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. 2021 Aug 4;6(35):22578–22588. doi: 10.1021/acsomega.1c02501

Natural Syringyl Mediators Accelerate Laccase-Catalyzed β-O-4 Cleavage and Cα-Oxidation of a Guaiacyl Model Substrate via an Aggregation Mechanism

Xu Chen 1, Xingyu Ouyang 1, Jiayi Li 1, Yi-Lei Zhao 1,*
PMCID: PMC8427646  PMID: 34514230

Abstract

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Laccase–mediator systems (LMSs) have been intensively investigated in lignin degradation. Although only natural metabolites are available for fungal lignin degradation, mediator molecules from metabolites have received substantially less attention than artificial organic–synthetic compounds. It remains unclear which metabolites can accelerate laccase-catalyzed reactions and how those natural mediators influence lignin degradation. In this work, we evaluated Trametes versicolor laccase-catalyzed reaction kinetics on a lignin guaiacyl subunit model (guaiacylglycerol-β-guaiacyl ether, G-β-GE) in the presence of a group of lignin syringyl subunit molecules: syringaldehyde, acetosyringone, and methyl syringate. We then compare their performance to a well-known synthetic mediator ABTS, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). Time-resolved UPLC-TOF-MS revealed that the syringyl mediators were more effective in accelerating the β-O-4 cleavage and Cα-oxidation of G-β-GE than ABTS under laccase-catalysis, despite the syringyl compounds possessing slower individual oxidation rates. In addition, the product profile of polymerization was also promoted dramatically, compared to that of the ABTS/laccase system. The LMS kinetic modeling suggested that mediator–substrate aggregation played a critical role in the laccase–mediator system; in which, the lignin syringyl and guaiacyl subunits likely form a π–π stacking van der Waals complex that can be oxidized faster than the syringyl or guaiacyl monomers by themselves. This syringyl–guaiacyl aggregation hypothesis postulates that the weak interactions in lignin biopolymers are able to accelerate the laccase-catalyzed biodegradation.

Introduction

Lignin is a complex three-dimensional natural polymer accounting for 15–30% of the weight of the lignocellulosic biomass and an underutilized feedstock for the pulp/paper industry as well as biofuel production.1,2 It consists of several types of phenylpropane monomers, including para-hydroxyphenyl (H, no methoxyl substitution on phenyl ring), guaiacyl (G, one methoxyl substitution), and syringyl (S, two methoxyl substitution) units. Typically, lignin contains G and S units, with the proportion of H units remaining relatively small.3,4 Lignin is formed by the aggregation of these subunits via various interunit linkages. The most abundant type of interunit linkage is β-O-4, which constitutes more than half of all interunit linkages.5,6 Differing combinations of subunits and interunit linkages make up the diversity of lignin, leading to an extremely complex three-dimensional network.7,8

Accordingly, lignin valorization is a significant challenge due to the complex and recalcitrant structure of lignin;9,10 one environment-friendly approach is to apply biocatalysts such as ligninases, a group of lignin-modifying enzymes including many peroxidases and laccase.11,12 Laccase (EC 1.10.3.2), a class of multicopper oxidase, adapts to a wide range of phenolic substrates and conditions, utilizing dioxygen molecules in the air as an electron acceptor to generate water.13,14 However, laccase possesses a relatively low redox potential (≤0.8 V), so it is limited to seizing the electron of the lignin phenolic subunit, <20% w/w of lignin.7 The lignin nonphenolic subunit cannot be directly oxidized due to its higher redox potential value (1.3 V) and the oxidation cannot automatically lead to a cleavage. In fact, when lignin is treated with laccase alone, it not only fails to break down the lignin molecule but also leads to lignin repolymerization.11,15 Intriguingly, laccase can oxidize nonphenolic lignin in the presence of a “mediator” molecule.16,17 These mediators are typically molecules with small molecular weights, which facilitate electron transfer and allow laccase to indirectly catalyze the oxidation of lignin’s nonphenolic subunit.18,19 The effect through which these small molecules effectively improve laccase catalysis is known as the laccase/mediator system (LMS).20

When examining the efficacy of an LMS compound, phenolic and nonphenolic lignin model compounds such as guaiacylglycerol-β-guaiacyl ether (G-β-GE) and veratrylglycerol-β-guaiacyl ether (VBG) are typically utilized to reduce the uncertainty induced by the complex structure of lignin.1,21−26 The interest in studying phenolic lignin subunits has been steadily rising. Since lignin contains a large number of nonphenolic subunits, the majority of research studies have focused on the use of laccase and artificial mediators, like ABTS or 1-hydroxybenzotriazole (HBT), to degrade nonphenolic β-O-4 lignin model compounds.27−31 Phenolic β-O-4 model compounds treated by laccase alone are prone to polymerization;32 organic–synthetic mediator, like ABTS or HBT, form coupling products between the mediator and model compounds to inhibit this polymerization.21,33 In short, nonphenolic lignin model compounds undergo cascaded radical reactions toward Cα oxidation,18,21,26−31,34 Cα–Cβ cleavage,18,25,26,28−30,34 and ether cleavage;21,28−31 phenolic lignin model compounds toward polymerization21,32,33,35−37 in major, and Cα-oxidations,38 Cα–Cβ cleavage,38 alkyl–aryl cleavage,38 aromatic ring cleavage39 simultaneously, mediators are fated to the grafting products.21,33,37 However, only a few reports have evaluated the efficacy of natural mediators in laccase-catalyzed reactions with phenolic lignin model compounds.40 As such, the LMS reactions and mechanisms of lignin model compounds with natural metabolite mediators remain unclear (Scheme 1).

Scheme 1. Chemical Structures of a Lignin Model Substrates (G-β-GE, Guaiacylglycerol-β-guaiacyl ether), an Artificial Mediator Molecule (ABTS, 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)), and Three Natural Syringyl Mediator Molecules (SA, Syringaldehyde; AS, Acetosyringone; MS, Methyl Syringate).

Scheme 1

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Diagram of G-β-GE laccase oxidation was also present (bottom).

In this study, we employed a linked guaiacyl subunit, guaiacylglycerol-β-guaiacyl ether (G-β-GE), as a lignin–mimic substrate (Scheme 1). It is a typical β-O-4 dimeric model compound, containing a phenolic group and a nonphenolic group, that is expected to mimic both the phenolic and nonphenolic subunits in lignin.21,33ABTS, syringaldehyde (SA), acetosyringone (AS), and methyl syringate (MS) were used as mediators representing organic synthetic (artificial) and natural mediator molecules, respectively. Laccase-alone and LMS reactions were monitored via a combination of UV–vis spectroscopy, high-performance liquid chromatography (HPLC), and ultraperformance liquid chromatography-time of flight mass spectrometry (UPLC-TOF-MS). The individual substrate and mediator molecules were also measured in laccase-alone reactions to determine their Michaelis–Menten parameters. Then, the resulting time-resolved consumptions of substrates and mediators were utilized for LMS kinetics analysis. Based on these findings, an updated reaction kinetic model was proposed to rationalize the substantial enhancement induced by the syringyl mediators.

Results and Discussion

Enhancement of G-β-GE Conversion

Laccase-catalyzed guaiacyl substrate (G-β-GE) consumption is illustrated in Figure 1A. In the absence of a mediator, the half-life (t1/2) was 7.2 h: [G-β-GE] = 1.0 mM and [Lac] = 1.7 μM at pH of 4.5 (acetate buffer) and 35 °C. The half-life decreased by 12.5% in the presence of 0.5 mM artificial mediator ABTS (t1/2 = 6.3 h). In the presence of 0.5 mM syringyl mediators, the t1/2 decreased to 4.7, 3.9, and 3.5 h for syringaldehyde (SA), acetosyringone (AS), and methyl syringate (MS), respectively, demonstrating that the syringyl family of natural mediators are significantly more effective than the well-known artificial mediator ABTS by about 25.4–44.4%.

Figure 1.

Figure 1

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Laccase-catalyzed reactions of substrate and mediators. (A) G-β-GB consumptions in the presence and absence of mediators ABTS, SA, AS, and MS (T. versicolor laccase, 1.7 μM, [G-β-GB] = 1.0 mM), measured via the UPLC-TOF-MS method. (B) Michaelis constant (KM) and the maximum initial velocity (Vmax) of G-β-GB, ABTS, SA, AS, and MS are measured via HPLC and UV–vis spectroscopic methods at [Lac] = 4.2 μM.

However, the natural syringyl mediators oxidize more slowly than ABTS (although still faster than the guaiacyl substrate). As shown in Figure 1B, the maximum initial velocity (Vmax) of syringyl mediators ranges from 3.9 to 9.6 μM/min at [Lac] = 4.2 μM, about 1 order of magnitude lower than ABTS (32.6 μM/min; also see Figures S1–S5). In addition, the Michaelis constant (KM) of the syringyl mediators was measured to be in a range of 53.6 to 81.8 μM; all three were larger than ABTS (36.1 μM). The guaiacyl substrate G-β-GE possessed a Vmax value of 5.9 μM/min and the largest KM value (246.1 μM), highlighting its need for a mediator and making it an ideal test substrate for LMS studies. Curiously, ABTS could be considered a more reactive mediator with a higher binding affinity (i.e., smaller KM value) for T. versicolor laccase, if only examining independent enzyme proficiency (kcat/KM): Vmax/KM/[Lac] = 95, 3.6 × 103, 2.9 × 102, 3.0 × 102, and 4.7 × 102 M–1·s–1 for G-β-GE, ABTS, SA, AS, and MS, respectively. Interestingly, the syringyl mediators appear superior to ABTS in accelerating the catalytic oxidation of G-β-GE by laccase.

Furthermore, UPLC-TOF-MS (Figure 1A) and HPLC (Figures S6–S11) chromatograms revealed that the G-β-GE peak areas fluctuated within the first 2 h of reaction, in both laccase-alone or laccase–mediator systems, deviating from the zero-order reaction by 10–20% if the curves were extrapolated with the 2–6 h data. These results indicate that a part of G-β-GE may be absorbed onto the laccase protein and that this adsorbed substrate is released back to the reaction system during substrate consumption, in agreement with the previous study by Lai and co-workers, who found that laccase can adsorb lignin and their affinity becomes higher by 8% in the presence of mediators.41 Moreover, it is also reported that lignocellulose and cellulase form nonproductive complexes.42 Thus, it is reasonable to assume that laccase and G-β-GE form nonproductive complexes when G-β-GE is highly excessive.

β-O-4 Cleavage and Cα Oxidation

UPLC-TOF-MS EIC traces in Figure 2A confirmed the mediator effect of ABTS and the three syringyl molecules. Over the first 5 h, mediator levels were first maintained (>95% for MS and >85% for ABTS, SA, and SA) but then decreased significantly after 6 h (Figure S12); indeed, ABTS, SA, AS, and MS started slow consumption at 3.2, 2.8, 2.4, and 1.5 h, respectively, when G-β-GE levels were 79.2, 78.0, 77.6, and 82.0%, respectively. Therefore, the early stage of G-β-GE consumption was considered to be a typical LMS reaction, in which the mediators act as a catalytic promoter; then, the mediator molecules became the second substrates taking part in the laccase-catalyzed reaction. Since the initial ratio of G-β-GE to mediators was 2:1, a typical LMS reaction takes place until the G-β-GE-to-mediator ratio is 1:1 in the reaction systems. The initial 6 h reaction course was analyzed via the LMS process in the following sections.

Figure 2.

Figure 2

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Comparison of laccase-alone, artificial mediator ABTS, and the syringyl natural mediators. (A) Delayed consumption of artificial and natural mediators. (B) Detectable products of β-O-4 cleavage and Cα oxidation. (C, D) Time-dependent relative yields of hydrolytic and methanolytic products of β-O-4 cleavage and (E) Cα oxidation (EIC, extracted ion chromatogram; r.t., retention time; see the detailed m/z and r.t. information in Table S4; Frg-OH and Frg-Ome structures are hypothetical).

Figure 2B plots small-molecule product profiles for the initial 6 h, in which three degradation-related signals are highlighted in panels C, D, and E. Figure 2C,D shows time-resolved yields of products hypothetically from β-O-4 cleavage, one from hydrolysis (Frg-OH, [M + Na]+m/z = 237.078 ± 0.003, retention time = 15–17 min) and another from methanolysis (Frg-Ome, [M + Na]+m/z = 251.095 ± 0.004, retention time = 20–22 min); although the two structures are hypothetical based on the high-resolution MS signals, similar chemicals have been reported in previous studies on HBT mediators29−31 and fungal ligninase (also see the UPLC-MS/MS evidence in Figure S13).43−45 The relative yields, compared to that of the laccase-alone reaction, over the first 6 h were determined to be 1.44-, 1.85-, 2.01-, and 1.86-fold in Frg-OH, and 1.19-, 1.79-, 2.12-, and 2.03-fold in Frg-Ome for ABTS, SA, AS, and MS, respectively (see Tables S1 and S2). Consistently, compared to ABTS, the β-O-4 cleavages in the presence of the syringyl mediators were dramatically enhanced (28.5, 39.6, and 29.2% in Frg-OH and 51.0, 78.4, and 70.8% in Frg-Ome, respectively).

Figure 2E shows the time-resolved yield of a G-β-GE oxidative product (PCα, [M + Na]+m/z = 341.106 ± 0.002, retention time = 43–45 min). A similar MS signal has been characterized as a Cα oxidative product in previous artificial-mediator studies.21,27,29−31,33 The oxidative product levels were negligible in the laccase-alone and ABTS–mediator reactions but significant in the syringyl mediators. The relative yields of AS and MS were increased by 34.4 and 34.1% over that of SA in the product profiles. Although the Cα oxidative product has a reduced molecular weight, the Cα oxidation would decrease its redox potential and suppress further degradation.43,44 Therefore, it is unlikely that the Frg-OH and Frg-Ome formations are related to the Cα-oxidation product (see Figure S14).

As shown in Table S3, the overall yields for the three low-molecular-weight (LMW) products were 4.6, 7.6, 9.3, 9.9, and 10.8% for the 5 h product profiles of the laccase-alone, laccase–ABTS, laccase–SA, laccase–AS, and laccase–MS systems, respectively. Thus, the formation of LMW products indicates that the syringyl mediators might be able to efficiently promote the most valuable degradation at the aliphatic linkage between phenolic and nonphenolic subunits in lignin valorization and significantly more so than the artificial mediator ABTS.

LMS Kinetic Model Updated with Substrate–Mediator Aggregation

We then conducted a kinetic modeling analysis based on the time-dependent substrate consumption experiments. Due to the limited available data, a simple two-step Michaelis model was used to fit the laccase-alone kinetics. As shown in Figure 3, the first step for the laccase-alone system was the formation of the Michaelis complex (ES) between laccase and G-β-GE. The second step was oxidation toward a S*+ radical cation that subsequently leads to various oxidative products.46 We focused the LMS kinetic modeling on the mediator’s role in substrate consumption rate and performed no additional fitting for rate constants of the complicated S*+ derivative reactions. In addition, we included unproductive protein–substrate and protein–mediator complexations to account for the G-β-GE fluctuation in the first 2 h.

Figure 3.

Figure 3

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Plausible mechanisms for laccase-catalyzed reaction. (A) Simple two-step Michaelis models for the laccase-alone system, the old laccase–mediator system, and the updated laccase–mediator system. (B) Fits of reaction progress with the old and the updated models for the time-resolved concentrations of substrate and mediators.

The classical mediator model proved unable to provide a reasonable rate constant of k5 between the activated mediator and substrate (see Table 1). Moreover, the coefficients of variation became as large as 1.1 × 103 even after substantial fitting effort. Inspired by the aggregation observed in the early stage, a mediator/substrate complex was instead used to model the two-stage Michaelis equation of the laccase–mediator system. In the aggregation mechanism, laccase, the mediator, and the substrate form a prereactive complex, with the aggregating mediator–substrate donating electrons to the oxidative state of laccase. The resulting MS*+ radical cation then decomposes to regenerate the catalytic mediator and release the S*+ radical cation. This updated LMS mechanism explains the fast oxidation of the guaiacyl substrate in the presence of lower kcat/KM syringyl mediators.

Table 1. Kinetics Parameters of the Simple Two-Step Michaelis Model Fitted via Dynafit Software Using Experimental Time-Resolved Concentrations of Substrates and Mediatorsa.

system rate constants CVa
laccase alone k1 (mM–1·min–1) k2 (min–1)    
  1.5 1.9   1.2–2.1
the old LMS model k3 (mM–1·min–1) k4 (min–1) k5 (mM–1·min–1)  
Lac-ABTS 4.3 7.9 5.3 ∼4.2
Lac-SA 2.1 1.8 0.34 ∼2.4
Lac-AS 2.2 1.3 2.8 × 102 ∼7.7 × 102
Lac-MS 2.4 2.3 3.8 × 102 ∼1.1 × 103
the updated LMS model k3′ (mM–2·min–1) k4′ (min–1) k5′ (min–1)  
Lac-ABTS 1.5 0.20 0.008 6.5–13.0
Lac-SA 2.3 0.32 0.015 1.2–2.7
Lac-AS 3.0 0.49 0.027 0.7–1.6
Lac-MS 1.7 0.72 0.029 0.6–1.9
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a

CV, coefficients of variation. Based on the Dynafit program, their maximal values shall be controlled within 100 for a plausible fitting model among multiple candidate kinetics models. It fails in the Lac-AS and Lac-MS systems if the old LMS model is used. The bold values (k4') represent the mediator efficacy in the updated LMS model.

As shown in Figure 3 and Table 1 (also Figures S15–S19), the laccase-alone kinetic parameters k1 and k2 were found to be 1.5 mM–1·min–1 and 1.9 min–1 for classical Michaelis equation, which is consistent with the kcat/KM values (1.47–19.45 mM–1·min–1, i.e., KM: 0.008–47 mM and kcat: 3.84–258.0 min–1) from the Brenda database.47 The LMS kinetic parameters k3′, k4, and k5′ were fitted in reasonable ranges of 1.5–3.0 mM–2·min–1, 0.20–0.72 min–1, and 0.008–0.029 min–1, respectively. The coefficients of variation were controlled to within <15.0, indicating that the experimental data supports the aggregation mechanism with higher reliability than previously assumed in the old LMS model, that is, a free-state activate mediator molecule M*+ oxidizes another free-state substrate in a solution (Figures S20 and S21). The old LMS model provided unreasonable rate constants as large as 380 mM–1·min–1 when fit to the experimental data, likely due to the low concentrations of activate mediator molecule M*+. Moreover, it is not physically meaningful to assume that the syringyl compounds, with lower redox potentials, deprive the guaiacyl compound, with a higher redox potential, of electrons—the syringyl subunit has one more electron-donating methoxy substitution on the phenolic subunit. It is thereby reasonable that the syringyl mediators play an on situ electron-transfer bridging role between laccase and the guaiacyl substrate in a prereactive aggregate at the laccase active site, rather than in an isolated free state in a solution after laccase-catalyzed oxidation. The oxidation of the aggregation complex could be also related to the enhancement of β-O-4 cleavage and Cα oxidation of G-β-GE, and the timeline relationship of mediator and substrate consumptions before the G-β-GE/mediator ratio reaches 1:1.

Preliminary structural modeling illustrated the π–π stacking and hydrogen-bonding interactions between the syringyl mediators and the G-β-GE molecules, rationalizing the syringyl substitution effect with different substituents of aldehyde, ketone, and ester. Figure 4 shows the lowest-energy conformers for their complexation, with BSSE-corrected binding energies of −17.4, −17.5, and −18.0 kcal/mol for SA, AS, and MS, respectively (Figure S22). The Cγ-hydroxyl group formed a hydrogen bond with the carbonyl oxygen atom of the syringyl subunits, with the H–O distances of 1.925 (SA), 1.902 (AS), and 1.896 Å (MS). Additionally, the distances between the guaiacyl phenolic, guaiacyl nonphenolic, and syringyl phenolic rings were 3.669 and 3.569 Å in SA, 3.683 and 3.599 Å in AS, and 3.704 and 3.547 Å in MS, respectively. Accordingly, the intermolecular π–π interactions between the nonphenolic subunit and the syringyl subunit were ranked MS > SA > AS, while the internal π–π interactions between the guaiacyl phenolic subunit and nonphenolic subunit were ranked SA > AS > MS. Moreover, we calculated the highest occupied molecular orbital (HOMO) of the three complexes to be −0.2827, −0.2813, and −0.2811 au (atomic unit) for SA, AS, and MS, respectively; the relative energies of AS and MS were 0.88 and 1.00 kcal/mol in reference to SA. Overall, the electron-donating properties are likely ranked in the same order (MS > AS > SA) as the HOMO energy level, as higher HOMO energy corresponds to lower ionization energy. In particular, the above frontier molecular orbital (FMO) analysis suggested that the electron density of the syringyl phenolic ring overlapped those of the guaiacyl nonphenolic ring significantly in the HOMOs, indicating that electron transfer is feasible between the mediator and substrate molecules when the oxidative copper center of laccase seizes electrons from the two phenolic subunits. These results validate that the following MS*+ degradation could break down the β-O-4 bonds or induce Cα oxidation via a two-electron process on the aliphatic link of the nonphenolic subunit. The β-O-4 cleavage via a one-electron process might lead to a phenoxyl radical stabilized by the two benzene rings in a sandwich geometry, and a cationic Cβ species that would interact with Frg-OH via hydrolysis or Frg-Ome via methanolysis (Figure S23).

Figure 4.

Figure 4

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Complexation of syringyl mediators and G-β-GE. (A) Syringaldehyde (SA), (B) acetosyringone (AS), and (C) methyl syringate (MS). The lowest-energy conformations were calculated via the ωB97-XD/6-31+G(d) level theory.

Depletion of Mediator Molecules: Conjugation and Oxidation

A variety of conjugative and oxidative relics were observed in the LMS experiments, particularly for the syringyl natural mediators. Their yields are illustrated in the time-resolved MS EIC peak areas in Figure 5, as are their possible chemical structures.

Figure 5.

Figure 5

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Conjugate compounds of G-β-GE and mediators as well as the oxidative relics of ABTS and the syringyl mediators. (A) EIC intensities of the two groups of compounds from mediators. (B) Product distributions of the four mediators after 6 h LMS reaction. (C) Possible chemical structures characterized from the previous literature (see the m/z and r.t. information in Table S4).

Although mediator consumption appears negligible in the early stages of the LMS reaction (Figures 2 and 3), a trace amount of conjugative and oxidative product was detected in the highly sensitive UPLC-TOF-MS measurement. As shown in Figure 5, the MS intensities of the conjugative and oxidative products of mediator molecules after the first 6 h were in the 104 magnitude range for the mediator–substrate conjugation and the 105 range for the mediator oxidation, 1–10% of the G-β-GE degradation (∼106 for PCα, Frg-OH, and Frg-Ome; Figure 2). Intriguingly, the mediator–substrate conjugation occurred between the syringyl mediator and oxidative product (PCα) of G-β-GE, with four-unit drop in the m/z value (MW: substrate + mediator – 4), while in ABTS a conjugative product from the original G-β-GE substrate (MW: substrate + mediator – 2) was detected in the product profile. This suggests that the syringyl mediator conjugation took place much more quickly with the Cα oxidative product, likely due to the complexation between PCα and mediators different from the aggregating structures in Figure 4: when Cα is converted to a sp2 carbon, the internal π–π interaction of G-β-GE is lost, and the mediator may bind the phenolic G subunit rather than the nonphenolic subunit PCα. A radical coupling product between G-β-GE and oxidized ABTS (ABTSox-G-β-GE) was also detected; which is consistent with the conjugative structures proposed in previous studies of artificial mediators,33 in which the orthogonal position of the guaiacyl phenolic ring was proposed as the connecting point to mediator molecules. Notably, Hilgers et al. observed a conjugate product between the oxidized ABTS and PCα, which was not detected in this work, probably due to a slower reaction rate at the relatively lower laccase concentration.33 Compared to ABTS, the syringyl mediators conjugated more rapidly: 4.90-, 4.66-, and 1.51-fold in MS, AS, and SA, respectively.

A MS signal with m/z of 169.053 ± 0.002 was determined to be 2,6-dimethoxy-1,4-benzoquinone (DMBQ), based on previous research on laccase-catalyzed syringaldehyde oxidation.48,49 DMBQ is a typical relic of syringyl subunit oxidation in the presence of laccase. The formation of DMBQ is similar to other peroxidase-catalyzed oxidation reactions;50 the oxidation is initiated by a free phenoxy radical from the syringyl compounds, followed by an electron translocation, a dioxide incorporation, and finally a decarboxylation toward DMBQ. Similarly, another MS signal was detected in the laccase–ABTS system ([M + H]+, m/z = 260.010 ± 0.001, and M = C9H9NO4S2), which was characterized as an oxidative product of ABTS.33Figure 5B plots the time-resolved oxidative products of the mediators, including ABTSox and DMBQ. The yields of DMBQ were most substantial in the laccase–SA system, followed by the laccase–AS system. Except for the intact mediator, the overall degradations of SA and AS mediators were severe, about 10.0 and 12.9% consumed in the 6 h LMS reaction, while MS and ABTS mediators were 8.5 and 7.8%, respectively.

Collectively, these results highlight the fact that the artificial mediator ABTS is slightly more stable in the laccase systems, remaining 92.2% after 6 h reaction, and portions of the syringyl mediators conjugate with an oxidative PCα product or oxidize to DMQB, with 90.9, 87.1, and 91.5% (SA, AS, and MS, respectively) remaining in the laccase–mediator systems.

Other Reactions of G-β-GE: Polymerization and Repolymerization

Figure 6 shows the overall product profiles of the four LMS systems at 2, 3, 4, and 5 h. The areas in the pie chart are presented with an equivalent amount of G-β-GE. Therefore, the degradation products of G-β-GE were counted as 1 equiv, and the mediator–substrate conjugative products as 1 equiv as well, but with dimers as 2 equiv and trimers as 3 equiv, etc. The response coefficient of EIC intensity for all species was assumed to be identical across all charts (see the overall mass balance at 0 h and t1/2 in Table S5).

Figure 6.

Figure 6

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Product profiles of the laccase-alone and LMS reactions. (A) Time-dependent yields of representative polymerization and repolymerization products. (B) Pie charts for the two groups of degradation and polymerization products. Some high-molecular-weight products from repolymerization cannot be detected within the instrumental m/z ranges in UPLC-TOF-MS and are classified as other products.

G-β-GE equivalents decreased from the start of LMS reaction to 46.0, 34.6, 22.9, and 21.9% after 5 h for ABTS, SA, AS, and MS, respectively. The equivalent amounts of G-β-GE degradation were 7.6, 9.3, 9.9, and 10.8% and those of G-β-GE polymerization were 26.9, 35.7, 23.5, and 21.8%, respectively. A significant number of products (10–20%) were detected with specific m/z values and retention times in the UPLC-TOF-MS measurement, but their chemical structure remains unknown. In the laccase-alone system, the remaining G-β-GE and degradation and polymerization products were 53.3, 4.6, and 33.3%, respectively, using the maximum equivalent amount of G-β-GE as 100%. Thereby, all four mediators can enhance the degradation and suppress the polymerization of the lignin model substrate G-β-GE. Among them, MS and AS performed most efficiently, with the highest yields of degradation. Repolymerization was still substantial in all laccase-catalyzed oxidation, and we observed that some dimers were formed in the early stages and later consumed (see Figure S24). Such a deep polymerization to trimer and tetramer products affects the UPLC-TOF-MS measurements, as large-weight molecules might stay in precipitation and not be soluble in the supernatants in the analytical instrument.

Conclusions

All three syringyl mediators were more LMS-effective than the artificial mediator ABTS, even though ABTS could be converted into the oxidative species more quickly. All of the mediator molecules, regardless of synthetic or natural origin, were consumed in the laccase-catalyzed reaction. Artificial mediators, like ABTS, might be slightly more resistant under oxidative conditions than natural syringyl mediators. In the mixture system of mediator and G-β-GE substrate, a mediator consumption trigger point was observed in all four LMS cases. This indicates that a specific complexation of substrate and mediator plays a critical role in the enhancement of laccase-catalyzed reactions. This study finds that the syringyl mediator can form a well-organized prereaction structure (molar ratio 1:1), while the ABTS–substrate interaction is much weaker and likely leads to several random complexes. Optimally then, in laccase–mediator systems, the mediator molecule should be specifically designed for a particular substrate and laccase.

Kinetic modeling supported the cocatalytic aggregation mechanism for the laccase–mediator system; if laccase-catalyzed mediator oxidation followed the same rate constant as the mediator-alone system, then the electron transfer rate between the oxidative mediator and G-β-GE substrate would be well outside the reasonable range. In the cases of acetosyringone and methyl syringate, the two syringyl mediators are oxidized much more slowly than ABTS but accelerate the laccase-catalytic reaction more effectively. With the assumption that the syringyl mediators and the G-β-GE substrate form a prereaction complex, all internal electron transfer rates enter a reasonable range. While it could be an exceptional mechanism for the syringyl–guaiacyl lignin in this study, future laccase–mediator studies should evaluate the “ensemble-effect” in this cocatalytic mechanism, which is popular in metal catalysis.51,52 The current kinetic modeling in this work focuses on the mediator effect based on substrate consumption rates, for lack of necessary mechanistic information, not yet extending to the formation of each product. In the cases of ABTS and syringaldehyde, it should be noted that both the old and updated LMS models work.

Although degradation is desirable in laccase-catalyzed lignin modification, polymerization and repolymerization were dominant in all five laccase-catalyzed reactions, including the laccase-alone system, with degradation still limited to 10–15% as has been seen in other studies.28,29 Both the degradation and polymerization were significantly accelerated with mediator molecules, particularly with methyl syringate. Notably, G-β-GE consumption was complete within 12 h in the laccase–SA, laccase–AS, and laccase–MS systems, compared to 27.3% of G-β-GE remaining in the laccase-alone system; however, a substantial amount of precipitate was observed in the methanol-quenched samples, attributed to severe repolymerization. In the late stage of the LMS reaction, the activated mediator molecules could graft onto the larger oligomers and polymers or form 5-5′ dehydroproducts. More intriguingly, the formation of the LMW compounds like Frg-OH and Frg-Ome implies that the phenolic guaiacyl subunit plays a critical role in the β-O-|4 cleavage, different from the nonphenolic veratryl subunit.21,28,29,31 Although the laccase-catalyzed radical formation and decay of the syringyl–guaiacyl π–π complex still occur and the β-O-|4 cleavage only generates a small amount of products, this novel aggregation LMS mechanism suggests that specific mediators are needed for each substrate, outlining future molecular design philosophies for this wide substrate spectrum enzyme.

Materials and Methods

Materials

Guaiacylglycerol-β-guaiacyl ether (G-β-GE) was provided by Tokyo Chemical Industry Co., Ltd. Mediator molecules ABTS, SA, AS, and MS were obtained by Shanghai Aladdin Bio-Chem Technology Co., Ltd. T. versicolor laccase was purchased from Sigma-Aldrich, with a labeled activity of 0.87 U/mg, and used without further purification. All other chemicals used were of analytical grade.

Enzyme Kinetic Measurement

Michaelis constants (KM) and maximum initial velocities (Vmax) of G-β-GE, ABTS, SA, AS, and MS were separately determined in the laccase-alone system without small molecules. Specifically, the substrates’ and mediators’ initial oxidation rates were individually measured over various concentration ranges in a 50 mM acetic acid–sodium acetate buffer (pH 4.5) at 35 °C: 12.5–500 μM for G-β-GE, 1.0–35 μM for ABTS, and 2.5–80 μM for SA, AS, and MS. G-β-GE consumption was monitored on an Elite P230II HPLC equipped with a UV detector (280 nm). ABTS, SA, AS, and MS consumptions were monitored with an Agilent 8453 UV–vis spectrophotometer (190–1100 nm range). Each reaction was performed in triplicate.

LMS Reactions of G-β-GE in the Presence of ABTS, SA, AS, or MS

G-β-GE, ABTS, SA, AS, and MS were individually dissolved in 50 mM acetic acid–sodium acetate buffer (pH 4.5) at a concentration of 10 mM, using 5% (v/v) N,N-dimethylformamide (DMF) as a cosolvent for improved solubility.27,28 Laccase was dissolved in 50 mM acetic acid–sodium acetate buffer (pH 4.5) at a concentration of 1.0 mg/mL. A typical reaction was prepared using 200 μL of laccase stock and 200 μL of G-β-GE stock, followed by 100 μL of pertinent mediator solution, and then diluted to a final volume of 2.0 mL with acetate buffer. The LMS mixtures were incubated at 35 °C and 150 rpm in an incubator shaker, and at 1, 2, 3, 4, 5, 6, and 12 h, 100 μL of the reaction mixture was transferred to a test tube, followed by 300 μL of methanol to quench enzymatic reactions.

G-β-GE Measurement of High-Performance Liquid Chromatography (HPLC)

For both the laccase-alone and LMS reactions, the methanol-quenched samples were centrifuged at 12 000 rpm for 3 min, and the supernatants were collected in glass vials for G-β-GE consumption determination by an Elite P230II HPLC equipped with a UV detector (280 nm). In a typical assay, 20 μL of supernatant was injected into an Agilent C18 reversed-phase column (150 mm × 4.6 mm, particle size 5 μm). The flow rate was set to 1.0 mL min–1 at 30 °C. Water and acetonitrile were used as A and B eluents, the former acidified with 0.1% acetic acid. The isocratic gradient was 0–20 min at 20% acetonitrile.

Product Profiles Characterized by Ultraperformance Liquid Chromatography-Time of Flight Mass Spectrometry (UPLC-TOF-MS)

The supernatants from the methanol-quenched samples were also collected in glass vials for subsequent product characterization on Agilent HPLC 1290-MS 6230. Chromatographic separation was achieved with an Agilent C18 reversed-phase column (150 × 4.6 mm2, particle size 5 μm) at 30 °C and an injection of 20 μL. As in the HPLC experiment, 0.1% acetic acid aqueous solution and acetonitrile were used as eluents A and B, respectively. Gradient elution was performed at a constant flow rate of 0.4 mL/min: 0–1.5 min at 15% B (isocratic), 1.5–47 min from 15 to 35% B (linear gradient), 47–48 min from 35 to 99% B (linear gradient), 48–53 min at 99% B (isocratic), 53–54 min from 99 to 5% B (linear gradient), and 54–59 min at 5% B (isocratic). In mass spectra acquisition, electrospray ionization (ESI) source in the positive ionization mode was applied. Each analysis was performed in triplicate.

Kinetic Model Fitting

Kinetic modeling was performed with Dynafit and COPASI software.53,54 Reaction rate constants were determined via global fitting for the G-β-GE consumption complete reaction progress curves. The underlying system of first-order ordinary differential equations was integrated using the Livermore solver for ordinary differential equation (LSODE) algorithm. The concentrations of these molecular species at time t were computed from their initial concentrations by solving an initial-value problem defined by a system of simultaneous first-order ordinary differential equations (ODEs).

Structural Modeling for Aggregation Complexes

The lowest-energy conformers for the substrate–mediator complexation were obtained using the conformer rotamer ensemble sampling tool (CREST), with the semiempirical tight-binding-based quantum chemistry method GFN2-xTB in the framework of meta-dynamics (MTD).55,56 The structures were then optimized with the empirical dispersion-included ωB97X-D functional57 in Gaussian 16,58 with basis sets of 6-31+G(d).59 The inter- and intramolecular interactions were studied by Frontier molecular orbital (FMO) composition analysis60 and independent gradient model (IGM) analysis61 via the Multiwfn 3.6 software package.62 Isosurface maps were produced using the VMD 1.9.3 program63 based on the outputs from the Multiwfn calculations.

Acknowledgments

This work was supported by grants from the National Key R&D Program of China (2018FYA0901200 and 2020YFA0907703) and the National Natural Science Foundation of China (31970041, 31770070, and 21377085). The authors thank the Center for High-Performance Computing at Shanghai Jiao Tong University for the computing resources. X.C. thanks Shenggan Luo for help with graphics.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.1c02501.

  • Detailed calculations of the degradation products (Tables S1–S3); the detailed assignments of the MS signals (Table S4); the mass balance before and after the reaction (Table S5); the UV–vis spectra and Michaelis–Menten plots (Figures S1–S5); the HPLC chromatogram (Figures S6–S11); the time-dependent mediator quantities (Figure S12); proposed structures of low-molecular-weight products (Figure S13); the proposed reaction diagram (Figure S14); the kinetic fitting details (Figures S15–S21); computational details (Figures S22 and S23); and pie charts of the gradient profiles (Figure S24) (PDF)

Author Contributions

Y.-L.Z. conceived the project. X.C. carried out the experiments. X.C. and Y.-L.Z. conducted the data analysis and kinetics modeling. X.O. and J.L. performed the quantum calculations. X.C. and Y.-L.Z. wrote up the manuscript.

The authors declare no competing financial interest.

Supplementary Material

ao1c02501_si_001.pdf (2.8MB, pdf)

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