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

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Journal of Molecular Catalysis B: Enzymatic 97 (2013) 203– 208

Contents lists available at ScienceDirect

Journal of Molecular Catalysis B: Enzymatic

jo ur nal home p age: www.elsev ier .com/ locate /molcatb

spectroscopic characterization of a phenolic natural mediator in theaccase biocatalytic reaction

ndrea Martoranaa, Lorenzo Soraceb, Harry Boerc, Rafael Vazquez-Duhaltd,iccardo Basosia,∗, Maria Camilla Barattoa,∗∗

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Via A. Moro 2, 53100 Siena, ItalyDepartment of Chemistry and INSTM RU, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, ItalyVTT Technical Research Centre of Finland, P.O. BOX 1000, FI-02044 Espoo, FinlandInstituto de Biotecnologia, Universidad Nacional Autonoma de Mexico, Apartado Postal 150-3, Cuernavaca, Moorelos 62210, Mexico

r t i c l e i n f o

rticle history:eceived 25 March 2013eceived in revised form 21 August 2013ccepted 22 August 2013vailable online 4 September 2013

a b s t r a c t

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 Coriolopsisgallica laccase catalytic reaction. A stable radical species is formed and an intense and well-structuredESR spectrum was detected and fully characterized at S-, X- and W-bands. The presence of by-productsgenerated as the result of by-reactions has been investigated and analyzed through NALDI-TOF MS, per-

eywords:cetosyringoneadical intermediateaccaseultifrequency ESRALDI-TOF MS

forming the experiments versus time. The superior radical stability of such phenoxy radical, due to sterichindrance in ortho to the phenol group and the great delocalization of the unpaired electron on theacetyl substituent, makes acetosyringone particularly interesting for biotechnological applications. Thisrepresents a good example for the development of new stable laccase mediator molecules.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

Several studies have been carried out using 3,5-dimethoxy--hydroxyacetophenone, also called acetosyringone, as natural

accase mediator. Acetosyringone and some other natural lignin-erived phenols, such as syringaldehyde, are promising eco-riendly mediators for dye degradation in terms of both efficiencynd velocity of oxidation [1,2].

Among the dimethoxy phenols, acetosyringone resulted therst compound showing a considerable radical stability and couldepresent a good example for the production of new mediatorolecules. The large amount of free radical produced has allowed

he identification and full characterization of the radical interme-iate generated during the catalytic reaction mediated by laccase.

n the past, evidence of such a stable radical was observed at the

-band ESR (Electron Spin Resonance) [3,4], but it was producedy horseradish peroxidase and ligninase in the presence of H2O2.n this work, for the first time, the radical was generated by laccase

∗ Corresponding author. Tel.: +39 0577 234240; fax: +39 0577 234239.∗∗ Corresponding author. Tel.: +39 0577 234247; fax: +39 0577 234239.

E-mail addresses: [email protected] (R. Basosi),[email protected] (M.C. Baratto).

381-1177/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.molcatb.2013.08.013

and O2 and an accurate determination of the magnetic parameters,through a multifrequency (S-, X-, W-band) ESR approach, has beenachieved.

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 oxygenas co-substrate. During its catalytic process, laccase removes asingle electron from the substrate generating free radicals, withthe concomitant reduction of oxygen to water [5]. Due to sterichindrance factors and to its redox potential between 0.5 and 0.8 V,laccase is unable to oxidize non-phenolic lignin units which have ahigh-redox potential (>1.5 V) and it can only oxidize phenolic unitsat the substrate surface. Furthermore it is known that fatty acidspresent in lignocellulosic pulps are responsible for “pitch prob-lems” which can be overcome using laccase, modifying liphophilicextractives [6–9]. To overcome the accessibility problem and toextend the ability of non-phenolic lignin unit oxidation, laccase isoften used in the presence of an oxidizing small molecule, calledmediator, which acts as a sort of electron shuttle between theenzyme 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 oxidizesother molecules, extending the range of substrates susceptibleto the enzymatic action. In this way, laccases are able to oxidize

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OH

Cu2+

Cu2+

Cu 2+

Cu2+

OH O

O

O

OH

OH

O

O

O

O

O

O

O

H+

O2

H+

T1

T2

T3

OH

Cu2+

Cu2+

Cu2+

Cu2+

OOH

Cu2+

Cu2+

Cu 2+

Cu2+

T3

+.

H2O

H2O

Sub(red)

Sub(ox)

H2O

heno

cil

sccaasalmtecgrccrr

oifous

wmforaap

t[i

Scheme 1. The role of the mediator, represented by the p

ompounds with a redox potential higher than 0.8 V and the directnteraction between the enzyme and the target substrate is noonger necessary.

An ideal redox mediator (i) must be a good laccase sub-trate, (ii) must have a stable oxidized form and (iii) its redoxonversion must be cyclic [15]. For industrial applications, lac-ase mediators should be environmentally friendly and availablet low cost [1,9,16–20]. Several compounds have been useds laccase mediators. Generally they are products of chemicalynthesis such as 2,2′-azino-bis(3-etilenbenzotiazolin-6-sulphoniccid) (ABTS), 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), vio-uric acid (VIOL) and 1-hydroxybenzotriazole (HBT) [21–23]. Their

ost relevant disadvantage is their high cost, toxicity and forma-ion of many by-products. In this regard, the use of new and moreffective mediators among natural phenolic compounds that areheaper, non-toxic and more eco-friendly, has recently arousedreat interest. In particular, lignin-derived phenols have beeneported as efficient mediators and among them, dimethoxyphenolompounds were described as the fastest and most efficient lac-ase natural mediators. However, until now, no information aboutadical structures of natural mediators generated during catalyticeactions was present in the literature.

Laccase activity involving phenols is enhanced by the presencef electron-donating substituents on the benzene ring that decreasets electrochemical potential. Moreover, two features are importantor improving the stability of phenoxy radicals: (i) steric hindrancef the OH group on the phenol, leading to a great density of thenpaired electron on the oxygen atom in the radical form and (ii) aubstituent group able to delocalize the unpaired electron [24,25].

It is known that laccase can act through two different path-ays: an electron transfer mechanism (ET route) or a H-abstractionechanism (HAT route) and in both pathways a radical species is

ormed as final product. In Scheme 2, the two different pathwaysf 3,5-dimethoxy-4-hydroxyacetophenone (also named acetosy-ingone) oxidation and the delocalization of the unpaired electronre shown. Until now, the only structural characterization of medi-tor radicals was performed on ABTS and VIOL [26] and on arecursor radical such as 4-methyamino benzoic acid [27].

In the present work we report a multifrequency ESR study onhe acetosyringone oxidation, catalyzed by laccase from C. gallica28] combined with two-dimensional X-band ESR measurementsn order to obtain a more complete description of the radical

lic compound, during the substrate oxidation by laccase.

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.

2. Materials and methods

Laccase from C. gallica was produced and purified as describedpreviously [29]. The enzyme preparation used in this study showed80% purity estimated by SDS-PAGE electrophoresis and an activityof 11200 U/mL (313 U/mg of protein) determined with syringal-dazine as substrate [30].

3,5-dimethoxy-4-hydroxyacetophenone was purchased fromSigma Aldrich and used without further purification.

The catalytic constants of acetosyringone transformation bylaccase from C. gallica were estimated by measuring the transfor-mation rate in 1 mL reaction mixture containing 0.22 nM of purifiedlaccase and acetosyringone ranging from 0.01 to 3.0 mM in 50 mMsuccinate buffer (pH 4.5) containing 20% ACN. The reaction progresswas 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 arraydetector (Perkin-Elmer 235C). The decrease in the concentrationof substrates was determined by measuring the decrease in peakarea 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 obtainedusing an S-band bridge (v = 3.8 GHz) SB-1111 Jagmar (Poland). Lowtemperatures were controlled with a Bruker ER 4111VT variabletemperature unit. CW X-band (9.87 GHz) measurements were per-formed on a Bruker E500 Elexsys Series using the Bruker ER 4122SHQE cavity and an Oxford helium continuous flow cryostat (ESR900). W-band (94.17 GHz) experiments were recorded on a BrukerElexsys E600 spectrometer operating in continuous wave equippedwith 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 aqueoussolution containing 15 �M of purified laccase from C. gallica and30 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

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A. Martorana et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 203– 208 205

O

O O

OH

O

O O

OHO

O O

O

O

O O

O

O

O

O

O

O

O

O

..

.

O O

O

O

O OH

+

HAT route

ET routeMed

ox

Medrid

- H+

Med Med

d by l

mi

1w

(sgatt1ws

3

rctT

Fdaa

O

Scheme 2. Mechanism of oxidation of acetosyringone mediate

ixture, the samples were quickly placed into 1 mm ID quartz cap-llary tubes and then placed inside standard suprasil ESR tubes.

For the TF-ESR measurements the spectra were recorded for.30 min per spectrum using 5 s of delay time until the intensityas close to zero.

Nano Assisted Laser/Desorption Ionization Time of FlightNALDI-TOF) mass spectra were acquired using an Autoflex II masspectrometer (Bruker Daltonics, Germany) using a 337 nm nitro-en laser as the ionization source at a repetition rate of 50 Hz. Thecceleration voltage was 20 kV in the reflector mode. For all spec-ra, 150 shots in the positive mode were accumulated. A NALDITM

arget (Bruker Daltonics, Germany) was used, directly spotting �L of the sample solution diluted ten times with a solution ofater/acetonitrile 1/1 (10 mg/mL) on the target and then the mea-

urement was performed.

. Results and discussion

Upon incubation of C. gallica laccase with acetosyringone at

oom temperature in acetate buffer solution, an intense purpleolour appeared. Therefore, the process was initially followed spec-rophotometrically throughout the reaction time as shown in Fig. 1.he kinetic trend analysis shows two absorption bands at 528

ig. 1. UV–vis spectral changes at 400 nm and 528 nm recorded in quartz cuvetteuring oxidation of acetosyringone in buffer solution at pH 6. Spectra recorded with

cycle time of 5 min within 5 h of running time. In the inset: the time traces for thecetosyringone oxidation reaction.

accase (top) and delocalization of unpaired electron (bottom).

and 400 nm with an isosbestic point at 444 nm showing that twospecies are linearly related. The signal at 400 nm decreases quicklyto zero, suggesting that this band is relative to a transient species.At the same time, the band at 528 nm increases with time toreach a plateau, which indicates the formation of the new prod-uct. In the inset the time traces for the reaction has been reportedand the kinetic constants for the acetosyringone oxidation are:kcat = 50 min−1 and KM = 0.74 mM.

Experiments performed under the same conditions with thesame 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 intermediateformed during the catalytic reaction, the system has been fullyinvestigated through multifrequency ESR spectroscopy (3.8 GHz, S-band; 9.8 GHz, X-band; 94.17 GHz, W-band), as shown in Fig. 2. Allthe instrumental conditions are reported in the caption. ESR spec-tra simulations were performed by Easyspin software package [31](ESI: ESR spectra simulation).

In Fig. 2A, the S-band ESR spectrum of the radical at room tem-perature is shown. The spectrum is characterized by a giso = 2.0045and a linewidth of 1.5 mT and it was simulated considering two dif-ferent sets of protons as main hyperfine coupling: two equivalentaromatic hydrogens with A = 0.17 mT and six equivalent hydrogenswith A = 0.14 mT belonging to the methoxy groups. The X-band ESRspectrum at room temperature reported in Fig. 2B shows a verystructured spectrum centred at giso = 2.0045. The set of couplingconstants used for the two frequencies were the same. However,due to the improved resolution of the X-band spectrum, it wasnecessary to include in this case a superhyperfine coupling ofA = 0.029 mT, relative to the three equivalent hydrogens of theacetyl group. This unambiguously indicated that all the hydrogenatoms present in the molecule are involved in the delocalizationof the unpaired electron, providing a good stabilization of the rad-ical intermediate. The well-structured pattern exhibited at S andX-band spectra of acetosyringone radical in liquid solution col-lapsed to an anisotropic, unresolved spectrum when the samplewas 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 recordedon a frozen solution (Fig. 2C). At this frequency it is indeed oftenpossible to resolve the g-tensor (gx, gy, gz) anisotropies, providing

a molecular fingerprint for the organic radical assignment [32]. Inour 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

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206 A. Martorana et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 203– 208

Fig. 2. Multifrequency CW ESR spectra of acetosyringone (black line) formed viaenzymatic oxidation, paired with its best simulation (red line). (A) S-band spectrumrecorded 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,

Table 1g- and hf-tensor magnetic parameters for acetosyringone radical. cavity.

ga AHaromatb AHOCH3

b AHCOCH3b

Isoc 2.0045 0.175 0.144 0.029x 2.0064y 2.0053z 2.0020

a The g-tensor principal values (gx,y,z) are given with a maximum error of ±0.0001.b hf-tensor are given in mT (estimated error of ±0.003 mT). The g- and hf-tensor

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 andgy = 2.0053 ± 0.0001 and gz = 2.0020 ± 0.0001. These values werethen 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 ESRspectra of the acetosyringone radical were recorded as shown inFig. 3. The t = 0 spectrum showed the highest intensity, followed byan exponential decay which led after 8 min to a plateau, lasting forabout 3 h [33]. The strong signal intensity and the great stability ofthe radical intermediate allowed the recording of the spectra with-out loss of resolution and the superhyperfine structure is visible inall spectra.

The greatest disadvantage in the oxidation of phenolic com-pounds catalyzed by laccase is the production of by-products whichmay lead to polymerization processes [34]. In order to explore thepresence of by-reactions, the catalytic process was also followedby NALDI-TOF MS, performing the experiments over period of 15days, as reported in Fig. 4.

As shown in Fig. 4A, after only 15 min of reaction time, theoxidation process yields a base peak assigned to two and threemonomeric units. The first step is the hydrogen abstraction fromthe phenolic group producing the phenoxy radical [M]•+, with m/z195 as the most intensive signal and the relative sodium adduct atm/z 218. The radical is further coupled with other neutral moleculesin a head-to-tail fashion to give a radical quinoid-type intermediatewith double and triple m/z 390 and 585 [nM]•+. Following this reac-tion mechanism, the variation of the terminal structure presumablyis focused on the phenolic group, while the acetyl group remainsunchanged. Another signal, probably relative to the deacetylated

dimer [M−COCH3]•+, is present at m/z 347. After 3 h (ESI: FigureS2B), phenoxy radical signal is still present with great intensitytogether with the sodium adduct, but the signals at m/z 390 and

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 referencesto colour in this figure legend, the reader is referred to the web version of this article.)

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A. Martorana et al. / Journal of Molecular Catalysis B: Enzymatic 97 (2013) 203– 208 207

F a laccN

5pbaa

rcgsdrfhortv

4

isopgmao

A

CdciCf

A

i0

[[

[

[[

[[[

[

[[

[[

[

[

[

[

[

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ig. 4. NALDI-TOF positive mode mass spectra of mixture acetosyringone/C. gallicALDI-TOF measurements).

85 disappear completely and only the deacetylated dimer peak isresent at m/z 347.2. After 6 h the radical is quenched and replacedy the ion [M+H]+ at m/z 197, while another signal at m/z 289ppears. These same signals were observed in spectra recordedfter 24–72 h (Fig. 4B) and 15 days of incubation.

The spectral modification during the experiment shows that theadical intermediate is still visible by MS spectrometry for 3 h, thusonfirming the result of the time dependent ESR spectra and thereat stability of this radical. The presence of new peaks in the MSpectra after a long period of incubation might be due either toismutation of some unstable by-products generated during theeaction or to the fact that intermediates are still good substratesor laccase so that the oxidation proceeds as long as the final productas a phenolic group capable of being oxidized. After purificationf the reaction mixture, more than 93% of the final product is rep-esented by acetosyringone itself, confirming the great stability ofhe radical intermediate and that by-products are produced in aery limited amount (ESI: Purification and 1H NMR).

. Conclusions

In conclusion, when acetosyringone is used as laccase mediator,t generates a very stable radical intermediate. The two methoxyubstituents in ortho position to the phenol group enable a rapidxidation by laccase and provide a good steric hindrance in thatortion of molecule where the radical is formed. The acetyl groupives a further delocalization for the unpaired electron. This abilityakes acetosyringone particularly interesting for biotechnological

pplications and it opens the way for the design and developmentf new stable laccase mediator molecules.

cknowledgements

This work is supported by PRIN 2009 STNWX3 MIUR, ItalianSGI Consortium and BISCOL (Bioprocessing for Substainable Pro-uction of Coloured Textiles) European project (CIP-Eco-innovationall n.256112). L.S. acknowledges the financial support Ente CaR-Fi. Careful reading and revising of the manuscript by Les Brooks,hemistry Professor Emeritus, Sonoma State University, is grate-

ully acknowledged.

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.molcatb.2013.8.013.

[

[[

ase in 0.1 M acetate buffer pH 4.5 after 15 min (A) and 72 h (B) of incubation (ESI:

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