Microbial degradation of lignin: Role of lignin peroxidase, manganese peroxidase, and laccase

Lignin peroxidase (LiP), laccase (LA) and manganese peroxidase (MnP) of white-rot basidiomycetes such as Phanerochaete chrysosporium, Coliorus versicolor, Phlebia radiata and Pleurotus eryngii catalyze oxidative degradation of lignin substructure model compounds and synthetic lignins (DHPs). Side chain- and aromatic ring cleavage products of both phenolic and non-phenolic substrates oxidized by LiP were isolated and characterized by NMR and MS. The cleavage mechanism was elucidated by using 18O, 2H, and 13C labeled lignin substructure dimers with 18O2 and H2 18O. Recent studies suggested that LiP is capable of oxidizing lignin directly at the protein surface via a long-range electron transfer process. LA and MnP, which oxidize phenolic but not non-phenolic moieties, generally degrade lignin stepwise from phenolic moieties. However, recent studies indicated that MnP and LA can degrade both phenolic and non-phenolic aromatic moieties of lignin with some special mediators.

In further investigation 4) we identified an alternative Cα-Cβ cleavage reaction of a β-O-4 model compound, 4ethoxy-3-methoxyphenylglycerol-β - 18 O-guaiacyl ether, to give 2-guaiacoxyethanol and benzyl alcohol probably via benzaldehyde, in ligninolytic cultures of P. chrysosporium. GC-MS analyses of the isolated products showed that 18 O of the ether oxygen of the substrate was not retained in the 2-guaiacoxyethanol product. When 4-ethoxy-3-methoxyphenylglycerol(γ-13 C)-β -guaiacyl ether used as substrate, the 2-guaiacoxyethanol product was labeled with 13 C at the 2-position but not the 1-position.
Mechanism of aromatic ring cleavage of lignin substructure model compounds by LiP. Kirk and Chang 16) compared white-rotted lignin polymer (isolated and purified from white-rotted wood) with nondegraded lignin, using a variety of chemical and physical methods. Among their conclusions was that aromatic rings had been cleaved while still in the polymer.
The mechanism of aromatic ring cleavage of lignin by fungi, however, remained unsolved until 1985. Umezawa and Higuchi 3)-5),15) synthesized 4-ethoxy-3methoxyphenylglycerol-β -guaiacyl[U-ring 13 C,OCD 3 ] ether, and 4-ethoxy-3-methoxyphenylglycerol-βsyringyl[U-ring 13 C,OCD 3 ] ether as substrate to elucidate the mechanism of aromatic ring cleavage of the model compounds. The compounds were incubated with ligninolytic cultures of P. chrysosporium in the presence of H 2 18 O. We isolated and identified for the first time β,γand α,β-cyclic carbonates, formate and oxalate esters of arylglycerol from the reaction mixtures as aromatic ring cleavage products (Fig. 4). 15) We finally identified a muconate ester of arylglycerol as an initial ring cleavage product of the dimers by LiP. 6) The cleavage mechanism of the aromatic ring was further elucidated by experiments using 2 H, 13 17) found that most of the initial stage of degradation reaction of β-O-4 lignin substructure model dimers was catalyzed by LiP. A synthetic lignin (DHP: dehydrogenation polymer of coniferyl alcohol prepared using horseradish peroxidase, M. W. >2200) was prepared and subjected to degradation with LiP to elucidate the mechanism of lignin degradation by this enzyme. 18) As the case of the degradation of β-O-4 lignin substructure model dimers by LiP, the cyclic carbonates and formate ester of arylglycerols, and arylglycerol were isolated from degradation products of the DHP by LiP ; the chemical structures of the products were identified by GC-MS. These results indicated that the lignin polymer is really degraded by the LiP of white-rot fungi.
Active sites of LiP to substrates. Doyle and his group 19) recently found that Trp 171 of LiP protein is hydroxylated at the Cβ position. They found that the hydroxylation process in both wild type and recombinant LiP isozyme H 8 is autocatalytic and that Trp 171 may be implicated in catalysis. Site directed mutagenesis of recombinant enzymes with Trp 171 substituted by Phe (W171F) or Ser (W171S) lost all activity for veratryl alcohol (VA; a LiP substrate) but not for two dye substrates. The result suggested two distinct substrate interaction sites in LiP, a heme-edge site, and a novel site centered around Trp 171 which is required for the oxidation of VA. Stop-flow kinetic studies strongly suggested that an electron-transfer pathway exists within the enzyme protein leading from the heme to a surface site in close proximity to Trp 171.
Johjima et al. 20) confirmed that the binding site of LiP for VA is Trp 171 by using three different chemically modified LiPs against VA acting as a reducing substrate, a reducing reagent for the rapid conversion of LiPIII back to native LiP, and as an enzyme-bound redox mediator. They 21) further studied the binding properties of LiP for synthetic lignin (DHP) by resonant mirror biosensor techniques, and found that among several ligninolytic enzymes only LiP specifically binds to DHP. Kinetic analysis showed that the binding is reversible, and LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. The His-Asp proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.
Tien's group 22) studied on the active site of LiP with respect to substrate size using either fungal or recombinant wild type, as well as mutated, recombinant LiPs. A nonphenolic tetrameric lignin model that contains β-O-4 linkages was used as substrate. Both natural and recombinant LiPs oxidized the tetrameric model forming four products, tetrameric, trimeric, dimeric, and monomeric carbonyl compounds. The result indicated that LiP is able to attack any of Cα-Cβ linkages in the tetrameric compound and that the substrate-binding sites is thus well exposed. Mutation of a Trp residue (W171S) completely inhibited the oxidation of the tetramer model. These results are consistent with LiP having an exposed active site capable of directly interacting with the lignin polymer without the need for low molecular weight mediators, such as VA.
Manganese peroxidase (MnP). Following the discovery of LiP in P. chrysosporium, 9),10) manganese peroxidase (MnP) secreted from the same fungus was found as another lignin degrading enzyme by Gold's group, 23), 24) and Crawford's group, 25) respectively, and subsequent investigations have shown that MnP is distributed in almost all white-rot fungi. 35) Ten extracellular peroxidase isozymes were purified from the culture of P. chrysosporium. 26 28) It has been proposed that chelated Mn 3+ acts as lowmolecular weight, diffusible redox-mediator that attacks the phenolic lignin structure. Further investigations 29)-31) showed that the chelated Mn 3+ system generates reactive intermediates (peroxy radicals) from unsaturated fatty acids such as linoleic acid and their derivatives (lipids). The MnP-lipid system is strong enough to degrade Cα-Cβ and β-aryl ether bonds in not only phenolic but also nonphenolic lignin model dimmers.
Hammel and his group 32) found that wood block cultures and defined liquid medium cultures of Ceriporiopsis subvermispora rapidly depolymerized and mineralized a 14 C-labeled, polyethylene glycollinked high molecular weight β-O-4 lignin model compound that represents major nonphenolic structure of lignin. The fungus cleaved the model between Cα and Cβ to release benzylic fragments. The fungal degradation on the model and methylated lignin was significantly faster in the presence of Tween 80, a source of unsaturated fatty acids.
Wariishi et al. 33) also found that MnP catalyzes substantial depolymerization of DHP by purified MnP of P. chrysosporium in the presence of malonic acid as the chelator. Both guaiacyl-and guaiacyl-syringyl lignin models were degraded substantially.
However, identification of cleavage products of side chain and aromatic ring of lignin substructure models and DHP by MnP, and the chemical degradation mechanism have scarcely been investigated.
Versatile peroxidase (VP). Versatile peroxidases (VP) that can oxidize Mn 2+ as well as phenolic and non-phenolic aromatic compounds have been isolated from Pleurotus and Bjerkandera. 34 by cultivating wood-rotting fungi in an agar medium containing several phenolic compounds, such as gallic acid, tannic acid, and hydroquinone, that white-rot fungi produced a large darkened zone around the mycerial mat, but no zone of darkening was associated with the growth of brown-rot fungi. Davidson et al. 37) subsequently investigated the reaction using 210 species of wood-rotting fungi, and concluded that the white-rotting type coincides with Bavendamm's reaction in general, and that the reaction is helpful in identifying fungi. The enzyme responsible for Bavendamm's reaction was extensively studied in the next 10 years, and characterized to be laccase (LA). 38) LA, p-diphenol oxidase (EC 1.10.3.2) has been isolated and characterized as a blue, copper containing oxidase from a lac tree (Rhus spp) and several fungi. White rot fungi constitutively produce laccase during primary metabolism. 39) 1. Degradation of β-1 model compounds. Kawai et al. 40) found that phenolic β-1 model compounds are degraded by LiP of P. chrysosporium and LA of C. versicolor via similar pathways. 1-(3,5-Dimethoxy-4hydroxyphenyl)-2-(3,5-dimethoxy-4-ethoxyphenyl)propane-1,3-diol (1, Fig. 6) was converted by LA of C. versicolor to 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(3,5-dimethoxy-4-ethoxyphenyl)-3-hydroxypropanone (2)by Cα oxidation, 2-(3,5-dimethoxy-4-ethoxyphenyl)-3-hydroxypropanal (5), 2,6-dimethoxy-p-hydroquinone (4) and its benzoquinone (3) by alkyl-phenyl cleavage (Fig. 6). Their experiment further showed that 18  Recently yellow laccase as well as blue laccase have been isolated from solid-state and submerged culture of Panus tigrinus. The yellow laccase had no blue maxima in the absorption spectrum, but catalyzed oxidation of VA and a non-phenolic β-1 dimer. The yellow laccase was suggested to be formed as a result of blue laccase modification by products of lignin degradation, which might play a role as natural electron-transfer mediators for the oxidation of nonphenolic substances. 41) 2. Degradation of β-O-4 model compounds. Kirk et al. 42) worked on degradation of the lignin model compound syringylglycerol-β -guaiacyl ether by Polyporus versicolor and Stereum frustulatum. They found that the benzyl alcohol group of the substrate was oxidized to a carbonyl group, giving α-guaiacoxyacetosyringone by whole culture of S. frustulatum and the culture filtrate of P. versicolor. The alkylphenyl carbon-to-carbon bond in both syringylglycerol-β -guaiacyl ether and αguaiacoxyacetosyringone was cleaved by culture filtrate of P. versicolor with formation of guaiacoxyacetaldehyde and guaiacoxyacetic acid, respectively. The syringyl moieties of both parent compounds were converted to 2,6-dimethoxy-p-benzoquinone by culture filtrate of. P. versicolor. Laccase also effected all the above reactions.
Kawai et al. 43) recently investigated the degradation of syringylglycerol-β -guaiacyl ether by LA of C. versicolor. They showed that the substrate is mainly converted to the α-carbonyl dimer, 2,6-dimethoxyhydroquinone, and glyceraldehyde 2-guaiacyl ether by alkyl-phenyl cleavage, and to guaiacol by O-Cβ cleavage. Syringaldehyde and guaiacoxyethanol as direct Cα-Cβ cleavage products of the substrate were not found. Subsequent investigation to identify the pathway to give guaiacol showed that α-carbonyl dimmer used as substrate is cleaved between Cα and Cβ to give syringic acid and guaiacol as shown in Fig. 7. The result indicated that phenolic β-O-4 compound is degraded not only by alkyl-phenyl cleavage, which has been proposed as a major LA-mediated degradative reaction, but also by Cα-Cβ -cleavage of the Cα-carbonyl dimmer previously 3. Syringyl polymer. Syringyl lignin model polymer (MW>2200) was degraded by LA of C. versicolor. 46) The polymer was depolymerized partially to form 2,6dimethoxy-p-hydroquinone, 2,6-dimethoxy-p-benzoquinone, and syringaldehyde. NMR spectra of the degraded substrate suggested that the LA catalyzed the oxidation of benzylic hydroxyl groups to ketones at the polymer stage.
4. Aromatic ring cleavage. Kawai et al. 47) found that 4,6-di-t-butylguaiacol is converted by LA of C. versicolor to a ring cleavage product, the muconolactone derivative, which was previously identified by Gierer and Imsgard 48) as a product in alkaline-oxygen oxidation of the same substrate. The experiment showed that 18  Hydrogen peroxide is only required for the conversion of native LiP and MnP into two electron-deficient reactive species (compound I). Compound I of LiP abstracts stepwise two electrons from the aromatic ring of lignin substrate to yield aryl cation radicals or aryl cations, which are attacked by O 2 or nucleophiles such as H 2 O and R-OH, respectively. The subsequent reactions of the cation radicals and cations are not controlled by the enzyme just as in the non-enzyme-directed coupling of phenoxy radicals of monolignol in lignin biosynthesis. Thus, the role of LiP, LA, and probably MnP in lignin biodegradation could be explained by the following unifying view. +Mediators → Phenoxy radicals of phenolic units and aryl cation radicals or cation radicals of non-phenolic units Non-enzymatic reaction 1) Homolytic or heterolytic cleavage of side chains (Cα-Cβ, alkyl-phenyl), and aromatic rings 2) O 2 attack on carbon-centered radical intermediates 3) Nucleophilic attack on aryl cations and Cα cations by H 2 O and R-OH → Degradation products Recent molecular investigations 49) on ligninolytic enzymes have shown that P. chrysosporium has two gene families including ten LiP-type and three MnP-type genes coding different isoenzymes expressed during secondary metabolism. Many ligninolytic peroxidase genes from other white-rot fungi, and two VP genes from Pleurotus eryngii have been cloned.
Biochemical and biotechnological approaches to lignin biodegradation open up a new field in biomass conversion, such as biopulping 50)-53) biobleaching, and treatment of Kraft bleaching effluents and related pollutants by lignin degrading basidiomycetes and their enzymes. 54) A review article 49) is referred to for molecular biology and engineering of lignin biodegradation.