mediator systems: Their reactivity towards phenolic lignin

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Laccase/mediator systems: Their reactivity towards phenolic lignin structures Roelant J. Hilgers, Jean-Paul Vincken, Harry Gruppen, and Mirjam A. Kabel ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03451 • Publication Date (Web): 04 Jan 2018 Downloaded from http://pubs.acs.org on January 7, 2018

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ACS Sustainable Chemistry & Engineering

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Laccase/mediator systems: Their reactivity towards phenolic lignin

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structures

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Roelant Hilgers, Jean-Paul Vincken, Harry Gruppen and Mirjam A. Kabel*

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Laboratory of Food Chemistry, Wageningen University and Research, the Netherlands

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Corresponding author:

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Dr. ir. Mirjam Kabel

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Laboratory of Food Chemistry

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Wageningen UR

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Bornse Weilanden 9

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6708 WG Wageningen

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The Netherlands

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Tel: +31 (0)317 483209

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E-mail: [email protected]

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ACS Paragon Plus Environment

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Abstract

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Laccase-mediator systems (LMS) have been widely studied for their capacity to oxidize

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the non-phenolic subunits of lignin (70-90% of the polymer). The phenolic subunits

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(10-30% of the polymer), which can also be oxidized without mediators, have received

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considerably less attention. Consequently, it remains unclear to what extent the presence

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of a mediator influences the reactions of the phenolic subunits of lignin. To get more

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insight in this, UHPLC-MS was used to study the reactions of a phenolic lignin dimer

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(GBG), initiated by a laccase from Trametes versicolor, alone or in combination with

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the mediators HBT and ABTS. The role of HBT was negligible, as its oxidation by

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laccase occurred slowly in comparison to that of GBG. Laccase and laccase/HBT

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oxidized GBG at a comparable rate, resulting in extensive polymerization of GBG. In

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contrast, laccase/ABTS converted GBG at a higher rate, as GBG was oxidized both

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directly by laccase, but also by ABTS radical cations, which were rapidly formed by

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laccase. The laccase/ABTS system resulted in Cα oxidation of GBG and coupling of

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ABTS to GBG, rather than polymerization of GBG. Based on these results, we propose

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reaction pathways of phenolic lignin model compounds with laccase/HBT and

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laccase/ABTS.

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Keywords:

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hydroxybenzotriazole

Oxidation;

radical

coupling;

Trametes

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versicolor;

ABTS;

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Introduction

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Lignin is one of the most abundant polymers in nature as part of plant cell walls, and

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currently considered as sustainable precursor for chemicals or materials.1-4 It is built up

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from sinapyl alcohol, coniferyl alcohol and p-coumaroyl alcohol (S, G and H units,

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respectively), which couple via radical polymerization to form a variety of C-C and C-O

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inter-unit linkages. The β-O-4 linkage is the most abundant one, accounting for

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approximately 45 to 94 % of the total inter-unit linkages, in lignins mildly isolated from

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plant materials.3 Lignin contains phenolic and non-phenolic subunits, which account for

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10-30 and 70-90% of the polymer, respectively.5

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Although the valorization of lignin is still underexploited,3 three main routes are

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considered relevant. The first is via lignin polymerization, which may result in

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improved binders and adhesives.6 Second, lignin depolymerization may lead to high-

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value low-molecular-weight aromatics.1-2 A third option is the functionalization of

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lignin via grafting of specific molecules onto lignin.7 A green alternative for lignin

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valorization is via biocatalysis and, in particular, laccase is known as one of the key

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activities towards lignin. Various lignin modifications are reported for laccase, such as

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polymerization,4,

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poorly understood, however, how to direct lignin modification by laccase towards one

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of these modifications. Thus, for an effective use of laccase in lignin valorization, it is

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essential to have a better understanding on how to control lignin modification by

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laccase.

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Laccases (E.C. 1.10.3.2) are oxidases that couple the reduction of molecular oxygen to

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the one-electron oxidation of a wide variety of aromatic substrates. The most powerful

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depolymerization,11-13 Cα oxidation14 and demethylation.15 It is

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laccases are produced by white-rot fungi, and can have redox potentials up to 800 mV

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vs. NHE.3 With respect to laccase activity towards lignin, it is important to distinguish

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the phenolic and non-phenolic subunits in lignin. The phenolic subunits can directly be

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oxidized by laccase, as the redox potentials of such subunits are sufficiently low. In

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contrast, the non-phenolic parts have redox potentials up to 1500 mV vs. NHE, and are

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therefore, recalcitrant to oxidation by laccase alone.16 Nevertheless, when laccase is

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combined with a so-called mediator, oxidation of non-phenolic lignin structures is

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possible as well.17 In such a laccase/mediator system (LMS), the mediator is first

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oxidized by the laccase, after which it can oxidize non-phenolic substrates via different

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mechanisms, such as electron transfer (ET) or radical hydrogen atom transfer (HAT).18

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As a LMS is required for the oxidation of the non-phenolic subunits in lignin, and the

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phenolic subunits can be oxidized by laccase alone, research on lignin modification by

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LMS has mainly focused on the reactivity of non-phenolic lignin subunits,14, 17-21 and

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several reaction pathways have been described for such incubations.22-24 Nevertheless,

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as lignin also contains a considerable amount of phenolic subunits, it is important to

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understand how these subunits react when incubated with LMS.

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Research on lignin conversion by laccase and LMS is often performed using lignin

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model compounds. Phenolic lignin model compounds have been used, but most often in

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the absence of mediators.25-27 A few studies have been published on the reactions of

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phenolic lignin model compounds in LMS incubations, and mainly polymerization was

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shown to occur. Conclusions about the degree of polymerization reached were obtained,

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but no insights about the structure of the (initially) formed reaction products could be

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provided.28-29 Consequently, it remains unclear how the phenolic subunits of lignin are

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modified by LMS and to what extent mediators play a role in such modifications.

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In the current research, we used the phenolic β-O-4 linked lignin model compound, 1-

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(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol,

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(guaiacylglycerol-β-guaiacyl ether, GBG), to mimic the phenolic subunits in lignin. We

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employed RP-UHPLC-PDA-ESI-MSn, to study detailed reaction pathways of GBG

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initiated by laccase (from Trametes versicolor) with or without a mediator. 2,2'-Azino-

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bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1-hydroxybenzotriazole

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(HBT) were used as mediators, as these are the most commonly used mediators in

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literature. MALDI-TOF-MS was used to investigate the formation of larger reaction

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products.

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Materials and Methods

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Materials

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Guaiacylglycerol-β-guaiacyl ether (GBG; Figure 1) was obtained from TCI chemicals

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(Toyo, Japan) and 2,5-dihydroxybenzoic acid (DHB) was obtained from Bruker

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Daltonics (Bremen, Germany). Laccase from Trametes versicolor and all other

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chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA). The laccase was

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partially purified, after which the activity was determined spectrophotometrically by

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oxidation of ABTS17 (1 U = 1 µmol ABTS oxidized per minute) (see S2 for details).

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Water was prepared using a Milli-Q water purification system (Merck Millipore,

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Billerica, MA).

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Incubation of GBG with laccase and laccase/mediator systems

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GBG was dissolved at 0.5 mM in sodium phosphate buffer (50 mM, pH 4) with or

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without an equimolar concentration of ABTS or HBT. Laccase was added to reach a

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final substrate and mediator concentration of 0.4 mM and a laccase activity of 0.1 U

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mL-1. The mixtures were incubated at 40 °C and 400 rpm in a thermomixer (Eppendorf,

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Hamburg, Germany). After 1, 2, 5, 10, 15, 20, 30 and 60 min, and after 24 h, 50 µL of

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the sample was transferred to another tube, and 5 µL sodium azide (20 mM) was added

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to stop the reaction.30 As no azide-related products were detected, and reaction products

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were stable in the presence of an excess of azide, the addition of azide was considered

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not to influence the reaction product profile (results not shown). The resulting samples

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were diluted 10 times in MilliQ water and were centrifuged (10000×g, 5 min, 20 ºC)

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prior to analysis by RP-UHPLC-PDA-MSn. A detailed description of the RP-UHPLC-

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PDA-MSn analysis can be found in S3 and S4 of the Supporting Information.

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Incubation of GBG with ABTS radical cations

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GBG was dissolved at 0.5 mM in sodium phosphate buffer (50 mM, pH 4). ABTS (2

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mM) was incubated with 0.20 U mL-1 laccase (2 min, 40 °C) and directly centrifuged

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over an Amicon Ultra-4 centrifugal filter (Merck Millipore) with a normalized

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molecular weight limit of 10 kDa. The dark green filtrate was incubated with the GBG

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solution at a ratio of 1:4 (2 min, 40 °C). As a control, the same incubation was done

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using ABTS that was not treated with laccase.

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Sample preparation for Matrix-Assisted Laser Desorption Ionization – Time Of

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Flight Mass Spectrometry (MALDI-TOF)

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The samples were prepared as described in 2.3, with the adaptation that the reaction was

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stopped after 7, 15 and 60 min. Samples were diluted 2 times in methanol, and a

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saturated solution of DHB was used as matrix. In a second experiment, similar

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incubations with laccase and laccase/HBT were stopped after 2, 5, 10, 15 and 20 min.

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For details about MALDI-TOF settings see S6.

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Results & Discussion

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Reaction products of GBG incubated with laccase

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GBG (Figure 1) was, first, incubated with laccase without addition of mediators. From

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the UHPLC-MS chromatograms, two main reaction products were observed upon

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incubation of GBG, both having their largest intensities during the first 20 min (Figure

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2, Table 1 and Figure S1). In addition, remaining GBG was detected (only trace

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amounts after 2 min). Using accurate mass determination the reaction products were

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found to correspond with molecular formulas C34H38H12 and C51H56O18 (Table 1).

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Based on their molecular formulas and the fact that the fragmentation patterns showed

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high similarities with GBG (Table 1 and Figure 3), these products were annotated as a

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dimer of GBG (molecular weight (Mw)=2×GBG-2H) and a trimer of GBG

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(Mw=3×GBG-4H; Table 1), formed via radical coupling. Recently, oligomerization of

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GBG was reported by others, and it was shown that coupling between two GBG radicals

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occurs via C-C bond formation.31 The same was suggested for the subsequent formation

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of a trimer, although no experimental evidence was given. Therefore, we have specified

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the coupling position in the dimer, but not of the second coupling in the trimer in Figure

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3. In addition to the dimer and trimer, only traces of other reaction products were

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observed (Table S1). After 24 h, precipitation was observed and no peaks were detected

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in the chromatograms, suggesting ongoing polymerization of the initially formed dimer

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and trimer. Thus, the activity of laccase alone was found to result in polymerization of

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GBG. This conclusion is in line with results on laccase activity towards phenolic model

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compounds reported by others.28, 31

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Reaction products of GBG incubated with a laccase/HBT system

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When GBG was incubated with laccase in combination with HBT, the reaction products

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formed were very comparable to those formed in the presence of laccase alone (Figure

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2, Table 1 and Figure S1). The same dimer and trimer were identified based on retention

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time, accurate mass and fragmentation pattern (Figure 2 and Table 1). Also the amounts

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were comparable, based on UV280 peak area. In addition to the dimer and trimer, a small

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peak (hardly visible) corresponding to the molecular formula C23H23N3O7 was observed.

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This, in combination with a fragmentation pattern similar to that of GBG (Table 1 and

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Figure 3), suggested the formation of a GBG-HBT radical coupling product (GBG-HBT

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CP, Figure 3). After prolonged incubation times (1 h) C23H21N3O7 was detected, which,

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most likely, corresponded to further oxidation of GBG-HBT CP to GBG-HBT CPox

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(Figure 3). It was recently shown for the first time that N-OH type mediators, such as

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HBT, couple to lignin upon addition of laccase.7 Our results confirm that such coupling

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can occur at the phenolic moieties of lignin, but also indicate that the extent of coupling

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between GBG and HBT is small as compared to GBG oligomerization. The proposed

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structures of all coupling products are depicted in Figure 3, together with their proposed

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MS2 fragmentation pathways. After 24 h, no GBG-related peaks were detected anymore

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and precipitation was observed. Approximately 45% of the original HBT was still

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present, as determined from UV280 peak areas (data not shown). Part of it was converted

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to benzotriazole (BT), which is inactive as a mediator.32 This conversion of HBT to BT

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has been described by others32-33 and also occurred in a control reaction with only HBT

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and laccase without GBG present (data not shown). Most likely, BT is formed upon a

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reaction of HBT radicals with aromatic amino acids of the enzyme.33

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Reaction products of GBG incubated with a laccase/ABTS system

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When GBG was incubated with laccase and ABTS, a larger variety of reaction products

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was detected compared to the incubations described above (Figure 2, Table 1 and Figure

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S1). Molecules with molecular formulas C17H18O6 and C35H35N4O12S4 were detected,

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especially after short reaction times (1-15 min). These were annotated as Cα oxidized

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GBG and a radical coupling product between oxidized GBG and ABTS (GBGox and

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GBG-ABTS CPIox in Table 1 and Figure 3), respectively. A relatively small peak

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corresponding to a coupling of non-oxidized GBG and ABTS (GBG-ABTS CPI) has

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been observed as well, but inconsistently. Likely, the latter coupling product is prone to

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react further. The structure and fragmentation pattern of GBG-ABTS CPI can be found

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in Figure S4. In addition, reaction products with molecular formulas C26H27N3O9S2,

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C26H25N3O9S2 were found. Based on these formulas and their fragmentation patterns,

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these products were annotated as GBG-ABTS CPII and GBG-ABTS CPIIox,

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respectively (Table 1 and Figure 3). Similar coupling products, between a phenolic

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compound and part of the ABTS molecule, have been reported for hydroxybenzoic

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acids34 and catechin,35 using NMR and mass spectrometry, respectively. To our

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knowledge, this is the first time that such coupling products are shown for lignin model

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compounds. The main MS2 fragments (m/z 242 and 214 in negative mode) match with

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the fragments reported for the catechin-ABTS products (m/z 244 and 216 in positive

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mode).35 As the adducts identified with NMR spectroscopy29 resulted from C-N

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coupling (rather than O-N coupling), the adducts in Figure 3 are also shown with a C-N

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bond between the GBG and ABTS moiety. In addition to these coupling products, a

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major peak was detected corresponding to C9H9NO4S2. As this molecule was hardly

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formed in a control reaction with only ABTS and laccase (data not shown), it was

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considered to be an ABTS cleavage product (ABTS CLP) formed upon coupling of

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GBG and ABTS. Lastly, a reaction product was detected with molecular formula

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C25H25N4O8S4. This could correspond to a radical coupling product of guaiacol and

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ABTS. It should be mentioned that for the formation of such a product cleavage of

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GBG to form guaiacol is required, either before or during coupling with ABTS. Besides

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the detection of C25H25N4O8S4, no indications were found for cleavage of GBG. In

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contrast to the incubation with laccase alone and laccase with HBT, no GBG was

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detected at all incubation times, suggesting a fast conversion of GBG into reaction

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products. The dimer and trimer found for incubations with laccase alone and

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laccase/HBT were absent at all incubation times when ABTS was used. After 24 h,

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approximately 29% of the initial amount ABTS was still present (data not shown).

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Whereas others reported polymerization as the main result of laccase/ABTS activity

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towards phenolic substrates,28-29 our results suggest that GBG preferably couples to

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ABTS, rather than to another GBG molecule.

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Reaction pathways of laccase and laccase/HBT with GBG

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To get more insight into the course of the reactions occurring during incubation of GBG

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with laccase and the laccase/mediator systems, product formation was monitored at 9

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different time points within the first hour. Hereto, peak areas from extracted ion

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chromatograms of the reaction products (Table 1) relative to the time point with the

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highest area, were plotted as function of incubation time (Figure 4). For clarity, only

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GBG-related reaction products are shown.

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Figure 4A and B show that, under the conditions used, a fast conversion of GBG to

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oligomeric products occurred. Already in the first minute, 67 and 69% of the GBG was

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converted by laccase and laccase/HBT, respectively. For both laccase and laccase/HBT

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it was shown that the dimer and the trimer gradually decreased in abundance and

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disappeared within one hour. The decrease of the dimer in the first 20 min occurred

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together with an increase in higher oligomers, indicating further polymerization (Figure

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S2). In the incubation with laccase/HBT, the coupling product GBG-HBT CP increased

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during the first 30 min, and then decreased again. The decrease of GBG-HBT CP

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coincided with an increase in its oxidized equivalent GBG-HBT CPox, confirming that

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GBG-HBT CP is oxidized further to GBG-HBT CPox. For such a conversion from

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alcohol to ketone at Cα, a benzylic radical should be formed. This may have occurred

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via radical hydrogen abstraction by a HBT radical. As a phenolic lignin structure itself

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could also act as HAT-type mediator,36 radical hydrogen abstraction may also have

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occurred by a phenoxyl radical of a GBG-related structure. If the latter indeed occurred,

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the oxidant probably was an oligomeric GBG radical, as all GBG was converted before

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conversion of GBG-HBT CP to GBG-HBT CPox took place (Figure 4).

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These findings indicate that upon incubation with both laccase and laccase/HBT,

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extensive polymerization of GBG takes place. Radical coupling between GBG and HBT

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occurred, but only to a small extent. The coupling product was further oxidized, and is

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probably prone to further polymerization. The rate of conversion of GBG and its

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oligomers does not seem to be influenced by the presence of HBT, suggesting that the

244

role of HBT is negligible when laccase/HBT is used on phenolic substrates. A

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schematic overview of the proposed reactions is depicted in Figure 5.

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Reaction pathways of laccase/ABTS with GBG

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Similar to the incubations with laccase and laccase/HBT, the reaction of GBG with

248

laccase/ABTS was monitored over time. Figure 4C shows that all GBG was converted

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within the first minute of incubation. Based on this and on the annotations discussed

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above, it can be stated that the laccase/ABTS system differs from laccase and

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laccase/HBT in two ways: (i) GBG is converted faster (higher rate), and (ii) GBG is

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converted into different reaction products (other pathways).

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A possible explanation for the higher conversion rate might be the rapid formation of

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ABTS radical cations (ABTS·+), which might undergo a redox reaction with GBG.

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ABTS·+ has been shown to induce oxidative polymerization of creosol, a phenolic

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monomeric lignin model compound29 and to undergo a redox reaction with vanillyl

257

alcohol.37 To check whether a similar interaction between GBG and ABTS·+ occurred,

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GBG was incubated with laccase-free ABTS·+. GBG was indeed converted upon

259

addition of ABTS·+, resulting in several reaction products: GBGox, GBG dimer,

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oxidized GBG dimer and GBG-ABTS CPI (Figure S3). The annotation of the latter two

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products can be found in Table S2 and Figure S4. As no GBG conversion was observed

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in a control reaction with neutral ABTS (Figure S3B), it was concluded that the higher

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GBG conversion rate by the laccase/ABTS system is caused by the fact that GBG is

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oxidized via two mechanisms: directly by laccase and via a redox reaction with

265

ABTS·+. It cannot be excluded that also the dication ABTS++ (formed via

266

disproportionation of ABTS·+) is involved in the oxidation of GBG. Nevertheless, as it

267

has been shown with cyclic voltammetry that vanillyl alcohol can rapidly be oxidized

268

by ABTS·+,37 we assume that the same applies to GBG.

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Figure S3 also shows that Cα oxidized GBG is formed upon addition of ABTS·+. The

270

fact that Cα oxidized products were not observed in incubations with laccase alone

271

implies that Cα oxidation is caused by ABTS·+ rather than laccase. Figure 4C shows

272

that the concentrations of GBGox, GBG-ABTS CPIox are highest after 1 min of

273

incubation, and that their concentration decreased relatively fast during the first 20 min.

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GBG-ABTS CPII was also highest after 1 min, but its decrease was slower. In contrast,

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275

the concentrations of GBG-ABTS CPIIox and ABTS CLP gradually increased during the

276

first hour of incubation, suggesting that they were formed upon conversion of the other

277

reaction products. After 24 h, most reaction products from Figure 4C had disappeared,

278

except for ABTS CLP and GBG-ABTS CPIIox. The areas of these peaks had even

279

increased with 33 and 23%, respectively (data not shown). These results indicate that

280

GBG formed coupling products with ABTS in the presence of laccase, after which a

281

part of the ABTS moiety was cleaved off. The ultimate product formed after coupling of

282

ABTS and GBG was GBG-ABTS CPIIox. To our knowledge, coupling of ABTS to

283

lignin and lignin model compounds has been suggested by others,8, 28 but the structure

284

of such lignin-ABTS coupling products has not been elaborated so far.

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Based on the above, an overview of the course of the reactions leading to the formation

286

of GBG-ABTS CPIIox was constructed (Figure 6). We suggest that after oxidation,

287

GBG· undergoes two follow-up reactions: (i) Radical coupling with ABTS·+ to form an

288

intermediate product GBG-ABTS CPI, or (ii) A second oxidation (by ABTS·+) to form

289

GBGox. In the latter case, GBGox can be further oxidized (by laccase or ABTS·+) to

290

GBGox·, which then also couples to ABTS·+ to form GBG-ABTS CPIox. GBG-ABTS

291

CPI and GBG-ABTS CPIox are subsequently converted to GBG-ABTS CPII and GBG-

292

ABTS CPIIox, upon which ABTS CLP was split off as a byproduct. The exact

293

mechanism behind this is unknown, but it is suggested to involve an oxidation step, as

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the intermediates (GBG-ABTS CPI and GBG-ABTS CPIox) did not react further after

295

inactivation of the laccase (Figure 4). Eventually, the formed GBG-ABTS CPII can be

296

oxidized to the ultimate reaction product GBG-ABTS CPIIox,

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Formation of larger oligomeric reaction products

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To investigate whether larger reactions products were formed upon incubation with

299

laccase or LMS, MALDI-TOF spectra were recorded. After 1 hour of incubation with

300

laccase and laccase/HBT, GBG oligomers up to DP 8 and DP 7 were detected,

301

respectively (Figure 7). As precipitation of reaction products occurred in both

302

incubations, and the mixture still contained small amounts of insoluble material after

303

dilution in methanol, it cannot be excluded that insoluble oligomers of higher DP were

304

formed. Besides the GBG oligomers, no other peaks were detected, suggesting that

305

incubation of GBG with laccase or laccase/HBT (almost) solely resulted in

306

oligomerization of GBG. In contrast, a completely soluble reaction mixture was

307

obtained after incubation with laccase/ABTS, in which all of the GBG oligomers were

308

absent. Instead, reaction products with m/z 657, 977, 989 and 1275 were detected.

309

Assuming that these m/z values, like all GBG oligomers, originate from Na+ adducts, it

310

seems plausible that m/z 657 and 977 correspond to a GBG dimer with two oxidized Cα

311

groups (Mw=2×GBG-6H) and a GBG trimer with one oxidized Cα group

312

(Mw=3×GBG-6H), respectively. Although these products were not detected using

313

UHPLC-MS, a GBG dimer and oxidized dimer were detected upon incubation with

314

ABTS·+ (Figure S3). It can therefore not be excluded that GBG is converted into

315

(oxidized) oligomers in the laccase/ABTS system. Another indication for this is that all

316

GBG had reacted during the first minute, but approximately 45% of the ABTS was still

317

present after 1 hour. Since an equimolar ratio of GBG and ABTS was used, it is

318

impossible that all GBG coupled to ABTS, and that GBG-ABTS CPIIox is the only

319

reaction product. Furthermore, polymerization of a phenolic model compound with

320

laccase and ABTS has been shown before, using SEC28 and MALDI-TOF.29

321

The role of the mediator in LMS incubations of GBG

15



322

Lignin contains 70-90% non-phenolic and 10-30% phenolic subunits.5 As the phenolic

323

lignin structures can be oxidized by laccase alone, the question remains whether and

324

how the mediator plays a role in the reaction rate and pathway when phenolic structures

325

are incubated with LMS. Based on the results discussed above, it can be concluded that

326

the role of HBT is very limited, in contrast to ABTS, which strongly influenced both

327

rate and pathway. Whereas the efficiency of a mediator towards non-phenolic lignin

328

structures has been reported to depend on its stability as a radical, and to its potency to

329

abstract a radical from a non-phenolic substrate38 (via electron transfer or radical

330

hydrogen atom transfer), we hypothesized that the role of the mediator towards phenolic

331

substrates is dominated by the rate of its oxidation. The large difference in redox

332

potentials, i.e. 1.08 V vs. NHE for HBT and 0.69 V vs. NHE for ABTS/ABTS·+,38

333

suggests that the oxidation of HBT by laccase occurs much slower than that of ABTS to

334

ABTS·+. This was confirmed by oxygen consumption measurements of both mediators

335

and GBG with laccase (Figure S5). Oxygen consumption of ABTS is 1.9 times faster

336

than that of GBG, whereas no substantial oxygen consumption of HBT with laccase was

337

detected within the time frame used. So, although oxidized HBT may probably undergo

338

a (redox) reaction with GBG, the conversion of GBG by laccase alone is already

339

complete before a substantial amount of HBT is oxidized. Consequently, the role of

340

HBT is negligible in this system. In contrast, the fast conversion of ABTS to ABTS·+

341

allows ABTS·+ to compete with GBG radicals in coupling reactions and to induce Cα

342

oxidations before GBG radicals start to polymerize. These results indicate that the

343

influence of the mediator on the conversion of a phenolic lignin structure is highly

344

dependent on its oxidation rate by laccase. It should be noted that the formation of

345

substrate-mediator adducts may also be influenced by the bulkiness and presence of

16


Page 16 of 28

Page 17 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


346

reactive groups on the mediator, but the comparison between HBT and ABTS does not

347

give any insights in this.

348

Conclusions

349

The mediators HBT and ABTS strongly differed regarding their influence on GBG

350

conversion by laccase. The influence of HBT was negligible, but ABTS strongly increased

351

the conversion rate of GBG and altered the followed reaction pathways. Whereas laccase

352

and laccase/HBT incubations resulted in extensive polymerization of GBG, incubation with

353

the laccase/ABTS system mainly resulted in Cα oxidation and coupling between GBG and

354

ABTS. This difference in influence between HBT and ABTS can be explained by the much

355

higher oxidation rate of ABTS by laccase, as compared to that of HBT. Extrapolating to

356

polymeric lignin, our results suggest that the use laccase/ABTS will most likely result in a

357

large degree of ABTS grafting on the phenolic lignin subunits, whereas such grafting

358

reactions are less likely to occur with laccase/HBT. As the GBG-ABTS adducts were found

359

to be relatively stable, this suggests that, once ABTS has been grafted on phenolic lignin

360

subunits, further polymerization by laccase is blocked. These insights can be helpful for

361

choosing the optimal mediator in a LMS, in which the mediator should not only be selected

362

based on its efficiency in oxidation of non-phenolic lignin subunits, but also on its

363

reactivity with the phenolic subunits.

364

Supporting Information: Detailed experimental information (laccase purification, LC-MS

365

and MALDI-TOF-MS), LC-MS data of minor reaction products, LC-MS chromatograms of

366

all time points, MALDI-TOF spectra (2-20 min), LC-MS results of GBG oxidation by

367

ABTS·+ and O2 consumption measurements.

368

17



369

References

370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433

1. Yao, B.; Ji, Y., Lignin biodegradation with laccase-mediator systems. Front. Energy Res. 2014, 2, 12. 2. Bugg, T. D.; Rahmanpour, R., Enzymatic conversion of lignin into renewable chemicals. Curr. Opin. Chem. Biol. 2015, 29, 10-17. 3. Munk, L.; Sitarz, A. K.; Kalyani, D. C.; Mikkelsen, J. D.; Meyer, A. S., Can laccases catalyze bond cleavage in lignin? Biotechnol. Adv. 2015, 33 (1), 13-24. 4. Areskogh, D.; Li, J.; Gellerstedt, G. R.; Henriksson, G., Investigation of the molecular weight increase of commercial lignosulfonates by laccase catalysis. Biomacromolecules 2010, 11 (4), 904-910. 5. Lundquist, K.; Parkås, J., Different types of phenolic units in lignins. BioResources 2011, 6 (2), 920-926. 6. Huber, D.; Ortner, A.; Daxbacher, A.; Nyanhongo, G. S.; Bauer, W.; Guebitz, G. M., Influence of oxygen and mediators on laccase-catalyzed polymerization of lignosulfonate. ACS Sustain. Chem. Eng. 2016, 4 (10), 5303-5310. 7. Munk, L.; Punt, A.; Kabel, M.; Meyer, A. S., Laccase catalyzed grafting of–N–OH type mediators to lignin via radical–radical coupling. RSC Adv. 2017, 7 (6), 3358-3368. 8. Bourbonnais, R.; Paice, M.; Reid, I.; Lanthier, P.; Yaguchi, M., Lignin oxidation by laccase isozymes from Trametes versicolor and role of the mediator 2, 2'-azinobis (3-ethylbenzthiazoline-6sulfonate) in kraft lignin depolymerization. Appl. Environ. Microbiol. 1995, 61 (5), 1876-1880. 9. Moya, R.; Saastamoinen, P.; Hernández, M.; Suurnäkki, A.; Arias, E.; Mattinen, M.-L., Reactivity of bacterial and fungal laccases with lignin under alkaline conditions. Bioresour. Technol. 2011, 102 (21), 10006-10012. 10. Prasetyo, E. N.; Kudanga, T.; Østergaard, L.; Rencoret, J.; Gutiérrez, A.; José, C.; Santos, J. I.; Nieto, L.; Jiménez-Barbero, J.; Martínez, A. T., Polymerization of lignosulfonates by the laccase-HBT (1hydroxybenzotriazole) system improves dispersibility. Bioresour. Technol. 2010, 101 (14), 5054-5062. 11. Fernaud, J. H.; Carnicero, A.; Perestelo, F.; Cutuli, M. H.; Arias, E.; Falcón, M., Upgrading of an industrial lignin by using laccase produced by Fusarium proliferatum and different laccase-mediator systems. Enzyme Microb. Technol. 2006, 38 (1), 40-48. 12. Shleev, S.; Persson, P.; Shumakovich, G.; Mazhugo, Y.; Yaropolov, A.; Ruzgas, T.; Gorton, L., Interaction of fungal laccases and laccase-mediator systems with lignin. Enzyme Microb. Technol. 2006, 39 (4), 841-847. 13. Xia, Z.; Yoshida, T.; Funaoka, M., Enzymatic synthesis of polyphenols from highly phenolic lignin-based polymers (lignophenols). Biotechnol. Lett. 2003, 25 (1), 9-12. 14. Bourbonnais, R.; Paice, M.; Freiermuth, B.; Bodie, E.; Borneman, S., Reactivities of various mediators and laccases with kraft pulp and lignin model compounds. Appl. Environ. Microbiol. 1997, 63 (12), 4627-4632. 15. Bourbonnais, R.; Paice, M. G., Demethylation and delignification of kraft pulp by Trametes versicolor laccase in the presence of 2, 2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate). Appl. Microbiol. Biotechnol. 1992, 36 (6), 823-827. 16. Couto, S. R.; Herrera, J. L. T., Industrial and biotechnological applications of laccases: a review. Biotechnol. Adv. 2006, 24 (5), 500-513. 17. Bourbonnais, R.; Paice, M. G., Oxidation of non-phenolic substrates: an expanded role for laccase in lignin biodegradation. FEBS Lett. 1990, 267 (1), 99-102. 18. Baiocco, P.; Barreca, A. M.; Fabbrini, M.; Galli, C.; Gentili, P., Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccase–mediator systems. Org. Biomol. Chem. 2003, 1 (1), 191-197. 19. Muheim, A.; Fiechter, A.; Harvey, P. J.; Schoemaker, H. E., On the mechanism of oxidation of non-phenolic lignin model compounds by the laccase-ABTS couple. Holzforschung 1992, 46 (2), 121126. 20. Eggert, C.; Temp, U.; Dean, J. F.; Eriksson, K.-E. L., A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase. FEBS Lett. 1996, 391 (1), 144-148. 21. Astolfi, P.; Brandi, P.; Galli, C.; Gentili, P.; Gerini, M. F.; Greci, L.; Lanzalunga, O., New mediators for the enzyme laccase: mechanistic features and selectivity in the oxidation of non-phenolic substrates. New J Chem. 2005, 29 (10), 1308-1317. 22. Kawai, S.; Nakagawa, M.; Ohashi, H., Aromatic ring cleavage of a non‐phenolic β‐O‐4 lignin model dimer by laccase of Trametes versicolor in the presence of 1‐hydroxybenzotriazole. FEBS lett. 1999, 446 (2-3), 355-358. 23. Kawai, S.; Asukai, M.; Ohya, N.; Okita, K.; Ito, T.; Ohashi, H., Degradation of a non-phenolic β-O-4 substructure and of polymeric lignin model compounds by laccase of Coriolus versicolor in the presence of 1-hydroxybenzotriazole. FEMS Microbiol. Lett. 1999, 170 (1), 51-57. 24. Christopher, L. P.; Yao, B.; Ji, Y., Lignin biodegradation with laccase-mediator systems. Front. Energy Res. 2014, 2, 12. 25. Kawai, S.; Umezawa, T.; Shimada, M.; Higuchi, T., Aromatic ring cleavage of 4, 6-di (tertbutyl) guaiacol, a phenolic lignin model compound, by laccase of Coriolus versicolor. FEBS lett. 1988, 236 (2), 309-311.

18


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434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466

26. Kawai, S.; Umezawa, T.; Higuchi, T., Degradation mechanisms of phenolic β-1 lignin substructure model compounds by laccase of Coriolus versicolor. Arch. Biochem. Biophys. 1988, 262 (1), 99-110. 27. Areskogh, D.; Li, J.; Nousiainen, P.; Gellerstedt, G.; Sipilä, J.; Henriksson, G., Oxidative polymerisation of models for phenolic lignin end-groups by laccase. Holzforschung 2010, 64 (1), 21-34. 28. Rittstieg, K.; Suurnäkki, A.; Suortti, T.; Kruus, K.; Guebitz, G. M.; Buchert, J., Polymerization of guaiacol and a phenolic β‐O‐4‐substructure by Trametes hirsuta laccase in the presence of ABTS. Biotechnol. Progr. 2003, 19 (5), 1505-1509. 29. Potthast, A.; Rosenau, T.; Koch, H.; Fischer, K., The reaction of phenolic model compounds in the laccase-mediator system (LMS) investigations by matrix assisted laser desorption ionization time-offlight mass spectrometry (MALDI-TOF-MS). Holzforschung 1999, 53 (2), 175-180. 30. Johannes, C.; Majcherczyk, A., Laccase activity tests and laccase inhibitors. J. Biotechnol. 2000, 78 (2), 193-199. 31. Ramalingam, B.; Sana, B.; Seayad, J.; Ghadessy, F.; Sullivan, M., Towards understanding of laccase-catalysed oxidative oligomerisation of dimeric lignin model compounds. RSC Adv. 2017, 7 (20), 11951-11958. 32. Li, K.; Helm, R. F.; Eriksson, K. E. L., Mechanistic studies of the oxidation of a non‐phenolic lignin model compound by the laccase/1‐hydroxybenzotriazole redox system. Biotechnol. Appl. Biochem. 1998, 27 (3), 239-243. 33. Sealey, J.; Ragauskas, A. J., Investigation of laccase/N-hydroxybenzotriazole delignification of kraft pulp. J. Wood Chem. Technol. 1998, 18 (4), 403-416. 34. Matsumura, E.; Yamamoto, E.; Numata, A.; Kawano, T.; Shin, T.; Murao, S., Structures of the laccase-catalyzed oxidation products of hydroxy-benzoic acids in the presence of ABTS [2, 2′-Azino-di(3-ethylbenzothiazoline-6-sulfonic Acid)]. Agric. Biol. Chem. 1986, 50 (5), 1355-1357. 35. Osman, A.; Wong, K.; Fernyhough, A., ABTS radical-driven oxidation of polyphenols: Isolation and structural elucidation of covalent adducts. Biochem. Biophys. Res. Commun. 2006, 346 (1), 321329. 36. Cañas, A. I.; Camarero, S., Laccases and their natural mediators: biotechnological tools for sustainable eco-friendly processes. Biotechnol. Adv. 2010, 28 (6), 694-705. 37. Bourbonnais, R.; Leech, D.; Paice, M. G., Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. Biochim. Biophys. Acta 1998, 1379 (3), 381-390. 38. Fabbrini, M.; Galli, C.; Gentili, P., Comparing the catalytic efficiency of some mediators of laccase. J. Mol. Catal. B: Enzym. 2002, 16 (5), 231-240.

467

19



468

Figures and Tables

469 470 471

Figure 1. Molecular structures of guaiacylglycerol-β-guaiacyl ether (GBG), 1-hydroxybenzotriazole (HBT) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS).

472

20


Page 20 of 28

Page 21 of 28

100

Relative Intensity (%)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


GBG dimer

A

GBG trimer 0 100

GBG dimer HBT

0 100

GBG-HBT CP

B

GBG trimer

ABTS CLP

C

GBG-ABTS CPII ABTS GBG-ABTS CPIIox Unknown 0 5

473 474 475 476

10

15

20

25

30

35

40

45

Time (min)

Figure 2. RP-UHPLC-MS chromatograms (negative mode) of GBG incubated for 5 min with laccase (A), laccase/HBT (B) and laccase/ABTS (C). Chromatograms of other time points can be found in the Supporting Information.

21



477 478 479 480 481 482

Page 22 of 28

Table 1. Compounds detected with UHPLC-PDA-ESI-ITMS and UHPLC-PDA-ESI-FTMS after incubation of GBG with laccase from T. versicolor in the presence or absence of ABTS and HBT. MS2 fragments and λmax were determined using UHPLC-PDA-ESI-ITMS. All other values were obtained using UHPLC-PDA-ESI-FTMS. Relative intensities of MS2 fragments are shown between brackets.* For ABTS, only data from UHPLC-PDA-ESI-ITMS were used, as FTMS only showed in-source fragmentation. N.D. = Not detected. Observed/ Mass MS2 fragments calculated error mass (ppm) Reaction products of GBG incubated with laccase alone C17H20O6 [M+Na]+ 320.12556/ -1.34 295 (55), 302 (16), 201 (8), 147 320.12599 (6), 219 (5), 176 (4) C34H38O12 [M-H]638.23720/ 1.36 589 (100), 483 (62), 513 (28), 435 638.23633 (26), 329 (20), 465 (12), 541 (12), 359 (8) C51H56O18 [M-H]956.34775/ 1.13 907 (100), 889 (60), 919 (30), 859 956.34666 (20) Reaction products of GBG incubated with laccase/HBT C6H5N3O [M-H]135.04329/ 0.23 106 (100), 78 (4) 135.04326 91 (63), 80 (40), 53 (18), 107 (8) [M+H]+

RT (min)

Tentative annotation

20.8

GBG

34.8

GBG dimer

44.7

GBG trimer

3.9

HBT

7.2

BT

C6H5N3

[M+H]+

20.9

GBG

C17H20O6

[M+Na]+

25.1

C23H23N3O7

[M-H]-

C23H21N3O7

[M-H]-

34.4

GBG-HBT CP GBG-HBT CPox GBG dimer

C34H38O12

[M-H]-

44.7

GBG trimer

5.6

ABTS CLP

8.7 17.0

Unknown Unknown

Unknown C25H25N4O8S4

[M-H][M-2H]-

329.99756 637.05667/ 637.05553

21.4

ABTS

C18H18N4O6S4

[M-2H]2-

514*

22.7

GBG-ABTS CPII GBG-ABTS CPIox

C26H27N3O9S2

[M-H]-

C35H35N4O12S4

[M-3H]2-

589.11946/ 589.118876 831.11369/ 831.11289

C17H18O6

[M-2H][M-H]-

31.9

24.4

28.3

GBGox

Molecular formula

Ionization

119.04832/ 119.04835 320.12556/ 320.12599 453.15375/ 453.15360 451.13838/ 451.13795 638.23720/ 638.23633

278 278

N.D.

306, 276, 269

-0.23

N.D.

-1.34

295, 302, 201, 219, 147, 176

260, 280 278

0.33

404 (100), 328 (38), 298 (30)

330

0.95

420 (100), 310 (27), 326 (15)

N.D.

589 (100), 483 (62), 513 (28), 435 (26), 329 (20), 465 (12), 541 (12), 359 (8) C51H56O18 [M-H]956.34775/ 1.13 907 (100), 889 (60), 919 (30), 859 956.34666 (20) Reaction products of GBG incubated with laccase/ABTS C9H9NO4S2 [M-H]258.99757/ 1.05 229 (37) 258.99730

318.11017/ 318.11034

λmax (nm)

1.36

1.79

302 (100), 238 (29), 222 (9) 363 (100), 378 (95), 228 (34), 605 (31), 405 (26), 257 (25), 335 (18)

278

N.D.

256, 292, 285 278 305

N.D.

342

0.99

242 (100), 214 (61), 228 (10)

550

0.96

572 (100), 228 (43), 256 (42)

305, 278

-0.52

N.D. 287 (100), 193 (71)

[M+H]+

280, 311

167 (100), 149 (70), 271 (68), 151 (59), 195 (44), 269 (32), 177 (26), 289 (22)

[M+Na]+

30.7

GBG-ABTS CPIIox

C26H25N3O9S2

[M-H]-

587.10366/ 587.10322

22


0.76

311 (100), 218 (16), 146 (11), 187 (6) 242, 214, 556, 228

585, 448, 328

Page 23 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


I

I

III

IV III

295 = II + III 201 = I + IV 219 = I 147 = I

II

GBG (320 Da)

404 = II + III 328 = I 298 = I + III

II

242 = V 214 = V + VI 228 = V + VII

VII VI

V

GBG-ABTS CPII (589 Da)

GBG-HBT CP (453 Da)

I

I

III

III

III

287 = III 193 = I

326 = III 420 = I

242 = V 214 = V + VI 556 = III 228 = V + VII

VII VI

V

GBGox (318 Da)

GBG-HBT Cpox (451 Da)

GBG-ABTS CPIIox (587 Da)

II III

I

II

I

III

III I

II IX VIII

V

II I

VII

III III

I

II

GBG dimer (638 Da)

GBG trimer (956 Da)

589 = II + III 483 = I + III 513 = I 435 = I + II + III + III 329 = I + I + III + III 465 = I + II + III 541 = II + II + III + III 359 = I + I + III

907 = I + III 889 = I + II + III 919 = II + II 859 = I + II + III + III

GBG-ABTS CPIox (831 Da) 572 = VIII* 256 = VIII* + IX 228 = V + VII X

ABTS CLP (259 Da) 229 = X*

483 484 485 486 487 488 489 490 491 492

Figure 3. Proposed structures of reaction products of GBG after incubation with laccase and LMS. The dotted lines represent the proposed fragmentation pattern, resulting in the MS2 fragments reported in Table 1. The fragmentation pattern of GBG and GBG-ABTS CPIox originate from the parent ions [M+Na]+ and [M-3H]2-, respectively. All other patterns originate from the parent ion [M-H]-. In the coupling products and oligomers, the positions of coupling could not be identified based on MS2 spectra, and are therefore based on literature.31,34-35 The position of the second interunit bond in the GBG trimer is not specified, as there is no experimental evidence for either C-C or C-O linkage. Fragmentations with an * (in GBG-ABTS CPIox and ABTS CLP) are suggested to be radical fragmentations.

23



100

100

A 50 0 100

B 50

Abundance (% of max)

Abundance (% of max)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

60 40 20 0

0

494 495 496 497 498 499 500 501 502

C

80

0

493

Page 24 of 28

10

20

30

40

50

60

time (min)

0

10

20

30

40

time (min)

Figure 4. Normalized MS peak areas in time of reaction products formed upon incubation of GBG with laccase alone (A), laccase/HBT (B) and laccase/ABTS (C). Areas were obtained from extracted ion chromatograms: GBG (m/z 343) ( ● ), GBG dimer (m/z 637) ( ● ), GBG trimer (m/z 955) ( ● ), GBG-HBT CP (m/z 452) ( ● ), GBG-HBT CPox (m/z 450) ( ○ ), GBGox (m/z 319 and 341) ( ○ ), GBGABTS CPIox (m/z 414) ( ● ), GBG-ABTS CPII (m/z 588) ( ● ), GBG-ABTS CPIIox (m/z 586) ( ○ ) and ABTS CLP (m/z 258) ( ● ). For the abundance (Y-axis), the time point with the largest peak area was set to 100%. For the other time points, the abundance was calculated as the peak area relative to the area of this largest peak. Amounts withdrawn per time point and injected to UHPLC-MS analysis were the same for all time points.

24


50

60

Page 25 of 28 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


503

lac

lac 1

Further polymerization

2 lac slow

lac slow lac


lac

504 505 506 507 508 509 510 511 512

Figure 5. Overview of proposed reactions occurring after incubation of GBG with laccase alone or a laccase/HBT system. The thick arrows represent the major reaction pathway. The reactions represented by thin arrows do occur, but slower and/or to a smaller extent. In case of laccase alone, all GBG reacts via route 1. In case of a laccase/HBT system, GBG reacts via route 1 and 2, but route 1 is favored due to the slow oxidation of HBT. Oxidation of GBG-HBT CP is proposed to occur via radical hydrogen abstraction by either a HBT radical (HBT·) or an oligomeric GBG radical ((GBG)n·). The C-C coupling in the dimer is based on Ramalingam et al.31 For clarity, reductions of O2 to H2O by laccase are not shown in the figure.

513

25



lac ABTS·+

ABTS

lac

lac or

GBG

GBG·

ABTS·+ ABTS

GBGox

ABTS·+ ABTS

·

ABTS·+

GBGox·

radical coupling

or ABTS·+ ABTS

radical coupling

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 28

ABTS +

GBG· ABTS·+ GBG-ABTS CPI

GBG-ABTS CPIox

ABTS

lac

lac ABTS CLP

lac or ABTS·+ ABTS

GBGox GBG-ABTS CPII

514 515 516 517 518 519 520 521

GBG-ABTS CPIIox

Figure 6. Overview of proposed reactions occurring after incubation of GBG with a laccase/ABTS system. GBG can be oxidized directly by laccase, or indirectly via ABTS·+, which is formed by laccase. The GBG radicals may react further to GBGox or may undergo radical coupling with ABTS·+. After coupling of GBG and ABTS, further rearrangements take place in which part of the ABTS molecule is cleaved from the reaction product. The insert shows the proposed mechanism of GBGox formation from GBG·. For clarity, reductions of O2 to H2O by laccase are not shown in the figure.

26


Page 27 of 28

522 1.5

II I II

B

III

1.0

IV

0.5

V

0 1.5

C

IX VII VIII 1000

X 1500

Peak

IV VI

0.5 0 500

524 525 526

III

V

0.5 0 1.5

1.0

523

A

I

1.0

Intensity (× 104)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2000

2500

I II III IV V VI VII VIII IX X

Tentative annotation GBG trimer GBG tetramer GBG pentamer GBG hexamer GBG heptamer GBG octamer GBG dimer (2 OX) GBG trimer (OX) Unknown Unknown

Ionization m/z observed [M+Na]+ 979.2 [M+Na]+ 1297.3 [M+Na]+ 1615.4 [M+Na]+ 1933.5 [M+Na]+ 2251.6 [M+Na]+ 2569.6 [M+Na]+ 657.1 [M+Na]+ 977.2 Unknown 989.1 Unknown 1275.2

3000

m/z

Figure 7. MALDI-TOF spectra (left) of GBG after 60 min incubation with laccase alone (A), laccase/HBT (B) and laccase/ABTS (C) measured in positive mode, and corresponding tentative peak annotations (right). OX = Cα hydroxyl oxidized to ketone.

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TOC/Abstract graphic (For Table of Contents Use Only)


Stable

phenolic lignin dimer 529 530

Synopsis:

531

This paper contributes to a more fundamental understanding of lignin modification by laccasemediator systems.

532 533 534

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mediator systems: Their reactivity towards phenolic lignin

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