accurately
Upon incubation of C. gallica laccase with acetosyringone at
room temperature in acetate buffer solution, an intense purple
colour appeared. Therefore, the process was initially followed spec-
trophotometrically throughout the reaction time as shown in Fig. 1.
The kinetic trend analysis shows two absorption bands at 528
Fig. 1. UV–vis spectral changes at 400 nm and 528 nm recorded in quartz cuvette
during oxidation of acetosyringone in buffer solution at pH 6. Spectra recorded with
a cycle time of 5 min within 5 h of running time. In the inset: the time traces for the
acetosyringone oxidation reaction.

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A. Martorana et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 203–208
Table 1
g- and hf-tensor magnetic parameters for acetosyringone radical. cavity.
b
b
b
ga
AHaromat
0.175
AHOCH3
AHCOCH3
Isoc
x
y
z
2.0045
2.0064
2.0053
2.0020
0.144
0.029
a
The g-tensor principal values (gx,y,z) are given with a maximum error of 0.0001.
hf-tensor are given in mT (estimated error of 0.003 mT). The g- and hf-tensor
b
values fit S-, X-, and W-band ESR spectra equally well (Fig. 2).
c
Isotropic value is referred to room temperature spectra.
Fig. 3. Two-dimensional surface plot of the acetosyringone X-band ESR spectra,
recorded at 9.87 GHz, 0.63 mW microwave power, 0.01 mT modulation amplitude,
90 s per spectrum using 5 s of delay time.
determine the three different g values: gx = 2.0066 0.0001 and
gy = 2.0053 0.0001 and gz = 2.0020 0.0001. These values were
then used to refine the S-band and X-band simulations. The mag-
netic parameters are reported in Table 1.
In order to study the radical stability, the time dependent ESR
spectra of the acetosyringone radical were recorded as shown in
Fig. 3. The t = 0 spectrum showed the highest intensity, followed by
an exponential decay which led after 8 min to a plateau, lasting for
about 3 h [33]. The strong signal intensity and the great stability of
the radical intermediate allowed the recording of the spectra with-
out loss of resolution and the superhyperfine structure is visible in
all spectra.
The greatest disadvantage in the oxidation of phenolic com-
pounds catalyzed by laccase is the production of by-products which
may lead to polymerization processes [34]. In order to explore the
presence of by-reactions, the catalytic process was also followed
by NALDI-TOF MS, performing the experiments over period of 15
days, as reported in Fig. 4.
As shown in Fig. 4A, after only 15 min of reaction time, the
oxidation process yields a base peak assigned to two and three
monomeric units. The first step is the hydrogen abstraction from
the phenolic group producing the phenoxy radical [M]•+, with m/z
195 as the most intensive signal and the relative sodium adduct at
m/z 218. The radical is further coupled with other neutral molecules
in a head-to-tail fashion to give a radical quinoid-type intermediate
with double and triple m/z 390 and 585 [nM]•+. Following this reac-
tion mechanism, the variation of the terminal structure presumably
is focused on the phenolic group, while the acetyl group remains
unchanged. Another signal, probably relative to the deacetylated
dimer [M−COCH3]•+, is present at m/z 347. After 3 h (ESI: Figure
S2B), phenoxy radical signal is still present with great intensity
together with the sodium adduct, but the signals at m/z 390 and
Fig. 2. Multifrequency CW ESR spectra of acetosyringone (black line) formed via
enzymatic oxidation, paired with its best simulation (red line). (A) S-band spectrum
recorded at 3.87 GHz, 0.5 mW microwave power, 0.08 mT modulation amplitude,
298 K; (B) X-band spectrum recorded at 9.87 GHz, 0.63 mW microwave power,
0.01 mT modulation amplitude, 298 K; (C) W-band spectrum recorded at 94.17 GHz,
0.05 mW microwave power, 0.1 mT modulation amplitude, 80 K. The asterisk (*)
indicates an unavoidable impurity in the cavity. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)

A. Martorana et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 203–208
207
Fig. 4. NALDI-TOF positive mode mass spectra of mixture acetosyringone/C. gallica laccase in 0.1 M acetate buffer pH 4.5 after 15 min (A) and 72 h (B) of incubation (ESI:
NALDI-TOF measurements).
585 disappear completely and only the deacetylated dimer peak is
present at m/z 347.2. After 6 h the radical is quenched and replaced
by the ion [M+H]+ at m/z 197, while another signal at m/z 289
appears. These same signals were observed in spectra recorded
after 24–72 h (Fig. 4B) and 15 days of incubation.
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This work is supported by PRIN 2009 STNWX3 MIUR, Italian
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