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.     

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.     

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.     

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.     

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.     

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.     

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.     

The C-C bond cleavage of a lignin model compound, 1,2-diarylpropane-1,3-diol, with a heme-enzyme model catalyst tetraphenylporphyrinatoiron(III)chloride in the presence of tert-butylhydroperoxide

The catalytic C-C bond cleavage of a lignin model compound was investigated by use of tetraphenylporphyrinatoiron(III)chloride as a model for enzymic degradation of lignin. The C-C bond of the lignin model compound 1,2-bis(4-ethoxy-3-methoxyphenyl) propane-1,3-diol was oxidatively cleaved by catalysis of iron-porphyrins in the presence of tert-butylhydroperoxide or iodosylbenzene at a room temperature. The products formed after complete oxidation of the substrate were identified as 4-O-ethylvanillin, alpha-hydroxy-4-ethoxy-3-methoxyacetophenone, 4-O-ethylvanillic acid, 4-ethoxy-3-methoxyphenylglycol, 4-ethoxy-3-methoxy-alpha-(4-ethoxy-3-methoxyphenyl)-beta-hydroxypropi ophenone and formaldehyde.     

Degradation of benzyl ether bonds of lignin by ruminal microbes

We examined microbial activity in the rumen to cleave benzyl ether bonds of lignin model compounds that fluoresced when the bonds were cleaved. 4-Methylumbelliferone veratryl ether dimer was degraded completely within 8 h even in the presence of fungicidal antibiotics, but no significant degradation occurred with bactericidal antibiotics. Degradation of a phenolic beta-O-4 trimer incorporating 4-methylumbelliferone by a benzyl ether linkage was stimulated by ruminal microbes, although its corresponding non-phenolic model compound, 1-(4-ethoxy-3-methoxyphenyl)-1-O-(4-methylumbelliferyl)-2-(2-methoxyp henoxy)-3-propanol, was not degraded. A coniferyl dehydrogenation polymer bearing fluorescent beta-O-4 benzyl ether that contains both phenolic and non-phenolic benzyl ether bonds was partially degraded (about 20%) in 48 h. These results suggest that ruminal microbes decompose benzyl ether linkages of lignin polymers under anaerobic conditions.     

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.     

Engineering of a manganese-binding site in lignin peroxidase isozyme H8 from Phanerochaete chrysosporium

A Mn(2+)-binding site was created in the recombinant lignin peroxidase isozyme H8 from Phanerochaete chrysosporium. In fungal Mn peroxidase, the Mn-binding site is composed of Glu35, Glu39, and Asp179. We generated a similar site in lignin peroxidase by generating an anionic binding site. We generated three mutations: Asn182Asp, Asp183Lys, and Ala36Glu. Its activity, veratryl alcohol, and Mn(2+) oxidation were compared to those of native recombinant enzyme and to fungal Mn peroxidase isozyme H4, respectively. The mutated enzyme was able to oxidize Mn(2+) and still retain its ability to oxidize veratryl alcohol. Steady-state results indicate that the enzyme's ability to oxidize veratryl alcohol was lowered slightly. The K(m) for Mn(2+) was determined to be 1.57 mM and the k(cat) = 5.45 s(-1). These results indicate that the mutated lignin peroxidase is less effective in Mn(2+) oxidation that the wild type fungal enzyme. The pH optima of veratryl alcohol and Mn oxidation were altered by the mutation. They are one unit of pH value higher than those of recombinant H8 and wild type fungal Mn peroxidase isozyme H4.     

Benzoquinones from Ardisia japonica with inhibitory activity towards human protein tyrosine phosphatase 1B (PTP1B)

From the whole plant of Ardisia japonica, four [1,4]benzoquinones were isolated by means of bioassay-directed fractionation of the EtOH extract. Apart from the two known compounds maesanin (1) and its congener 2, two new benzoquinones, i.e., 5-ethoxy-2-hydroxy-3-[(10Z)-pentadec-10-en-1-yl][1,4]benzoquinone (3) and 5-ethoxy-2-hydroxy-3-[(8Z)-tridec-8-en-1-yl][1,4]benzoquinone (4), were identified. All compounds showed significant in vitro bioactivities against the PTP1B enzyme, with IC50 values in the range of ca. 3-19 microM.     

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.     

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.     

Formation of pyrogallol ether during oxidative destruction of oak lignin with air oxygen

Formation of pyrogallol ether during thermal oxidation of oak (Quercus iberica L.) lignin in the presence of air oxygen was investigated. A model reaction demonstrated that the substance identified by us as pyrogallol ether (1,2-dioxy-3-methoxybenzene) is derived from syringic aldehyde.     

Arylglycerol-gamma-Formyl Ester as an Aromatic Ring Cleavage Product of Nonphenolic beta-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.     

Production and characterization of recombinant lignin peroxidase isozyme H2 from Phanerochaete chrysosporium using recombinant baculovirus.

Recombinant Phanerochaete chrysosporium lignin peroxidase isozyme H2 (pI 4.4) was produced in insect cells infected with a genetically engineered baculovirus containing a copy of the cDNA clone lambda ML-6. The recombinant enzyme was purified to near homogeneity and is capable of oxidizing veratryl alcohol, iodide, and, to a lesser extent, guaiacol. The Km of the recombinant enzyme for veratryl alcohol and H2O2 is similar to that of the fungal enzyme. The guaiacol oxidation activity or any other activity is not dependent upon Mn2+. The purified recombinant peroxidase is glycosylated with N-linked carbohydrate(s). The recombinant lignin peroxidase eluted from an anion exchange resin similar to that of native isozyme H1 rather than H2. However, the pI of the recombinant enzymes is different from both H1 and H2 isozymes. Further characterization of native isozymes H1 and H2 from the fungal cultures revealed identical N-terminus residues. This indicates that isozymes H1 and H2 differ in post-translational modification.