Biodegradation mechanism of lignin by white-rot basidiomycetes

Journal of Biotechnology, 30 (1993) 1-8 © 1993 Elsevier Science Publishers B.V. All rights reserved 0168-1656/93/$06.00

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BIOTEC 00897

Biodegradation mechanism of lignin by white-rot basidiomycetes Takayoshi Higuchi Wood Research Institute, Kyoto University, Uji, Kyoto, Japan (Received April 1981; revision accepted 26 January 1992)

Summary White-rot basidiomycetes such as Coriolus versicolor, Phanerochaete chrysosporium and Phlebia radiata have been found as typical lignin degrader. During the past 15 years chemistry and biochemistry of lignin biodegradation by white-rot basidiomycetes have considerably advanced by means of chemical analysis of the biodegraded lignin and studies of the degradation mechanism of lignin substructure model compounds by ligninolytic cultures of these basidiomycetes and their enzymes. Excellent reviews (Kirk and Farrell, 1987; Schoemaker, 1990; Umezawa and Higuchi, 1991) concerning microbial and enzymatic degradation of lignin have recently appeared. We (Higuchi and Nakatsubo, 1980; Higuchi, 1990) synthesized major lignin substructure model compounds linked by /3-0-4, /3-5, /3-1, /3-/3' and 5-5' bonds and used as substrates for cultures of P. chrysosporium and C. versicolor and their enzymes, lignin peroxidase and laccase. The degradation products were extracted with ethyl acetate and identified by NMR and GC-MS to elucidate degradation pathways of the lignin substructure model compounds. The results are classified to the cleavage of side chain and aromatic ring opening of the compounds.

White-rot basidiomycetes; Lignin peroxidase; Laccase; Lignin biodegradation; Aromatic ring opening; Phanerochaete chrysosporium

Correspondence to: T. Higuchi, Wood Research Institute, Kyoto University, Uji, Kyoto 611, Japan.

Cleavage of propyl side chains of/3-1 and ~ - 0 - 4 compounds Kirk and Nakatsubo (1983) showed that a deuterated non-phenolic diarylpropane-l,3-diol, one of the major lignin substructure compound, was degraded by a ligninolytic culture of Phanerochaete chrysosporium to give phenylglycol, a-hydroxyacetophenone and benzaldehyde with retention of hydrogen atoms at C,~ and Ct3. They also showed by the ligninolytic culture experiment with tSo that the benzyl hydroxyl oxygen atom of the phenylglycol was derived from molecular oxygen. Subsequently Tien and Kirk (1983) and Glenn et al. (1983) first discovered the enzyme lignin peroxidase which catalyzes C~-C¢ cleavage of propyl side chain of/3-1 compounds in agreement with in vivo experiment. Hence, Habe et al. (1985) synthesized deuterated 1,2-diarylpropane-l,3-diols as substrate for experiments with lignin peroxidase of P. chrysosporium, and found the formation of the previously identified products with retention of the deuterium at C~ and Ct3 of the side chain. The result indicated that hydrogen abstraction was not involved in the C~-Ct~ bond cleavage as illustrated in Fig. 1. We (Yokota et al., 1990; Kawai et al., 1988a) further found that phenolic /3-1 compounds were degraded by lignin peroxidase of P. chrysosporium and laccase of C. versicolor to give C~-C¢ cleavage products in addition to alkyl-phenyl cleavage products and C~ carbonyl compounds which are known to occur commonly by the mediation of laccase. Degradation of arylglycerol-/3-aryl ether (/3-0-4) lignin substructure which is the most frequent interphenylpropane linkage (40-60% in lignin) has been studied

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Fig. 1. Degradation pathways of deuterated /3-1 models (1, 1') by lignin peroxidase of P. chrysosporium. D, deuterium. 2, 4-methoxyphenylglycol; 3, 4-methoxybenzaldehyde; 4, 1-(4-methoxyphenyl)-2-hydroxyethanone; 5, 1-(4-methoxyphenyl)-2-(4-methoxyphenyl)propane-l-one-3-ol.

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Fig. 2. Degradation of a,/3-dideuterated arylglycerol-/3-aryl ether model by lignin peroxidase of P.

chrysosporium. 6, 4-ethoxy-3-methoxyphenylglycerol-/3-vanillin-3,-benzyldiether; 7, 4-ethoxy-3-methoxyphenylglycerol-3,-benzyl ether; 8, 4-ethoxy-3-methoxybenzaldehyde; 9, benzyloxyacetaldehyde; 10, vanillin.

for many years by several investigators. Tien and Kirk (1983), and Glenn et al. (1983), showed that lignin peroxidase mediates C,-C~ cleavage o f / 3 - 0 - 4 lignin substructure model compounds as well as C,-C~ cleavage of /3-1 model compounds mentioned above. To elucidate the degradation mechanism we prepared a,/3-dideuterated 4ethoxy-3-methoxyphenylglycerol-/3-vanillin--/-benzyldiether as substrate and used for lignin peroxidase experiments. Our results showed that the substrate was converted by lignin peroxidase to 4-ethoxy-3-methoxybenzaldehyde and benzyloxyacetaldehyde, and vanillin by C~-Ct~ cleavage followed by O-C~ cleavage, and 7-benzyl ether of 4-ethoxy-3-methoxyphenylglycerol by O-C4 cleavage, respectively. The results also showed that deuterium at C~ and C~ of the 7-benzyl ether of 4-ethoxy-3-methoxyphenylglycerol, of 4-ethoxy-3-methoxybenzaldehyde and of benzyloxyacetaldehyde were almost quantitatively retained after the C,-Ct~ and O-C4 bond cleavages. These results are in agreement with reaction mechanism of one electron oxidation of aromatic rings of non-phenolic aromatic compounds by lignin peroxidase to give unstable cation radicals which undergo spontaneous transformation as illustrated in Fig. 2. The C,-Ct3 bond of the propyl side chain is homolytically cleaved via an aryl cation radical of the substrate to give benzyl cation, and guaiacoxyethyl radical which is attacked by dioxygen to form an unstable hemiketal. The O-C~ bond of the hemiketal is subsequently cleaved to give vanillin and benzaldehyde. We (Umezawa and Higuchi, 1985a) further found alternative C~-C~ cleavage reaction of / 3 - 0 - 4 model compounds to give 2-guaiacoxyethanol and benzyl alcohol by ligninolytic culture of P. chrysosporium. Isotopic investigations using arylglycerol-/3-[180]guaiacyl ether and arylglcerol [y13C]guaiacyl ether as substrate

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Fig. 3. Mechanism of guaiacoxyethanol formation from a / 3 - 0 - 4 lignin substructure model by ligninolytic culture of P. chrysosporium. 11, 4-ethoxy-3-methoxybenzyl alcohol; 12, 2-guaiacoxyethanol.

showed that the guaiacyl group at the/3-position of the substrate is rearranged to the adjacent y-position, and that the rearranged intermediate is cleaved between C a and C~ to give guaiacoxyethanol as illustrated in Fig. 3. For degradation of p h e n o l i c / 3 - 0 - 4 compounds by laccase of C. versicolor we found that syringylglycerol-/3-guaiacyl ether was mainly converted to the a-carbonyl dimer, 2,6-dimethoxyhydroquinone, and glyceraldehyde-2-guaiacyl ether by alkylphenyl cleavage, and to guaiacol by O-C# cleavage. We further found that the a-carbonyl dimer was cleaved between C a and C~ to give syringic acid and guaiacol. However, syringic acid formed was rapidly decarboxylated to give 2,6-dimethoxyhydroquinone and not accumulated. Very recently we found that lignin peroxidase of P. chrysosporium also mediates the degradation of p h e n o l i c / 3 - 0 - 4 compounds in a similar way to that by laccase.

Aromatic ring opening of lignin model compounds We (Umezawa and Higuchi, 1985b) identified for the first time an aromatic ring opening product of a / 3 - 0 - 4 lignin substructure model compound by ligninolytic cultures of P. chrysosporium; the/3,y-cyclic carbonate. Subsequently we (Umezawa et al., 1986a; Umezawa and Higuchi, 1987a) identified several esters of arylglycerol as products of aromatic ring opening of / 3 - 0 - 4 model dimers by the fungus; a,/3-cyclic carbonate, y-formate, methyl oxalate, and cis, cis-muconate. Our tracer experiments (Umezawa et al., 1986b) using 1,3-dihydroxy-l-(4-ethoxy-3-methoxyphenyl)-2-[U-ring-13C](2-methoxyphenoxy)propane and 1,3-dihydroxy-l-(4ethoxy-3-methoxyphenyl)-2-[U-ring-13C](2,6-dimethoxyphenyl)propane as substrates confirmed that the esters of arylglycerol were all labeled with 13C and ring opening products as shown in Fig. 4. The aromatic ring opening products were also identified as lignin peroxidase degradation products o f / 3 - 0 - 4 model compounds.

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Fig. 4. Formation of aromatic ring opening products from arylglycerol-/3-aryl-U-I3C,OCD3 ethers by lignin peroxidase of P. chrysosporium. 13,/3,y-cyclic carbonate; 14, y-formate; 15,/3-methyl oxalate.

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(13) Fig. 5. Mechanisms of aromatic ring opening of f l - O - 4 lignin substructure models by ]ignin peroxidase of e. chrysosporium. 16, cis, ci3 mueonate.

GC-MS analyses of the ring opening products (Umezawa and Higuchi, 1987b) formed under H2180 or 180 2 showed that one of the carbonyl oxygen atom of mucanote, and of the methyl oxalate is derived from H 2 0 and the other from 0 2. The carbonyl oxygen atoms of the cyclic carbonates and formate were derived from H2 O. Furthermore, mass spectrometric analyses (Umezawa and Higuchi, 1986) of the degradation products of 1,3-dihydroxy-l-(4-ethoxy-3-methoxyphenyl)-2-(2[OCeH3]-methoxyphenoxy) propane by the enzyme showed that the methyl group of the methyl ester of the oxalate was derived from the methoxyl group of the B ring of the substrate. Thus the results clearly indicated that demethoxylation of the substrate is not prerequest for ring opening, and that the ring opening by the enzyme is entirely different from ring opening catalyzed by conventional oxygenases. Based on these experimental results we (Umezawa and Higuchi, 1987b) proposed a general mechanism for aromatic ring opening of / 3 - 0 - 4 lignin substructure model compounds by lignin peroxidase (Fig. 5). One electron oxidation of the B-ring forms the corresponding cation radicals which are attacked nucleophilically: The remaining radical is attacked by dioxygen or radical species derived from dioxygen. These ring opening products of / 3 - 0 - 4 model compounds were also formed by C. c,ersicolor and C. hirsutus (Yoshihara et al., 1988) which produce lignin peroxidase. Recently we (Kawai et al., 1988b) found that 4,6-di-t-butylguaiacol is converted to a ring opening product, the muconolactone derivative by laccase of C. cersicolor. Our experiment showed that 180 from 180 2 but not from H2180 is incorporated into the muconolactone. We, therefore, proposed the pathway A for ring opening of 4,6-di-t-butylguaiacol by laccase (Fig. 6). All these studies on the side chain cleavage and ring opening of lignin model compounds indicated that both lignin peroxidase and laccase, which catalyzes one electron oxidation of either phenolic or non-phenolic compounds, are involved in the initial degradation of lignin substructure model compounds.

.k~OCH 3 OH pathway A laccase (17} A A J o~/t.J~ ~OCH3~.J~ ]-OCH3 r +

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pathway B Fig. 6. Mechanism for degradationof 4,6-di-t-butylguaiaeolby laccaseof butylguaiacol; 18, muconolactone derivative.

C. uersicolor.

17, 4,6-di-t-

Very recently we (Hattori and Higuchi, 1991) found that non-phenolic syringyl and biphenyl compounds were degraded by lignin peroxidase to give aromatic ring opening products but the corresponding phenolic compounds did not give the ring opening products. The results indicated that lignin peroxidase preferentially catalyzes the opening of non-phenolic rings but not phenolic rings.

Degradation of lignin by lignin peroxidase Finally a DHP (MW 2200) prepared from (/3-0-4)(/3-/3') lignin substructure trimer and coniferyl alcohol was subjected to lignin peroxidase oxidation (Umezawa and Higuchi, 1989). The degradation products were extracted and acetylated. GC-MS analysis of the acetate showed the cyclic carbonates, formate, 4-ethoxy-3methoxybenzaldehyde, arylglycerol, and a-ketoarylglycerol as products. The results indicate that lignin peroxidase catalyzes the cleavage of C~-C~ bond, O-C4 bond and aromatic ring opening of synthetic lignin (DHP), which confirms the involvement of lignin peroxidase in the initial degradation of lignin.

Role of veratryl alcohol in the degradation of lignin by lignin peroxidase Harvey et al. (1986) suggested that veratryl alcohol acts as a radical mediater in oxidation of anisyl alcohol by lignin peroxidase. We (Hattori and Higuchi, 1991) recently found that the enzymatic degradation of/3-1 compound is accelerated by the addition of veratryl alcohol. To elucidate

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Fig. 7. Lineweaver-Burk plots for lignin peroxidase activities for veratryl alcohol oxidation in the presence of different concentrations (0-0.5 mM) of 1,2-bis(4-methoxyphenyl)propane-l,3-diol. ©, 0 mM; za, 0.2 mM; D, 0.5 mM. The reaction mixture contained li~nin peroxidase (1.7 nkat) and H202 (0.5 mM).

the role of veratryl alcohol in the degradation of /3-1 compound by lignin peroxidase Lineweaver-Burk plots were prepared for the enzyme activities in various concentrations of /3-1 compound as shown in Fig. 7. The result showed that the inhibition pattern observed for the oxidation of veratryl alcohol by/3-1 compound is neither a competitive nor a non-competitive, but a mixed type of the two. The results suggest that veratryl alcohol acts in two ways, as a protector against inactivation of lignin peroxidase and a radical mediator. /3-1 and veratryl alcohol seem to compete for the same binding site of lignin peroxidase. Our experiment also suggested that the cation radical of veratryl alcohol mediates possibly one electron oxidation o f / 3 - 1 compound.

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