Lignin Substructure Model Compounds Research Articles

Alkadienyl and alkenyl itaconic acids (ceriporic acids G and H) from the selective white-rot fungus Ceriporiopsis subvermispora: a new class of metabolites initiating ligninolytic lipid peroxidation

New ceriporic acids-alkadienyl and alkenyl itaconic acids having a bis-allyl (3-[(Z,Z)-hexadec-7,10-dienyl]-itaconic acid; ceriporic acid G) and a monoene (3-[(Z)-octadec-9-enyl]-itaconic acid; ceriporic acid H) structure in their side chains-were isolated from the cultures of the selective lignin-degrading fungus Ceriporiopsis subvermispora. The new metabolites ceriporic acid G and H were synthesized by a cross-aldol condensation and a Grignard substitution reaction, respectively. Ceriporic acid G triggered the manganese peroxidase (MnP)-catalyzed lipid peroxidation and decomposed a recalcitrant non-phenolic lignin substructure model compound. Except for simple fatty acids, this is the first report of a fungal metabolite that induced ligninolytic lipid peroxidation.

Alkoxyl- and carbon-centered radicals as primary agents for degrading non-phenolic lignin-substructure model compounds

Lignin degradation by white-rot fungi proceeds via free radical reaction catalyzed by oxidative enzymes and metabolites. Basidiomycetes called selective white-rot fungi degrade both phenolic and non-phenolic lignin substructures without penetration of extracellular enzymes into the cell wall. Extracellular lipid peroxidation has been proposed as a possible ligninolytic mechanism, and radical species degrading the recalcitrant non-phenolic lignin substructures have been discussed. Reactions between the non-phenolic lignin model compounds and radicals produced from azo compounds in air have previously been analysed, and peroxyl radical (PR) is postulated to be responsible for lignin degradation (Kapich et al., FEBS Lett., 1999, 461, 115-119). However, because the thermolysis of azo compounds in air generates both a carbon-centred radical (CR) and a peroxyl radical (PR), we re-examined the reactivity of the three radicals alkoxyl radical (AR), CR and PR towards non-phenolic monomeric and dimeric lignin model compounds. The dimeric lignin model compound is degraded by CR produced by reaction of 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH), which under N(2) atmosphere cleaves the α-β bond in 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-propanediol to yield 4-ethoxy-3-methoxybenzaldehyde. However, it is not degraded by the PR produced by reaction of Ce(4+)/tert-BuOOH. In addition, it is degraded by AR produced by reaction of Ti(3+)/tert-BuOOH. PR and AR are generated in the presence and absence of veratryl alcohol, respectively. Rapid-flow ESR analysis of the radical species demonstrates that AR but not PR reacts with the lignin model compound. Thus, AR and CR are primary agents for the degradation of non-phenolic lignin substructures.

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 18O 2 and H218O. 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. (Communicated by Yasuyuki YAMADA, M.J.A.)

The closed system pyrolysis of β-O-4 lignin substructure model compounds

Lignin substructure model compounds having a β-O-4 linkage were synthesized. These were guaiacylglycerol-β-guaiacyl ether (GGE) and guaiacylglycerol-β-syringyl ether (GSE) which model guaiacyl and syringyl units respectively. Closed system microscale pyrolysis of GGE and GSE was carried out at 300 °C both in the presence and absence of water vapour. The laboratory degradation of GSE occurred predominantly by breaking the Cβ–O bond of the β-O-4 linkage to form the ring B fragment of the model compound. This was followed by demethylation of the aromatic methoxyl groups. Char formation was another significant process during pyrolysis. There are structural features of the char formed during the heating of GGE which indicate incorporation of the remaining fragment of the model compound (ring A coupled with the propyl segment of the aryl ether linkage).

Degradation of a non-phenolic β- O-4 substructure and of polymeric lignin model compounds by laccase of Coriolus versicolor in the presence of 1-hydroxybenzotriazole

A β- O-4 lignin substructure model compound, 2-(2,6-dimethoxy-4-formylphenoxy)-1,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)propane, was oxidized by Coriolus versicolor laccase in the presence of 1-hydroxybenzotriazole to form four products: 2,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)propanone, 1-(4-ethoxy-3-methoxyphenyl)-3-hydroxypropanone, 4-ethoxy-3-methoxybenzoic acid, and 2,6-dimethoxy- p-benzoquinone. The results show that three types of the reactions, β-ether cleavage, Cα-Cβ cleavage, and Cα-oxidation are catalyzed by laccase–1-hydroxybenzotriazole couple. Furthermore, depolymerization of a polymeric guaiacyl lignin model compound prepared from [β- 14C]coniferyl alcohol was also caused by this system.

Degradation of a non-phenolic β-O-4 substructure and of polymeric lignin model compounds by laccase ofCoriolus versicolorin the presence of 1-hydroxybenzotriazole

A β-O-4 lignin substructure model compound, 2-(2,6-dimethoxy-4-formylphenoxy)-1,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)propane, was oxidized by Coriolus versicolor laccase in the presence of 1-hydroxybenzotriazole to form four products: 2,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)propanone, 1-(4-ethoxy-3-methoxyphenyl)-3-hydroxypropanone, 4-ethoxy-3-methoxybenzoic acid, and 2,6-dimethoxy-p-benzoquinone. The results show that three types of the reactions, β-ether cleavage, Cα-Cβ cleavage, and Cα-oxidation are catalyzed by laccase–1-hydroxybenzotriazole couple. Furthermore, depolymerization of a polymeric guaiacyl lignin model compound prepared from [β-14C]coniferyl alcohol was also caused by this system.

Biodegradation mechanism of lignin by white-rot basidiomycetes

White-rot basidiomycetes such as Coriolus versicolor, Phanerochaete chrysosporium and Phlebia radiata have been found as typical lignin degrader. During the past 15 years chemistry and biochemistry of lignin biodegradation by white-rot basidiomycetes have considerably advanced by means of chemical analysis of the biodegraded lignin and studies of the degradation mechanism of lignin substructure model compounds by ligninolytic cultures of these basidiomycetes and their enzymes. Excellent reviews (Kirk and Farrell, 1987; Schoemaker, 1990; Umezawa and Higuchi, 1991) concerning microbial and enzymatic degradation of lignin have recently appeared. We (Higuchi and Nakatsubo, 1980; Higuchi, 1990) synthesized major lignin substructure model compounds linked by β-O-4, β-5, β-1, β-β′ and 5-5′ bonds and used as substrates for cultures of P. chrysosporium and C. versicolor and their enzymes, lignin peroxidase and laccase. The degradation products were extracted with ethyl acetate and identified by NMR and GC-MS to elucidate degradation pathways of the lignin substructure model compounds. The results are classified to the cleavage of side chain and aromatic ring opening of the compounds.

Degradation of Lignin Substructure Model Compounds by Copper (II)-Amine Complex as a Model Catalyst of Fungal Laccase

Degradation of Lignin Substructure Model Compounds by Copper (II)-Amine Complex as a Model Catalyst of Fungal Laccase

Degradation of non-phenolic β- o-4 lignin substructure model compounds by Acinetobacter sp.

Acinetobacter sp. utilized non-phenolic β- o-4-model compounds2-methoxy-4-formylphenoxyacetic acid and veratrylglycerol-β-guaiacyl ether (VGE) as sole carbon source. Vanillin, vanillic acid, protocatechuic acid and catechol were detected in the 2-methoxy-4-formylphenoxyacetic acid amended culture. Veratryl alcohol, veratraldehyde, veratric acid, vanillic acid, protocatechuic acid, catechol and guaiacol were identified from veratrylglycerol-β-guaiacyl ether culture. Acinetobacter sp. produced catechol 1,2-dioxygenase and protocatechuate 3,4-dioxygenase that cleaved catechol and protocatechuic acid, respectively.

Characterization of an extracellular lignin peroxidase of the lignocellulolytic actinomycete Streptomyces viridosporus.

Previously we reported production of an extracellular lignin-inducible peroxidase by Streptomyces viridosporus (M. Ramachandra, D.L. Crawford, and A.L. Pometto III, Appl. Environ. Microbiol. 53:2754-2760, 1987). This peroxidase was shown to oxidize 3,4-dihydroxyphenylalanine, 2,4-dichlorophenol, homoprotocatechuic acid, caffeic acid, and N,N,N',N'-tetramethylphenylenediamine and was found in higher than normal levels in strains enhanced for lignocellulose degradation. In the present study, we used a pure extracellular enzyme preparation with high peroxidase isoform P3 activity to oxidize lignin substructure model compounds of both the 1,2-diaryl propane and arylglycerol-beta-aryl ether types and containing C alpha-carbonyl and C alpha-hydroxyl groups. The reactions were monitored by gas chromatography-mass spectrometry and high-pressure liquid chromatography techniques. In the presence, but not the absence, of hydrogen peroxide, the enzyme preparation catalyzed C alpha-C beta bond cleavage in the side chains of the diaryl ethers 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)propane-1,3-diol (I) and 1-(4-hydroxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)propan-1-one (II) and the diaryl ethane 1-(4-methoxyphenyl)-2-(phenyl)ethan-1-one (III). Rapid hydrogen peroxide consumption was observed when the enzyme preparation was added to either milled corn lignin or lignocellulose. Additional characterizations showed that this enzyme is a heme protein (Soret band, 408 nm) and a major component of the ligninolytic system of S. viridosporus T7A. This is the first report of a lignin peroxidase in a bacterium. We have designated this new lignin peroxidase as ALiP-P3.

Degradation of a non-phenolic arylglycerol β-aryl ether by Streptomyces cyaneus

A non-phenolic β- O-4 lignin substructure model compound, 1,3-dihydroxy-2-(2-methoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane, was degraded by streptomyces cyaneus in liquid shaken cultures. 4-Ethoxy-3-methoxybenzoic acid could be identified by TLC and GC/MS as the main product, indicating the cleavage of the CαCβ bond of the aryl glycerol-β-aryl ether. In addition, 4-ethoxy-3-methoxybenzaldehyde and guaiacol were identified in culture extracts as further degradation products.

Degradation mechanisms of phenolic β-1 lignin substructure model compounds by laccase of Coriolus versicolor

Phenolic β-1 lignin substructure model compounds, 1-(3,5-dimethoxy-4-hydroxy-phenyl)-2-(3,5-dimethoxy-4-ethoxyphenyl)propane-1,3-diol ( I) and 1-(3,5-dimethoxy-4-ethoxyphenyl)-2-(3,5-dimethoxy-4-hydroxyphenyl)propane-1,3-diol ( II) were degraded by laccase of Coriolus versicolor. Substrate I was converted to 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-(3,5-dimethoxy-4-ethoxyphenyl)-3-hydroxypropanone ( III), 1-(3,5-dimethoxy-4-ethoxyphenyl)-2-hydroxyethanone ( IV), syringaldehyde ( V), 1-(3,5-dimethoxy-4-ethoxyphenyl)-3-hydroxypropanal ( VI), 2,6-dimethoxy- p-hydroquinone ( VII), and 2,6-dimethoxy- p-benzoquinone ( VIII). Furthermore, incorporations of 18O of 180 2 into ethanone ( IV) and 18O of H 2 18O into hydroquinone ( VII) and benzoquinone ( VIII) were confirmed. Substrate II gave 1-(3,5-dimethoxy-4-hydroxyphenyl)ethane-1,2-diol ( IX), 1-(3,5-dimethoxy-4-hydroxyphenyl)-2-hydroxyethanone ( X), and 3,5-dimethoxy-4-ethoxybenzaldehyde ( XI). Also 18O of H 2 18O was incorporated into glycol ( IX) and ethanone ( X). Based on the structures of the degradation products and the isotopic experiments, it was established that three types of reactions occurred via phenoxy radicals of substrates caused by laccase: (i) C α-C β cleavage (between C1 and C2 carbons); (ii) alkyl-aryl cleavage (between C1 carbon and aryl group); and (iii) Cα (C1) Oxidation.

Degradation of a non-phenolic .BETA.-O-4 lignin substructure model compound by Coriolus hirsutus.

Degradation of a non-phenolic .BETA.-O-4 lignin substructure model compound by Coriolus hirsutus.

Mechanism of aromatic ring cleavage of β- O-4 lignin substructure models by lignin peroxidase

This investigation examined the aromatic ring cleavage of β- O-4 lignin substructure model compounds by lignin peroxidase of Phanerochaete chrysosporium. Based on tracer experiments using H 2 18O and 18O 2, mechanisms of the aromatic ring cleavage of the β- O-4 lignin models were proposed. The mechanisms involve one-electron oxidation of the β- O-4 lignin models by the enzyme followed by attack of nucleophiles and radical coupling with O 2.

Arylglycerol-γ-Formyl Ester as an Aromatic Ring Cleavage Product of Nonphenolic β- O -4 Lignin Substructure Model Compounds Degraded by Coriolus versicolor

4-Ethoxy-3-methoxyphenylglycerol-gamma-formyl ester (compound IV) was identified as a degradation product of both 4-ethoxy-3-methoxyphenylglycerol-beta-syringaldehyde ether (compound I) and 4-ethoxy-3-methoxyphenylglycerol-beta-2,6-dimethoxyphenyl ether (compound II) by a ligninolytic culture of Coriolus versicolor. An isotopic experiment with a C-labeled compound (compound II') indicated that the formyl group of compound IV was derived from the beta-phenoxyl group of beta-O-4 dimer as an aromatic ring cleavage fragment. However, compound IV was not formed from 4-ethoxy-3-methoxyphenylglycerol-beta-guaiacyl ether (compound III). gamma-Formyl arylglycerol (compound IV) could be a precursor of 4-ethoxy-3-methoxyphenylglycerol (compound VI), because 3-(4-ethoxy-3-methoxyphenyl)-1-formyloxy propane (compound VII) was cleaved to give 3-(4-ethoxy-3-methoxyphenyl)-1-propanol (compound VIII) by C. versicolor. 4-Ethoxy-3-methoxyphenylglycerol-beta,gamma-cyclic carbonate (compound V), previously found as a degradation product of compound III by Phanerochaete chrysosporium (T. Umezawa, and T. Higuchi, FEBS Lett., 25:123-126, 1985), was also identified from the cultures with compound I, II, and III and degraded to give the arylglycerol (compound VI). An isotopic experiment with C-labeled compounds II' and III' indicated that the carbonate carbon of compound V was derived from the beta-phenoxyl groups of beta-O-4 substructure.

Metabolism of a non-phenolic .BETA.-O-4 lignin substructure model compound by Coriolus versicolor.

A non-phenolic β-O-4 lignin substructure model, 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (I), was metabolized by a ligninolytic culture of Coriolus versicolor. Based on the identification of the metabolic products (II∼XI), the following reactions were found to occur in the culture; a) oxidation (III) and reduction (II) at the benzyl (Cα') position of the substrate (I), b) β-ether cleavage to give arylglycerols (IV, V), and c) Cα-Cβ cleavage of the arylglycerols and/or arylglycerol moiety of the substrate (I). In addition, β-deoxy diol (VI) and γ-formylglycerol (VII) were obtained as degradation products from substrate (I).

Metabolism of non-phenolic diarylpropane lignin substructure model compound by Coriolus versicolor

Metabolism of non-phenolic diarylpropane lignin substructure model compound by Coriolus versicolor

Lignin-Degrading Enzyme from the Hymenomycete Phanerochaete chrysosporium Burds

The extracellular fluid of ligninolytic cultures of the wood-decomposing basidiomycete Phanerochaete chrysosporium Burds. contains an enzyme that degrades lignin substructure model compounds as well as spruce and birch lignins. It has a molecular size of 42,000 daltons and requires hydrogen peroxide for activity.

Chemical mechanism of an important cleavage reaction in the fungal degradation of lignin

Previous research established that lignin substructure model compounds of the non-phenolic β-1 type (R-C αHOH-C βHR′-C γH 2OH, in which R,R′ = ▪ O-alkyl) are cleaved between C α and C β by Phanerochaete chrysosporium. The C β-derived product is a racemic phenylglycol (R′-CHOH-CH 2OH) in which the benzyl hydroxyl oxygen atom is derived from molecular oxygen. Investigations described here have established, through the use of an isotope-trapping technique and the same organism, that the C α-derived product is the aromatic aldehyde (R-C αHO); this aldehyde is rapidly reduced in cultures to the corresponding alcohol. A β-1 substrate without the C γH 2OH moiety is also cleaved, showing that the C γ-group is not required. A β-1 substrate with a C α-carbonyl group is only slowly degraded (to polar products), probably not via C α-C β cleavage. Mass spectrometric analysis of the products formed from deuterated β-1 substrates established that hydrogen (deuterium) atoms at C α and at C β are not lost during C α-C β cleavage. These results establish that neither C α-oxidation, C β-hydroxylation nor formation of a C α-C β double bond occurs prior to C α-C β cleavage, and that cleavage does not involve C γ. A concerted mechanism is indicated, in which C β-hydroxylation, with a hydroxyl derived from O 2, occurs simultaneously with, or after, C α-C β cleavage.

Further study discounts role for singlet oxygen in fungal degradation of lignin model compounds

This study reexamined our contention that singlet oxygen (1O2) plays a role in the fungal degradation of lignin (BBRC 102(1981)484). Cultures of Phanerochaete chrysosporium and a photochemical 1O2-generating system (riboflavin/light/O2) cleaved a lignin substructure model compound, 1,2-bis(4-methoxyphenyl)propane-1,3-diol (I), by indistinguishable mechanisms. However, the rate of cleavage of I in D2O was the same as in H2O in the photochemical 1O2-generating system, indicating that 1O2 was not involved. Furthermore, products formed from I in a chemical system for generating 1O2 (H2O2 + NaOCl) differed from those produced by cultures or the photochemical system. It is concluded that 1O2 is not responsible for cleavage of I or related compounds in the fungal cultures or in the photochemical system.