Chlorination and cleavage of lignin structures by fungal chloroperoxidases

Two fungal chloroperoxidases (CPOs), the heme enzyme from Caldariomyces fumago and the vanadium enzyme from Curvularia inaequalis, chlorinated 1-(4-ethoxy-3-methoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane, a dimeric model compound that represents the major nonphenolic structure in lignin. Both enzymes also cleaved this dimer to give 1-chloro-4-ethoxy-3-methoxybenzene and 1,2-dichloro-4-ethoxy-5-methoxybenzene, and they depolymerized a synthetic guaiacyl lignin. Since fungal CPOs occur in soils and the fungi that produce them are common inhabitants of plant debris, CPOs may have roles in the natural production of high-molecular-weight chloroaromatics and in lignin breakdown.     

An extracellular H2O2-requiring enzyme preparation involved in lignin biodegradation by the white rot basidiomycete Phanerochaete chrysosporium

An H₂O₂-requiring enzyme system was found in the extracellular medium of ligninolytic cultures of Phanerochaete chrysosporium. The enzyme system generated ethylene from 2-keto-4-thiomethyl butyric acid (KTBA), and oxidized a variety of lignin model compounds including the diarylpropane 1-(4′-ethoxy-3′-methoxyphenyl) 1,3-dihydroxy-2-(4″-methoxyphenyl)propane (I), a β-ether dimer 1-(4′-ethoxy-3′-methoxyphenyl)glycerol-β-guaiacyl ether (IV) and an olefin 1-(4′-ethoxy-3′-methoxy-phenyl)1,2-propene (VI). The products found were equivalent to the metabolic products previously isolated from intact ligninolytic cultures. In addition, the enzyme system partially degraded ¹⁴C-ring labeled lignin. The enzyme was not found in high nitrogen (N) cultures, nor in cultures of a ligninolytic mutant strain which is incapable of metabolizing lignin.     

Degradation of the γ-Carboxyl-Containing Diarylpropane Lignin Model Compound 3-(4′-Ethoxy-3′-Methoxyphenyl)-2-(4″-Methoxyphenyl)Propionic Acid by the Basidiomycete Phanerochaete chrysosporium

The white-rot basidiomycete Phanerochaete chrysosporium metabolized 3-(4′-ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)propionic acid (V) in low-nitrogen, stationary cultures, conditions under which ligninolytic activity is expressed. The ability of several fungal mutant strains to degrade V reflected their ability to degrade [¹⁴C]lignin to ¹⁴CO₂. 1-(4′-Ethoxy-3′-methoxyphenyl)-2-(4″-methoxyphenyl)-2- hydroxyethane (VII), anisyl alcohol, and 4-ethoxy-3-methoxybenzyl alcohol were isolated as metabolic products, indicating an initial oxidative decarboxylation of V, followed by α, β cleavage of the intermediate (VII). Exogenously added VII was rapidly converted to anisyl alcohol and 4-ethoxy-3-methoxybenzyl alcohol. When the degradation of V was carried out under ¹⁸O₂, ¹⁸O was incorporated into the β position of the diarylethane product (VII), indicating that the reaction is oxygenative.     

p-Benzoquinone monoketals,novel degradation products of β-O-4 lignin model compounds by Coriolus versicolor and lignin peroxidase of Phanerochaete chrysosporium

2-(4-Ethoxy-3-methoxyphenyl)-3-hydroxymethyl-6,10-dimethoxy-1,4-dioxaspiro[4,5]deca-6,9-diene-8-one (III) and its isomer IV were identified as catabolites of 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (I) by the culture of Coriolus versicolor. Compound III was also produced from 4-ethoxy-3-methoxyphenylglycerol-β-syringic acid ether (II) by lignin peroxidase of Phanerochaete chrysosporium. An isotopic experiment showed that molecular oxygen was incorporated into the quinone oxygen of III in the degradation of II by lignin peroxidase.     

Aromatic ring cleavage of a non-phenolic beta-O-4 lignin model dimer by laccase of Trametes versicolor in the presence of 1-hydroxybenzotriazole

The novel cleavage products, 2,3-dihydroxy-1-(4-ethoxy-3-methoxyphenyl)-1-formyloxypropane (II) and 1-(4-ethoxy-3-methoxyphenyl)-1,2,3-trihydroxypropane-2,3-cyclic carbonate (III) were identified as products of a non-phenolic beta-O-4 lignin model dimer, 1,3-dihydroxy-2-(2,6-dimethoxylphenoxy)-1-(4-ethoxy-3-methoxypheny l)propane (I), by a Trametes versicolor laccase in the presence of 1-hydroxybenzotriazole (1-HBT). An isotopic experiment with a 13C-labeled lignin model dimer, 1,3-dihydroxy-2-(2,6-[U-ring-13C] dimethoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl)propane (I-13C) indicated that the formyl and carbonate carbons of products II and III were derived from the beta-phenoxy group of beta-O-4 lignin model dimer I as aromatic ring cleavage fragments. These results show that the laccase-1-HBT couple could catalyze the aromatic ring cleavage of non-phenolic beta-O-4 lignin model dimer in addition to the beta-ether cleavage, Calpha-Cbeta cleavage, and Calpha-oxidation.     

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-γ-formyl ester (compound IV) was identified as a degradation product of both 4-ethoxy-3-methoxyphenylglycerol-β-syringaldehyde ether (compound I) and 4-ethoxy-3-methoxyphenylglycerol-β-2,6-dimethoxyphenyl ether (compound II) by a ligninolytic culture of Coriolus versicolor. An isotopic experiment with a ¹³C-labeled compound (compound II′) indicated that the formyl group of compound IV was derived from the β-phenoxyl group of β-O-4 dimer as an aromatic ring cleavage fragment. However, compound IV was not formed from 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether (compound III). γ-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-β,γ-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 ¹³C-labeled compounds II′ and III′ indicated that the carbonate carbon of compound V was derived from the β-phenoxyl groups of β-O-4 substructure.     

Degradation of the gamma-Carboxyl-Containing Diarylpropane Lignin Model Compound 3-(4'-Ethoxy-3'-Methoxyphenyl)-2-(4'-Methoxyphenyl)Propionic Acid by the Basidiomycete Phanerochaete chrysosporium

The white-rot basidiomycete Phanerochaete chrysosporium metabolized 3-(4'-ethoxy-3'-methoxyphenyl)-2-(4'-methoxyphenyl)propionic acid (V) in low-nitrogen, stationary cultures, conditions under which ligninolytic activity is expressed. The ability of several fungal mutant strains to degrade V reflected their ability to degrade [C]lignin to CO(2). 1-(4'-Ethoxy-3'-methoxyphenyl)-2-(4'-methoxyphenyl)-2- hydroxyethane (VII), anisyl alcohol, and 4-ethoxy-3-methoxybenzyl alcohol were isolated as metabolic products, indicating an initial oxidative decarboxylation of V, followed by alpha, beta cleavage of the intermediate (VII). Exogenously added VII was rapidly converted to anisyl alcohol and 4-ethoxy-3-methoxybenzyl alcohol. When the degradation of V was carried out under O(2), O was incorporated into the beta position of the diarylethane product (VII), indicating that the reaction is oxygenative.     

Triterpenes and lignans from the leaves of Styrax tonkinensis

Two triterpenes (1 and 2) and eight lignans (3–10) were isolated from the ethyl acetate-soluble fraction of the leaves of Styrax tonkinensis (Pierre) Craib ex Hartw (Styracaceae). Their structures were established as ursolic acid (1), pomolic acid (2), 3,3′-bis(3,4-dihydro-6-methoxy-2H-1-benzopyran) (3), rac-(8α,8′β)-4,4′-dihydroxy-3,3′-dimethoxylignan-9,9′-diyldiacetate (4), (-)-secoisolariciresinol (5), (+)-pinoresinol (6), 4,4′-dihydroxy-3,3′-dimethoxy-9-ethoxy-9,9′-epoxylignan (7), (2S,3R, 4R)-4-[1-ethoxy-1-(4-hydroxy-3-methoxy)phenyl]methyl-2-(4-hydroxy-3-methoxy)phenyl-3-hydroxymethyl-tetrahydrofuran (8), (-)-neo-olivil-(9-O-9″)-seco-isolariciresinol (9) and isolariciresinol (10) based on MS, ¹H-and ¹³C-NMR spectral data. All these compounds (1–10) were firstly isolated from this plant, and compounds 2–5 and 7–9 were reported from the Styrax genus for the first time. Furthermore, the chemotaxonomic significance of the isolated compounds was discussed.     

N-Acetyldopamine derivatives from Periostracum Cicadae

Five new N-acetyldopamine (NADA) derivatives (1–5) and one known NADA quinone methide (6) were isolated from Periostracum Cicadae (the cast-off shell of the cicada Cryptotympana pustulata Fabricius), which is known as chantui in China and is used in traditional Chinese medicine to treat soreness of the throat, hoarseness, itching, and spasms. By combined analysis of one-dimensional and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry, CD spectra, and chemical evidence, the structures of the isolated compounds were established as (R)-N-(2-(3,4-dihydroxyphenyl)-1-ethoxy-2-oxoethyl)acetamide (1), (1R,2R)-N-(1,2-diethoxy-2-(3,4-dihydroxyphenyl)-ethyl)acetamide (2), (R)-N-(1-acetamido-2-ethoxy-2-(3,4-dihydroxyphenyl)-ethyl)acetamide (3), (1R,2R)-N-(2-(3,4-dihydroxyphenyl)-2-ethoxy-1-methoxyethyl)acetamide (4), (1S,2S)-N-(2-(3,4-dihydroxyphenyl)-2-ethoxy-1-methoxyethyl)acetamide (5), and (R)-N-(2-(3,4-dihydroxyphenyl)-2-methoxyethyl)acetamide (6).     

Studies on Lignin

In comparison with the ethanolysis, the mercaptolysis of pine wood and pine ethanol lignin has been studied. The delignification was found to be almost complete when wood powder was cooked with the ethanolic hydrogen chloride containing 10% of ethyl mercaptan; while the same cooking without the mercaptan caused only 50% of delignification. Addition of 2% of mercaptan resulted more than 90% of delignification. As already reported briefly, from the mercaptolysis oil of pine ethanol lignin, 2-ethoxy-1-(4-hydroxy-3-methoxyphenyl)-propanone- (1), a thioether corresponding to Hibbert’s α-ethoxypropiovanillone, was isolated.     

The molecular composition of lignin in spruce decayed by white-rot fungi (Phanerochaete chrysosporium and Trametes versicolor) using pyrolysis-GC–MS and thermochemolysis with tetramethylammonium hydroxide

Pyrolysis-gas chromatography-mass spectrometry (Py-GC–MS) and off-line thermochemolysis with tetramethylammonium hydroxide followed by GC–MS were used in the molecular characterisation of lignin in spruce wood decayed by Phanerochaete chrysosporium and Trametes versicolor. Mono-methoxyphenols were the main pyrolysis products from the undegraded lignin. Py-GC–MS provided qualitative evidence that 2-methoxy-4-(prop-2-enal)phenol and trans-2-methoxy-4-(1-hydroxy-prop-2-enyl)phenol content decreased whereas 1,2-dihydroxybenzene increased in intensity relative to other products upon fungal decay. Comparison of methylated phenols from thermochemolysis revealed that ratio of methyl 3,4-dimethoxybenzoate to 3,4-dimethoxybenzaldehyde increased from 0.69 in control spruce to 2.3 after decay by P. chrysosporium and 3.7 following growth of T. versicolor. The results indicate that white-rot fungi cleave alkyl side chains of β-O-4 linked mono-methoxyphenylpropane structures between the α–β carbon atoms to give lignin residues enriched in carboxylic acids as well as demethylating methoxy groups attached to aromatic nuclei to give dihydroxybenzene products. Py-GC–MS and thermochemolysis are complementary methods for tracking demethylation of aromatic nuclei and oxidation of alkyl side chains caused by white-rot fungi.     

Oxygenation of 4-Alkoxyl Groups in Alkoxybenzoic Acids by Polyporus dichrous

The degradation of several alkyl ethers of vanillic acid, of 3-ethoxy-4-hydroxybenzoic acid, and of syringic acid, by the lignin-decomposing fungus Polyporus dichrous included (i) 4-dealkylation (e.g., 3-ethoxy-4-isopropoxybenzoic acid was in part dealkylated to 3-ethoxy-4-hydroxybenzoic acid), (ii) hydroxylation of the 4-alkoxyl groups (e.g., 3-ethoxy-4-isopropoxybenzoic acid was oxidized in part to 2-[4-carboxy-2-ethoxyphenoxy]-propane-1-ol), and (iii) reduction of carboxyl groups (older cultures) (e.g., 3-ethoxy-4-isopropoxybenzoic acid was reduced to 3-ethoxy-4-isopropoxybenzaldehyde and 3-ethoxy-4-isopropoxybenzyl alcohol). Some ethers (e.g., tri-O-methyl gallic acid and glycerol-beta-[4-carboxy-2-ethoxyphenyl]-ether) were not affected. The dealkylations and hydroxylations indicate that the fungus has a relatively nonspecific mechanism for oxygenating various 4-alkoxyl groups of alkoxybenzoic acids; no evidence for oxygenation of 3-alkoxyl groups was obtained. Hydroxylation products were generally degraded further, probably via dealkylation. The vanillic acid and 3-ethoxy-4-hydroxybenzoic acid formed by dealkylations were readily metabolized. Although the isopropyl ether of syringic acid was hydroxylated to 2-(4-carboxy-2, 6-dimethoxyphenoxy)-propane-1-ol, neither this compound nor the parent isopropyl ether was dealkylated; syringic acid itself was only slowly and incompletely metabolized. The relationship of these results to lignin degradation is discussed.     

Effects of fungal elicitor on lignin biosynthesis in cell suspension cultures of soybean

Soybean (Glycine max L.) cells cultured in B5 medium produce extremely low amounts of lignin. However, modification in the growth medium, by lowering the concentration of NO⁻₃ and PO²⁻₄, results in the lignification of these cells without affecting levels of cell wall-esterified 4-coumaric and ferulic acid. The production of an extracellular, macromolecular complex by the cultured soybean cells (Moore TS Jr 1973 Plant Physiol 51: 529-536) allows a rapid, nondestructive solubilization of the lignin which can be estimated by reaction with phloroglucinol in free solution. This system has been used to study the effects of fungal elicitor on the synthesis of lignin in soybean cells. The inclusion of very low levels of an elicitor fraction from the cell walls of Phytophthora megasperma in the medium in which lignification of the soybean cells occurs suppressed both the accumulation of extracellular lignin and phloroglucinol staining of the cell walls without affecting the levels of bound hydroxycinnamic acids. The activity profiles of phenylalanine ammonia-lyase (EC 4.3.1.5) and isoenzymes of 4-coumarate:CoA ligase (EC 6.2.1.12) were compared in lignifying and elicitor-treated cell cultures as was the activity of chalcone synthase, an enzyme of flavonoid biosynthesis. The measured activities of these enzymes in cell cultures treated with elicitor were considerably lower than in untreated cells.     

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

Abstract A lignin substructure model, 1-(4-ethoxy-3,5-dimethoxyphenyl)-2-(4-ethoxy-3-methoxyphenyl)-propane-1,3-diol(I), was actively metabolized by a white-rot fungus Coriolus versicolor in low nitrogen stationary cultures favouring the ligninolytic activity in the fungus. Cleavage of the dimer I between C_α and C_β of the propanoid side chain was the major degradative reaction by the fungus.     

New alkaloids from Aegle marmelos

Four new alkaloids, O-(3,3-dimethylallyl)-halfordinol, N-2-ethoxy-2-(4-methoxyphenyl)ethylcinnamamide, N-2-methoxy-2-[4-(3′,3′-dimethyl     

Bioactive compounds from the edible mushroom Cortinarius caperatus

Six compounds, 5-(1-hydroxyethyl)dihydrofuran-2(3H)-one (1), 4-ethoxy-4-oxobutanoic acid (2), 4-ketononanoic acid (3), methyl(2-acetylaminoethyl)sulfoxide (4), methyl benzoate (5) and p-hydroxybenzoic acid (6) were isolated from the fruiting bodies of Cortinarius caperatus. Compounds 1, 3, 5, and 6 inhibited growth of Flammulina velutipes mycelia. Compounds 2, 3, 5, and 6 exhibited growth regulatory activities toward rice seedlings, while compounds 3, 5, and 6 regulated the growth of lettuce. Compound 4 was first isolated from a natural source. In addition, the activity of compound 6 against rice was compared with those of its analogs.     

Lignin Peroxidase Activity Is Not Important in Biological Bleaching and Delignification of Unbleached Kraft Pulp by Trametes versicolor

The discovery in 1983 of fungal lignin peroxidases able to catalyze the oxidation of nonphenolic aromatic lignin model compounds and release some CO₂ from lignin has been seen as a major advance in understanding how fungi degrade lignin. Recently, the fungus Trametes versicolor was shown to be capable of substantial decolorization and delignification of unbleached industrial kraft pulps over 2 to 5 days. The role, if any, of lignin peroxidase in this biobleaching was therefore examined. Several different assays indicated that T. versicolor can produce and secrete peroxidase proteins, but only under certain culture conditions. However, work employing a new lignin peroxidase inhibitor (metavanadate ions) and a new lignin peroxidase assay using the dye azure B indicated that secreted lignin peroxidases do not play a role in the T. versicolor pulp-bleaching system. Oxidative activity capable of degrading 2-keto-4-methiolbutyric acid (KMB) appeared unique to ligninolytic fungi and always accompanied pulp biobleaching.     

Purification and characterization of an intracellular peroxidase from Streptomyces cyaneus.

An intracellular peroxidase (EC 1.11.1.7) from Streptomyces cyaneus was purified to homogeneity. The enzyme had a molecular weight of 185,000 and was composed of two subunits of equal size. It had an isoelectric point of 6.1. The enzyme had a peroxidase activity toward o-dianisidine with a Km of 17.8 microM and a pH optimum of 5.0. It also showed catalase activity with a Km of 2.07 mM H2O2 and a pH optimum of 8.0. The purified enzyme did not catalyze C alpha-C beta bond cleavage of 1,3-dihydroxy-2-(2-methoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl) propane, a nonphenolic dimeric lignin model compound. The spectrum of the peroxidase showed a soret band at 405 nm, which disappeared after reduction with sodium dithionite, indicating that the enzyme is a hemoprotein. Testing the effects of various inhibitors on the enzyme activity showed that it is a bifunctional enzyme having catalase and peroxidase activities.     

Purification and characterization of an intracellular peroxidase from Streptomyces cyaneus.

An intracellular peroxidase (EC 1.11.1.7) from Streptomyces cyaneus was purified to homogeneity. The enzyme had a molecular weight of 185,000 and was composed of two subunits of equal size. It had an isoelectric point of 6.1. The enzyme had a peroxidase activity toward o-dianisidine with a Km of 17.8 microM and a pH optimum of 5.0. It also showed catalase activity with a Km of 2.07 mM H2O2 and a pH optimum of 8.0. The purified enzyme did not catalyze C alpha-C beta bond cleavage of 1,3-dihydroxy-2-(2-methoxyphenoxy)-1-(4-ethoxy-3-methoxyphenyl) propane, a nonphenolic dimeric lignin model compound. The spectrum of the peroxidase showed a soret band at 405 nm, which disappeared after reduction with sodium dithionite, indicating that the enzyme is a hemoprotein. Testing the effects of various inhibitors on the enzyme activity showed that it is a bifunctional enzyme having catalase and peroxidase activities.     

Degradation of nonphenolic lignin by the laccase/1-hydroxybenzotriazole system

Phenolic and nonphenolic (permethylated) synthetic [14C]lignins were depolymerized by Trametes villosa laccase in the presence of a radical mediator, 1-hydroxybenzotriazole (HOBT). Gel permeation chromatography of the treated lignins showed that approximately 10% of their substructures were cleaved. The system also cleaved a beta-O-4-linked model compound, 1-(4-ethoxy-3-methoxy-ring-[14C]phenyl)-2-(2-methoxyphenoxy)-propane- 1,3-diol, and a beta-1-linked model, 1, 2-bis-(3-methoxy-4-[14C]methoxyphenyl)-propane-1,3-diol, that represent nonphenolic substructures in lignin. High performance liquid chromatography of products from the oxidized models showed that they were produced in sufficient yields to account for the ability of laccase/HOBT to depolymerize nonphenolic lignin.