A non-phenolic lignin model dimer, 1-(4-ethoxy-3-methoxyphenyl)-2-phenoxypropane-1,3-diol, was oxidized by a lipid peroxidation system that consisted of a fungal manganese peroxidase, Mn(II), and unsaturated fatty acid esters. The reaction products included I-(4ethoxy-3-methoxyphenyl)-1-oxo-2-phenoxy-3-hydroxypropane and 1-(4-ethoxy-3-methoxyphenyl)-l-oxo-3-hydroxypropane, indicating that substrate oxidation occurred via benzylic hydrogen abstraction. The peroxidation system depolymerized both exhaustively methylated (non-phenolic) and unmethylated (phenolic) synthetic lignins efficiently. It may therefore enable white-rot fungi to accomplish the initial delignification of wood.

The enzymatic mechanism of monolignol polymerization in lignin biosynthesis is not known, although it has been the subject of significant interest for more than 60 years. Peroxidase had been considered to be the exclusive plant enzyme involved in the oxidative polymerization of lignin precursors. Recently, laccase and laccase‐like oxidase activities have been associated with lignification. Laccase is bound to lignifying plant cell walls and can polymerize lignin precursors in vitro. Strong circumstantial evidence from different species implicates this enzyme in the polymerization of lignin precursors. Lignin has a complex structure and it has been difficult to analyze the heterogeneity of lignin by chemical and physical techniques. If lignin precursors are polymerized by enzymes that differ in their catalytic properties, then lignin heterogeneity could be produced by differential expression of multiple enzymes during plant development. When laccase genes are correctly identified, these ideas can be tested in genetic experiments where gain or loss of function can be predicted by the presence or absence of the functional gene.

Many ligninolytic fungi appear to lack lignin peroxidase (LiP), the enzyme generally thought to cleave the major, recalcitrant, nonphenolic structures in lignin. At least one such fungus, Ceriporiopsis subvermispora, is nevertheless able to degrade these nonphenolic structures. Experiments showed that wood block cultures and defined liquid medium cultures of C. subvermispora rapidly depolymerized and mineralized a 14 C-labeled, polyethylene glycol-linked, high-molecular-weight ␤-O-4 lignin model compound (model I) that represents the major nonphenolic structure of lignin. The fungus cleaved model I between C ␣ and C ␤ to release benzylic fragments, which were shown in isotope trapping experiments to be major products of model I metabolism. The C ␣-C ␤ cleavage of ␤-O-4 lignin structures to release benzylic fragments is characteristic of LiP catalysis, but assays of C. subvermispora liquid cultures that were metabolizing model I confirmed that the fungus produced no detectable LiP activity. Three results pointed, instead, to the participation of a different enzyme, manganese peroxidase (MnP), in the degradation of nonphenolic lignin structures by C. subvermispora. (i) The degradation of model I and of exhaustively methylated (nonphenolic), 14 C-labeled, synthetic lignin by the fungus in liquid cultures was almost completely inhibited when the Mn concentration of the medium was decreased from 35 M to approximately 5 M. (ii) The fungus degraded model I and methylated lignin significantly faster in the presence of Tween 80, a source of unsaturated fatty acids, than it did in the presence of Tween 20, which contains only saturated fatty acids. Previous work has shown that nonphenolic lignin structures are degraded during the MnP-mediated peroxidation of unsaturated lipids. (iii) In experiments with MnP, Mn(II), and unsaturated lipid in vitro, this system mimicked intact C. subvermispora cultures in that it cleaved nonphenolic ␤-O-4 lignin model compounds between C ␣ and C ␤ to release a benzylic fragment.

Fungi that cause brown rot of wood are essential biomass recyclers and also the principal agents of decay in wooden structures, but the extracellular mechanisms by which they degrade lignocellulose remain unknown. To test the hypothesis that brown-rot fungi use extracellular free radical oxidants as biodegradative tools, Gloeophyllum trabeum was examined for its ability to depolymerize an environmentally recalcitrant polyether, poly(ethylene oxide) (PEO), that cannot penetrate cell membranes. Analyses of degraded PEOs by gel permeation chromatography showed that the fungus cleaved PEO rapidly by an endo route.13 C NMR analyses of unlabeled and perdeuterated PEOs recovered from G. trabeum cultures showed that a major route for depolymerization was oxidative COC bond cleavage, a reaction diagnostic for hydrogen abstraction from a PEO methylene group by a radical oxidant. Fenton reagent (Fe(II)͞ H 2 O 2 ) oxidized PEO by the same route in vitro and therefore might account for PEO biodegradation if it is produced by the fungus, but the data do not rule out involvement of less reactive radicals. The reactivity and extrahyphal location of this PEO-degrading system suggest that its natural function is to participate in the brown rot of wood and that it may enable brown-rot fungi to degrade recalcitrant organopollutants.

Lignin model dimers are valuable tools for the elucidation of microbial ligninolytic mechanisms, but their low molecular weight (MW) makes them susceptible to nonligninolytic intracellular metabolism. To address this problem, we prepared lignin models in which unlabeled and ␣-14 C-labeled ␤-O-4-linked dimers were covalently attached to 8,000-MW polyethylene glycol (PEG) or to 45,000-MW polystyrene (PS). The watersoluble PEG-linked model was mineralized extensively in liquid medium and in solid wood cultures by the white rot fungus Phanerochaete chrysosporium, whereas the water-insoluble PS-linked model was not. Gel permeation chromatography showed that P. chrysosporium degraded the PEG-linked model by cleaving its lignin dimer substructure rather than its PEG moiety. C ␣-C ␤ cleavage was the major fate of the PEG-linked model after incubation with P. chrysosporium in vivo and also after oxidation with P. chrysosporium lignin peroxidase in vitro. The brown rot fungus Gloeophyllum trabeum, which unlike P. chrysosporium lacks a vigorous extracellular ligninolytic system, was unable to degrade the PEG-linked model efficiently. These results show that PEG-linked lignin models are a marked improvement over the low-MW models that have been used in the past.