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Article
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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Page 1 of 28 ACS Sustainable Chemistry & Engineering
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4 1 Laccase/mediator systems: Their reactivity towards phenolic lignin
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5 Roelant Hilgers, Jean-Paul Vincken, Harry Gruppen and Mirjam A. Kabel*
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17 6 Laboratory of Food Chemistry, Wageningen University and Research, the Netherlands
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23 8 Corresponding author:
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26 9 Dr. ir. Mirjam Kabel
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29 10 Laboratory of Food Chemistry
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32 11 Wageningen UR
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38 13 6708 WG Wageningen
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41 14 The Netherlands
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44 15 Tel: +31 (0)317 483209
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47 16 E-mail: mirjam.kabel@wur.nl
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ACS Sustainable Chemistry & Engineering Page 2 of 28
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4 19 Abstract
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7 20 Laccase-mediator systems (LMS) have been widely studied for their capacity to oxidize
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9 21 the non-phenolic subunits of lignin (70-90% of the polymer). The phenolic subunits
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11 22 (10-30% of the polymer), which can also be oxidized without mediators, have received
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13 23 considerably less attention. Consequently, it remains unclear to what extent the presence
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24 of a mediator influences the reactions of the phenolic subunits of lignin. To get more
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18 25 insight in this, UHPLC-MS was used to study the reactions of a phenolic lignin dimer
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20 26 (GBG), initiated by a laccase from Trametes versicolor, alone or in combination with
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22 27 the mediators HBT and ABTS. The role of HBT was negligible, as its oxidation by
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24 28 laccase occurred slowly in comparison to that of GBG. Laccase and laccase/HBT
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26 29 oxidized GBG at a comparable rate, resulting in extensive polymerization of GBG. In
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30 contrast, laccase/ABTS converted GBG at a higher rate, as GBG was oxidized both
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31 31 directly by laccase, but also by ABTS radical cations, which were rapidly formed by
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33 32 laccase. The laccase/ABTS system resulted in Cα oxidation of GBG and coupling of
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35 33 ABTS to GBG, rather than polymerization of GBG. Based on these results, we propose
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37 34 reaction pathways of phenolic lignin model compounds with laccase/HBT and
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39 35 laccase/ABTS.
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42 36 Keywords: Oxidation; radical coupling; Trametes versicolor; ABTS;
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37 hydroxybenzotriazole
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4 38 Introduction
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7 39 Lignin is one of the most abundant polymers in nature as part of plant cell walls, and
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9 40 currently considered as sustainable precursor for chemicals or materials.1-4 It is built up
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11 41 from sinapyl alcohol, coniferyl alcohol and p-coumaroyl alcohol (S, G and H units,
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13 42 respectively), which couple via radical polymerization to form a variety of C-C and C-O
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43 inter-unit linkages. The β-O-4 linkage is the most abundant one, accounting for
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18 44 approximately 45 to 94 % of the total inter-unit linkages, in lignins mildly isolated from
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20 45 plant materials.3 Lignin contains phenolic and non-phenolic subunits, which account for
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22 46 10-30 and 70-90% of the polymer, respectively.5
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25 47 Although the valorization of lignin is still underexploited,3 three main routes are
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27 48 considered relevant. The first is via lignin polymerization, which may result in
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29 49 improved binders and adhesives.6 Second, lignin depolymerization may lead to high-
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50 value low-molecular-weight aromatics.1-2 A third option is the functionalization of
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34 51 lignin via grafting of specific molecules onto lignin.7 A green alternative for lignin
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36 52 valorization is via biocatalysis and, in particular, laccase is known as one of the key
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38 53 activities towards lignin. Various lignin modifications are reported for laccase, such as
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40 54 polymerization,4, 8-10
depolymerization,11-13 Cα oxidation14 and demethylation.15 It is
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42 55 poorly understood, however, how to direct lignin modification by laccase towards one
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56 of these modifications. Thus, for an effective use of laccase in lignin valorization, it is
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57 essential to have a better understanding on how to control lignin modification by
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49 58 laccase.
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52 59 Laccases (E.C. 1.10.3.2) are oxidases that couple the reduction of molecular oxygen to
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54 60 the one-electron oxidation of a wide variety of aromatic substrates. The most powerful
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4 61 laccases are produced by white-rot fungi, and can have redox potentials up to 800 mV
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6 62 vs. NHE.3 With respect to laccase activity towards lignin, it is important to distinguish
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8 63 the phenolic and non-phenolic subunits in lignin. The phenolic subunits can directly be
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64 oxidized by laccase, as the redox potentials of such subunits are sufficiently low. In
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13 65 contrast, the non-phenolic parts have redox potentials up to 1500 mV vs. NHE, and are
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15 66 therefore, recalcitrant to oxidation by laccase alone.16 Nevertheless, when laccase is
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17 67 combined with a so-called mediator, oxidation of non-phenolic lignin structures is
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19 68 possible as well.17 In such a laccase/mediator system (LMS), the mediator is first
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21 69 oxidized by the laccase, after which it can oxidize non-phenolic substrates via different
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23 70 mechanisms, such as electron transfer (ET) or radical hydrogen atom transfer (HAT).18
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71 As a LMS is required for the oxidation of the non-phenolic subunits in lignin, and the
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28 72 phenolic subunits can be oxidized by laccase alone, research on lignin modification by
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30 73 LMS has mainly focused on the reactivity of non-phenolic lignin subunits,14, 17-21 and
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32 74 several reaction pathways have been described for such incubations.22-24 Nevertheless,
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34 75 as lignin also contains a considerable amount of phenolic subunits, it is important to
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36 76 understand how these subunits react when incubated with LMS.
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39 77 Research on lignin conversion by laccase and LMS is often performed using lignin
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78 model compounds. Phenolic lignin model compounds have been used, but most often in
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44 79 the absence of mediators.25-27 A few studies have been published on the reactions of
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46 80 phenolic lignin model compounds in LMS incubations, and mainly polymerization was
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48 81 shown to occur. Conclusions about the degree of polymerization reached were obtained,
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50 82 but no insights about the structure of the (initially) formed reaction products could be
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52 83 provided.28-29 Consequently, it remains unclear how the phenolic subunits of lignin are
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84 modified by LMS and to what extent mediators play a role in such modifications.
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4 85 In the current research, we used the phenolic β-O-4 linked lignin model compound, 1-
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6 86 (4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol,
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8 87 (guaiacylglycerol-β-guaiacyl ether, GBG), to mimic the phenolic subunits in lignin. We
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88 employed RP-UHPLC-PDA-ESI-MSn, to study detailed reaction pathways of GBG
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13 89 initiated by laccase (from Trametes versicolor) with or without a mediator. 2,2'-Azino-
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15 90 bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 1-hydroxybenzotriazole
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17 91 (HBT) were used as mediators, as these are the most commonly used mediators in
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19 92 literature. MALDI-TOF-MS was used to investigate the formation of larger reaction
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21 93 products.
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27 95 Materials and Methods
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30 96 Materials
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33 97 Guaiacylglycerol-β-guaiacyl ether (GBG; Figure 1) was obtained from TCI chemicals
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35 98 (Toyo, Japan) and 2,5-dihydroxybenzoic acid (DHB) was obtained from Bruker
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37 99 Daltonics (Bremen, Germany). Laccase from Trametes versicolor and all other
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100 chemicals were purchased from Sigma Aldrich (St. Louis, MO, USA). The laccase was
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42 101 partially purified, after which the activity was determined spectrophotometrically by
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44 102 oxidation of ABTS17 (1 U = 1 µmol ABTS oxidized per minute) (see S2 for details).
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46 103 Water was prepared using a Milli-Q water purification system (Merck Millipore,
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48 104 Billerica, MA).
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51 105 Incubation of GBG with laccase and laccase/mediator systems
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4 106 GBG was dissolved at 0.5 mM in sodium phosphate buffer (50 mM, pH 4) with or
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6 107 without an equimolar concentration of ABTS or HBT. Laccase was added to reach a
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8 108 final substrate and mediator concentration of 0.4 mM and a laccase activity of 0.1 U
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109 mL-1. The mixtures were incubated at 40 °C and 400 rpm in a thermomixer (Eppendorf,
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13 110 Hamburg, Germany). After 1, 2, 5, 10, 15, 20, 30 and 60 min, and after 24 h, 50 µL of
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15 111 the sample was transferred to another tube, and 5 µL sodium azide (20 mM) was added
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17 112 to stop the reaction.30 As no azide-related products were detected, and reaction products
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19 113 were stable in the presence of an excess of azide, the addition of azide was considered
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21 114 not to influence the reaction product profile (results not shown). The resulting samples
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23 115 were diluted 10 times in MilliQ water and were centrifuged (10000×g, 5 min, 20 ºC)
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116 prior to analysis by RP-UHPLC-PDA-MSn. A detailed description of the RP-UHPLC-
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28 117 PDA-MSn analysis can be found in S3 and S4 of the Supporting Information.
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31 118 Incubation of GBG with ABTS radical cations
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34 119 GBG was dissolved at 0.5 mM in sodium phosphate buffer (50 mM, pH 4). ABTS (2
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36 120 mM) was incubated with 0.20 U mL-1 laccase (2 min, 40 °C) and directly centrifuged
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38 121 over an Amicon Ultra-4 centrifugal filter (Merck Millipore) with a normalized
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40 122 molecular weight limit of 10 kDa. The dark green filtrate was incubated with the GBG
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42 123 solution at a ratio of 1:4 (2 min, 40 °C). As a control, the same incubation was done
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124 using ABTS that was not treated with laccase.
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47 125 Sample preparation for Matrix-Assisted Laser Desorption Ionization – Time Of
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126 Flight Mass Spectrometry (MALDI-TOF)
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127 The samples were prepared as described in 2.3, with the adaptation that the reaction was
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55 128 stopped after 7, 15 and 60 min. Samples were diluted 2 times in methanol, and a
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4 129 saturated solution of DHB was used as matrix. In a second experiment, similar
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6 130 incubations with laccase and laccase/HBT were stopped after 2, 5, 10, 15 and 20 min.
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8 131 For details about MALDI-TOF settings see S6.
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4 133 Results & Discussion
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7 134 Reaction products of GBG incubated with laccase
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10 135 GBG (Figure 1) was, first, incubated with laccase without addition of mediators. From
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12 136 the UHPLC-MS chromatograms, two main reaction products were observed upon
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14 137 incubation of GBG, both having their largest intensities during the first 20 min (Figure
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16 138 2, Table 1 and Figure S1). In addition, remaining GBG was detected (only trace
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139 amounts after 2 min). Using accurate mass determination the reaction products were
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21 140 found to correspond with molecular formulas C34H38H12 and C51H56O18 (Table 1).
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23 141 Based on their molecular formulas and the fact that the fragmentation patterns showed
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25 142 high similarities with GBG (Table 1 and Figure 3), these products were annotated as a
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27 143 dimer of GBG (molecular weight (Mw)=2×GBG-2H) and a trimer of GBG
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29 144 (Mw=3×GBG-4H; Table 1), formed via radical coupling. Recently, oligomerization of
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145 GBG was reported by others, and it was shown that coupling between two GBG radicals
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34 146 occurs via C-C bond formation.31 The same was suggested for the subsequent formation
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36 147 of a trimer, although no experimental evidence was given. Therefore, we have specified
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38 148 the coupling position in the dimer, but not of the second coupling in the trimer in Figure
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40 149 3. In addition to the dimer and trimer, only traces of other reaction products were
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42 150 observed (Table S1). After 24 h, precipitation was observed and no peaks were detected
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151 in the chromatograms, suggesting ongoing polymerization of the initially formed dimer
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152 and trimer. Thus, the activity of laccase alone was found to result in polymerization of
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49 153 GBG. This conclusion is in line with results on laccase activity towards phenolic model
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51 154 compounds reported by others.28, 31
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54 155 Reaction products of GBG incubated with a laccase/HBT system
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4 156 When GBG was incubated with laccase in combination with HBT, the reaction products
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6 157 formed were very comparable to those formed in the presence of laccase alone (Figure
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8 158 2, Table 1 and Figure S1). The same dimer and trimer were identified based on retention
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159 time, accurate mass and fragmentation pattern (Figure 2 and Table 1). Also the amounts
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13 160 were comparable, based on UV280 peak area. In addition to the dimer and trimer, a small
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15 161 peak (hardly visible) corresponding to the molecular formula C23H23N3O7 was observed.
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17 162 This, in combination with a fragmentation pattern similar to that of GBG (Table 1 and
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19 163 Figure 3), suggested the formation of a GBG-HBT radical coupling product (GBG-HBT
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21 164 CP, Figure 3). After prolonged incubation times (1 h) C23H21N3O7 was detected, which,
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23 165 most likely, corresponded to further oxidation of GBG-HBT CP to GBG-HBT CPox
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166 (Figure 3). It was recently shown for the first time that N-OH type mediators, such as
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28 167 HBT, couple to lignin upon addition of laccase.7 Our results confirm that such coupling
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30 168 can occur at the phenolic moieties of lignin, but also indicate that the extent of coupling
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32 169 between GBG and HBT is small as compared to GBG oligomerization. The proposed
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34 170 structures of all coupling products are depicted in Figure 3, together with their proposed
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36 171 MS2 fragmentation pathways. After 24 h, no GBG-related peaks were detected anymore
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172 and precipitation was observed. Approximately 45% of the original HBT was still
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41 173 present, as determined from UV280 peak areas (data not shown). Part of it was converted
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43 174 to benzotriazole (BT), which is inactive as a mediator.32 This conversion of HBT to BT
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45 175 has been described by others32-33 and also occurred in a control reaction with only HBT
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47 176 and laccase without GBG present (data not shown). Most likely, BT is formed upon a
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49 177 reaction of HBT radicals with aromatic amino acids of the enzyme.33
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52 178 Reaction products of GBG incubated with a laccase/ABTS system
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4 179 When GBG was incubated with laccase and ABTS, a larger variety of reaction products
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6 180 was detected compared to the incubations described above (Figure 2, Table 1 and Figure
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8 181 S1). Molecules with molecular formulas C17H18O6 and C35H35N4O12S4 were detected,
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182 especially after short reaction times (1-15 min). These were annotated as Cα oxidized
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13 183 GBG and a radical coupling product between oxidized GBG and ABTS (GBGox and
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15 184 GBG-ABTS CPIox in Table 1 and Figure 3), respectively. A relatively small peak
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17 185 corresponding to a coupling of non-oxidized GBG and ABTS (GBG-ABTS CPI) has
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19 186 been observed as well, but inconsistently. Likely, the latter coupling product is prone to
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21 187 react further. The structure and fragmentation pattern of GBG-ABTS CPI can be found
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23 188 in Figure S4. In addition, reaction products with molecular formulas C26H27N3O9S2,
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189 C26H25N3O9S2 were found. Based on these formulas and their fragmentation patterns,
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28 190 these products were annotated as GBG-ABTS CPII and GBG-ABTS CPIIox,
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30 191 respectively (Table 1 and Figure 3). Similar coupling products, between a phenolic
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32 192 compound and part of the ABTS molecule, have been reported for hydroxybenzoic
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34 193 acids34 and catechin,35 using NMR and mass spectrometry, respectively. To our
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36 194 knowledge, this is the first time that such coupling products are shown for lignin model
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195 compounds. The main MS2 fragments (m/z 242 and 214 in negative mode) match with
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41 196 the fragments reported for the catechin-ABTS products (m/z 244 and 216 in positive
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43 197 mode).35 As the adducts identified with NMR spectroscopy29 resulted from C-N
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45 198 coupling (rather than O-N coupling), the adducts in Figure 3 are also shown with a C-N
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47 199 bond between the GBG and ABTS moiety. In addition to these coupling products, a
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49 200 major peak was detected corresponding to C9H9NO4S2. As this molecule was hardly
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201 formed in a control reaction with only ABTS and laccase (data not shown), it was
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54 202 considered to be an ABTS cleavage product (ABTS CLP) formed upon coupling of
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4 203 GBG and ABTS. Lastly, a reaction product was detected with molecular formula
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6 204 C25H25N4O8S4. This could correspond to a radical coupling product of guaiacol and
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8 205 ABTS. It should be mentioned that for the formation of such a product cleavage of
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206 GBG to form guaiacol is required, either before or during coupling with ABTS. Besides
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13 207 the detection of C25H25N4O8S4, no indications were found for cleavage of GBG. In
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15 208 contrast to the incubation with laccase alone and laccase with HBT, no GBG was
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17 209 detected at all incubation times, suggesting a fast conversion of GBG into reaction
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19 210 products. The dimer and trimer found for incubations with laccase alone and
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21 211 laccase/HBT were absent at all incubation times when ABTS was used. After 24 h,
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23 212 approximately 29% of the initial amount ABTS was still present (data not shown).
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213 Whereas others reported polymerization as the main result of laccase/ABTS activity
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28 214 towards phenolic substrates,28-29 our results suggest that GBG preferably couples to
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30 215 ABTS, rather than to another GBG molecule.
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33 216 Reaction pathways of laccase and laccase/HBT with GBG
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36 217 To get more insight into the course of the reactions occurring during incubation of GBG
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38 218 with laccase and the laccase/mediator systems, product formation was monitored at 9
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40 219 different time points within the first hour. Hereto, peak areas from extracted ion
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42 220 chromatograms of the reaction products (Table 1) relative to the time point with the
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221 highest area, were plotted as function of incubation time (Figure 4). For clarity, only
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222 GBG-related reaction products are shown.
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49 223 Figure 4A and B show that, under the conditions used, a fast conversion of GBG to
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51 224 oligomeric products occurred. Already in the first minute, 67 and 69% of the GBG was
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53 225 converted by laccase and laccase/HBT, respectively. For both laccase and laccase/HBT
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55 226 it was shown that the dimer and the trimer gradually decreased in abundance and
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4 227 disappeared within one hour. The decrease of the dimer in the first 20 min occurred
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6 228 together with an increase in higher oligomers, indicating further polymerization (Figure
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8 229 S2). In the incubation with laccase/HBT, the coupling product GBG-HBT CP increased
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230 during the first 30 min, and then decreased again. The decrease of GBG-HBT CP
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13 231 coincided with an increase in its oxidized equivalent GBG-HBT CPox, confirming that
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15 232 GBG-HBT CP is oxidized further to GBG-HBT CPox. For such a conversion from
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17 233 alcohol to ketone at Cα, a benzylic radical should be formed. This may have occurred
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19 234 via radical hydrogen abstraction by a HBT radical. As a phenolic lignin structure itself
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21 235 could also act as HAT-type mediator,36 radical hydrogen abstraction may also have
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23 236 occurred by a phenoxyl radical of a GBG-related structure. If the latter indeed occurred,
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237 the oxidant probably was an oligomeric GBG radical, as all GBG was converted before
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28 238 conversion of GBG-HBT CP to GBG-HBT CPox took place (Figure 4).
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30 239 These findings indicate that upon incubation with both laccase and laccase/HBT,
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32 240 extensive polymerization of GBG takes place. Radical coupling between GBG and HBT
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34 241 occurred, but only to a small extent. The coupling product was further oxidized, and is
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36 242 probably prone to further polymerization. The rate of conversion of GBG and its
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243 oligomers does not seem to be influenced by the presence of HBT, suggesting that the
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41 244 role of HBT is negligible when laccase/HBT is used on phenolic substrates. A
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43 245 schematic overview of the proposed reactions is depicted in Figure 5.
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46 246 Reaction pathways of laccase/ABTS with GBG
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49 247 Similar to the incubations with laccase and laccase/HBT, the reaction of GBG with
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51 248 laccase/ABTS was monitored over time. Figure 4C shows that all GBG was converted
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53 249 within the first minute of incubation. Based on this and on the annotations discussed
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55 250 above, it can be stated that the laccase/ABTS system differs from laccase and
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4 251 laccase/HBT in two ways: (i) GBG is converted faster (higher rate), and (ii) GBG is
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6 252 converted into different reaction products (other pathways).
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9 253 A possible explanation for the higher conversion rate might be the rapid formation of
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11 254 ABTS radical cations (ABTS·+), which might undergo a redox reaction with GBG.
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13 255 ABTS·+ has been shown to induce oxidative polymerization of creosol, a phenolic
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256 monomeric lignin model compound29 and to undergo a redox reaction with vanillyl
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18 257 alcohol.37 To check whether a similar interaction between GBG and ABTS·+ occurred,
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20 258 GBG was incubated with laccase-free ABTS·+. GBG was indeed converted upon
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22 259 addition of ABTS·+, resulting in several reaction products: GBGox, GBG dimer,
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24 260 oxidized GBG dimer and GBG-ABTS CPI (Figure S3). The annotation of the latter two
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26 261 products can be found in Table S2 and Figure S4. As no GBG conversion was observed
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262 in a control reaction with neutral ABTS (Figure S3B), it was concluded that the higher
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31 263 GBG conversion rate by the laccase/ABTS system is caused by the fact that GBG is
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33 264 oxidized via two mechanisms: directly by laccase and via a redox reaction with
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35 265 ABTS·+. It cannot be excluded that also the dication ABTS++ (formed via
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37 266 disproportionation of ABTS·+) is involved in the oxidation of GBG. Nevertheless, as it
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39 267 has been shown with cyclic voltammetry that vanillyl alcohol can rapidly be oxidized
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268 by ABTS·+,37 we assume that the same applies to GBG.
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269 Figure S3 also shows that Cα oxidized GBG is formed upon addition of ABTS·+. The
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270 fact that Cα oxidized products were not observed in incubations with laccase alone
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49 271 implies that Cα oxidation is caused by ABTS·+ rather than laccase. Figure 4C shows
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51 272 that the concentrations of GBGox, GBG-ABTS CPIox are highest after 1 min of
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53 273 incubation, and that their concentration decreased relatively fast during the first 20 min.
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55 274 GBG-ABTS CPII was also highest after 1 min, but its decrease was slower. In contrast,
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4 275 the concentrations of GBG-ABTS CPIIox and ABTS CLP gradually increased during the
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6 276 first hour of incubation, suggesting that they were formed upon conversion of the other
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8 277 reaction products. After 24 h, most reaction products from Figure 4C had disappeared,
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278 except for ABTS CLP and GBG-ABTS CPIIox. The areas of these peaks had even
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12
13 279 increased with 33 and 23%, respectively (data not shown). These results indicate that
14
15 280 GBG formed coupling products with ABTS in the presence of laccase, after which a
16
17 281 part of the ABTS moiety was cleaved off. The ultimate product formed after coupling of
18
19 282 ABTS and GBG was GBG-ABTS CPIIox. To our knowledge, coupling of ABTS to
20
21 283 lignin and lignin model compounds has been suggested by others,8, 28 but the structure
22
23 284 of such lignin-ABTS coupling products has not been elaborated so far.
24
25
26 285 Based on the above, an overview of the course of the reactions leading to the formation
27
28
286 of GBG-ABTS CPIIox was constructed (Figure 6). We suggest that after oxidation,
29
30
31 287 GBG· undergoes two follow-up reactions: (i) Radical coupling with ABTS·+ to form an
32
33 288 intermediate product GBG-ABTS CPI, or (ii) A second oxidation (by ABTS·+) to form
34
35 289 GBGox. In the latter case, GBGox can be further oxidized (by laccase or ABTS·+) to
36
37 290 GBGox·, which then also couples to ABTS·+ to form GBG-ABTS CPIox. GBG-ABTS
38
39 291 CPI and GBG-ABTS CPIox are subsequently converted to GBG-ABTS CPII and GBG-
40
41
292 ABTS CPIIox, upon which ABTS CLP was split off as a byproduct. The exact
42
43
44 293 mechanism behind this is unknown, but it is suggested to involve an oxidation step, as
45
46 294 the intermediates (GBG-ABTS CPI and GBG-ABTS CPIox) did not react further after
47
48 295 inactivation of the laccase (Figure 4). Eventually, the formed GBG-ABTS CPII can be
49
50 296 oxidized to the ultimate reaction product GBG-ABTS CPIIox,
51
52
53 297 Formation of larger oligomeric reaction products
54
55
56
57
58
14
59
1
2
3
4 298 To investigate whether larger reactions products were formed upon incubation with
5
6 299 laccase or LMS, MALDI-TOF spectra were recorded. After 1 hour of incubation with
7
8 300 laccase and laccase/HBT, GBG oligomers up to DP 8 and DP 7 were detected,
9
10
301 respectively (Figure 7). As precipitation of reaction products occurred in both
11
12
13 302 incubations, and the mixture still contained small amounts of insoluble material after
14
15 303 dilution in methanol, it cannot be excluded that insoluble oligomers of higher DP were
16
17 304 formed. Besides the GBG oligomers, no other peaks were detected, suggesting that
18
19 305 incubation of GBG with laccase or laccase/HBT (almost) solely resulted in
20
21 306 oligomerization of GBG. In contrast, a completely soluble reaction mixture was
22
23 307 obtained after incubation with laccase/ABTS, in which all of the GBG oligomers were
24
25
26
308 absent. Instead, reaction products with m/z 657, 977, 989 and 1275 were detected.
27
28 309 Assuming that these m/z values, like all GBG oligomers, originate from Na+ adducts, it
29
30 310 seems plausible that m/z 657 and 977 correspond to a GBG dimer with two oxidized Cα
31
32 311 groups (Mw=2×GBG-6H) and a GBG trimer with one oxidized Cα group
33
34 312 (Mw=3×GBG-6H), respectively. Although these products were not detected using
35
36 313 UHPLC-MS, a GBG dimer and oxidized dimer were detected upon incubation with
37
38
314 ABTS·+ (Figure S3). It can therefore not be excluded that GBG is converted into
39
40
41 315 (oxidized) oligomers in the laccase/ABTS system. Another indication for this is that all
42
43 316 GBG had reacted during the first minute, but approximately 45% of the ABTS was still
44
45 317 present after 1 hour. Since an equimolar ratio of GBG and ABTS was used, it is
46
47 318 impossible that all GBG coupled to ABTS, and that GBG-ABTS CPIIox is the only
48
49 319 reaction product. Furthermore, polymerization of a phenolic model compound with
50
51
320 laccase and ABTS has been shown before, using SEC28 and MALDI-TOF.29
52
53
54
321 The role of the mediator in LMS incubations of GBG
55
56
57
58
15
59
1
2
3
4 322 Lignin contains 70-90% non-phenolic and 10-30% phenolic subunits.5 As the phenolic
5
6 323 lignin structures can be oxidized by laccase alone, the question remains whether and
7
8 324 how the mediator plays a role in the reaction rate and pathway when phenolic structures
9
10
325 are incubated with LMS. Based on the results discussed above, it can be concluded that
11
12
13 326 the role of HBT is very limited, in contrast to ABTS, which strongly influenced both
14
15 327 rate and pathway. Whereas the efficiency of a mediator towards non-phenolic lignin
16
17 328 structures has been reported to depend on its stability as a radical, and to its potency to
18
19 329 abstract a radical from a non-phenolic substrate38 (via electron transfer or radical
20
21 330 hydrogen atom transfer), we hypothesized that the role of the mediator towards phenolic
22
23 331 substrates is dominated by the rate of its oxidation. The large difference in redox
24
25
26
332 potentials, i.e. 1.08 V vs. NHE for HBT and 0.69 V vs. NHE for ABTS/ABTS·+,38
27
28 333 suggests that the oxidation of HBT by laccase occurs much slower than that of ABTS to
29
30 334 ABTS·+. This was confirmed by oxygen consumption measurements of both mediators
31
32 335 and GBG with laccase (Figure S5). Oxygen consumption of ABTS is 1.9 times faster
33
34 336 than that of GBG, whereas no substantial oxygen consumption of HBT with laccase was
35
36 337 detected within the time frame used. So, although oxidized HBT may probably undergo
37
38
338 a (redox) reaction with GBG, the conversion of GBG by laccase alone is already
39
40
41 339 complete before a substantial amount of HBT is oxidized. Consequently, the role of
42
43 340 HBT is negligible in this system. In contrast, the fast conversion of ABTS to ABTS·+
44
45 341 allows ABTS·+ to compete with GBG radicals in coupling reactions and to induce Cα
46
47 342 oxidations before GBG radicals start to polymerize. These results indicate that the
48
49 343 influence of the mediator on the conversion of a phenolic lignin structure is highly
50
51
344 dependent on its oxidation rate by laccase. It should be noted that the formation of
52
53
54 345 substrate-mediator adducts may also be influenced by the bulkiness and presence of
55
56
57
58
16
59
1
2
3
4 346 reactive groups on the mediator, but the comparison between HBT and ABTS does not
5
6 347 give any insights in this.
7
8
9 348 Conclusions
10
11
12 349 The mediators HBT and ABTS strongly differed regarding their influence on GBG
13
14 350 conversion by laccase. The influence of HBT was negligible, but ABTS strongly increased
15
16 351 the conversion rate of GBG and altered the followed reaction pathways. Whereas laccase
17
18
352 and laccase/HBT incubations resulted in extensive polymerization of GBG, incubation with
19
20
21 353 the laccase/ABTS system mainly resulted in Cα oxidation and coupling between GBG and
22
23 354 ABTS. This difference in influence between HBT and ABTS can be explained by the much
24
25 355 higher oxidation rate of ABTS by laccase, as compared to that of HBT. Extrapolating to
26
27 356 polymeric lignin, our results suggest that the use laccase/ABTS will most likely result in a
28
29 357 large degree of ABTS grafting on the phenolic lignin subunits, whereas such grafting
30
31
358 reactions are less likely to occur with laccase/HBT. As the GBG-ABTS adducts were found
32
33
34 359 to be relatively stable, this suggests that, once ABTS has been grafted on phenolic lignin
35
36 360 subunits, further polymerization by laccase is blocked. These insights can be helpful for
37
38 361 choosing the optimal mediator in a LMS, in which the mediator should not only be selected
39
40 362 based on its efficiency in oxidation of non-phenolic lignin subunits, but also on its
41
42 363 reactivity with the phenolic subunits.
43
44
45 364 Supporting Information: Detailed experimental information (laccase purification, LC-MS
46
47 365 and MALDI-TOF-MS), LC-MS data of minor reaction products, LC-MS chromatograms of
48
49
366 all time points, MALDI-TOF spectra (2-20 min), LC-MS results of GBG oxidation by
50
51
52 367 ABTS·+ and O2 consumption measurements.
53
54
368
55
56
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58
17
59
1
2
3
4 369 References
5
370 1. Yao, B.; Ji, Y., Lignin biodegradation with laccase-mediator systems. Front. Energy Res. 2014,
6 371 2, 12.
7 372 2. Bugg, T. D.; Rahmanpour, R., Enzymatic conversion of lignin into renewable chemicals. Curr.
8 373 Opin. Chem. Biol. 2015, 29, 10-17.
374 3. Munk, L.; Sitarz, A. K.; Kalyani, D. C.; Mikkelsen, J. D.; Meyer, A. S., Can laccases catalyze
9 375 bond cleavage in lignin? Biotechnol. Adv. 2015, 33 (1), 13-24.
10 376 4. Areskogh, D.; Li, J.; Gellerstedt, G. R.; Henriksson, G., Investigation of the molecular weight
11 377 increase of commercial lignosulfonates by laccase catalysis. Biomacromolecules 2010, 11 (4), 904-910.
378 5. Lundquist, K.; Parkås, J., Different types of phenolic units in lignins. BioResources 2011, 6 (2),
12 379 920-926.
13 380 6. Huber, D.; Ortner, A.; Daxbacher, A.; Nyanhongo, G. S.; Bauer, W.; Guebitz, G. M., Influence
14 381 of oxygen and mediators on laccase-catalyzed polymerization of lignosulfonate. ACS Sustain. Chem.
15 382 Eng. 2016, 4 (10), 5303-5310.
383 7. Munk, L.; Punt, A.; Kabel, M.; Meyer, A. S., Laccase catalyzed grafting of–N–OH type mediators
16 384 to lignin via radical–radical coupling. RSC Adv. 2017, 7 (6), 3358-3368.
17 385 8. Bourbonnais, R.; Paice, M.; Reid, I.; Lanthier, P.; Yaguchi, M., Lignin oxidation by laccase
18 386 isozymes from Trametes versicolor and role of the mediator 2, 2'-azinobis (3-ethylbenzthiazoline-6-
387 sulfonate) in kraft lignin depolymerization. Appl. Environ. Microbiol. 1995, 61 (5), 1876-1880.
19 388 9. Moya, R.; Saastamoinen, P.; Hernández, M.; Suurnäkki, A.; Arias, E.; Mattinen, M.-L.,
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21 390 2011, 102 (21), 10006-10012.
391 10. Prasetyo, E. N.; Kudanga, T.; Østergaard, L.; Rencoret, J.; Gutiérrez, A.; José, C.; Santos, J. I.;
22 392 Nieto, L.; Jiménez-Barbero, J.; Martínez, A. T., Polymerization of lignosulfonates by the laccase-HBT (1-
23 393 hydroxybenzotriazole) system improves dispersibility. Bioresour. Technol. 2010, 101 (14), 5054-5062.
24 394 11. Fernaud, J. H.; Carnicero, A.; Perestelo, F.; Cutuli, M. H.; Arias, E.; Falcón, M., Upgrading of an
395 industrial lignin by using laccase produced by Fusarium proliferatum and different laccase-mediator
25 396 systems. Enzyme Microb. Technol. 2006, 38 (1), 40-48.
26 397 12. Shleev, S.; Persson, P.; Shumakovich, G.; Mazhugo, Y.; Yaropolov, A.; Ruzgas, T.; Gorton, L.,
27 398 Interaction of fungal laccases and laccase-mediator systems with lignin. Enzyme Microb. Technol. 2006,
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29 401 lignin-based polymers (lignophenols). Biotechnol. Lett. 2003, 25 (1), 9-12.
30 402 14. Bourbonnais, R.; Paice, M.; Freiermuth, B.; Bodie, E.; Borneman, S., Reactivities of various
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32 405 15. Bourbonnais, R.; Paice, M. G., Demethylation and delignification of kraft pulp by Trametes
33 406 versicolor laccase in the presence of 2, 2′-azinobis-(3-ethylbenzthiazoline-6-sulphonate). Appl. Microbiol.
34 407 Biotechnol. 1992, 36 (6), 823-827.
408 16. Couto, S. R.; Herrera, J. L. T., Industrial and biotechnological applications of laccases: a review.
35 409 Biotechnol. Adv. 2006, 24 (5), 500-513.
36 410 17. Bourbonnais, R.; Paice, M. G., Oxidation of non-phenolic substrates: an expanded role for
37 411 laccase in lignin biodegradation. FEBS Lett. 1990, 267 (1), 99-102.
412 18. Baiocco, P.; Barreca, A. M.; Fabbrini, M.; Galli, C.; Gentili, P., Promoting laccase activity
38 413 towards non-phenolic substrates: a mechanistic investigation with some laccase–mediator systems. Org.
39 414 Biomol. Chem. 2003, 1 (1), 191-197.
40 415 19. Muheim, A.; Fiechter, A.; Harvey, P. J.; Schoemaker, H. E., On the mechanism of oxidation of
416 non-phenolic lignin model compounds by the laccase-ABTS couple. Holzforschung 1992, 46 (2), 121-
41 417 126.
42 418 20. Eggert, C.; Temp, U.; Dean, J. F.; Eriksson, K.-E. L., A fungal metabolite mediates degradation
43 419 of non-phenolic lignin structures and synthetic lignin by laccase. FEBS Lett. 1996, 391 (1), 144-148.
420 21. Astolfi, P.; Brandi, P.; Galli, C.; Gentili, P.; Gerini, M. F.; Greci, L.; Lanzalunga, O., New
44 421 mediators for the enzyme laccase: mechanistic features and selectivity in the oxidation of non-phenolic
45 422 substrates. New J Chem. 2005, 29 (10), 1308-1317.
46 423 22. Kawai, S.; Nakagawa, M.; Ohashi, H., Aromatic ring cleavage of a non‐phenolic β‐O‐4 lignin
47 424 model dimer by laccase of Trametes versicolor in the presence of 1‐hydroxybenzotriazole. FEBS lett.
425 1999, 446 (2-3), 355-358.
48 426 23. Kawai, S.; Asukai, M.; Ohya, N.; Okita, K.; Ito, T.; Ohashi, H., Degradation of a non-phenolic
49 427 β-O-4 substructure and of polymeric lignin model compounds by laccase of Coriolus versicolor in the
50 428 presence of 1-hydroxybenzotriazole. FEMS Microbiol. Lett. 1999, 170 (1), 51-57.
429 24. Christopher, L. P.; Yao, B.; Ji, Y., Lignin biodegradation with laccase-mediator systems. Front.
51 430 Energy Res. 2014, 2, 12.
52 431 25. Kawai, S.; Umezawa, T.; Shimada, M.; Higuchi, T., Aromatic ring cleavage of 4, 6-di (tert-
53 432 butyl) guaiacol, a phenolic lignin model compound, by laccase of Coriolus versicolor. FEBS lett. 1988,
433 236 (2), 309-311.
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4 434 26. Kawai, S.; Umezawa, T.; Higuchi, T., Degradation mechanisms of phenolic β-1 lignin
435 substructure model compounds by laccase of Coriolus versicolor. Arch. Biochem. Biophys. 1988, 262
5 436 (1), 99-110.
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7 438 polymerisation of models for phenolic lignin end-groups by laccase. Holzforschung 2010, 64 (1), 21-34.
439 28. Rittstieg, K.; Suurnäkki, A.; Suortti, T.; Kruus, K.; Guebitz, G. M.; Buchert, J., Polymerization of
8 440 guaiacol and a phenolic β‐O‐4‐substructure by Trametes hirsuta laccase in the presence of ABTS.
9 441 Biotechnol. Progr. 2003, 19 (5), 1505-1509.
10 442 29. Potthast, A.; Rosenau, T.; Koch, H.; Fischer, K., The reaction of phenolic model compounds in
443 the laccase-mediator system (LMS) investigations by matrix assisted laser desorption ionization time-of-
11 444 flight mass spectrometry (MALDI-TOF-MS). Holzforschung 1999, 53 (2), 175-180.
12 445 30. Johannes, C.; Majcherczyk, A., Laccase activity tests and laccase inhibitors. J. Biotechnol.
13 446 2000, 78 (2), 193-199.
447 31. Ramalingam, B.; Sana, B.; Seayad, J.; Ghadessy, F.; Sullivan, M., Towards understanding of
14 448 laccase-catalysed oxidative oligomerisation of dimeric lignin model compounds. RSC Adv. 2017, 7 (20),
15 449 11951-11958.
16 450 32. Li, K.; Helm, R. F.; Eriksson, K. E. L., Mechanistic studies of the oxidation of a non‐phenolic
17 451 lignin model compound by the laccase/1‐hydroxybenzotriazole redox system. Biotechnol. Appl. Biochem.
452 1998, 27 (3), 239-243.
18 453 33. Sealey, J.; Ragauskas, A. J., Investigation of laccase/N-hydroxybenzotriazole delignification of
19 454 kraft pulp. J. Wood Chem. Technol. 1998, 18 (4), 403-416.
20 455 34. Matsumura, E.; Yamamoto, E.; Numata, A.; Kawano, T.; Shin, T.; Murao, S., Structures of the
456 laccase-catalyzed oxidation products of hydroxy-benzoic acids in the presence of ABTS [2, 2′-Azino-di-
21 457 (3-ethylbenzothiazoline-6-sulfonic Acid)]. Agric. Biol. Chem. 1986, 50 (5), 1355-1357.
22 458 35. Osman, A.; Wong, K.; Fernyhough, A., ABTS radical-driven oxidation of polyphenols: Isolation
23 459 and structural elucidation of covalent adducts. Biochem. Biophys. Res. Commun. 2006, 346 (1), 321-
460 329.
24 461 36. Cañas, A. I.; Camarero, S., Laccases and their natural mediators: biotechnological tools for
25 462 sustainable eco-friendly processes. Biotechnol. Adv. 2010, 28 (6), 694-705.
26 463 37. Bourbonnais, R.; Leech, D.; Paice, M. G., Electrochemical analysis of the interactions of laccase
464 mediators with lignin model compounds. Biochim. Biophys. Acta 1998, 1379 (3), 381-390.
27 465 38. Fabbrini, M.; Galli, C.; Gentili, P., Comparing the catalytic efficiency of some mediators of
28 466 laccase. J. Mol. Catal. B: Enzym. 2002, 16 (5), 231-240.
29
467
30
31
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34
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37
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47
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4 468 Figures and Tables
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19 469
20
21 470 Figure 1. Molecular structures of guaiacylglycerol-β-guaiacyl ether (GBG), 1-hydroxybenzotriazole
22 471 (HBT) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS).
23
472
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
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42
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47
48
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55
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20
59
1
2
3
4 100 GBG dimer A
5
6 Relative Intensity (%)
GBG trimer
7 0
8 100
GBG dimer
9
B
10 HBT GBG-HBT CP
11 GBG trimer
0
12 100
ABTS CLP C
13 GBG-ABTS CPII
14 ABTS GBG-ABTS CPIIox
15 Unknown
0
16 5 10 15 20 25 30 35 40 45
17 473 Time (min)
18
19 474 Figure 2. RP-UHPLC-MS chromatograms (negative mode) of GBG incubated for 5 min with
20 475 laccase (A), laccase/HBT (B) and laccase/ABTS (C). Chromatograms of other time points can be
21 476 found in the Supporting Information.
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
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44
45
46
47
48
49
50
51
52
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54
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56
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58
21
59
1
2
3
4 477 Table 1. Compounds detected with UHPLC-PDA-ESI-ITMS and UHPLC-PDA-ESI-FTMS after
5 478 incubation of GBG with laccase from T. versicolor in the presence or absence of ABTS and HBT.
6 479 MS2 fragments and λmax were determined using UHPLC-PDA-ESI-ITMS. All other values were
480 obtained using UHPLC-PDA-ESI-FTMS. Relative intensities of MS2 fragments are shown between
7
481 brackets.* For ABTS, only data from UHPLC-PDA-ESI-ITMS were used, as FTMS only showed
8 482 in-source fragmentation. N.D. = Not detected.
9
10 RT Tentative Molecular Ionization Observed/ Mass MS2 fragments λmax
11 (min) annotation formula calculated error (nm)
mass (ppm)
12
Reaction products of GBG incubated with laccase alone
13 20.8 GBG C17H20O6 [M+Na]+ 320.12556/ -1.34 295 (55), 302 (16), 201 (8), 147 278
14 320.12599 (6), 219 (5), 176 (4)
15 34.8 GBG dimer C34H38O12 [M-H]- 638.23720/ 1.36 589 (100), 483 (62), 513 (28), 435 278
16 638.23633 (26), 329 (20), 465 (12), 541 (12),
359 (8)
17
44.7 GBG trimer C51H56O18 [M-H]- 956.34775/ 1.13 907 (100), 889 (60), 919 (30), 859 N.D.
18 956.34666 (20)
19 Reaction products of GBG incubated with laccase/HBT
20 3.9 HBT C6H5N3O [M-H]- 135.04329/ 0.23 106 (100), 78 (4) 306,
21 135.04326 276,
[M+H]+ 91 (63), 80 (40), 53 (18), 107 (8) 269
22
23 7.2 BT C6H5N3 [M+H]+ 119.04832/ -0.23 N.D. 260,
24 119.04835 280
25 20.9 GBG C17H20O6 [M+Na]+ 320.12556/ -1.34 295, 302, 201, 219, 147, 176 278
26 320.12599
25.1 GBG-HBT C23H23N3O7 [M-H]- 453.15375/ 0.33 404 (100), 328 (38), 298 (30) 330
27 CP 453.15360
28 31.9 GBG-HBT C23H21N3O7 [M-H]- 451.13838/ 0.95 420 (100), 310 (27), 326 (15) N.D.
29 CPox 451.13795
30 34.4 GBG dimer C34H38O12 [M-H]- 638.23720/ 1.36 589 (100), 483 (62), 513 (28), 435 278
31 638.23633 (26), 329 (20), 465 (12), 541 (12),
359 (8)
32 44.7 GBG trimer C51H56O18 [M-H]- 956.34775/ 1.13 907 (100), 889 (60), 919 (30), 859 N.D.
33 956.34666 (20)
34 Reaction products of GBG incubated with laccase/ABTS
35 5.6 ABTS CLP C9H9NO4S2 [M-H]- 258.99757/ 1.05 229 (37) 256,
36 258.99730 292,
285
37 8.7 Unknown Unknown [M-H]- 329.99756 302 (100), 238 (29), 222 (9) 278
38 17.0 Unknown C25H25N4O8S4 [M-2H]- 637.05667/ 1.79 363 (100), 378 (95), 228 (34), 605 305
39 637.05553 (31), 405 (26), 257 (25), 335 (18)
40 21.4 ABTS C18H18N4O6S4 [M-2H]2- 514* N.D. 342
41
42 22.7 GBG-ABTS C26H27N3O9S2 [M-H]- 589.11946/ 0.99 242 (100), 214 (61), 228 (10) 550
CPII 589.118876
43 24.4 GBG-ABTS C35H35N4O12S4 [M-3H]2- 831.11369/ 0.96 572 (100), 228 (43), 256 (42) 305,
44 CPIox 831.11289 278
45 [M-2H]- N.D.
46 28.3 GBGox C17H18O6 [M-H]- 318.11017/ -0.52 287 (100), 193 (71) 280,
47 318.11034 311
[M+H]+ 167 (100), 149 (70), 271 (68), 151
48 (59), 195 (44), 269 (32), 177 (26),
49 289 (22)
50 [M+Na]+
51 311 (100), 218 (16), 146 (11), 187
(6)
52 30.7 GBG-ABTS C26H25N3O9S2 [M-H]- 587.10366/ 0.76 242, 214, 556, 228 585,
53 CPIIox 587.10322 448,
54 328
55
56
57
58
22
59
1
2
3
4 I I
5
6 IV III
295 = II + III 404 = II + III 242 = V
7 III VII VI
II 201 = I + IV II 328 = I 214 = V + VI
8 219 = I 298 = I + III 228 = V + VII
9 147 = I
V
10
11
12 GBG (320 Da) GBG-HBT CP (453 Da) GBG-ABTS CPII (589 Da)
13
14
I I
15
III III III
16 287 = III 326 = III VII VI 242 = V
17 193 = I 420 = I 214 = V + VI
18 556 = III
228 = V + VII
19 V
20
21 GBGox (318 Da) GBG-HBT Cpox (451 Da) GBG-ABTS CPIIox (587 Da)
22
23 III
II
I
24
25 I II
III
III
26
I
27 II
28 IX
29
VIII
30 V
31 II
I
32 III
VII
33 III
34 I
II
35 GBG dimer (638 Da) GBG trimer (956 Da) GBG-ABTS CPIox (831 Da)
36
37 589 = II + III 907 = I + III 572 = VIII*
38 483 = I + III 889 = I + II + III 256 = VIII* + IX
513 = I 919 = II + II 228 = V + VII
39 435 = I + II + III + III 859 = I + II + III + III
40 329 = I + I + III + III X
41 465 = I + II + III
42 541 = II + II + III + III
43 359 = I + I + III
44 ABTS CLP (259 Da)
45
46 483
229 = X*
47
48 484 Figure 3. Proposed structures of reaction products of GBG after incubation with laccase and LMS.
49 485 The dotted lines represent the proposed fragmentation pattern, resulting in the MS2 fragments
486 reported in Table 1. The fragmentation pattern of GBG and GBG-ABTS CPIox originate from the
50
487 parent ions [M+Na]+ and [M-3H]2-, respectively. All other patterns originate from the parent ion
51 488 [M-H]-. In the coupling products and oligomers, the positions of coupling could not be identified
52 489 based on MS2 spectra, and are therefore based on literature.31,34-35 The position of the second inter-
53 490 unit bond in the GBG trimer is not specified, as there is no experimental evidence for either C-C or
54 491 C-O linkage. Fragmentations with an * (in GBG-ABTS CPIox and ABTS CLP) are suggested to be
55 492 radical fragmentations.
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57
58
23
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1
2
3
4 100 100
A
Abundance (% of max)
5
Abundance (% of max)
6 50 80 C
7
60
8 0
9 100
40
10 B
11 50 20
12
13 0 0
0 10 20 30 40 50 60 0 10 20 30 40 50 60
14
time (min) time (min)
15 493
16 494 Figure 4. Normalized MS peak areas in time of reaction products formed upon incubation of GBG
17 495 with laccase alone (A), laccase/HBT (B) and laccase/ABTS (C). Areas were obtained from extracted
18 496 ion chromatograms: GBG (m/z 343) ( ● ), GBG dimer (m/z 637) ( ● ), GBG trimer (m/z 955) ( ● ),
19 497 GBG-HBT CP (m/z 452) ( ● ), GBG-HBT CPox (m/z 450) ( ○ ), GBGox (m/z 319 and 341) ( ○ ), GBG-
20 498 ABTS CPIox (m/z 414) ( ● ), GBG-ABTS CPII (m/z 588) ( ● ), GBG-ABTS CPIIox (m/z 586) ( ○ ) and
21 499 ABTS CLP (m/z 258) ( ● ). For the abundance (Y-axis), the time point with the largest peak area
22 500 was set to 100%. For the other time points, the abundance was calculated as the peak area relative
23 501 to the area of this largest peak. Amounts withdrawn per time point and injected to UHPLC-MS
502 analysis were the same for all time points.
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1
2
3
4 503
5
6
7
8 lac Further
lac
9 polymerization
10 1
11
12
2
13
14 lac
15 slow
16
17
18 lac
slow
19
lac
20
21
22 Further lac
23 polymerization
24
25
26 504
27
28 505 Figure 5. Overview of proposed reactions occurring after incubation of GBG with laccase alone or
29 506 a laccase/HBT system. The thick arrows represent the major reaction pathway. The reactions
30 507 represented by thin arrows do occur, but slower and/or to a smaller extent. In case of laccase alone,
508 all GBG reacts via route 1. In case of a laccase/HBT system, GBG reacts via route 1 and 2, but
31
509 route 1 is favored due to the slow oxidation of HBT. Oxidation of GBG-HBT CP is proposed to
32 510 occur via radical hydrogen abstraction by either a HBT radical (HBT·) or an oligomeric GBG
33 511 radical ((GBG)n·). The C-C coupling in the dimer is based on Ramalingam et al.31 For clarity,
34 512 reductions of O2 to H2O by laccase are not shown in the figure.
35
36 513
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1
2
3
4
lac
5
6 ABTS ABTS·+
7
8
9
10
11 lac lac
12
or or
13
ABTS·+ ABTS ABTS·+ ABTS ABTS·+ ABTS
14 GBG GBG· GBGox GBGox·
15
radical coupling
radical coupling
16
17 ABTS·+ ABTS +·
18
19
20
21
22
23
24
GBG·
25
26
ABTS·+
27 GBG-ABTS CPI GBG-ABTS CPIox
28 ABTS
29 lac lac
30
31 ABTS CLP
32
33
34
35 lac
36 or
37 ABTS·+ ABTS
38
39
40
41
42
43
44
45
46
GBGox
47
48 GBG-ABTS CPII GBG-ABTS CPIIox
514
49
50 515 Figure 6. Overview of proposed reactions occurring after incubation of GBG with a laccase/ABTS
51 516 system. GBG can be oxidized directly by laccase, or indirectly via ABTS·+, which is formed by
52 517 laccase. The GBG radicals may react further to GBGox or may undergo radical coupling with
53 518 ABTS·+. After coupling of GBG and ABTS, further rearrangements take place in which part of the
54 519 ABTS molecule is cleaved from the reaction product. The insert shows the proposed mechanism of
55 520 GBGox formation from GBG·. For clarity, reductions of O2 to H2O by laccase are not shown in the
521 figure.
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57
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59
1
2
3
4 522
5 1.5 Peak Tentative Ionization m/z
6
1.0
I A
III annotation observed
7 II IV
0.5 V I GBG trimer [M+Na]+ 979.2
8
Intensity (× 104)
VI
0 II GBG tetramer [M+Na]+ 1297.3
9 I
1.5 II III GBG pentamer [M+Na]+ 1615.4
10
1.0
III B IV GBG hexamer [M+Na]+ 1933.5
11 IV V GBG heptamer [M+Na]+ 2251.6
12 0.5
V
0 VI GBG octamer [M+Na]+ 2569.6
13
1.5 VII GBG dimer (2 OX) [M+Na]+ 657.1
14 IX C
1.0 VIII GBG trimer (OX) [M+Na]+ 977.2
15 VII VIII IX Unknown Unknown 989.1
X
16 0.5
X Unknown Unknown 1275.2
17 0
500 1000 1500 2000 2500 3000
18 523 m/z
19
20 524 Figure 7. MALDI-TOF spectra (left) of GBG after 60 min incubation with laccase alone (A),
21 525 laccase/HBT (B) and laccase/ABTS (C) measured in positive mode, and corresponding tentative
22 526 peak annotations (right). OX = Cα hydroxyl oxidized to ketone.
23
527
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4 528 TOC/Abstract graphic (For Table of Contents Use Only)
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11 Further polymerization
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15
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18
19 phenolic Stable
20
lignin dimer
21
22 529
23
530 Synopsis:
24
25 531 This paper contributes to a more fundamental understanding of lignin modification by laccase-
26
532 mediator systems.
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28 533
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30 534
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59

LAC ABTS Systems

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