Can laccases catalyze bond cleavage in lignin?

    Can laccases catalyze bond cleavage in lignin? Line Munk, Anna K. Sitarz, Dayanand C. Kalyani, J. Dalgaard Mikkelsen, Anne S. Meyer PII: DOI: Reference:

S0734-9750(14)00195-5 doi: 10.1016/j.biotechadv.2014.12.008 JBA 6878

To appear in:

Biotechnology Advances

Received date: Revised date: Accepted date:

15 August 2014 6 December 2014 25 December 2014

Please cite this article as: Munk Line, Sitarz Anna K., Kalyani Dayanand C., Mikkelsen J. Dalgaard, Meyer Anne S., Can laccases catalyze bond cleavage in lignin?, Biotechnology Advances (2015), doi: 10.1016/j.biotechadv.2014.12.008

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Can laccases catalyze bond cleavage in lignin? Line Munk#, Anna K. Sitarz#, Dayanand C. Kalyani, J. Dalgaard Mikkelsen, Anne S. Meyer*

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Center for BioProcess Engineering, Dept. of Chemical and Biochemical Engineering,

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#These two authors contributed equally.

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E-mail address: [email protected] (A.S. Meyer).

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* Corresponding author. Tel.: +45 4525 2800.

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Building 229, Technical University of Denmark, DK-2800 Kgs. Lyngby, Denmark

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ACCEPTED MANUSCRIPT Abstract Modification of lignin is recognized as an important aspect of the successful refining of

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lignocellulosic biomass, and enzyme-assisted processing and upcycling of lignin is receiving

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significant attention in the literature. Laccases (EC 1.10.3.2) are taking the centerstage of this attention, since these enzymes may help degrading lignin, using oxygen as the oxidant. Laccases

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can catalyze polymerization of lignin, but the question is whether and how laccases can directly

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catalyze modification of lignin via catalytic bond cleavage. Via a thorough review of the available literature and detailed illustrations of the putative laccase catalyzed reactions, including the possible

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reactions of the reactive radical intermediates taking place after the initial oxidation of the phenolhydroxyl groups, we show that i) Laccase activity is able to catalyze bond cleavage in low

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molecular weight phenolic lignin model compounds; ii) For laccases to catalyze inter-unit bond

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cleavage in lignin substrates, the presence of a mediator system is required. Clearly, the higher the

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redox potential of the laccase enzyme, the broader the range of substrates, including o- and pdiphenols, aminophenols, methoxy-substituted phenols, benzenethiols, polyphenols, polyamines, which may be oxidized. In addition, the currently available analytical methods that can be used to detect enzyme catalyzed changes in lignin are summarized, and an improved nomenclature for unequivocal interpretation of the action of laccases on lignin is proposed.

Keywords: lignocellulose, biocatalysis, mediators, oxidation

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ACCEPTED MANUSCRIPT 1. Introduction Lignin is a complex, hydrophobic biopolymer built of phenylpropanoid units. Lignin is present in

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plant cell walls and thus in lignocellulosic biomass feedstocks used in the pulp/paper industry and

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in cellulosic biofuel production. Lignin typically comprises 20–32 % by weight of the lignocellulosic biomass (Chen et al., 2011), and modification or removal of the lignin prior to

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enzymatic hydrolysis and fermentation in lignocellulose-to-ethanol schemes would be advantageous

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because: i) lignin tends to adsorb cellulolytic enzymes to its surface (Yang et al., 2012), ii) lignin degradation byproducts have been shown to inhibit the activity of cellulolytic enzymes (Berlin et

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al., 2006). In both cases the yield of fermentable sugars is decreased. Various attempts have been made to design integrated pretreatment systems for removing the lignin from the pretreated biomass

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(Koo et al., 2012; Pedersen et al., 2010; Yang et al., 2012). Recently, particular attention has been

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drawn to the development of environmentally friendly technologies using oxidoreductive enzymes

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for treatment of lignin, including pretreatment of lignocellulose for de-lignification, in order to improve the processing and conversion of the biomass (Gutiérrez et al., 2012; Kudanga and Le Roes-Hill, 2014; Rico et al., 2014). Laccases (EC.1.10.3.2, in the expanded CAZY database including auxiliary redox enzymes now referred to as AA1s (Levasseur et al., 2013)) principally catalyze the oxidation of diphenol hydroxyls, and can also act on hydroxyl groups of monophenols and related compounds, using oxygen as the electron acceptor; hence, during one round of the overall laccase catalyzed reaction, transferring 4 electrons, one molecule of O2 is reduced to two molecules of H2O (www.brenda-enzymes.org). Accordingly, laccase activity does not require the presence of H2O2 such as peroxidases, e.g. lignin peroxidases (EC 1.11.1.14) and manganese peroxidase (EC 1.11.1.13). Hydrogen peroxide addition is currently not feasible in enzymatic lignocellulose or lignin conversion, which is why laccase catalysis is in focus. Laccase modifications, e.g. depolymerization of lignin polymers, could, for example, lead to the synthesis of

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ACCEPTED MANUSCRIPT lignin-derived cyclic oxygenates (CyclOx), used to reduce bunker fuel consumption in the marine sector. When 10 % cyclic oxygenate is added to standard diesel fuel, soot emissions can be reduced

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by 50 % (Herreros et al., 2014).

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This review presents an overview of the current knowledge relating to the action of laccase on lignin and lignin-like compounds. The goal is to provide an in-depth review of the ability of

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laccases, alone or during action via mediators, to cleave ether and/or carbon-carbon bonds in lignin.

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Additionally, we summarize the methodologies available for detecting changes occurring during enzyme catalyzed actions on lignin. We also introduce an unequivocal nomenclature to help clarify

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the interpretation of the current literature and experimental data relating to the action of laccase on

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2. Lignin structure and sources

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lignin.

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Lignin is a highly complex phenylpropanoid polymer mainly synthesized from three monomeric precursors (Fig.1). When incorporated into the lignin polymers, the monomeric units are known as p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units. Owing to numerous plant physiology studies, the general structure of lignin, defined by linkages between subunits in lignin, and the pathways for lignin synthesis are well described (Ralph et al., 2007). The composition of lignin is influenced by the origin and growth conditions of the plant and can even differ among cell types within the same plant (Moura et al., 2010). Lignins are commonly divided into three types according to the ratio of the three monomeric units (Djikanovic et al., 2012). Softwood (gymnosperm) lignins are mainly composed of G-units with minor amounts of H-units, whereas hardwood (angiosperm - dicots) lignins are composed of G- and S-units in approximately equal ratios (Espiñeira et al., 2011). The high amount of S-units in hardwood lignins tends to give a more linear structure by comparison to softwood lignins. Grass lignins (angiosperm - monocots) contain

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ACCEPTED MANUSCRIPT all units (G-, S- and H-units), but the ratios between the three units vary within this group (Buranov and Mazza, 2008). Additionally, grass lignins are characterized by a high acetylation degree and a

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high occurrence of ferulic acid and p-coumaric acid (del Río et al., 2012; Martínez et al., 2008). To

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some extent, the combinations of H, G and S-units affects the formation of inter-unit linkages in lignin because of the differences in their substitutions. Although occurrence of the monomeric units

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can be related to the biomass type, no such correlation between occurrence of specific inter-unit

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linkages and type of biomass is evident from the published literature. Table 1 provides an overview of the occurrence of the typical inter-unit linkages in three major biomass types (softwood,

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hardwood, grasses). Inter-unit linkages are named by numbering the 9 C-atoms in the phenylpropanoid units, with the aromatic carbons marked from 1-6 and the aliphatic carbons

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marked as α, β, and γ (Fig. 1). The linkage names indicate between which two C-atoms of two

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phenylpropanoid units the coupling has occurred, while the position in the second phenylpropanoid

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unit is marked with a prime (Henriksson, 2009). The specific linkage connecting the two C-atoms in each unit is highlighted by the illustrations in Table 1. The most abundant type of inter-unit linkage is β-O-4’, which often comprises more than 50 % of all inter-unit linkages in all types of native lignin (softwood, hardwood, grasses), (Branau and Mazza, 2008; Zakzeski et al., 2010). Since coupling is favored at the β-position of monolignols, other β-couplings, besides β-O-4’, are commonly found in form of β-5’, β-β’, and β-1’. Coupling of oligomers results in formation of the inter-unit linkages such as 4-O-5’ and 5-5’, which also serve as potential branching points in the polymer (Ralph et al., 2007). Development of more advanced NMR techniques for analysis of interunit linkages have led to the discovery of new couplings, such as spirodienone, which is related to β-1’ bonds, and dibenzodioxocine, which arises from a 5-5’ unit coupled to a third phenylpropanoid monomer (Crestini et al., 2011). Lignins with relatively high numbers of C-C linkages compared to ether linkages are often referred to as condensed lignins. Condensed lignins are frequently more

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ACCEPTED MANUSCRIPT rigid and less prone to degradation, since the energy required to break C-C linkages such as β-5’ and ether linkages such as β-O-4 is 125-127 kcal/mol and 54-72 kcal/mol, respectively

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(Parthasarathi et al., 2011).

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In addition to variations in native structures, lignin in biomass is exposed to different types of industrial processing, which often result in structural modification. Reactive groups, such as

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hydroxyl and ether groups, are oxidized to carbonyl groups or react to give more condensed and

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non-phenolic sub-structures (Chakar and Ragauskas, 2004; Parthasarathi et al., 2011; Shevchenko et al., 1999). To provide an insight into the characteristics of some of the technical and native-like

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lignins resulting from various industrial processes, some frequently used lignin sources are listed in Table 2. Synthetic lignins are commonly known as dehydrogenation polymers (DHPs) and are

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synthesized from the primary lignin precursors, or similar structures that may be radioactively

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labeled, in the presence of a catalyst e.g. horseradish peroxidase.

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Native and industrially modified lignins are frequently used in scientific studies to explore the effect of laccase in relation to biomass conversion, whereas synthetic lignins, such as DHP, are employed in such studies much less frequently (Gutiérrez et al., 2012; Qui and Chen, 2012; Rico et al., 2014).

3. Laccase and its potential in lignin degradation The degradation of lignin is seen as a complex process involving different classes of enzymes, e.g. peroxidases, oxidoreductases, oxidases, which have recently been classified in the CAZy database as Auxiliary Activities (AA) family (Levasseur et al., 2013). One of those enzymes is laccase (benzenediol: oxygen oxidoreductases; EC 1.10.3.2; AA1). Laccase belongs to the multicopper oxidase family, a group of enzymes that is widespread in numerous fungi, plants, and bacteria (Virk et al., 2012). Phenol oxidases such as laccases use O2 as the final electron acceptor rather than H2O2, a fact which differentiates laccases from other lignin modifying enzymes such as lignin

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ACCEPTED MANUSCRIPT peroxidase, versatile peroxidases, and manganese peroxidase (AA2), which are all dependent on H2O2. This fact makes laccases more industrially applicable with regards to economy in potential

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industrial applications, which translates into less corroded tanks and sustainability. Laccases contain

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four copper ions, which can be classified into three groups according to their spectroscopic features: type 1 (T1) or blue copper, type 2 (T2) or ‘‘normal’’ copper and type 3 (T3), an

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antiferromagnetically coupled binuclear copper pair. The T2 and T3 centers are close together and

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form a trinuclear cluster. T1 is the site where substrate oxidation takes place. Electrons are then shuttled along a pathway containing cysteine and histidine residues to the trinuclear cluster which is

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the site of oxygen reduction (Shleev et al., 2005; Sitarz et al., 2014). The 3D crystallographic structure of the Trametes versicolor laccase, the most widely studied fungal laccase, has shown that

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the catalytically important copper ions are located in different domains with the T1 copper situated

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in a shallow cleft on the surface of the enzyme, whereas the T2 and the T3 copper sites, which have

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one (T2Cu) and two (T3αCu and T3βCu) copper atoms, respectively, that form a trinuclear copper cluster (T2/T3), are positioned centrally at the interface between two protein structural domains in the enzyme that provide the ligand residues for the coordination of the copper atoms (Sitarz et al. 2014). The positions of the copper ions concur with the structural results available for other fungal laccases (e.g. the laccase derived from Melanocarpus albomyces (for which the crystal structure was published by Hakulinen et al., 2002)). The redox potentials of laccases range from 0.5 to 0.8 V vs. NHE (Normal Hydrogen Electrode) (Table 3) depending on the type of organism from which the enzyme has been isolated. So far, laccases from white-rot basidiomycetes fungi have been reported to have the highest redox potential (0.8 V vs. NHE), which is an advantage when considering the enzyme for biotechnological applications, since a higher redox potential enables the enzyme to abstract electrons from the nonphenolic lignin subunits, i.e. units where no hydroxyl group remains on the benzene ring since the

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ACCEPTED MANUSCRIPT hydroxyl has formed e.g. a radical-radical coupling linkage, e.g. as for one of the units in 8-O-4′ coniferyl alcohol ether. Laccase activity on phenol oxidation is known to be proportional to the

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redox potential difference between the T1Cu and the substrate. Some recently reported molecular

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evolution data (based on random mutagenesis) for laccases produced mutations in the region close to the T1Cu, which improved phenol oxidation catalysis (Maté et al., 2010). Although it remains

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unclear how laccases influence lignin modification, it is possible that catalytic efficacy of fungal

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laccases on lignin can be enhanced by targeting mutations close to the T1 copper. Laccase has not always been considered as an enzyme that could contribute to lignin breakdown, mostly due to the

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following two characteristics:

i) Its redox potential is too low in order to oxidize non-phenolic units of lignin (ca. 80 % of the

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total lignin units) and

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ii) P. chrysosporium, a model organism for fungal lignin degradation, was suspected not to be able

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to express laccase (de Jong et al., 1994; Hattaka, 1994; Kirk and Farrell, 1987). The turning point for the idea that laccase may be used as a main enzyme or a part of an enzyme cocktail that could contribute to lignin degradation has been mainly initiated by the following events described in the following publications: i) Research by Ander and Eriksson as early as 1976, whose genetic studies on Sporotrichum pulverulentum showed that a laccase-less mutant was not able to degrade kraft lignin, whereas a laccase-positive revertant regained the ability of the wild type S. pulverulentum to degrade kraft lignin and all the major components of wood. ii) Kawai et al., (1988a) proved the ability of laccase from Coriolus versicolor to degrade phenolic lignin substructure model compounds. iii) Research by Bourbonnais and Paice (1990) revealed that the redox potential of laccase can be modulated by the addition of small molecular weight compounds (known as mediators), which

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ACCEPTED MANUSCRIPT enabled laccase from Trametes versicolor to catalyze oxidation and degradation of non-phenolic lignin units.

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These important findings for establishing that laccase plays an important role in lignin degradation

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were additionally supported by:

i) Demonstrations that white-rot fungi, which possess a number of aggressive lignin degraders,

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seem to operate without expressing LiP activity (Périé and Gold, 1991; Rüttmann-Johnson et

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al., 1993; Srebotnik et al., 1994).

ii) Eggert et al., (1997) and Bermek et al., (1998) described a laccase-less mutant of P.

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cinnabarinus that naturally lacks the ability to produce LiP and MnP, and therefore is totally devoid of the chance to metabolize 14C ring-labeled DHP to 14CO2 and bleach kraft pulp,

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respectively. Moreover, when the purified laccase was added to the experimental setup in an

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equivalent amount to that found in the wild type fungus, a laccase-less mutant of P.

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cinnabarinus was able to restore its ability to decompose 14C ring-labeled DHP to 14CO2 iii) Kim et al., (1986) discovered that a mutant of Pleurotus which lacked laccase activity degraded lignin poorly,

iv) Youn et al., (1995) proved that laccase is able to degrade several lignin model compounds and may be visualized cytochemically in degraded wood cell walls v) Kües and Rühl (2011) analyzed a fungal genome and confirmed the role of laccases in lignin degradation in Basidiomycota, due to the revelation of the existence of multigenic families of MCOs genes. vi) Xie et al., (2014) gave genetic evidence for some laccases playing a role in lignocellulose and plastic degradation as well as detoxification of phenolic substrates. All these experimental results confirm the likely role of laccase in lignin degradation and therefore laccase can be seen as a green alternative for lignin modification and for upcycling of lignin.

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ACCEPTED MANUSCRIPT However, the reasons for the large range in catalytic efficacy on lignin among fungal laccases having reasonably high amino acid sequence homology are unclear. In addition, as discussed below,

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most of the current understanding of laccase action on lignin has been obtained on variously treated

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lignins, lignin derivatives, or model compounds. This coupled with the limited quantitative analytical methodology on intact water-insoluble lignin are significant barriers to be overcome to

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improve the understanding of laccase action on intact biomass.

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4. Equivocal nomenclature for the description of observed alterations in lignin structures. Studies concerning laccase treatment of lignin tend to use different nomenclature to describe the

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changes in phenylpropanoid units of lignin that occur due to the enzymatic catalysis. Varying nomenclature occasionally makes it difficult to comprehend to what extent laccase acts on lignin.

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Fig. 2 illustrates how different plausible events that may take place in lignin as result of laccase

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catalysis can be described by the prudent use of specific words. In this review, we only focus on

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laccase-catalyzed changes in lignin, but we believe that the terminology proposed will cover changes that occur in lignin structures during any oxidoreductive enzyme action on lignin. The change in a lignin structure is initiated when lignin is oxidized by abstraction of a single electron from a phenylpropanoid subunit. The abstraction of an electron activates the lignin surface by creating an active radical, thus making the lignin locally more reactive (Fig. 2) (Suurnäkki et al., 2010). Activation of the lignin structure may induce different reactive events such as bond cleavage, modification, and/or coupling (Fig. 2). Since each of these events lead to different further reactions, it is important that the terms are used carefully, and we propose that the nomenclature used to describe laccase and laccase.mediator action on lignin follows the terminology used in Fig. 2. Lignin is rich in conjugated bond systems, so electrons can delocalize to areas where the structure is most stable and this phenomenon may lead to modifications seen as changes in functional groups (Fig. 2) (Gutiérrez et al., 2012).

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ACCEPTED MANUSCRIPT Another outcome of activation may occur when the radical form of a lignin subunit reacts with a low molecular weight compound, e.g. a mediator or phenylpropanoid unit, which causes the low

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molecular weight compound to attach to the surface of the lignin through radical coupling (Fig 2). If

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the coupling reaction results in the distribution of attached monomeric molecules (reaction b, Fig. 2) on the surface of lignin, it is referred to as grafting. Grafting affects the properties of lignin

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including solubilization (Moldes et al., 2008). If the coupling reactions continue as a radical

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polymerization process (reaction a, Fig. 2) of e.g. phenylpropanoid units or oligomers, the reaction is regarded as polymerization, and the average molecular weight of the lignin polymer increases

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(Barneto et al., 2012; Shleev et al., 2006). Although the same mechanism is responsible for both grafting and polymerization, we distinguish between these incidents (Fig. 2). In contrast to the

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events described above, the activated lignin subunits may not be stabilized by the mentioned

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modifications or couplings, but instead go through bond cleavage. If activated phenylpropanoid

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units result in cleavage of bonds within lignin, it is likely that a number of consecutive cleavages will facilitate opening or even fragmentation of lignin, resulting in a decrease in the average molecular weight of the lignin polymer i.e. depolymerization (Fig. 2) (Rico et al., 2014). In the literature

apparently related words such as delignification, degradation, and

depolymerization, which might seem to describe the same action of laccase and/or LMS on lignin polymers, appear to be used indiscriminately. However, it must be emphasized that delignification of lignin is associated with removal of lignin from the biomass, which gives rise to increased pulp brightness and decreased kappa number (an indication of residual lignin content). We would like to stress that these changes do not directly prove that lignin is degraded into smaller subunits, associated with C-C and/or ether bond cleavage. Increased pulp brightness and decreased kappa number are equally likely to be caused by enhanced solubility of the lignin polymer due to radical grafting of sulfonated mediators onto the lignin structure (Moldes and Vidal, 2011). Grafting of

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ACCEPTED MANUSCRIPT sulfonated mediators onto the lignin structure has been demonstrated to enable dissolution of lignin at pH 2.4 (Lund and Ragauskas, 2001). Consequently, there should be a clear distinction between

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the act of delignification and the act of actual bond cleavage in lignin. We suggest, therefore, that

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depolymerization is used as the proper description for the fragmentation of lignin associated with bond cleavage, yielding a smaller, truncated lignin polymer. The fragmentation of lignin associated

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with bond cleavage contributes to the overall degradation of lignin.

5. Laccase action in relation to lignin degradation

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5.1 Effect of laccase on lignin model compounds

Due to the complexity of native lignin structures, the results from early studies regarding the action

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of laccase on lignin are derived from experiments with lignin model compounds (Zakzeski et al.,

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2010). Consequently, the findings from research using lignin model compounds will also be

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included in this assessment of laccase action on lignin polymers. Although single aromatic ring structures have been used as lignin model compounds for laccase action in some of the early literature (e.g. Kawai et al., 1988b), currently used lignin model compounds are soluble, low molecular weight organic compounds comprising of minimum 2-3 aromatic rings that represent substructures and linkages similar to those found in native lignin (Rochefort et al., 2004). Phenolic compounds are typical substrates of laccases because their redox potentials (0.5 to 1.0 V vs. normal hydrogen electrode (NHE)) are low enough to allow electron abstraction. The oxidative action of laccase has been demonstrated on phenolic β-O-4’ (representing the majority of the interunit linkages in native lignin) and β-1’ lignin model dimers by catalyzing the formation of phenoxy radicals (Fig 2.). Initially the dimers undergo Cα-oxidations, which either results in ketone formation at the Cα-atom or fragmentation of the dimers either through Cα-Cβ cleavage or through alkyl-aryl cleavage (Cα-C1 cleavage) (Kawai et al., 1988a; Youn et al., 1995). In addition, Kawai et

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ACCEPTED MANUSCRIPT al. (1988b) demonstrated aromatic ring cleavage in a phenolic monomer, indicating that laccase has the potential to cleave a variety of bonds in phenolic subunits of lignin. To be representative of

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more condensed lignin structures, Crestini and Argyropoulos (1998), studied 5-5 (and 5-α) phenolic

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model compounds, which resulted in bond cleavage through side-chain reactions, but no cleavage

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of the actual 5-5’ bond. So far, no studies have been able to demonstrate the action of laccase alone on non-phenolic model compounds, and it is therefore generally acknowledged that laccase alone is

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5.2 Effect of laccase on lignin polymers

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only able to cleave bonds in phenolic compounds (Fig 2).

The relevance of investigating small lignin model compounds to understand macromolecular lignin

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degradation has been questioned. In contrast to lignin model compounds, native and industrial

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lignin polymers are complex, high molecular weight polymers, which have low solubility (native

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lignin) or are highly modified, which may imply less reactive functional groups and more condensed structures (technical lignin). These inevitable differences between model compounds and lignin, native as well processed, may have huge influence on the proneness of lignin towards oxidation catalyzed by laccase. In addition, native lignin is comprised of 80-90 % non-phenolic subunits, while the remaining 10-20 % is phenolic subunits (Schoemarker et al., 1985). Since the ability of laccase to catalyze oxidation of a lignin substrate is closely related to the presence of phenolic groups in a substrate, the degree of modification of a native or industrial lignin polymer caused by laccase catalysis alone may be limited. However, the possible degree of modification of lignin caused by laccase alone is difficult to clarify based on the present literature because only a limited number of studies focus on unraveling what really happens during catalysis of a lignin polymer by laccase alone and to what extent the lignin polymer can be modified. In early studies, oxidation, demethylation and carboxyl group formation in milled wood lignin have been observed

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ACCEPTED MANUSCRIPT (Ishihara, 1980). Otherwise, the majority of studies demonstrate almost exclusively by size exclusion, that treatment with laccase alone results in polymerization of various lignin polymers as

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seen in Table 4a. Only in the case of modified lignin polymers with high phenolic ratios (30 % mol)

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or carbohydrate bonded DHP (33 % mol), was laccase shown to decrease the lignin polymer molecular weight (Kondo et al., 1990; Xia et al., 2003). Overall, these studies give good evidence

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that the sole action (without the use of a mediator) of laccase does modify the phenolic subunits of

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lignin polymer to some extent, and modification predominantly occurs in such a way that it induces polymerization (Kondo et al., 1990; Mattinen et al., 2011; Shleev et al., 2006). Still, taking into

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account the findings from lignin model compounds and the fact that some modified lignin polymers have been shown to decrease in molecular weight after laccase treatment, it cannot be excluded that

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the activation of lignin by laccase may also induce bond cleavage of the weakest bonds, e.g. ether

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bonds (β-O-4’, 4-O-5’), if the conditions for favoring bound cleavage are present. Cleavage of

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bonds with higher binding dissociation energies, which are more common in condensed lignin, e.g. β-5’ and 5-5’, seems to be less plausible.

5.3 Laccases and laccase mediator systems (LMS) The direct action of laccase alone is, in principle, restricted to phenolic subunits, since non-phenolic subunits have higher redox potential (>1.5V vs. NHE) and therefore they cannot be oxidized by laccase alone. The restriction of laccase action to phenolic subunits can, however, be overcome by use of a mediator (Rochefort et al., 2004). A mediator is a molecule that acts as an electron carrier between laccase and the substrate to be oxidized. Thus, the LMS becomes a stronger catalyst than laccase alone by expanding the oxidation ability of the enzyme. Once the mediator is oxidized by laccase by electron abstraction, it diffuses away from the catalytic pocket of the enzyme and in turn oxidizes e.g. a non-phenolic subunit of lignin. Mediators are believed to expand the reach of laccase

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ACCEPTED MANUSCRIPT due to their ability to diffuse into the plant cell wall because of their comparatively smaller size. Additionally, the oxidation of non-phenolic subunits is possible due to oxidized mediators

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becoming strong oxidizing intermediates, whose redox potentials exceed those of non-phenolic

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subunits of lignin (Rochefort et al., 2004). Several compounds have been described as laccase mediators and they are usually divided into natural and synthetic compounds (Christopher et al.,

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2014). Mediators of natural origin are compounds that are lignin degradation products, plant

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phenolics present in plants as secondary metabolites or secondary, extracellular, fungal metabolites (4-hydroxybenzylic alcohol, p-cinnamic acid, sinapic acid, etc.). The existence of natural mediators

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makes it difficult to distinguish between the sole action of laccase and the action in combination with a mediator. When studying the sole action of laccase, it is difficult to exclude the possibility of

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interference from natural mediators, which are easily present as phenolic compounds released from

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(2,2’-azinobis(3-ethylbenzthiazoline-6-sulphonate)),

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hydroxybenzotriazole,),

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e.g. lignin. Synthetic mediators, on the other hand, are co-added with laccases. HBT (1-

(2,2,6,6-tetramethylpiperidine 1-oxyl), and violuric acid are amongst the most commonly used synthetic mediators. Not all molecules acting as electron carriers should be characterized as redox mediators since their oxidized intermediates are electrochemically unstable (Morozova et al., 2007b). The redox potential of a mediator seems to play a negligible role in the catalytic efficiency of oxidation. Rather, the effectiveness of a mediator is likely to depend on the chemical reactivity of the radical formed after the initial oxidation step (González et al., 2009). Because the oxidoreductive capability of both laccases (Table 3) and mediators varies, the oxidoreductive capability of the LMS varies with the chosen laccase and mediator combination.

5.4 Effect of LMS on lignin model compounds

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ACCEPTED MANUSCRIPT The elucidation of recently discovered LMS has extended the substrate activity of laccase to include oxidation of non-phenolic lignin model compounds, as LMS are capable of catalyzing the formation

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of other radicals than just phenoxy radicals. Based on the treatment with Trametes versicolor

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laccase and HBT of a non-phenolic β-O-4’ lignin model dimer, Kawai et al., (2002) suggested that a β-aryl radical cation and a benzylic radical are the intermediates that undergo series of random

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reactions resulting in; Cβ-ether cleavage, Cα-Cβ cleavage, Cα-oxidation, and aromatic ring cleavages.

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These findings are a good indication that LMS are able to modify and plausibly cleave bonds of the non-phenolic subunits of lignin polymers that are rich in ether bonds (i.e. native-like lignin).

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Additionally, Crestini et al., (2003) demonstrated that the presence of a HBT mediator strongly favors oxidation reaction pathways over coupling reactions. Although there is good evidence for the

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enhancing ability of mediators, they do not appear to facilitate the degradation of non-phenolic 5-5’

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(and α-5’) model dimers, as laccase does not either cleave or modify these model compounds in

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presence of ABTS or HBT (Crestini and Argyropoulos, 1998). The question thus remains whether mediators are able to extend the catalyzing ability of laccase towards more condensed lignins. If not, the utilization of LMS may be less relevant for condensed lignin polymers

5.5 Effect of LMS on lignin polymers It is a general understanding that the action of laccase fundamentally catalyzes polymerization, while LMS catalyze depolymerization (Bourbonnais et al., 1995; Shleev et al., 2006; Hernández Fernaud et al., 2006), a perception that is generally supported by the overview in Table 4a. Shleev et al., (2006) observed that changes in molecular weight varied between the two most commonly applied mediators, ABTS and HBT, which indicates that these two mediators may act through different mechanisms. Hernández Fernaud et al., (2006) obtained similar results with the same mediators, although the laccase and substrate identity varied. The results of Shleev et al. (2006) and Hernández Fernaud et al. (2006) thus suggest that the presence of a mediator and the type of

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ACCEPTED MANUSCRIPT mediator influence the course of action during catalysis. Therefore, it may be possible to control the outcome of laccase treatment of lignin using mediators. In addition, both studies discussed above

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were executed on kraft lignin, which suggests that in spite of a more condensed lignin structure,

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LMS are sufficiently capable of cleaving bonds to induce depolymerization. In contrast to results demonstrating decrease in average molecular weight (depolymerization) of lignins using LMS,

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studies with LMS have also shown that the oxidizing capability of some mediators, e.g. TEMPO

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and acetosyringone, leads to results suggesting polymerization of lignin (Moya et al., 2011). Polymerization of lignin using LMS has been demonstrated with both ABTS and HBT as mediators

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(Prasetyo et al., 2010). The fact that studies concerning LMS treatment are so contradictory regarding polymerization and depolymerization of lignin emphasizes the complex context in which

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be unilateral.

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LMS are operating, and the reaction course following activation of lignin subunits does not seem to

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5.6 Does LMS catalyze polymerization or depolymerization? As the overviews in Table 4a and 4b illustrate, the effect of LMS treatment on native or technical lignin is equivocal, especially when it comes to concluding whether the catalysis assists polymerization or depolymerization of lignin in biomass. Li et al., (2007) have suggested that a competition between depolymerization and depolymerization occurs when the β-O-4’ are broken during steam explosion of aspen wood. Such a balance is undoubtedly affected by the surrounding conditions and the presence of mediators. It is likely that a similar competition between reactions routes, illustrated in Fig 2, occurs after activation of lignin subunits. It also seems that the type of radical created by LMS may influence the course of action towards depolymerization or polymerization (Crestini et al., 2003). The existence of competition between reaction pathways is supported by Moldes et al., (2008) and Barneto et al. (2012), who have investigated the effect of various LMS on removal of residual lignin in pulp. Both studies suggest that competition exists

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ACCEPTED MANUSCRIPT between mediated oxidation of lignin and oxidative coupling of mediators (grafting) or polymerization, and the resulting course of action may depend on the mediator identity. The

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suggestion of competition between reaction routes is a good explanation for the inconsistent results

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regarding polymerization and depolymerization. Additionally, the results illustrate that the choice of mediator is an important factor, but whether the reaction conditions favor a specific course of action

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has not yet been clarified.

5.7 Effect of LMS on biomass

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Many studies conducted with LMS concern treatment of an entire biomass or pulp (Table 4b). This impedes the elucidation of the actual role of laccase and LMS in lignin degradation. It is commonly

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seen that these studies address the effect of laccase treatment on lignocellulosic biomass by

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demonstrating an increase in the enzymatic conversion to sugar (Sitarz et al., 2013; Kalyani et al.,

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2012; Qui and Chen, 2012) or by demonstrating a decrease in kappa number and increase in brightness in pulp biomass (Babot et al., 2011; Zhang et al., 2012). However, relating these changes to the degradation action of laccases on lignin should be done with caution. An increase in the extent of conversion to sugar with laccase addition may occur due circumstances other than degradation of lignin caused by the presence of laccase. For example, if laccase renders potential inhibitory components of hydrolytic enzymes harmless or interacts with lignin in such a way that it prevents the cellulolytic enzymes from absorbing onto lignin, laccase addition could cause an increase in free or active hydrolytic enzyme (Palonen and Vikari, 2004; Moilanen et al., 2011). Likewise, lignin modifications or coupling of small polar molecules, i.e. small sulfonated compounds, onto lignin (grafting), may decrease kappa number and klason lignin, which can be seen as an indication of lignin removal by laccase or LMS. However, changes in kappa number and klason lignin do not provide any knowledge or information about the action or role of enzymes in

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ACCEPTED MANUSCRIPT cleavage of bonds in lignin and are a sign of improved solubility of lignin caused by coupling of sulfonated mediators to its fibers (Moldes and Vidal, 2011) rather than proof of enzymatic

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degradation of lignin.

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However, newer studies with LMS treatments by Du et al., (2013) and Rico et al., (2014), provide more informative results with regards to the direct effect of LMS on lignin. Rico et al., (2014)

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treated a eucalyptus feedstock with M. thermophila laccase and a methyl syringate mediator. The

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results were analyzed by Py-GC/MS and 2D NMR and revealed several modifications to the lignin polymer:

Formation of Cα-oxidized syringyl lignin units

ii)

Removal of guaiacyl and syringyl units

iii)

Lower numbers of aliphatic side-chains per phenylpropane unit, which are mainly

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i)

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involved in β-O-4’ and β-β’ inter-unit linkages, suggesting Cα -Cβ cleavage (Table 4b). Du et al., (2013), provided similar results, where kraft pulp was treated with either M. thermophila

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laccase - methyl syringate mediator or P. cinnabarius - HBT mediator and analyzed by SEC, 1H NMR and Py−GC/MS.

6. Contemporary analytical techniques for enzyme catalyzed changes in lignin It is evident that large amounts of lignin will be produced from future lignocellulosic ethanol processes and that is why enzymatic modification of lignin is also an important technical issue in upcycling of this heterogeneous polymer. Following that thread, it is important to understand exactly how lignin is degraded. Targeted techniques and methodologies are required in order to enable an unequivocal and trustworthy evaluation of the enzyme catalyzed changes in lignin regardless of the research center assessing the experiments. A strong impact is expected from the application of current analytical techniques such as two-dimensional (2D) or three-dimensional

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ACCEPTED MANUSCRIPT (3D) Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared (FTIR) spectroscopy, Size Exclusion Chromatography (SEC), and Pyrolysis Gas Chromatography/Mass Spectroscopy (Py-

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GC/MS), which may all provide useful information for quantitative and qualitative evaluation of the

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enzymatic action on the lignin polymer, highlighting the degradation products of the complex lignin structure (Gutiérrez et al., 2012; Martínez et al., 2011; Prasetyo et al., 2010). Based on the results

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obtained using 2D and 3D NMR, FTIR, SEC and Py-GC/MS, one is thus able to elucidate (predict)

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the structural changes that lignin undergoes during enzymatic catalysis. Methods such as acidolysis, hydrogenolysis, nitrobenzene oxidation, cupric (II) oxidation, permagenate oxidation, ozonation,

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thioacetolysis, derivatization followed by reductive cleavage (Yuan et al., 2013) are not in the scope of this review due to their involvement in selective cleavage of the lignin backbone, resulting in a

6.1 2D NMR

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lack of effective evaluation of the enzymatic modifications that the lignin polymer could undergo.

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2D NMR is a spectroscopic technique that combines the sensitivity of 1H NMR with the higher resolution of 13C NMR. This technique can be used to provide detailed structural information about the lignin polymer (the ratio between the different lignin units as well as the percentage of the main inter-unit linkages) from different types of biomasses (Heikkinen et al., 2003). Additionally, the initiation of multidimensional NMR techniques; 2D or 3D heteronuclear single quantum correlation (HSQC), has extended the prospect of lignin structural analysis considerably, where even structures of minor or unknown components can be elucidated (Rencoret et al., 2011; Crestini et al., 2011; Sette et al., 2011). 2D HSQC NMR is a powerful technique for lignin characterization but its weak points are that only soluble lignin fragments can be analyzed and that 4-O-5’, and 5-5’ subunits cannot be detected (Yuan et al., 2013) . To solve this problem this technique could be used in

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ACCEPTED MANUSCRIPT combination with some of the destructive analytical methods (that are mentioned but are not in the

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scope of this review).

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6.2. Py-GC/MS

Py-GC/MS has emerged as a highly sensitive technique for characterizing the chemical structure of

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lignin (del Río et al., 2005; Laskar et al., 2013). The use of pyrolysis in combination with mass

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spectrometry (Py-GC/MS) has proved to be of particular interest in the study of lignocellulose modification and has already been used to characterize different patterns of wood decay by fungi

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(del Río et al., 2002; Martínez et al., 2011). Py-GC/MS has also been used to attempt to elucidate whether or not chemical modifications observed in wheat straw were related to lignocellulose-

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degrading enzymes produced by Streptomyces (Hernandez et al., 2001). The combination of nuclear

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magnetic resonance spectroscopy (2D NMR and 1H and

13

C NMR) and Py-GC/MS, is currently

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regarded as one of the best tools for analysis of lignin polymers, giving a complete picture of the chemical features in the lignin structure (Colombini et al., 2009; Salanti et al., 2010; Rencoret et al., 2011). This type of analytical combinations are highly needed in the elucidation of laccase action on lignin. In principle, the combination of Py-GC/MS with advanced NMR spectroscopy might allow structural-unit

based

quantitative

measurements

of

lignin,

but

structural-analysis-based

quantification of lignin is still not feasible or very much in its infancy. Further developments in the analytical area of lignin research are highly required. At present many studies employs lignin samples derived from different biomass treatments or even from Kraft cooking as substrates to study laccase action on lignin. It is uncertain whether these substrates properly reflect the lignin structures in situ, and hence whether the action of laccases and laccase-mediator systems work in the same manner on intact biomass as on these differently treated isolated lignin substrates. New

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ACCEPTED MANUSCRIPT analytical methods allowing quantitative measurements on lignin would provide a significantly

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improved foundation for understanding laccase action on lignin in biomass.

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6.3. FTIR and SEC

FTIR and SEC are also used to measure chemical changes of lignin; however they do not provide

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exact differentiation between the specific lignin subunits (Ibarra et al., 2007; Ahmad et al., 2010;

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Prasetyo et al., 2010; Maijala et al., 2012). No universal method has been established for analyzing enzymatic delignification, and many conclusions are based on the way the results are interpreted.

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For example, contradicting studies by Gutiérrez et al., (2012) and Prasetyo et al., (2010) are worth mentioning, where the final outcome differed depending on the person handling the data. Gutiérrez

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et al., (2012) showed a decrease in the aromatic region in 2D HSQC NMR spectra for elephant

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grass and eucalypt wood after laccase treatment from T. villosa with HBT as the mediator. These

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results were supported by a decrease in lignin number with a high content of oxidized S units in the residual lignin and improved cellulose hydrolysis and ethanol production. Prasetyo et al., (2010) also recorded a corresponding decrease in the aromatic region in 2D HSQC NMR spectra after treatment with the same LMS, however, seemingly contradictory FTIR, SEC, and 13C NMR spectra with Py-GC/MS chromatograms suggested that new ether and C-C aryl-aryl linkages were formed as a result of the strong polymerization, which clearly is not in accordance with the findings of Gutiérrez et al., (2012). Although different substrates were used, this exemplifies the need for consistent methodologies when elucidating the effects and mechanisms involved in laccase treatment of native or technical lignin. Nevertheless, the use of the modern lignin analytical techniques mentioned in this section could ensure that problematic and time-consuming protocols are avoided and the chemical structure and mode of enzymatic action on lignin polymer can be evaluated.

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7. Conclusions

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Lignin is a very complex and recalcitrant polymer synthesized by coupling of phenylpropanoid

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units, and therefore its degradation is a challenge in the bioindustry. Since there is a lot of evidence in the literature that laccase plays an important role in lignin degradation, we have summarized the

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current knowledge and attempted to answer the question; is laccase able to solely, without a use of a

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mediator, cleave bonds that exist in lignin? Based on our knowledge and the literature, the answer is no. It appears that laccase is able to cleave bonds in phenolic model compounds of lignin (e.g.

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between C1 and C2 carbon, known as Cα-Cβ cleavage, and between C1 and aryl group known as alkyl-aryl cleavage) (Kawai et al., 1988a) without use of a mediator. However, laccase is only able

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to cleave bonds in non-phenolic subunits of lignin (Rico et al., 2014) in the presence of a mediator.

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For the successful enzymatic, oxidative catalysis of phenolic compounds one requirement needs to

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be met: the redox potential of laccase must be higher than that of a phenolic subunit in lignin. Therefore, the higher the redox potential of the laccase, the broader the range of substrates that may be oxidized. Future challenges for the molecular evolution of laccases may include tuning the redox potential of laccase to allow oxidation of non-phenolic subunits in lignin without the use of a mediator. There is general consensus in the available literature that laccases act on lignin polymers by oxidizing lignin subunits to reactive radical intermediates. It is also clear that this action results in modification of the lignin. However, the exact attack mechanisms and the enzyme features of laccases providing for efficacy in relation to lignin modification are presently unclear The lack of certainty surrounding the mode of action of laccase may be influenced by the fact that there is no specific method available to evaluate the degree of the degradation of the lignin polymer. Furthermore, there is a lack of the congruent use of nomenclature that is clearly able to describe the changes that the lignin polymer undergoes. Therefore, the choice of methodologies and subsequent

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ACCEPTED MANUSCRIPT interpretation of analytic results is a key factor in understanding the effects of laccase action on lignin. Equally important is the consequent employment of representative reference enzymes,

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mediators and well-characterized substrates, which may facilitate the understanding of the

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mechanisms of action of laccase, interacting with lignocellulosic biomass.

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Prasetyo EN, Kudanga T, Østergaard L, Rencoret J, Gutiérrez A, del Río JC, et al. Polymerization of lignosulfonates by the laccase-HBT (1-hydroxybenzotriazole) system improves dispersibility. Bioresour. Technol. 2010;101:5054-62.

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Qiu W, Chen H. Enhanced the enzymatic hydrolysis efficiency of wheat straw after combined steam explosion and laccase pretreatment. Bioresour. Technol. 2012;118:8-12.

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Ralph J, Brunow G, Boerjan W. Lignins. Encyclopedia of Life Sciences: John Wiley & Sons, Ltd; 2007. p. 1-10.

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Rencoret J, Gutiérrez A, Nieto L, Jiménez-Barbero J, Faulds CB, Kim H, et al. Lignin Composition and Structure in Young versus Adult Eucalyptus globulus Plants. Plant Physiol. 2011;155:66782.

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Rico A, Rencoret J, del Río JC, Martinez ÁT, Gutiérrez A. Pretreatment with laccase and a phenolic mediator degrades lignin and enhances saccharification of Eucalyptus feedstock. Biotechnol. Biofuels. 2014;7:6.

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Rochefort D, Leech D, Bourbonnais R. Electron transfer mediator systems for bleaching of paper pulp. Green Chem. 2004;6:14-24.

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Sitarz AK, Mikkelsen JD, Højrup P, Meyer AS. Identification of a laccase from Ganoderma lucidum CBS 229.93 having potential for enhancing cellulase catalyzed lignocellulose degradation. Enzyme Microb. Technol. 2013;53:378-85.

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Sitarz AK, Mikkelsen JD, Meyer AS. Structure, functionality, and tuning up of laccases for lignocellulose and other industrial applications. Crit. Rev. Biotechnol. DOI:10.3109/07388551

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Srebotnik E, Jensen KA, Hammel KE. Fungal degradation of recalcitrant nonphenolic lignin structures without lignin peroxidase. Proc Natl Acad Sci U S A. 1994;91:12794-7.

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Srinivasan C, Dsouza TM, Boominathan K, Reddy CA. Demonstration of Laccase in the White Rot Basidiomycete Phanerochaete chrysosporium BKM-F1767. Appl. Environ. Microbiol. 1995;61:4274-7.

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Tuomela M, Vikman M, Hatakka A, Itävaara M. Biodegradation of lignin in a compost environment: A review. Bioresour. Technol. 2000;72:169-83.

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ACCEPTED MANUSCRIPT Yang H, Wang K, Song X, Xu F, Sun R-C. Enhance enzymatic hydrolysis of tripoloid poplar following stepwise acid treatment and alkaline fractionation. Proc. Biochem. 2012;47:619-25.

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Youn H-D, Hah YC, Kang S-O. Role of laccase in lignin degradation by white-rot fungi. FEMS Microbiol. Lett. 1995;132:183-8.

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Yuan T-Q, Xu F, Sun R-C. Role of lignin in a biorefinery: Separation characterization and valorization. J. Chem. Technol. Biotechnol. 2013;88:346-52.

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Zakzeski J, Bruijnincx PCA, Jongerius AL, Weckhuysen BM. The Catalytic Valorization of Lignin for the Production of Renewable Chemicals. Chem. Rev. 2010;110:3552-99.

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Zhang L, Gellerstedt G. NMR observation of a new lignin structure, a spiro-dienone. Chem. Commun. 2001:2744-5.

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Zheng Z, Li H, Li L, Shao W. Biobleaching of wheat straw pulp with recombinant laccase from the hyperthermophilic Thermus thermophilus. Biotechnol. Lett. 2012;34:541-7.

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ACCEPTED MANUSCRIPT Acknowledgement

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This work was partially supported by the Danish National Advanced Technology Foundation via the Technology Platform ‘Biomass for the 21st century—B21st’.

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ACCEPTED MANUSCRIPT Figure caption Figure 1. The three major lignin precursors. Figure 2. Proposal for plausible changes that may occur during action of laccase on lignin. Each

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box signifies events that should be attributed to a specific type of event. The arrows indicate how one event may induce the next. Specific reactions are illustrated using a phenolic β-1 model dimer

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(adapted from Kawai et al., 1988b). Proposed changes and nomenclature also account for laccase-

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mediator-systems (LMS). However, for simplicity only phenolic compounds and changes related to

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their structure are presented (see paragraph 5.4 for changes in non-phenolic model compounds).

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Fig. 1.

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Activation of lignin structure

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Electron withdrawal from subunit in lignin → Radical formation → Increase in reactivity

Bond cleavage

Coupling

Change in functional group:

Attachment of phenylpropanoids and/or mediators

AC CE P

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D

MA

Modification

also: acetylation and demethylation

(b)

(a)

Depolymerization

Grafting

Polymerization

Consecutive cleavage of linkages within the lignin polymer

Single attachment

Continuing attachment

→ Decrease in average molecular weight → Release of lignin substructures → Changes in properties of lignin e.g. solubility

→ Increase in average molecular weight

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Fig. 2.

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Structure

Softwood

Hardwood

Grasses

The most common bond in all lignin types.

45-50 %

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β-O-4’ β-aryl ether

60-80 %

69-94 %

9-13 %

3-11 %

2-6 %

PT ED

β-5’ 125-128 kcal/mol

β-1’ diphenyl ethane

4-15 %

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82 kcal/mol

3-12 %

11-14 %

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β-β’ pinoresinol

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54-69 kcal/mol

phenylcoumaran

Comments

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Bond name and BDE

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Table 1. Inter-unit linkages with bond dissociation energies (BDE) and their frequency in native lignin.

1-9 %

1-7 %

low

Most susceptible to break down during lignin processing.

Most common C-C bond in lignin. High ratio of S-units impedes formation

Formation is reduced when the γ-position of S-units is acetylated.

May occur in spirodienone structures.

65-166 kcal/mol

4-O-5’ Diphenylether 78-83 kcal/mol

3.5-8 %

6-9 %

Associated with branching in the lignin polymer.

References Zakzeski et al., 2010,; Wong, 2009; Martínez et al., 2008; Wang et al., 2013; Parthasarathi et al., 2011.

Zakzeski et al., 2010; Buranov and Mazza, 2008; del Río et al, 2011; del Río et al., 2012; Parthasarathi et al., 2011.

Zakzeski et al., 2010; Martínez et al., 2008; Elder, 2014.

Zhang and Gellerstedt, 2001; Zakzeski et al., 2010; Sette et al., 2011; Parthasarathi et al., 2011.

Zakzeski et al., 2010; Buranov and Mazza, 2008; Wong, 2009; Wang et al., 2013; Ralph et al., 2007; Parthasarathi et al., 2011. 38

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5-5’ Biphenyl

19-27 %

3-9 %

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Associated with branching in the lignin polymer. Can lead to the formation of dibenzodioxocine by coupling with a monolignol.

-

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115-118 kcal/mol

5-7 %

0-2 %

2-3 %

3-5 %

3-5 %

Associated with branching in the lignin polymer.

Zakzeski et al., 2010; del Río et al., 2012; Wang et al., 2013; Lange et al., 2013; Elder, 2013.

Zakzeski et al., 2010; del Río et al., 2012.

CE

-

PT ED

72 kcal/mol

Spirodienone

3-4 %

MA

Dibenzodioxocine (α-β-O-4-4’)

NU

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High ratio of S-units impedes formation of 5-5 linkages

Wong, 2009; Zakzeski et al., 2010; Lange et al., 2013; Wang et al., 2013; Ralph et al., 2007; Parthasarathi et al., 2011.

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In cases of unavailable information in literature, the fields are marked “-“.

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Table 2. Various lignin sources with monomer molecular formulas and weights derived from typical lignin processes (Zakzeski et al.,

180*

Lignosulphonate process

C9H7.5O2.5(OCH3)0.39(SO3H)0.6;

Organosolv process

C9H8.53O2.45(OCH3)1.04;

Steam explosion process

C9H8.53O2.45(OCH3)1.04;

Cellulolytic treatment

C9H8.53O2.45(OCH3)1.04;

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~188*

~188*

Kraft lignin: Sulfonated (S), Soluble in alkali, increased phenolic hydroxyl groups and biphenyl structures. Residual Lignin: Lignin fragments bound to carbohydrates in the kraft pulp.

Moya et al., 2011; Tuomela et al., 2000; Desina and Cres12; Vishtal and Kraslawski, 2011, Chakar and Ragauskas, 2004.

Soluble in water and other strongly polar solvents, sulfonated (S, SO3).

Zakzeski et al., 2010; Desina and Crestini, 2012; Vishtal and Kraslawski, 2011.

Wood components including lignin are separated through treatment with organic solvents.

Insoluble in water, may contain phosphorous groups

Moya et al., 2011; Vishtal and Kraslawski, 2011.

High temperature steam treatment followed by a rapid pressure release.

Reduction of β-O-4 linkages, increase in C-C linkages.

Li et al., 2007.

Cellulase enzyme treatment followed by solvent extraction.

Similar to native structure, macromolecular, residues of carbohydrate and protein.

Ikeda et al., 2001; Tuomela et al., 2000.

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References

Treatment at elevated temperature and pressure in presence of NaOH and Na2SO4.

Treatment at elevated temperature and pressure in the presence of sulfite salts and/or SO2.

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188*

nd*

Lignin characteristics

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C9H8.5O2.1S0.1(OCH3)0.8(CO2H)0.2;

Conditions

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Kraft lignin process

Monomer molecular formula and weight of lignin

PT ED

Process

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2010; Aresta et al., 2012).

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nd*

Ikeda et al., 2001.

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* Monomer molecular weight; nd: not determined

Similar to native structure, macromolecular, residues of carbohydrate.

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Ball milling

Wood is grounded in a ball mill and lignin is extracted with a dioxane-water mixture.

C9H8.83O2.37(OCH3)0.96;

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Table 3. Ranking of selected laccases according to their redox potential Redox potential [mV] 340

Rhus vernicifera

430

Myceliphthora thermophila

465

NU

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Zuccini (Caburbita pepo)

Melanocarpus albomyces

Pleurotus ostreatus

Neurospora crassa Trametes hirsuta

Alcalde, 2007 Alcalde, 2007 Kiiskinen et al., 2004

510

Alcalde, 2007

650

Morozova et al., 2007

710

Alcalde, 2007

780

Alcalde, 2007

780

Shleev et al., 2004

790

Alcalde, 2007

AC

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Trametes versicolor

PT ED

Rhizoctania solani

Alcalde, 2007

470

MA

Scytalidium thermophilum

Reference

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Laccase

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Table 4a.Overview of scientific studies concerning the effect of laccase (lac) and/or laccase mediator systems (LMS) on lignin polymers. P represents

Lac: P

Myceliophthora thermophila

-

Lac: P LMS: DP κ↓

Trametes versicolor

ABTS

LMS: DP

Phanerochaete cinnabarius

PT

Substrate

References

Lignosulphonates

Areskogh et al., 2010

Kraft lignin

Bourbonnais et al., 1995

3-HAA

DHP

Eggert et al., 1996

violuric acid, syringaldehyde pyrocatechol , guaiacol vanillin, polyphenon 60

Eucalyptus kraft lignin

Gouveia et al., 2012

-

Spruce milled wood lignin

Grönqvist et al., 2005

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Mediators

SC

Laccase(s)

PT ED

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Outcome

MA

polymerization and DP represents depolymerization.

Myceliophthora thermophila

Lac: P

Trametes hirsuta

Lac: Modify LMS: DP (ABTS) LMS: slight P (HBT)

Fusarium proliferatum

ABTS, HBT

Agave kraft lignin

Hernández Fernaud et al., 2006

Lac: P (DHP) Lac: DP (DHPglucoside)

Coriolus versicolor

-

DHP DHP-Glucoside

Kondo et al., 1990

Lac: P ↑ 5-5’, 5-O-4’

Trametes hirsuta; Melanocarpus albomyces; Thielavia arenaria; Chaetomium thermophilum

-

DHP

Maijala et al., 2012

AC

CE

Lac: P LMS:P

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Lac:P

Melanocarpus albomyces

-

Lac:P LMS:P

Melanocarpus albomyces; Streptomyces ipomoea

LMS:P

Trametes villosa; Trametes hirsuta

Lac: P LMS: DP

Trametes hirsuta;

Lac: P

Trametes hirsute; Melanocarpus albomyces; Thielavia arenaria

Lac: DP

Rhus vernicifera

PT

-

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Trametes hirsuta

Mattinen et al., 2011

Acetosyringone

Softwood and hardwood kraft lignin Spruce and birch organosolve lignin

Moya et al., 2011

Lignosulfonate

Prasetyo et al., 2010

Synthetic kraft lignin Pine pulp lignin

Shleev et al., 2006

Birch and mixed hardwood organosolv lignin Pine and eucalypt steam exp. lignin

van de Pas et al., 2011

Lignophenol polymers

Xia et al., 2003

NU

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DHP

PT ED

HBT

CE

ABTS, HBT

AC

Mattinen et al., 2008

MA

Lac:P

Flax soda lignin Spruce enzymatic mild acidolysis lignin Eucalyptus dioxane lignin

-

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Table 4b. Overview of scientific studies concerning the effect of laccase and/or laccase mediator systems on biomass substrates with lignin. Laccase(s)

Mediators

Lac and LMS: κ↓ Brightness↑ viscosity↓ S/G ratio↓ (after peroxide treatment)

Myceliophthora thermophila

Methyl syringate, Syringaldehyde

LMS: κ↑ Brightness↓ Relation to ortho/para -positions of mediators (before peroxide treatment)

Trametes villosa

LMS: Saccharification↑ Cα-Cβ cleavage

Trametes versicolor

LMS: DP κ↓ Pc-HBT: Cα-oxidation Mt-MeS: Cα-Cβ cleavage Lac and LMS: Klason Lignin↓ Saccharification↑ effect of Lac
Substrate

References

Eucalyptus pulp

Babot et al., 2011

p-coumaric acid, Ferulic acid Sinapic acid, Coniferaldehyde Sinapyl aldehyde, Vanillin Syringaldehyde, Acetovanillone Acetosyringone

Kenaf pulp Sisal pulp

Barneto et al., 2012

HBT

Corn stover

Chen et al, 2012

Phanerochaete cinnabarius Myceliophthora thermophila

HBT Methyl Syringate

Eucalyptus kraft pulp

Du et al., 2013

Trametes villosa

HBT

Eucalyptus feedstock Elephant grass feedstock

Gutiérrez et al, 2012

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PT ED

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Outcome

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HBT

Eucalyptus kraft pulp

Ibarra et al., 2007

-

Pretreated spruce (softwood) Pretreated giant reed (hardwood)

Moilanen et al., 2011

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Pycnoporus cinnabarinus

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LMS: κ↓ Brightness↑ viscosity↓ S/G ratio↓ Cα-oxidation

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Cerrena unicolor

Lac and LMS: κ↓ Brightness↑ (after peroxide treatment) coupling of mediators

Trametes villosa

HBT, Violuric acid, Promazine Syringaldehyde, Vanillin

Eucalyptus kraft pulp

Moldes et al., 2008

Lac and LMS: Klason Lignin↓ S/G ratio↑ Cα-oxidation Cα-Cβ cleavage Effect of Lac
Myceliophthora thermophila

Methyl Syringate

Eucalyptus feedstock

Rico et al., 2014

ABTS HBT Guaiacol

Wheat straw pulp

Zhang et al., 2012

MA PT ED

CE

AC

Lac and LMS: κ↓ Brightness↑ Effect of Lac
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Softwood: Saccharification↑ cellulase-lignin binding↓ Hardwood: Saccharification↓ cellulase-lignin binding↑

Thermus thermophilus

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