Akio Enoki

Degradation of lignin-related compounds, pure cellulose, and wood components by white-rot and brown-rot fungi

Eight of twelve white-rot fungi tested degraded the three lignin model dimers characterized by — -4, ß-linkages, and a phenyl glycol structure, respectively, a pure cellulose Substrate, and wood blocks, whereas eight of 12 brown-rot fungi tested failed to degrade the dimers or the pure cellulose. But the brown-rot fungi caused substantial weight losses of wood and degraded the cellulose component äs well äs the lignin component in the wood. Brown-rot fungi failed to degrade cellulose under the conditions in which ligninolytic Systems of the fungi were not produced, and cellulolytic Systems were necessarily produced by brown-rot fungi under the condilions in which ligninolytic Systems of the fungi were produced. The activity of white-rot fungi against the three dimers was in proportion to their ability to degrade wood. Poria subacida, a white-rot fungus, failed to degrade filter paper, but degraded the three dimers and removed about 70% of the lignin, only 1.8% of the cellulose, and 24.8% of the total weight loss of Japanese beech wood. Therefore the strain of P. subacida kept in our laboratory is useful for removing lignin from wood without concomitant loss of cellulose. Brown-rot fungi degraded the polysaccharides preferentially, whereas white-rot fungi degraded the lignin somewhat preferentially. Brown-rot fungi may generate a unique wood-component-degrading System that can split cellulose into fragments and modify the lignin cornponent.

?-Ether cleavage of the lignin model compound 4-ethoxy-3-methoxyphenylglycerol-?-guaiacyl ether and derivatives by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 4-ethoxy-3-methoxyphenyl-glycerol-β-guaiacyl ether (V) in low nitrogen, stationary cultures under which conditions the ligninolytic enzyme system is expressed. 4-Ethoxy-3-methoxyphenylglycerol XIII, guaicol and 4-ethoxy-3-methoxybenzyl alcohol (II) were isolated as metabolic products. Exogenously added XIII was rapidly converted to 4-ethoxy-3-methoxybenzyl alcohol indicating that it is an intermediate in the metabolism of V. P. chrysosporium also metabolized 1-(4′-ethoxy-3′-methoxyphenyl)-2-(2″-methoxyphenoxy)-3-hydroxypropane VI. The degradation pathway for this dimer also included initial β-ether cleavage and α-hydroxylation of the diol product 1-(4′-ethoxy-3′-methoxyphenyl) 2,3 dihydroxypropane (XI) to yield the triol XIII which was cleaved at the α, β bond to yield 4-ethoxy-3-methoxybenzyl alcohol. Finally P. chrysosporium also cleaved the dimer 1-(4′-ethoxy-3′-methoxyphenyl)-2-(2″-methoxyphenoxy)-1-hydroxypropane (VIII) at the β-ether linkage yielding 1-(4′-ethoxy-3′-methoxyphenyl) 1,2 dihydroxypropane (IX) which was subsequently cleaved at the α, β bond to yield II. All of the results indicate that oxidative β-ether cleavage is an important initial reaction in the metabolism of β-aryl ether lignin substructure dimeric compounds. Metabolities were identified after comparison with chemically synthesized standards by gas liquid chromatography-mass spectrometry.

Metabolism of the lignin model compounds veratrylglycerol-β-guaiacyl ether and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether by Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium metabolized the lignin model compounds veratylglycerol-β-guaiacyl ether I and 4-ethoxy-3-methoxy-phenylglycerol-β-guaiacyl ether V in stationary culture under an atmosphere of 100% oxygen and under nitrogen limiting conditions. 2-(o-methoxyphenoxy)-ethanol VII was identified as a product of the metabolism of both substrates. Veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol IV were identified as metabolites of I and V respectively. Metabolites were identified after comparison with chemically synthesized standards by mass spectrometry. These results indicate the existence of an enzyme system capable of directly cleaving the etherated dimers I and V at the α, β bond. The additional identification of 2-(o-methoxyphenoxy)-1,3 propanediol IX as a metabolic product indicates that cleavage of the alkyl-phenyl bond of these dimers or their metabolites also occurs.

A Phanerochaete chrysosporium mutant defective in lignin degradation as well as several other secondary metabolic functions

A pleiotropic mutant of Phanerochaete chrysosporium 104-2 lacking phenol oxidase and unable to form fruit bodies and a revertant strain 424-2 were isolated after UV mutagenesis. Strains 104-2 and 424-2 had no apparent dysfunction in primary metabolism with glucose as a carbon source. Unlike the wild type strain and strain 424-2, strain 104-2 was unable to evolve 14CO2 from 14C ring, side chain and 3-O-14C-methoxy labeled lignin. In addition, strain 104-2 was unable to evolve 14CO2 from a variety of lignin model compounds including 14C-4′-methoxy labeled veratrylglycerol-β-guaiacyl (V) ether, γ-14C-guaiacylglycerol-β-guaiacyl ether (VI), as well as 1-(14C-4′-methoxy, 3′-methoxyphenyl)1,2 propene (III) and 1-(14C-4′-methoxy-3′-methoxyphenyl) 1,2 dihydroxypropane (IV). The addition of peroxidase/H2O2 to cultures of strain 104-2 did not alter its capacity to degrade the labeled lignins. A variety of unlabeled lignin model compounds previously shown to be degraded by the wild type organism including β-aryl ether dimers and diaryl propane dimers were also not degraded by the mutant 104-2. The revertant strain 424-2 regained the capacity to degrade these compounds. The substrates described are degraded by oxygen requiring system(s) expressed during the secondary phase of growth, suggesting this pleiotropic mutant is possibly defective in the onset of postprimary metabolism. The inability of the mutant to produce the secondary metabolite veratryl alcohol and to elaborate enzymes in the veratryl alcohol biosynthetic pathway supports this hypothesis.

Degradation of the diarylpropane lignin model compound 1-(3′,4′-diethoxyphenyl)-1,3-dihydroxy-2-(4′'-methoxyphenyl)-propane and derivatives by the basidiomycete Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized 1-(3′,4′-diethoxyphenyl)-1,3(dihydroxy)-2-(4′-methoxyphenyl)-propane (XII) in low nitrogen stationary cultures, conditions under which the ligninolytic enzyme system is expressed. 3,4-Diethoxybenzyl alcohol (IV), 1,2(dihydroxy)-1-(4′-methoxyphenyl)ethane (XX) and anisyl alcohol were isolated as metabolic products indicating an initial α, β bond cleavage of this dimer. Exogenously added XX was rapidly converted to anisyl alcohol, indicating that XX is an intermediate in the metabolism of XII. Fungal cleavage of the α, β bond of 1-(3′-4′-diethoxyphenyl)-1-(hydroxy)-2-(4′-methoxyphenyl)ethane (XI) also occurred, indicating that a γ hydroxymethyl group is not a prerequisite for this reaction. P. chrysosporium also metabolized 1-(4′-ethoxy-3′-methoxyphenyl)-2,2(dihydroxy)-2-(4′-methoxyphenyl)propane-1-ol (XIII). The major products of the degradation of this triol included 4-ethoxy-3-methoxybenzyl alcohol (III) and 2-hydroxy-1-(4′-methoxyphenyl)-1-oxoethane (XXI). The nature of the products formed indicates that this triol is also cleaved directly at the α,β bond. The significant difference in the nature of the products formed from the diaryl propane (XII) and the triol (XIII), however, suggests that XIII is not an intermediate in the major pathway for the degradation of XII. Metabolites were identified after comparison with chemically synthesized standards by GLC-mass spectrometry.

Relationship between production of hydroxyl radicals and degradation of wood by the brown-rot fungus, Tyromyces palustris

In cultures of Tyromyces palustris containing glucose or wood as carbon source, we measured changes in production of a low-molecular-weight (15kDa) extracellular substance that catalyzes a redox reaction between O 2 and an electron donor to produce the hydroxyl radicals, OH. We also measured the activity of one-electron oxidation relative to degradation of wood, crystalline cellulose, and lignin substructure models in cultures. In the glucose cultures, activities of two cellulases (CMCase and Avicelase) were negligible, as was production of oxidant. In the wood cultures, CMCase and oxidant production, but not Avicelase activity, were high. These findings suggest that most of the one-electron oxidation in wood cultures is caused by hydroxyl radicals produced in a redox reaction between O 2 and certain electron donors catalyzed by the low-molecular-weight substance. The production of OH in T. palustris cultures was related to the rates of degradation of wood, crystalline cellulose, and lignin models in the cultures. On the basis of these results, we suggest that the hydroxyl radicals produced in the redox reaction between O 2 and an electron donor, mediated by the low-molecular-weight substance. play an important role in wood and crystalline cellulose degradation by T. palustris.

Vanillate hydroxylase from the white rot basidiomycete Phanerochaete chrysosporium

A soluble enzyme fraction from Phanerochaete chrysosporium catalyzed the oxidative decarboxylation of vanillic acid to methoxy-p-hydroquinone. The enzyme, partially purified by ammonium sulfate precipitation, required NADPH and molecular oxygen for activity. NADH was not effective. Optimal activity was displayed between pH 7.5–8.5. Neither EDTA, KCN, NaN3, nor o-phenanthroline (5 mM) were inhibitory. The enzyme was inducible with maximal activity displayed after incubation of previously grown cells with 0.1% vanillate for 30h.

An extracellular substance from the white-rot basidiomycete Phanerochaete chrysosporium for reducing molecular oxygen and ferric iron

An extracellular substance that had single-electron oxidation activity was isolated from wood-containing cultures of the white-rot basidiomycete Phanerochaete chrysosporium. The substance was partially purified by acetone precipitation, gel filtration chromatography on Sephadex G-50 and G-25, and DEAE Affi-Gel Blue gel chromatography. It contained about 18% protein, 20% neutral carbohydrate and 0.28% Fe(II) by weight. This substance was low molecular weight and could catalyze redox reactions between an electron donor, such as NADH, and O 2 to produce H 2 O 2 and to reduce H 2 O 2 to OH. It reduced Fe(III) to Fe(II) and strongly absorbed Fe(II). This extracellular substance seems to be involved in the initial degradation of cellulose and lignin in wood by the fungus.

RIBOFLAVIN‐PHOTOSENSITIZED OXIDATIVE DEGRADATION OF A VARIETY OF LIGNIN MODEL COMPOUNDS

Oxidation of several lignin model compounds with alkylated paraphenolic groups by photosensitizing riboflavin (RF). rose bengal (RB) and methylene blue (MB) was examined. Photosensitizing RF cleaved l–(3‘‐4’‐diethoxyphenyl)‐1.3 dihydroxy‐2‐(4‐methoxyphenyl)propane (I). 4‐ethoxy‐3‐methoxyphenylglycerol‐(3‐guaiacyl ether (II) and l‐(4‘‐ethoxy‐3’‐methoxyphenyI)‐1,3 dihydroxypropane (IV) at their respective Cα‐Cβ bonds. Riboflavin also oxidized 3.4‐diethoxy‐benzaldehyde (VI) to the corresponding acid, and hydroxylated the conjugated olefin l‐(4‘‐ethoxy‐3’‐methoxyphenyl)1.2 propene (III) to yield the initial product IV. In contrast, MB and RB hydroxylated III but had no effect on I, II, IV or VI under identical conditions. This suggested RF effected transformations via a hydrogen radical abstraction (Type I) rather than a 1O2 mediated reaction.

Alkyl-phenyl cleavage of the lignin model compounds guaiacylglycol and glycerol-?-guaiacyl ether by Phanerochaete chrysosporium

The white rot basidiomycete Phanerochaete chrysosporium metabolized guaiacylglycol-β-guaiacyl ether (I) in high nitrogen, shaking and stationary cultures. 2-(o-Methoxyphenoxy) ethanol (X), 2-(o-methoxyphenoxy) acetic acid (IX) and methoxy-phydroquinone (MHQ) were identified as products of the metabolism of (I). P. chrysosporium also metabolized guaiacylglycerol-β-guaiacyl ether (IV) in high nitrogen stationary cultures. 2-(o-Methoxyphenoxy)-1,3 propanediol (XII) and 3-hydroxy, 2-(o-methoxy-phenyxy) propionic acid (XIV) were identified as products of the metabolism of (IV). Finally, P. chrysosporium metabolized α-deoxyguaiacylglycol-β-guaiacyl ether (VI) and α-deoxyguaiacylglycerol-β-guaiacyl ether (VII) in limiting nitrogen cultures. 2-(o-Methoxyphenoxy) ethanol (X) and 2-(o-methoxyphenoxy)-1,3 propanediol (XII) were identified as products of the metabolism of VI and VII respectively indicating α hydroxylation of those substrates with subsequent alkyl-phenyl bond cleavage. Metabolites were identified after comparison with chemically synthesized standards by GLC-mass spectrometry.