Role of guaiacol in the degradation of arylglycerol-β-guaiacyl ether by Phanerochaete chrysosporium

Abstract It was found that guaiacol and 4-ethoxy-3-methoxy-phenylglycerol were produced via different pathways and that they were not counterpart compounds to each other in the cleavage of the β - O -4 bond of 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether by a white-rot fungus, Phanerochaete chrysosporium .     

Transformations of arylpropane lignin model compounds by a lignin peroxidase of the white-rot fungus Phanerochaete chrysosporium

Various lignin model compounds of the O-arylpropane type were oxidized with purified lignin peroxidase from the white-rot fungus Phanerochaete chrysosporium, and oxidation products were identified by gas-chromatography/mass-spectroscopy procedures. Our results are in accord with the theory that lignin peroxidase catalyzes one-electron oxidations of its substrates with formation of cation radicals, and that these radicals undergo degradative reactions that are predictable from a knowledge of cation radical and oxygen chemistry. Cation radicals formed from O-arylpropane model compounds appeared to undergo the following types of degradative transformations: addition of water to ring-centered radicals, followed by proton loss yielding quinones and alcohols; nucleophilic attack by hydroxy functions on propanoid moieties giving cyclic ketals as intermediates which decompose to yield side chain migration products; transfer of the charge of a radical from a ring to the associated alkyl moiety through an ether bond, with loss of a proton from the latter, forming a new carbon-centered radical. The new alkyl-centered radicals apparently were able to abduct dioxygen to form peroxyl radicals which decomposed giving a variety of oxidation products and probably superoxide anion. Specific examples of the above transformations are presented, and their relevance to lignin degradation is discussed.     

Removal of hazardous compounds by lignin peroxidase from Phanerochaete chrysosporium

Phenolic compounds, which are present in many industrial wastewaters, have become a cause for worldwide concern due to their persistence, toxicity and health risks. Enzymatic approaches to remove phenol have been tried for some years as they have several advantages compared with the conventional methods. This paper reports some studies on the use of the white rot fungus Phanerochaete chrysosporium which produces the enzyme lignin peroxidases for the removal of phenol, chlorophenol, and dyes. Batch studies in Erylenmeyer flasks showed complete removal of phenol (500 2 10^х kg/m³) in 30 h. It was also seen that phenol has a significant inhibitory effect on the biomass growth and the enzyme synthesis if added in the early stages of the growth. However, phenol was effectively removed when added after attaining the maximum enzyme activity. 90% of the dyes were removed in about three days, whereas only 62% of the added 4-chlorophenol was removed in about ten days.     

Biochemistry of the oxidation of lignin by Phanerochaete chrysosporium

The objective of this research was to identify the biochemical agents responsible for the oxidative degradation of lignin by the white-rot fungus Phanerochaete chrysosporium. 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 (H(2)O(2)) and possibly superoxide (O(2)(*)(-)) in lignin degradation. Involvement of hydroxyl radical (*OH) or singlet oxygen (1O(2)) 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 H(2)O(2) for activity.     

Degradation of synthetic lignin by the protoplasts of Phanerochaete chrysosporium in the presence of lignin peroxidase or manganese peroxidase

Lignin was mineralized in the experiments in which ¹⁴C-lignin was incubated with lignin peroxidase or manganese peroxidase in a tartrate buffer in the presence of cycloheximide-treated protoplasts obtained from the ligninolytic mycelia of Phanerochaete chrysosporium. The rate of lignin mineralization was dependent on the lignin peroxidase or manganese peroxidase concentration in the medium. In the experiments in which lignin was incubated with lignin peroxidase or manganese peroxidase, lignin was repolymerized irrespective of the presence of protoplasts mineralizing lignin, suggesting that an active degradation of lignin and repolymerization took place. Taking into account that lignin peroxidase and manganese peroxidase were the only extracellular enzymes in the experiments in which lignin was mineralized by the protoplasts, it is postulated that lignin peroxidase and/or manganese peroxidase can degrade lignin into small fragments which can then be further absorbed by the fungal cells and subsequently degraded to CO₂.     

Influence of lignin peroxidase concentration and localisation in lignin biodegradation by Phanerochaete chrysosporium

Summary The lignin mineralization rate in cultures of Phanerochaete chrysosporium increases with lignin peroxidase concentration up to 20 nkat ml^–1. At higher concentrations the rate of lignin mineralization decreases with increasing lignin peroxidase concentration. The amount of mycelium is not a limiting factor for lignin mineralization at high exocellular lignin peroxidase in association with the mycelium as pellets and no free exocellular enzyme induce a lignin mineralization rate equivalent to cultures reconstituted with washed pellets supplemented with 15 nkat ml^–1 of exogenous free enzyme. These results show that although lignin degradation by lignin peroxidase seems to be facilitated when lignin peroxidase is localised on the surface of the mycelium, free exocellular lignin peroxidase can also efficiently enhance mineralization of lignin by P. chrysosporium.     

Nutritional Regulation of Lignin Degradation by Phanerochaete chrysosporium

Previous studies have shown that a lignin-degrading system appears in cultures of the white rot fungus Phanerochaete chrysosporium in response to nitrogen starvation, apparently as part of secondary metabolism. We examined the influence of limiting carbohydrate, sulfur, or phosphorus and the effect of varying the concentrations of four trace metals, Ca, and Mg. Limitation of carbohydrate or sulfur but not limitation of phosphorus triggered ligninolytic activity. When only carbohydrate was limiting, supplementary carbohydrate caused a transient repression of activity. In carbohydrate-limited cultures, ligninolytic activity appeared when the supplied carbohydrate was depleted, and this activity was associated with a decrease in mycelial dry weight. The amount of lignin degraded depended on the amount of carbohydrate provided, which determined the amount of mycelium produced during primary growth. Carbohydrate-limited cultures synthesized only small amounts of the secondary metabolite veratryl alcohol compared with nitrogen-limited cultures. l-Glutamate sharply repressed ligninolytic activity in carbohydrate-starved cultures, but NH(4) did not. Ligninolytic activity was also triggered in cultures supplied with 37 muM sulfur as the only limiting nutrient. The balance of trace metals, Mg, and Ca was important for lignin degradation.     

Wood stimulates the demethoxylation of [O14CH3]-labeled lignin model compounds by the white-rot fungi Phanerochaete chrysosporium and Phlebia radiata

Mineralization of polymeric wood lignin and its substructures is a result of complex reactions involving oxidizing and reducing enzymes and radicals. The degradation of methoxyl groups is an essential part of this process. The presence of wood greatly stimulates the demethoxylation of a non-phenolic lignin model compound (a [O¹⁴CH₃]-labeled β-O-4 dimer) by the lignin-degrading white-rot fungi Phlebia radiata and Phanerochaete chrysosporium. When grown on wood, both fungi produced up to 47 and 40% ¹⁴CO₂ of the applied ¹⁴C activity, respectively, under air and oxygen in 8 weeks. Without wood, the demethoxylation of the dimer by both fungi was lower, varying between 0.5 and 35%. Addition of nutrient nitrogen together with glucose decreased demethoxylation when the fungi were grown on spruce wood under air. Because the evolution of ¹⁴CO₂ in the absence of wood was poor, the fungi may have preferably used wood as a carbon and nitrogen source. The amount of fungal mycelium, as determined by the ergosterol assay, did not show connection to demethoxylation. P. radiata also showed a high demethoxylation of [O¹⁴CH₃]-labeled vanillic acid in the presence of birch wood. The degradation of lignin and lignin-related substances should be studied in the presence of wood, the natural substrate for white-rot fungi.     

Degradation of azo compounds by ligninase from Phanerochaete chrysosporium: involvement of veratryl alcohol

Phanerochaete chrysosporium decolorized several polyaromatic azo dyes in ligninolytic culture. The oxidation rates of individual dyes depended on their structures. Veratryl alcohol stimulated azo dye oxidation by pure lignin peroxidase (ligninase, LiP) in vitro. Accumulation of compound II of lignin peroxidase, an oxidized form of the enzyme, was observed after short incubations with these azo substrates. When veratryl alcohol was also present, only the native form of lignin peroxidase was observed. Azo dyes acted as inhibitors of veratryl alcohol oxidation. After an azo dye had been degraded, the oxidation rates of veratryl alcohol recovered, confirming that these two compounds competed for ligninase during the catalytic cycle. Veratryl alcohol acts as a third substrate (with H2O2 and the azo dye) in the lignin peroxidase cycle during oxidations of azo dyes.     

黄孢原毛皮革菌降解苯胺的工艺研究

通过正交试验优化筛选了适合黄孢原毛皮革菌降解苯胺的适宜培养基和摇瓶培养降解条件。结果表明：其适宜降解的液体培养基组成为：蔗糖20g／L,可溶性淀粉20g／L,(NH4)2SO4l0g／L,Mn^2 lμmol／L,Tween-800．3％,蛋白胨30g／L。适宜降解的摇瓶培养条件为：接种量为20％、pH为7．0、温度为30℃、培养时间为12d．此条件下的苯胺最高降解率可达95．5％。     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Degradation of 2,4-dinitrotoluene by the lignin-degrading fungus Phanerochaete chrysosporium.

Under ligninolytic conditions, the white rot basidiomycete Phanerochaete chrysosporium mineralizes 2,4-dinitrotoluene (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell extracts. The multistep pathway involves the initial reduction of I to yield 2-amino-4-nitrotoluene (II). II is oxidized by MnP to yield 4-nitro-1,2-benzoquinone (XII) and methanol. XII is then reduced to 4-nitro-1,2-hydroquinone (V), and the latter is methylated to 1,2-dimethoxy-4-nitrobenzene (X). 4-Nitro-1,2-hydroquinone (V) is also oxidized by MnP to yield nitrite and 2-hydroxybenzoquinone, which is reduced to form 1,2,4-trihydroxybenzene (VII). 1,2-Dimethoxy-4-nitrobenzene (X) is oxidized by LiP to yield nitrite, methanol, and 2-methoxy-1,4-benzoquinone (VI), which is reduced to form 2-methoxy-1,4-hydroquinone (IX). The latter is oxidized by LiP and MnP to 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (VII). The key intermediate 1,2,4-trihydroxybenzene is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial reduction of a nitroaromatic group generates the peroxidase substrate II. Oxidation of II releases methanol and generates 4-nitro-1,2-benzoquinone (XII), which is recycled by reduction and methylation reactions to regenerate intermediates which are in turn substrates for peroxidase-catalyzed oxidation leading to removal of the second nitro group. Thus, this unique pathway apparently results in the removal of both aromatic nitro groups before ring cleavage takes place.     

A novel enzymatic decarboxylation of oxalic acid by the lignin peroxidase system of white-rot fungus Phanerochaete chrysosporium

Oxidation of veratryl alcohol by lignin peroxidase (LiP) was potently inhibited by oxalic acid. The inhibition analysis with Lineweaver-Burk plots clearly showed that the type of inhibition is non-competitive. The enzymatic oxidation of veratryl alcohol in the presence of 14C-oxalic acid yielded radioactive carbon dioxide. The results indicate that the apparent inhibition of LiP is caused by reduction of the veratryl alcohol cation radical intermediate back to the substrate level by oxalate, which is concomitantly oxidized to carbon dioxide.     

Degradation of 2,7-dichlorodibenzo-p-dioxin by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions, the white-rot basidiomycete Phanerochaete chrysosporium degraded 2,7-dichlorodibenzo-p-dioxin (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell-free extracts. The multistep pathway involves the degradation of I and subsequent intermediates by oxidation, reduction, and methylation reactions to yield the key intermediate 1,2,4-trihydroxybenzene (III). In the first step, the oxidative cleavage of the dioxin ring of I, catalyzed by LiP, generates 4-chloro-1,2-benzoquinone (V), 2-hydroxy-1,4-benzoquinone (VIII), and chloride. The intermediate V is then reduced to 1-chloro-3,4-dihydroxybenzene (II), and the latter is methylated to form 1-chloro-3,4-dimethoxybenzene (VI). VI in turn is oxidized by LiP to generate chloride and 2-methoxy-1,4-benzoquinone (VII), which is reduced to 2-methoxy-1,4-dihydroxybenzene (IV). IV is oxidized by either LiP or MnP to generate 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (III). The other aromatic product generated by the initial LiP-catalyzed cleavage of I is 2-hydroxy-1,4-benzoquinone (VIII). This intermediate is also generated during the LiP- or MnP-catalyzed oxidation of the intermediate chlorocatechol (II). VIII is also reduced to 1,2,4-trihydroxybenzene (III). The key intermediate III is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial oxidative cleavage of both C-O-C bonds in I by LiP generates two quinone products, 4-chloro-1,2-benzoquinone (V) and 2-hydroxy-1,4-benzoquinone (VIII). The former is recycled by reduction and methylation reactions to generate an intermediate which is also a substrate for peroxidase-catalyzed oxidation, leading to the removal of a second chlorine atom. This unique pathway results in the removal of both aromatic chlorines before aromatic ring cleavage takes place.     

Degradation of azo dyes by the lignin-degrading fungus Phanerochaete chrysosporium.

Under nitrogen-limiting, secondary metabolic conditions, the white rot basidiomycete Phanerochaete chrysosporium extensively mineralized the specifically 14C-ring-labeled azo dyes 4-phenylazophenol, 4-phenylazo-2-methoxyphenol, Disperse Yellow 3 [2-(4'-acetamidophenylazo)-4-methylphenol], 4-phenylazoaniline, N,N-dimethyl-4-phenylazoaniline, Disperse Orange 3 [4-(4'-nitrophenylazo)-aniline], and Solvent Yellow 14 (1-phenylazo-2-naphthol). Twelve days after addition to cultures, the dyes had been mineralized 23.1 to 48.1%. Aromatic rings with substituents such as hydroxyl, amino, acetamido, or nitro functions were mineralized to a greater extent than unsubstituted rings. Most of the dyes were degraded extensively only under nitrogen-limiting, ligninolytic conditions. However, 4-phenylazo-[U-14C]phenol and 4-phenylazo-[U-14C]2-methoxyphenol were mineralized to a lesser extent under nitrogen-sufficient, nonligninolytic conditions as well. These results suggest that P. chrysosporium has potential applications for the cleanup of textile mill effluents and for the bioremediation of dye-contaminated soil.     

Degradation of 2,4,5-trichlorophenol by the lignin-degrading basidiomycete Phanerochaete chrysosporium.

Under secondary metabolic conditions the white rot basidiomycete Phanerochaete chrysosporium rapidly mineralizes 2,4,5-trichlorophenol. The pathway for degradation of 2,4,5-trichlorophenol was elucidated by the characterization of fungal metabolites and oxidation products generated by purified lignin peroxidase (LiP) and manganese peroxidase (MnP). The multistep pathway involves cycles of peroxidase-catalyzed oxidative dechlorination reactions followed by quinone reduction reactions to yield the key intermediate 1,2,4,5-tetrahydroxybenzene, which is presumably ring cleaved. In the first step of the pathway, 2,4,5-trichlorophenol is oxidized to 2,5-dichloro-1,4-benzoquinone by either MnP or Lip. 2,5-Dichloro-1,4-benzoquinone is then reduced to 2,5-dichloro-1,4-hydroquinone. The 2,5-dichloro-1,4-hydroquinone is oxidized by MnP to generate 5-chloro-4-hydroxy-1,2-benzoquinone. The orthoquinone is in turn reduced to 5-chloro-1,2,4-trihydroxybenzene. Finally, the 5-chlorotrihydroxybenzene undergoes another cycle of oxidative dechlorination and reduction reactions to generate 1,2,4,5-tetrahydroxybenzene. The latter is presumably ring cleaved, with subsequent degradation to CO2. In this pathway, the substrate is oxidatively dechlorinated by LiP or MnP in a reaction which produces a quinone. The quinone intermediate is recycled by a reduction reaction to regenerate an intermediate which is again a substrate for peroxidase-catalyzed oxidative dechlorination. This pathway apparently results in the removal of all three chlorine atoms before ring cleavage occurs.     

Degradation of 2,4-dinitrotoluene by the lignin-degrading fungus Phanerochaete chrysosporium.

Under ligninolytic conditions, the white rot basidiomycete Phanerochaete chrysosporium mineralizes 2,4-dinitrotoluene (I). The pathway for the degradation of I was elucidated by the characterization of fungal metabolites and oxidation products generated by lignin peroxidase (LiP), manganese peroxidase (MnP), and crude intracellular cell extracts. The multistep pathway involves the initial reduction of I to yield 2-amino-4-nitrotoluene (II). II is oxidized by MnP to yield 4-nitro-1,2-benzoquinone (XII) and methanol. XII is then reduced to 4-nitro-1,2-hydroquinone (V), and the latter is methylated to 1,2-dimethoxy-4-nitrobenzene (X). 4-Nitro-1,2-hydroquinone (V) is also oxidized by MnP to yield nitrite and 2-hydroxybenzoquinone, which is reduced to form 1,2,4-trihydroxybenzene (VII). 1,2-Dimethoxy-4-nitrobenzene (X) is oxidized by LiP to yield nitrite, methanol, and 2-methoxy-1,4-benzoquinone (VI), which is reduced to form 2-methoxy-1,4-hydroquinone (IX). The latter is oxidized by LiP and MnP to 4-hydroxy-1,2-benzoquinone, which is reduced to 1,2,4-trihydroxybenzene (VII). The key intermediate 1,2,4-trihydroxybenzene is ring cleaved by intracellular cell extracts to produce, after reduction, beta-ketoadipic acid. In this pathway, initial reduction of a nitroaromatic group generates the peroxidase substrate II. Oxidation of II releases methanol and generates 4-nitro-1,2-benzoquinone (XII), which is recycled by reduction and methylation reactions to regenerate intermediates which are in turn substrates for peroxidase-catalyzed oxidation leading to removal of the second nitro group. Thus, this unique pathway apparently results in the removal of both aromatic nitro groups before ring cleavage takes place.     

Cleavage of nonphenolic beta-1 diarylpropane lignin model dimers by manganese peroxidase from Phanerochaete chrysosporium.

Purified manganese peroxidase (MnP) from Phanerochaete chrysosporium oxidizes nonphenolic beta-1 diarylpropane lignin model compounds in the presence of Tween 80, and in three- to fourfold lower yield in its absence. In the presence of Tween 80, 1-(3',4'-diethoxyphenyl)-1-hydroxy-2-(4'-methoxyphenyl)propane (I) was oxidized to 3,4-diethoxybenzaldehyde (II), 4-methoxyacetophenone (III) and 1-(3',4'-diethoxyphenyl)-1-oxo-2-(4'-methoxyphenyl)propane (IV), while only 3,4-diethoxybenzaldehyde (II) and 4-methoxyacetophenone (III) were detected when the reaction was conducted in the absence of Tween 80. In contrast to the oxidation of this substrate by lignin peroxidase (LiP), oxidation of substrates by MnP did not proceed under anaerobic conditions. When the dimer (I) was deuterated at the alpha position and subsequently oxidized by MnP in the presence of Tween 80, yields of 3,4-diethoxybenzaldehyde, 4-methoxyacetophenone remained constant, while the yield of the alpha-keto dimeric product (IV) decreased by approximately sixfold, suggesting the involvement of a hydrogen abstraction mechanism. MnP also oxidized the alpha-keto dimeric product (IV) to yield 3,4-diethoxybenzoic acid (V) and 4-methoxyacetophenone (III), in the presence and, in lower yield, in the absence of Tween 80. When the reaction was performed in the presence of 18O2, both products, 3,4-diethoxybenzoic acid and 4-methoxyacetophenone, contained one atom of 18O. Finally, MnP oxidized the substrate 1-(3',5'-dimethoxyphenyl)-1-hydroxy-2-(4'-methoxyphenyl)propane (IX) to yield 3,5-dimethoxybenzaldehyde (XI), 4-methoxyacetophenone (III) and 1-(3',5'-dimethoxyphenyl)-1-oxo-2-(4'-methoxyphenyl)propane (X). In sharp contrast, LiP was not able to oxidize IX. Based on these results, we propose a mechanism for the MnP-catalyzed oxidation of these dimers, involving hydrogen abstraction at a benzylic carbon, rather than electron abstraction from an aromatic ring.