Delignification of an unbleached hardwood kraft pulp by Phanerochaete chrysosporium

Summary Culture conditions affecting lignin degradation of an unbleached hardwood kraft pulp by Phanerochaete chrysosporium have been examined. Optimum pH and temperature for lignin degradation (about 33%) were 3.5 and 38°C, respectively. Optimum fungal growth was at a pH of 4.5 and a temperature of around 32°C. Addition of exogeneous glucose to the cultures lessened the degradation of pulp carbohydrates. Lignin degradation was stimulated by oxygen atmosphere and non-agitated cultures. Increased surface to volume ratio (decreased culture depth) enhanced lignin degradation (about 56% at a depth of 1.2 cm). Finally, the correlations: pulp yield vs. residual glucose, ligninase activity vs. mycelium, and extent of delignification vs. residual extracellular H₂O₂ were discussed in light of recent findings of ligninases responsible for ligninolysis.     

Relationship Between Lignin Degradation and Production of Reduced Oxygen Species by Phanerochaete chrysosporium

The relationship between the production of reduced oxygen species, hydrogen peroxide (H₂O₂), superoxide (O₂), and hydroxyl radical (·OH), and the oxidation of synthetic lignin to CO₂ was studied in whole cultures of the white-rot fungus Phanerochaete chrysosporium Burds. The kinetics of the synthesis of H₂O₂ coincided with the appearance of the ligninolytic system; also, H₂O₂ production was markedly enhanced by growth under 100% O₂, mimicking the increase in ligninolytic activity characteristic of cultures grown under elevated oxygen tension. Lignin degradation by whole cultures was inhibited by a specific H₂O₂ scavenger, catalase, implying a role for H₂O₂ in the degradative process. Superoxide dismutase also inhibited lignin degradation, suggesting that O₂ is also involved in the breakdown of lignin. The production of ·OH was assayed in whole cultures by a benzoate decarboxylation assay. Neither the kinetics of ·OH synthesis nor the final activity of its producing system obtained under 100% O₂ correlated with that of the lignin-degrading system. However, lignin degradation was inhibited by compounds which react with ·OH. It is concluded that H₂O₂, and perhaps O₂, are involved in lignin degradation; because these species are relatively unreactive per se, their role must be indirect. Conclusions about a role for ·OH in ligninolysis could not be reached.     

Ultrastructural Localization of Hydrogen Peroxide Production in Ligninolytic Phanerochaete chrysosporium Cells

Previous studies have shown that the hydroxyl radical derived from hydrogen peroxide (H₂O₂) is involved in lignin degradation by Phanerochaete chrysosporium. In the present study, the ultrastructural sites of H₂O₂ production in ligninolytic cells of P. chrysosporium were demonstrated by cytochemically staining cells with 3,3′-diaminobenzidine (DAB). Hydrogen peroxide production, as evidenced by the presence of oxidized DAB deposits, appeared to be localized in the periplasmic space of cells from ligninolytic cultures grown for 14 days in nitrogen-limited medium. When identical cells were treated with DAB in the presence of aminotriazole, periplasmic deposits of oxidized DAB were not observed, suggesting that the deposits resulted from the H₂O₂-dependent peroxidatic oxidation of DAB by catalase. Cells from cultures grown for 3 or 6 days in nitrogen-limited medium or for 14 days in nitrogen-sufficient medium had little ligninolytic activity and low specific activity for H₂O₂ production and did not contain periplasmic oxidized DAB deposits. The results suggest that in cultures grown in nitrogen-limited medium, there is a positive correlation between the occurrence of oxidized DAB deposits, the specific activity for H₂O₂ production in cell extracts, and ligninolytic activity.     

Effect of Agitation on Ligninase Activity and Ligninase Production by Phanerochaete chrysosporium

The white rot fungus Phanerochaete chrysosporium produces extracellular ligninases as part of its idiophasic ligninolytic system. Agitation has been widely reported to suppress both ligninase production and lignin degradation. Results show that mechanical inactivation of ligninase is possibly the reason why ligninase accumulation is low or absent in agitated shake-flask cultures. Agitation seems to affect the catalytic activity of ligninase and has no apparent effect on either the rate of ligninase production or the physiology of P. chrysosporium. The detergents Tween 20, Tween 40, Tween 60, Tween 80, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) are able to protect both purified ligninase and extant ligninase in culture fluids (free of biomass) against mechanical inactivation due to agitation. Addition of Tween 80 at the end of primary growth to agitated shake flasks containing either pelleted or immobilized mycelial cultures results in production and maintenance of high levels of ligninase activity over several days under conditions of high agitation. Possible mechanisms by which the detergents could protect ligninase are discussed.     

Biochemistry of the oxidation of lignin by Phangerochate chrysosporium

The objective of this research was to identify the biochemical agents responsible for the oxidative degradation of lignin by the white-rot fungus . We examined the hypothesis that activated oxygen species are involved, and we also sought the agent in ligninolytic cultures responsible for a specific oxidative degradative reaction in substructure model compounds. Results of studies of the production of activated oxygen species by cultures, of the effect of their removal on ligninolytic activity, and of their action on substructure model compounds support a role for hydrogen peroxide (H₂O₂) and possibly superoxide (O₂ ·^-) in lignin degradation. Involvement of hydroxyl radical (·OH) or singlet oxygen (¹O₂) is not supported by our data. The actual biochemical agent responsible for one important oxidative C-C bond cleavage reaction in non-phenolic lignin substructure model compounds, and in lignin itself, was found to be an enzyme. The enzyme is extracellular, has a molecular weight of 42,000 daltons, is azide-sensitive, and requires H₂O₂ for activity.     

Influence of Molecular Size and Ligninase Pretreatment on Degradation of Lignins by Xanthomonas sp. Strain 99

The purpose of this study was to examine the relationship between the molecular size of lignin in several preparations and extent of degradation (mineralization) by Xanthomonas sp. strain 99. The influence of ligninase pretreatment was also examined. Five synthetic lignins and one ¹⁴C-methylated spruce lignin were used. The extent of mineralization to ¹⁴CO₂ was greatest for the samples containing the most low-molecular-weight material, and the low-molecular-weight portions were preferentially (or perhaps solely) degraded. Pretreatment of the five synthetic lignins with crude ligninase increased their molecular size and decreased their degradability by the xanthomonad. Pretreatment of the methylated spruce lignin with crude ligninase caused both polymerization and depolymerization but resulted in a net decrease in bacterial degradability. Our results suggest that the xanthomonad can degrade lignins only up to a molecular weight of 600 to 1,000.     

Role of veratryl alcohol in lignin degradation by Phanerochaete chrysosporium

Several aromatic compounds increased initial lignin degradation rates in cultures of Phanerochaete chrysosporium. This activation was connected to increased H₂O₂ production and glucose oxidation rates. Veratryl alcohol, a natural secondary metabolite of P. chrysosporium, also activated the lignin-degrading system. In the presence of added veratryl alcohol the ligninolytic system appeared 6–8 h earlier than in reference cultures. This effect was only seen when lignin was added after the primary growth was completed because lignin itself also caused earlier appearance of the degradative system. In cultures which received no added lignin or veratryl alcohol the ligninolytic activity only appeared once the alcohol started to accumulate. The degradation patterns of veratryl alcohol and lignin were similar. The activity levels of lignin degradation and glucose oxidation could be regulated by veratryl alcohol concentration. It is suggested that either veratryl alcohol itself or a metabolite derived from it is actually responsible for the low levels of ligninolytic activity in glucose grown cultures.     

Free hydroxyl radical is not involved in an important reaction of lignin degradation by Phanerochaete chrysosporium Burds.

Hydroxyl radical (HO.) has been implicated in the degradation of lignin by Phanerochaete chrysosporium. This study assessed the possible involvement of HO. in degradation of lignin substructural models by intact cultures and by an extracellular ligninase isolated from the cultures. Two non-phenolic lignin model compounds [aryl-C(alpha)HOH-C(beta)HR-C(gamma)H2OH, in which R = aryl (beta-1) or R = O-aryl (beta-O-4)] were degraded by cultures, by the purified ligninase, and by Fenton's reagent (H2O2 + Fe2+), which generates HO.. The ligninase and the cultures formed similar products, derived via an initial cleavage between C(alpha) and C(beta) (known to be an important biodegradative reaction), indicating that the ligninase is responsible for model degradation in cultures. Products from the Fenton degradation were mainly polar phenolics that exhibited little similarity to those from the biological systems. Mass-spectral analysis, however, revealed traces of the same products in the Fenton reaction as seen in the biological reactions; even so, an 18O2-incorporation study showed that the mechanism of formation differed. E.s.r. spectroscopy with a spin-trapping agent readily detected HO. in the Fenton system, but indicated that no HO. is formed during ligninase catalysis. We conclude, therefore that HO. is not involved in fungal C(alpha)-C(beta) cleavage in the beta-1 and beta-O-4 models and, by extension, in the same reaction in lignin.     

Formation and Action of Lignin-Modifying Enzymes in Cultures of Phlebia radiata Supplemented with Veratric Acid

Transformation of veratric (3,4-dimethoxybenzoic) acid by the white rot fungus Phlebia radiata was studied to elucidate the role of ligninolytic, reductive, and demeth(ox)ylating enzymes. Under both air and a 100% O₂ atmosphere, with nitrogen limitation and glucose as a carbon source, reducing activity resulted in the accumulation of veratryl alcohol in the medium. When the fungus was cultivated under air, veratric acid caused a rapid increase in laccase (benzenediol:oxygen oxidoreductase; EC 1.10.3.2) production, which indicated that veratric acid was first demethylated, thus providing phenolic compounds for laccase. After a rapid decline in laccase activity, elevated lignin peroxidase (ligninase) activity and manganese-dependent peroxidase production were detected simultaneously with extracellular release of methanol. This indicated apparent demethoxylation. When the fungus was cultivated under a continuous 100% O₂ flow and in the presence of veratric acid, laccase production was markedly repressed, whereas production of lignin peroxidase and degradation of veratryl compounds were clearly enhanced. In all cultures, the increases in lignin peroxidase titers were directly related to veratryl alcohol accumulation. Evolution of ¹⁴CO₂ from 3-O¹⁴CH₃-and 4-O¹⁴CH₃-labeled veratric acids showed that the position of the methoxyl substituent in the aromatic ring only slightly affected demeth(ox)ylation activity. In both cases, more than 60% of the total ¹⁴C was converted to ¹⁴CO₂ under air in 4 weeks, and oxygen flux increased the degradation rate of the ¹⁴C-labeled veratric acids just as it did with unlabeled cultures.     

Methanol formation during lignin degradation by Phanerochaete chrysosporium

Summary Methanol formation during the degradation of synthetic lignin (DHP), spruce and birch milled wood lignin (MWL) by Phanerochaete chrysosporium Burds. was studied under different culture conditions. When 100-ml flasks with 15–20 ml volumes of culture media containing high glucose and low nitrogen concentrations were used the metabolism of methanol to formaldehyde, formic acid and CO₂ was repressed thereby facilitating methanol determination. In standing cultures with oxygen flushing the fungus converted up to 25% of the DHP-methoxyl groups to methanol and 0.5–1.5% to ¹⁴CO₂ within 22–24 h. Methanol formation from methoxyl-labelled DHP was strongly repressed by high nitrogen in the medium, by addition of glutamic acid and by culture agitation. These results indicate that methanol is formed only under ligninolytic conditions and during secondary metabolism. Methanol is most likely released both from the lignin polymer itself and from lignin degradation products. Methanol was also formed from MWL preparations with higher percentage yields produced from birch as compared to spruce MWL.Small amounts of methanol detected in cultures without lignin probably emanated from demethoxylation of veratryl alcohol synthesized de novo from glucose by the fungus during secondary metabolism. Catalase or superoxide dismutase added to the fungal culture prior to addition of lignin, did not decrease methanol formation. Horseradish peroxidase plus H₂O₂ in vitro caused 5–7% demethoxylation of O¹⁴CH₃-DHP in 22 h, while laccase gave smaller amounts of methanol (1.8%). Since addition of H₂O₂ gave similar results as peroxidase plus H₂O₂, it seems likely that the main effect of peroxidase demethoxylation emanates from the hydrogen peroxide.     

Recent advances in studies of the mechanisms of microbial degradation of lignins

Major advances in our understanding of the biochemical and enzymological mechanisms of lignin biodegradation have been made in the past three years. Research has principally involved two ligninolytic microorganisms, the white rot fungus Phanerochaete chrysosporium and the actinomycete Streptomyces viridosporus. Research has been centred on attempts to identify the microbial catalysts that mediate lignin decay in these two microbes. Emphasis has been on studies concerned with isolating specific lignin catabolic enzymes and/or reduced forms of oxygen involved in attacking the lignin polymer. The possibility that lignin degradation might be non-enzymatic and mediated by extracellular reduced oxygen species such as hydrogen peroxide (H₂O₂), superoxide (O₂∪c-|_.), hydroxyl radical (·OH) or singlet oxygen (¹O₂) has been investigated with both microorganisms. Using methods which have not always been unequivocal, the question of involvement of reduced oxygen species in lignin degradation by P. chrysosporium has been examined exhaustively. Evidence for the involvement of H₂O₂ is conclusive. However, there is little evidence to support the involvement of other extracellular reduced oxygen species, including ·OH, directly in the process of lignin degradation. Scavenger studies have been inconclusive because of questions of their specificity. If activated oxygen species are involved, the activated oxygen is probably held within the active site of an enzyme molecule. With S. viridosporus, scavenger studies also strongly indicate that extracellular reduced oxygen species are not involved in lignin degradation since scavengers generally do not significantly affect the ligninolytic system. The involvement of specific enzymes in lignin degradation by both P. chrysosporium and S. viridosporus has now been confirmed. With P. chrysosporium, ligninolytic enzymes recently discovered include extracellular non-specific peroxidases and oxygenases. They show numerous activities including dehydrogenative, peroxidatic, oxygenative and C_α?C_β cleavages of lignin side chains. At least one P. chrysosporium enzyme, a unique H₂O₂-requiring oxygenase, has been purified to homogeneity. Evidence has been presented to show that S. viridosporus also produces a ligninolytic enzyme complex involved in demethylation of lignin's aromatic rings and in the oxidation of lignin side chains and cleavage of β-tether linkages within the polymer. The combined activites of these enzymes generate water-soluble polymeric modified lignin fragments, which are then slowly degraded further by S. viridosporus. The β-ether cleaving enzyme complex is probably membrane associated, but it is not extracellular. These first isolations of ligninolytic enzymes have changed the course of basic research on lignin biodegradation. New research priorities are already emerging and include enzyme purifications, kinetic studies, enzyme reaction mechanism studies and screenings for more enzymes. In addition, genetic studies are being carried out with both P. chrysosporium and S. viridosporus. Genetic manipulations include not only classical mutagenesis techniques, but also recombinant DNA techniques such as protoplast fusion. This latter technique has already been used to generate overproducers of the ligninolytic enzyme complex in S. viridosporus and it has been successfully used to recombine mutant strains of P. chrysosporium.     

Role of Veratryl Alcohol in Regulating Ligninase Activity in Phanerochaete chrysosporium

Ligninase activity in Phanerochaete chrysosporium is stimulated by incubating cultures with various substrates for the enzyme, including veratryl (3,4-dimethoxybenzyl) alcohol, which is a secondary metabolite of this fungus. This study was designed to provide insight into the mechanism involved in this stimulation. Ligninase activity increased 2 to 4 h after the addition of exogenous veratryl alcohol to ligninolytic cultures. This increase was prevented by inhibitors of protein synthesis. Analysis of the extracellular proteins by high-performance anion-exchange liquid chromatography revealed increases in the amounts of some, but not all, ligninase species. The normal rapid decrease in ligninase activity in aging cultures was not prevented or retarded by veratryl alcohol, indicating that veratryl alcohol does not increase ligninase activity by protecting extant enzyme. We conclude that veratryl alcohol probably functions via an induction type of mechanism, affecting only certain ligninase species. Results with an isolated lignin indicate that lignin (or its biodegradation products) functions in the same way that veratryl alcohol does.     

Production of Ligninases and Degradation of Lignin in Agitated Submerged Cultures of Phanerochaete chrysosporium

Research on the extracellular hemeprotein ligninases of Phanerochaete chrysosporium has been hampered by the necessity to produce them in stationary culture. This investigation examined the effects of detergents on development of ligninase activity in agitated submerged cultures. Results show that addition of Tween 80, Tween 20, or 3-[(3-colamidopropyl)dimethylammonio]1-propanesulfonate to the cultures permits development of ligninase activity comparable to that routinely obtained in stationary cultures. The detergent-amended cultures express the entire ligninolytic system, assayed as the complete oxidation of [¹⁴C]lignin to ¹⁴CO₂. The detergent effect is evidently not merely in facilitating release of extant enzyme. Development of ligninolytic activity in the agitated cultures, as in stationary cultures, is idiophasic. Ion-exchange fast protein-liquid chromatography indicated that the heme protein profiles in agitated and stationary cultures are very similar. These findings should make it possible to scale up production of ligninolytic enzymes in stirred tank fermentors.     

Lignin-carbohydrate complex formed in isolated cell walls of callus

Cell walls of Pinus elliottii tissue cultures were isolated and incubated with coniferyl alcohol and H₂O₂. Lignin having physical and chemical properties similar to that prepared from wood was formed by the peroxidase attached to the walls. Fractions of the callus lignin isolated enzymatically or chemically contained bound carbohydrate. The lignin was also strongly bound to a protein containing hydroxyproline, probably extensin. This system may be analogous to the earliest stage of normal lignin formation in which monomers are transported from the protoplast into the primary wall and middle lamella, where peroxidase polymerizes monomers and catalyzes bonds to carbohydrate and protein.     

Factors affecting lignin degradation in lignocellulose by Phanerochaete chrysosporium

Cultural conditions affecting lignin degradation by Phanerochaete chrysosporium in various lignocellulosic materials were studied in comparison to an isolated lignin preparation. With shallow mycelial cultures, the degradation of lignin in wood proceeded more slowly in a 100% O₂-atmosphere than in an air atmosphere, indicating that pure oxygen was toxic to the fungus. The organism was able to degrade lignin efficiently even under 30% CO₂ and 10% O₂ concentrations. Evolution of ¹⁴CO₂ from labelled lignocellulosic materials was shown not to be representative of total lignin degradation. Addition of glucose to the culture did not affect lignin degradation measured by ¹⁴CO₂ evolution, whereas lignin degradation measured by Klason lignin method stopped completely (poplar) or slowed considerably (straw). Due to partial depolymerization of lignin to soluble products, measuring only the evolution of ¹⁴CO₂ results in an underestimation of the total amount of lignin bioaltered. The soluble products from all of the tested lignocellulosic materials and from the isolated lignin had an average molecular weight of about 1,000 and the products could be further fractionated by ion exchange chromatography. The relative amount of these products could be varied from 15 to 45% from the original lignin.     

The ligninolytic activities of Lentinus edodes and Phanerochaete chrysosporium

Summary Two important lignin-degrading fungi with existing or potential applications in the production of food, feed and/or fiber products from wood are Lentinus edodes (Berk.; Sing.=Lentinula edodes [Pegler]) and Phanerochaete chrysosporium (Burds). This study discusses their relative ability to degrade lignin and the factors controlling their ligninolytic activity (synthetic ¹⁴C-lignin¹⁴CO₂). Ligninolytic activity in P. chrysosporium is known to develop after the fungus ceases vegetative growth, and to require both O₂ and an exogenous carbon source such as glucose. It has an extracellular ligninase in high titer which is assayed by the oxidation of veratryl alcohol to veratraldehyde. Here, P. chrysosporium was found to have a high capacity for lignin degradation (it was not easily saturated with lignin). Certain inorganic elements, including Fe²⁺, Ca²⁺ and Mo⁶⁺, were found to stimulate its ligninolytic activity. Calcium addition was required, with 40 ppm Ca²⁺ giving the highest activity. As in P. chrysosporium, ligninolytic activity in L. edodes was found to require both O₂ and an exogenous carbon source. However, in contrast to P. chrysosporium, L. edodes was only moderately ligninolytic, had a lower capacity for lignin degradation (was more easily saturated with lignin), and showed maximal activity only during the vegetative growth period. Also in contrast to P. chrysosporium, ligninolytic activity in L. edodes was not stimulated by Ca²⁺. Instead, manganese was required, with 10 ppm Mn²⁺ giving optimal activity. An extracellular ligninase capable of oxidizing veratryl alcohol to veratraldehyde was not detected in L. edodes.     

Pretreatment of hybrid poplar by aqueous ammonia

Enzymatic hydrolysis of hybrid poplar treated by ammonia recycle percolation (ARP) was studied applying cellulase enzyme supplemented with additional xylanase or pectinase. The effect of xylanase addition was much more significant than pectinase addition. Conversion of ARP‐treated hybrid poplar to ethanol was carried out by simultaneous saccharification and fermentation (SSF) and SS and cofermentation (SSCF). The maximum ethanol yield observed from the SSCF experiment was 78% of theoretical maximum based on the total carbohydrate (glucan + xylan). The same feedstock was also treated by soaking in aqueous ammonia (SAA), a batch pretreatment process with lower severity than ARP. The test results indicated that relatively high severity is required to attain acceptable level of digestibility of hybrid poplar. In order to lower the severity of the pretreatment, addition of H₂O₂ was attempted in the SAA. Addition of H₂O₂ significantly enhanced delignification of hybrid poplar due to its oxidative degradation of lignin. Several different H₂O₂ feeding schemes and different temperature profiles were attempted in operation of the SAA to investigate the effects of H₂O₂ on degradation of lignin and carbohydrates in hybrid poplar. More than 60% of lignin in hybrid poplar was removed with stepwise‐increase of temperature (60–120°C after 4h of reaction). Increase of carbohydrate degradation was also observed under this condition. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

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.     

Improvement in the production of extracellular lignin peroxidases by Phanerochaete chrysosporium: effect of solid manganese(IV)oxide

Summary Addition of solid manganese(IV)oxide to cultures of Phanerochaete chrysosporium at the beginning of ligninolytic activity was shown to improve production, enzymatic activity, and stability of the ligninases produced. Darkening of mycelia incubated with shaking and N-limitation coincides with the onset of ligninolytic activity and is due to the deposition of amorphous MnO₂. By the addition of MnO₂, probably mimicking the naturally occuring deposition of MnO₂ on the mycelia of some white rot fungi, it was intended to protect ligninases against inactivation and damage by hydrogen peroxide via catalytic decomposition of H₂O₂ by MnO₂. Comparative analyses of protein fractions and of purified single proteins from both –MnO₂ and +MnO₂ cultures confirmed that the addition of MnO₂ to cultures leads to a quantitatively different pattern of ligninase isoenzymes. This was paralleled with a higher specific enzymatic activity toward several substrates of some proteins from +MnO₂ cultures. From pulse labelling experiments with [¹⁴C]amino acids it was concluded that the different protein pattern in both cultures may be post-translational. Following the time course of the protein pattern by repeated incubations, a clearcut difference in the build-up of haem proteins in both cultures was demonstrated. Ligninolytic activity of +MnO₂ and –MnO₂ cultures was measured using a ¹⁴C-labelled synthetic lignin, but no significant differences were found.Offprint requests to: H. W. Kern     

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 H₂¹⁸O and ¹⁸O₂, 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 O₂.