Ceriporic Acid B, an Extracellular Metabolite of Ceriporiopsis

Ceriporic Acid B, an Extracellular Metabolite of Ceriporiopsis subvermispora, Suppresses the Depolymerization of Cellulose by the Fenton Reaction...
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Biomacromolecules 2005, 6, 2851-2856

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Ceriporic Acid B, an Extracellular Metabolite of Ceriporiopsis subvermispora, Suppresses the Depolymerization of Cellulose by the Fenton Reaction Noor Rahmawati, Yasunori Ohashi, Takahito Watanabe, Yoichi Honda, and Takashi Watanabe* Laboratory of Biomass Conversion, Research Institute for Sustainable Humanosphere, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan Received May 26, 2005; Revised Manuscript Received July 5, 2005

The white rot fungus, Ceriporiopsis subVermispora, is able to degrade lignin in wood without intensive damage to cellulose. Since lignin biodegradation by white rot fungi proceeds by radical reactions, accompanied by the production of a large amount of Fe3+-reductant phenols and reductive radical species in the presence of iron ions, molecular oxygen, and H2O2, C. subVermispora has been proposed to possess a biological system which suppresses the production of a cellulolytic active oxygen species, •OH, by the Fenton reaction. In the present paper, we demonstrate that 1-nonadecene-2,3-dicarboxylic acid (ceriporic acid B), an extracellular metabolite of C. subVermispora, strongly inhibited •OH production and the depolymerization of cellulose by the Fenton reaction in the presence of iron ions, cellulose, H2O2, and a reductant for Fe3+, hydroquinone (HQ), at the physiological pH of the fungus. Introduction Iron ions and hydroxyl radicals (•OH) play important roles in wood decay by saprotrophic fungi. When wood degrading fungi colonize wood, their extracellular enzymes are not able to diffuse into the intact wood cell walls, because the enzymes are too large to penetrate the pores of the wood cell walls.1 The hydroxyl radical is a principal low molecular mass oxidant that erodes wood cell walls to enhance the accessibility of the extracellular enzymes of wood rot fungi to wood cell wall components. In 1974, similarities between wood treated with the hydroxyl radical and with brown rot fungi were reported.2 Subsequently, hydroxyl radicals were detected in incubations with the brown rot fungus Poria placenta through the use of electron spin resonance and spin trapping agents.3 The involvement of hydroxyl radicals in wood decay has also been demonstrated by chemiluminescence assay and the detection of hydroxylated derivatives after the incubation of wood rot fungi with aromatic marker compounds.4-6 Hydroxyl radicals are produced by the reaction of Fe2+ with H2O2 (Fenton reaction: Fe2+ + H2O2 f Fe3+ + OH+ •OH). In the Fenton system, catalysts for the reductive half-cycle (Fe3+ f Fe2+) accelerate hydroxyl radical formation. Wood rot fungi possess versatile enzymatic and nonenzymatic systems to accelerate the reductive half-cycle. With regard to the nonenzymatic system, the production of diffusible Fe3+-reducing metabolites has been extensively studied.7,8 Brown rot fungi decompose cellulose by the Fenton reaction. At the same time, they protect fungal hyphae * To whom correspondence should be addressed. Phone: +81-774-383640. Fax: +81-774-38-3643. E-mail: [email protected] (T. Watanabe).

by a pH gradient due to the accumulation of oxalate crystals on fungal hyphae.9 Hydroxyl radicals are also produced by white rot fungi.5 In white rot, lignin degradation proceeds by radical reactions driven by oxidative enzymes, such as peroxidases and laccase. This process produces large amounts of Fe3+ reductants, lignin-derived phenols,10 and some reductive radical intermediates, such as formate anion radicals from oxalate and semiquinone radicals. Reduction of O2 with the radicals produces O2•-, which in turn reduces Fe3+ or disproportionates into H2O2. By the combination of H2O2 production and Fe3+ reduction, the generation of •OH is accelerated (Figure 1). Thus, lignin biodegradation is related to the production of hydroxyl radicals by the Fenton reaction. In contrast to the brown rot and nonselective white rot fungi, selective lignin-degrading fungi like Ceriporiopsis subVermispora are able to decompose lignin in wood without extensive damage to cellulose.11,12 Wood decay by the biopulping fungus proceeds without the penetration of extracellular enzymes into the wood cell wall regions. This indicates that selective white rot fungi possess unknown extracellular systems that attenuate the production of hydroxyl radicals. In this context, it was an open question how selective white rot fungi suppress the Fenton reaction to achieve wood decay with a minimum loss of cellulose, in the presence of molecular oxygen, Fe ions, H2O2, ligninderived phenols, and radicals. By considering that the wood decay caused by these fungi proceeds without the penetration of extracellular enzymes into wood cell wall regions, it can be concluded that the control of the Fenton reaction by low molecular mass compounds plays a central role in the protection of cellulose. Previously, we reported that C. subVermisrpora produced a series of novel itaconic acid

10.1021/bm050358t CCC: $30.25 © 2005 American Chemical Society Published on Web 08/23/2005

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Figure 1. A proposed pathway for the generation of hydroxyl radicals (•OH) during lignin biodegradation.

Figure 2. Chemical structure of ceriporic acid B produced by C. subvermispora.

derivatives with a long alk(en)yl side chain at position C-3 of their core (ceriporic acids) (Figure 2)13-15 and that one of the metabolites, ceriporic acid B, strongly suppressed •OH production by the Fenton reaction through direct interaction with a redox cycle of iron ions.16 However, the suppressive effects of this metabolite on cellulose depolymerization have not yet been elucidated. In the present paper, we report that the fungal metabolite inhibited the depolymerization of cellulose by the Fenton reaction at the physiological pH of the fungus. Roles of this unique fungal metabolite in selective white rot are also discussed. Materials and Methods General Methods. FeSO4, FeCl3, 1,4-hydroquinone (HQ), hydrogen peroxide and ethylenediamine N,N,N′,N′-tetraacetic acid (EDTA) were obtained from Wako Pure Chemical Industries (Osaka, Japan). Cupriethylenediamine was purchased from Kanto Chemicals (Tokyo, Japan). Dissolving pulp from hardwood sulfite pulp (viscosity 597 mL/g, R-cellulose > 93%) was obtained from Nippon Paper Co. Ltd. (Tokyo, Japan). Unbleachead kraft pulp from hardwood (UKP; viscosity 1050 mL/g and κ number 20.3) was obtained from Oji Paper Co. Ltd. (Tokyo, Japan). Milli-Q water was used throughout the experiments. All of the chemicals used were of analytical reagent grade. 1-Nonadecene-2,3-dicarboxylic acid (ceriporic acid B) was synthesized as reported.14 Removal of Metal Ions from Pulp by Chelation. To suppress side reactions due to metal ions adsorbed in the original pulp, pulp samples were preliminarily treated with EDTA (0.5% on pulp) for 1 h at 80 °C at 5% of the pulp consistency. After treatments, the pulp was washed with 2 L of Milli-Q water. Treatments of Pulp with the Fenton System. Pulp was treated with Fenton’s reagent (FeSO4 and H2O2) or a solution of FeCl3, HQ, and H2O2 at 25 °C in a 10-mL test tube in a thermoshaker bath at 100 rpm in the dark. In the reactions

with ceriporic acid B, 0.5 mM iron ions and 2.5 mM of the metabolite were incubated at 25 °C for 30 min for complex formation in a 10-mL test tube before the treatment of the pulp. The reactions were carried out in a 20-mM succinate buffer (pH 3.0, 4.5, and 7.0) or sodium phosphate buffer (pH 7.0) containing 0.1 g of never-dried dissolving pulp or 0.5 g of UKP. Reactions were started by the addition of H2O2 (100 mM). The total volumes of the reaction solutions were 1.0 and 5.0 mL for never-dried dissolving pulp and UKP, respectively. Detailed reaction conditions are described in the legend of each figure. After the reaction, pulp was washed with water, air-dried, and subjected to determinations of the κ number and the degree of polymerization. Determinations of the K Number, Degree of Polymerization, and Production of Hydroxyl Radicals (•OH). The κ number of the pulp was determined according to DIN 54357. The degree of polymerization was determined by measuring intrinsic viscosity according to the method of Brown and Wikstrom.17 Hydroxyl radicals (•OH) produced were determined by the 2-deoxy-D-ribose assay.18 Results In the present study, two different cellulosic materials, never-dried dissolving pulp and unbleached kraft pulp (UKP), were used to evaluate the effects of ceriporic acid B on the depolymerization of cellulose and deliginification by the Fenton reaction. Chelators for Fe ions behave as prooxidants or antioxidants depending on the molar ratio between the chelator and the iron ions. It was suggested that a large excess of oxalate sequesters Fe ions by the formation of a complex composed of Fe ions and oxalate with the molar ratio 1:2 or 1:3, which acts to suppress the production of •OH by the Fenton system in the presence of 2,5-dimethoxybenzoquinone.19 It was also reported that an excess of 2,3-dihydroxybenzoic acid inhibits iron reduction, probably by the coordination of the hexadentate ligand to ferric iron to limit its reactivity to an Fe3+ reductant.20 As for ceriporic acid B, its inhibitory effects increased with an increase in the molar ratio of the ligand to Fe3+. The inhibitory effects reached a plateau at a molar ratio of Fe3+ to ceriporic acid B of 1:4.16 Therefore,

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Inhibition of Cellulose Degradation by Ceriporic Acid B

Table 1. Suppression of Hydroxyl Radical Production by Ceriporic Acid B in Fenton Reactions at Different pHsa production of hydroxyl radicals (absorbance at 532 nm) pH

Fe2+, H2O2

Fe2+, H2O2, ceriporic acid B

Fe3+, HQ, H2O2

Fe3+, H2O2, HQ, ceriporic acid B

3.0 4.5 7.0b 7.0c

0.264 0.271 0.194 0.239

0.021 0.100 0.027 0.052

0.524 0.468 0.183 0.225

0.027 0.041 0.046 0.038

a Experimental conditions are the same as those given in Figures 3 and 4, except that reactions were carried out in the presence of 2.5 mM 2-deoxyribose for 30 min. b Sodium phosphate buffer used. c Sodium succinate buffer used.

Figure 3. Effects of pH on the decrease in the degree of polymerization of cellulose (never-dried dissolving pulp) by the treatments with Fe2+ and H2O2 in the presence and absence of ceriporic acid B. Reactions were carried out at 25 °C for 6 h in sodium succinate (20 mM, pH 3.0, 4.5) or sodium phosphate (20 mM, pH 7.0) buffers. The complete system (2) contained 0.5 mM FeSO4, 100 mM H2O2, 0.1 g never-dried dissolving pulp, and 2.5 mM ceriporic acid B in the buffer. Control (9) contained never-dried dissolving pulp and H2O2 in the buffer. The Fenton system (b) contained FeSO4, H2O2, and neverdried dissolving pulp in the buffer. Total volume of the reaction mixture was 1.0 mL.

experiments were carried at a fixed molar ratio (1:5) of iron ions to ceriporic acid B throughout this study. Suppression of •OH Production and the Depolymerization of Cellulose (Never-Dried Dissolving Pulp) by Ceriporic Acid B in the Fenton Reaction. Previously, we reported that ceriporic acid B suppressed the production of hydroxyl radicals by the Fenton reaction.16 However, it has not been clarified if this metabolite exhibits its inhibitory effects in the presence of cellulose. Therefore, never-dried dissolving pulp was reacted with Fenton’s reagent, and the degree of polymerization and the production of hydroxyl radicals were determined at pH 3.0-7.0. At pH 7.0, sodium phosphate and succinate buffers were used, because phosphate binds to Fe2+ to decrease the reactivity with H2O2.21 In the reactions of Fe2+ and H2O2 with the never-dried dissolving pulp in the absence of ceriporic acid B (Figure 3), a decrease in the degree of polymerization was observed at all the pHs employed. At pH 7.0, the decrease in the degree of polymerization in the phosphate buffer was slightly smaller than that in the succinate buffer, but the differences were negligible when compared with the reactions in acidic media at pHs 3.0 and 4.5. When ceriporic acid B was preliminarily mixed with Fenton’s reagent, the metabolite strongly suppressed the depolymerization of cellulose. At the same time, the metabolite inhibited the production of •OH in the presence of cellulose (Table 1). The inhibitory effects of ceriporic acid B on cellulose depolymerization and the production of •OH were more remarkable in the reactions of Fe3+, H2O2, and HQ (Table 1 and Figure 4). This can be explained by the strong inhibition of Fe3+ reduction by ceriporic acid B.14 In the absence of ceriporic acid B, a rapid decline in the degree of polymerization of cellulose was observed at pHs 3.0 and 4.5, but depolymerization of cellulose at pH 7.0 was much

Figure 4. Effects of pH on the decrease in the degree of polymerization of cellulose (never-dried dissolving pulp) by the treatments with Fe3+, hydroquinone (HQ), and H2O2. Reactions were carried out at 25 °C for 6 h in sodium succinate (20 mM, pH 3.0, 4.5) or sodium phosphate (20 mM, pH 7.0) buffers. The complete system (2) contained 0.5 mM FeCl3, 0.25 mM HQ, 100 mM H2O2, 0.1 g neverdried dissolving pulp, and 2.5 mM ceriporic acid B in the buffer. Control (9) contained never-dried dissolving pulp and H2O2 in the buffer. The Fenton system (b) contained FeCl3, H2O2, HQ, and never-dried dissolving pulp in the buffer. Total volume of the reaction mixture was 1.0 mL.

less remarkable. This is mainly because autoxidation of ferrous aquo species is disfavored at the acidic pHs. Formation of unreactive ferric hydroxo species, and its hydrolysis products, is also disfavored under the acidic conditions. The effects of ceriporic acid B on cellulose degradation by the Fenton reaction was assayed for a longer period up to 24 h. Treatment of the never-dried dissolving pulp with the Fenton’s reagent (Fe2+ and H2O2) at pH 4.5 resulted in rapid decrease in the viscosity (Figure 5). The degree of polymerization of pulp decreased from 725 to 520 by the Fenton reaction after 24 h. However, when ceriporic acid B was preliminarily added to the Fe2+ ions, the decrease in the degree of polymerization was suppressed to 664. The effects of ceriporic acid B on cellulose depolymerization were also examined using a reaction system composed of Fe3+, HQ, and H2O2 (Figure 6). HQ was used as a reductant for Fe3+. The addition of ceriporic acid B to the reaction system strongly inhibited the depolymerization of cellulose, as observed in the direct reactions between Fe2+ and H2O2.

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Figure 5. Suppression of cellulose depolymerization (never-dried dissolving pulp) by ceriporic acid B in the reaction of Fe2+ with H2O2. Reactions were carried out at 25 °C in sodium succinate (20 mM, pH 4.5) buffer. The complete system (2) contained 0.5 mM FeSO4, 100 mM H2O2, 0.1 g never-dried dissolving pulp, and 2.5 mM ceriporic acid B in the buffer. Control (9) contained never-dried dissolving pulp and H2O2 in the buffer. The Fenton system (b) contained FeSO4, H2O2, and never-dried dissolving pulp in the buffer. Total volume of the reaction mixture was 1.0 mL.

Figure 6. Suppression of cellulose depolymerization (never-dried dissolving pulp) by ceriporic acid B in the reaction of Fe3+, hydroquinone, and H2O2. Reactions were carried out at 25 °C in sodium succinate (20 mM, pH 4.5) buffer. The complete system (2) contained 0.5 mM FeCl3, 0.25 mM HQ, 100 mM H2O2, 0.1 g never-dried dissolving pulp, and 2.5 mM ceriporic acid B in the buffer (pH 4.5). Control (9) contained never-dried dissolving pulp and H2O2 in the buffer. The Fenton system (b) contained FeCl3, H2O2, HQ, and neverdried dissolving pulp in the buffer. Total volume of the reaction mixture was 1.0 mL.

Effects of Ceriporic Acid B on the Delignification and Depolymerization of UKP by the Fenton Reaction. Fenton reactions were carried out using UKP in the presence and absence of ceriporic acid B. UKP contains residual lignin after kraft pulping. Therefore, the removal of lignin by the Fenton reaction was estimated by measuring the κ number of the pulp, in addition to the assay of the degree of polymerization. Suppression of cellulose depolymerization by ceriporic acid B was observed in both the reactions with Fe2+/H2O2 and with Fe3+/HQ/H2O2 (Figures 7 and 8). The inhibitory

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Figure 7. Suppression of UKP depolymerization by ceriporic acid B in the reaction of Fe2+ with H2O2. Reactions were carried out at 25 °C in sodium succinate (20 mM, pH 4.5) buffer. The complete system (2) contained 0.5 mM Fe2+, 100 mM H2O2, 0.1 g UKP, and 2.5 mM ceriporic acid B in the buffer. Control (9) contained UKP and H2O2 in the buffer. The Fenton system (b) contained FeSO4, H2O2, and UKP in the buffer. Total volume of the reaction mixture was 5.0 mL.

Figure 8. Suppression of UKP depolymerization by ceriporic acid B in the reaction of Fe3+, hydroquinone, and H2O2. Reactions were carried out at 25 °C in sodium succinate (20 mM, pH 4.5) buffer. The complete system (2) contained 0.5 mM FeCl3, 0.25 mM hydroquinone (HQ), 100 mM H2O2, 0.1 g UKP, and 2.5 mM ceriporic acid B in the buffer (pH 4.5). Control (9) contained UKP and H2O2 in the buffer. The Fenton system (b) contained FeCl3, H2O2, HQ, and UKP in the buffer. Total volume of the reaction mixture was 5.0 mL.

effects were more remarkable for the Fe3+/HQ/H2O2 system, as observed for the never-dried dissolving pulp. It was found that ceriporic acid B suppressed the degradation of cellulose. However, no significant differences were found in the reduction of the κ number between tests with and without ceriporic acid B in the two reaction systems, Fe2+/H2O2 and Fe3+/HQ/H2O2 (Table 2). We conclude that ceriporic acid B has inhibitory effects on cellulose depolymerization, regardless of the presence or absence of residual lignin in pulp. Discussion Hydroxyl radicals react with carbohydrates with a high rate constant around 109 M-1 sec-1. The oxidation starts from

Inhibition of Cellulose Degradation by Ceriporic Acid B Table 2. Effects of Ceriporic Acid B on the Decrease in the κ Number of UKP by the Treatments with Fe2+ and H2O2 or Fe3+, HQ, and H2O2a κ number Fe3+, Fe3+, H2O2, Fe2+, Fe3+, HQ, HQ, ceriporic Fe2+, H2O2, time/h UKP H2O2 ceriporic acid B H2O2 H2O2 acid B 0 6 12 24

20.3 20.0 19.9 19.9

20.3 19.7 19.7 19.5

20.3 19.9 19.5 19.4

20.3 19.8 19.8 19.6

20.3 19.6 19.2 19.0

20.3 19.7 19.5 19.1

a Experimental conditions are the same as those given in Figures 7 and 8.

hydrogen abstraction and proceeds by rearrangements of the resultant carbon-centered radicals, cleavage of the glycosidic bonds, and fragmentation via β-elimination.22,23 Hydroxyl radicals also react with lignin by electrophilic addition to a double bond and an aromatic nuclei to initiate a variety of consecutive processes including disproportionation, phenolic coupling, oxidative cleavage of ether bonds including demethoxylation, CR-Cβ cleavage, and hydroxylation of aromatic nuclei.24 In the reactions of Fenton’s reagents with wood, however, depolymerizations of cellulose and hemicelluloses are more remarkable than delignification, as found in wood decay caused by brown rot fungi. The reactions of UKP with Fenton’s reagents also showed that the extent of cellulose degradation was much stronger than that of delignification (Table 2, Figures 7 and 8). Chelating agents for iron ions have been extensively studied in relation to the oxidative damage of living tissues by hydroxyl radicals. For the promotion of •OH production by chelating agents, the redox potentials of the reductive halfcycle from Fe3+ to Fe2+ in the complex should be higher than that of the reductant in the reaction system. For instance, an Fe3+-EDTA complex (Fe3+-EDTA/Fe2+-EDTA, E°′ ) +0.12V/NHE) is readily reduced by natural reductants with lower redox potential including NADH (NAD+, H/NADH, E°′ ) -0.32 V/NHE), and O2- (O2/O2-, E°′ ) -0.33 V/NHE), while Fe3+-desferrioxamine (DFO) (Fe3+-DFO/ Fe2+-DFO, E°′ ) -0.45V/NHE) is not reduced by the reductants.25 Thus, •OH production by the Fenton system is strongly inhibited by DFO. The standard redox potential of Fe3+-DTPA/Fe2+-DTPA (E° ) -0.034 V/NHE, pH 6-8) is high enough to be reduced by O2-.26 However, DTPA decreases the reduction rate of Fe3+ because Fe3+-DTPA lacks a site accessible to reductants. Thus, the existence of accessible sites in iron complexes is also a key factor to control the Fenton reaction by chelators. In addition to these two factors, the reaction rate of Fe2+ with H2O2 also directly affects the overall production rate of •OH. The rate constant for free Fe2+ ions with H2O2 is below 100 M-1 s-1. However, chelation with ATP and ADP increases the rate constants to 6.6 × 103 and 1.1 × 104 M-1 s-1, respectively.25 Some chelates such as Quin2 promote both the reduction of Fe3+ and oxidation of Fe2+ by H2O2 to accelerate the overall production rate of •OH.27

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In contrast to the prooxidative iron chelators, ceriporic acid B, a metabolite of a selective white rot fungus, C. subVermispora, is a strong inhibitor of the iron redox reactions. It inhibits the reduction of Fe3+ ions by ascorbic acid, HQ, and cysteine.14 This suggests that the redox potential of the complex between Fe3+ and ceriporic acid B is lower than that of these reductants or that ceriporic acid B is able to sequester iron ions from the reductants. Since the inhibitory effects of ceriproric acid B on the Fenton reaction increased with an increase in the molar ratio between the metabolite and iron ions, sequestration is the most likely phenomenon to explain the inhibition.16 In the present paper, we demonstrated that the fungal metabolite inhibited the depolymerization of cellulose by the Fenton reaction in the presence of Fe ions, cellulose, and a reductant for Fe3+, HQ. The 2-deoxy-D-ribose assay strongly suggested that the suppression of cellulose depolymerization by the metabolite at pH 3.0-7.0 was due to a decrease in the production rate of •OH by the Fenton system. Thus, ceriporic acid B keeps iron ions in solutions in a redoxunreactive form, in the presence of cellulose. This indicates that the metabolite is able to protect cellulose in an oxidative environment where free radicals and phenols are produced in the presence of molecular oxygen and iron ions. Iron ions play a key role in fungal metabolism and growth. Since the bioavailability of iron is limited in nature, a number of microorganisms produce iron-chelating agents, such as siderophores, to aid in the assimilation of the ferric iron. On the other hand, some microorganisms produce iron-chelating metabolites to sequester Fe ions to facilitate metal tolerance. In wood decay, wood rot fungi secrete Fe3+-reducing extracellular metabolites to promote the production of hydroxyl radicals by the Fenton reaction to attack wood cell walls.2-9 The Fenton-based •OH -producing system plays a central role in wood decay by brown rot fungi. Interestingly, it was also reported that a brown rot fungus, Coniophora puteana, produced •OH on agar plates, but the production increased two- or threefold when the fungus was in contact with bacteria such as Bacillus subtilis.6 This phenomenon can be explained by a defense mechanism of the fungus. In contrast to these biological functions, ceriporic acid B is unique in its function to suppress iron redox reactions to attenuate the depolymerization of cellulose by the Fenton reaction. The extracellular fungal system should contribute to the prevention of cellulose damage during free radicalmediated lignin biodegradation, which might be advantageous for the synergism between selective white rot fungi and cellulose-degrading microorganisms. Conclusions Ceriporic acid B, an extracellular metabolite of C. subVermispora, strongly inhibited •OH production and the depolymerization of cellulose by the Fenton reaction in the presence of iron ions, cellulose, H2O2, and a reductant for Fe3+, hydroquinone (HQ), at the physiological pH of the fungus. In the field of pulp bleaching, we expect that the use of iron-redox regulators such as ceriporic acid B and its analogues would suppress the degradation of cellulose during bleaching sequences.

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Acknowledgment. This work was supported by a Grantin-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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References and Notes (1) Cowling, E. B.; Brown, W. Structural features of cellulosic materials in relation to enzymatic hydrolysis. ACS AdV. Chem. Ser. 1969, 95, 152-187. (2) Koenigs, J. W. Hydrogen peroxide and iron: a proposed system for decomposition of wood by brown-rot basidiomycetes. Wood Fibre 1974, 6, 66-80. (3) Illman, B. L.; Meinholtz, D. C.; Highley, T. L. Oxygen free radical detection in wood colonized by the brown rot fungus Postia placenta. Biodeterioration Res. 1989, 2, 497-509. (4) Backa, S.; Gierer, J.; Reitberger, T.; Nilsson, T. Hydroxyl radical activity in brown-rot fungi studied by a new chemiluminescent method, Holzforschung 1993, 46, 61-67. (5) Backa, S.; Gierer, J.; Reitberger, T.; Nilsson, T. Hydroxyl radical associated with the growth of white rot fungi. Holzforschung 1993, 47, 181-187. (6) Tornberg, K.; Olsson, S. Detection of hydroxyl radicals produced by wood-decomposing fungi. FEMS Microbiol. Ecol. 2002, 40, 1320. (7) Goodell, B.; Jellison, J.; Liu, J.; Daniel, G.; Paszcynski, A.; Fekete, F.; Krishnamurthy, S.; Jun, L.; Xu, G. Low molecular weight chelators and phenolic compounds isolated from wood decay fungi and their role in the fungal biodegradation of wood. J. Biotechnol. 1997, 53, 133-162. (8) Kerem, Z.; Jensen, K. A.; Hammel, J. K. E. Biodegradative mechanism of the brown rot basidiomycete Gloeophyllum trabeum: evidence for an extracellular hydroquinone-driven fenton reaction. FEBS Lett. 1999, 446, 49-54. (9) Wood, P. M. Pathways for production of Fenton’s reagent by woodrotting fungi. FEMS Microbiol. ReV. 1994, 13, 313-320. (10) Pracht, J.; Boenigk, J.; Isenbeck-Schro¨ter, M.; Keppler, F.; Scho¨ler, H. F. Abiotic Fe(III) induced mineralization of phenolic substances. Chemosphere 2001, 44, 613-619. (11) Messner, K.; Srebotnik, E. Biopulping: an overview of developments in an environmentally safe paper making technology. FEMS Microbiol. ReV. 1994, 13, 351-364. (12) Blanchette, R. A.; Krueger, E. W.; Haight, J. E.; Akhtar, M.; Akin, D. E. Cell wall alterations in loblolly pine wood decayed by the whiterot fungus, Ceriporiopsis subVermispora. J. Biotechnol. 1997, 53, 203-213. (13) Enoki, M.; Honda, Y.; Watanabe, T.; Kuwahara, M. A novel dicarboxylic acid produced by white-rot fungus Ceriporiopsis subVermispora. In Proceedings of the 44th Lignin Symposium; October 7-8, 1999, Gifu, Japan; pp 69-72. (14) Enoki, M.; Honda, Y.; Kuwahara, M.; Watanabe, T. Chemical synthesis, iron redox interactions and charge-transfer complex (CTC) formation of ceriporic acids from the selective lignin-degrading

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basidiomycete, Ceriporiopsis subVermispora. Chem. Phys. Lipids 2002, 120, 9-20. Amirta, R.; Fujimori, K.; Shirai, N.; Honda, Y.; Watanabe, T. Ceriporic acid C, a hexadecenylitaconate produced by a lignindegrading fungus, Ceriporiopsis subVermispora. Chem. Phys. Lipids 2003, 126, 121-131. Watanabe, T.; Teranishi, H.; Honda, Y.; Kuwahara, M. A selective lignin-degrading fungus, Ceriporiopsis subVermispora, produces alkylitaconates that inhibit the production of cellulolytic active oxygen species, hydroxyl radical in the presence of iron and H2O2. Biochem. Biophys. Res. Commun. 2002, 297, 918-923. Brown, W.; Wikstrom, R. A. viscosity-molecular weight relationship for cellulose in cadoxen and a hydrodynamic interpretation. Eur. Polymer J. 1965, 1, 1-10. Halliwell, B.; Grootveld, M..; Gutteridge, J. M. Methods for the measurement of hydroxyl radicals in biomedical systems: deoxyribose degradation and aromatic hydroxylation. Methods Biochem. Anal. 1988, 33, 59-90. Varela, E.; Tien, M. Effect of pH and oxalate on hydroquinonederived hydroxyl radical formation during brown rot wood degradation. Appl. EnViron. Microbiol. 2003, 69, 6025-6031. Qian, Y.; Goodel, B.; Felix, C. C. The effect of low molecular chelators on iron chelation and free radical generation as studied by ESR measurement. Chemosphere 2002, 48, 21-28. Benitez, F. J.; Acero, J. L.; Real, F. J.; Rubio, F. J.; Leal, A. I. The role of hydroxyl radicals for the decomposition of p-hydroxyphenylacetic acid in aqueous solutions. Water Res. 2001, 35, 1338-1343. Zegota, H.; Sonntag, C. Radiation chemistry of carbohydrates, XV OH radical induced scission of the glycosidic bond in disaccharides. Z. Naturforsch. B 1977, 32, 1060-1067. Park, J. S.; Wood, P. M.; Gilbert, B. C.; Whitwood, A. C. EPR evidence for hydroxyl and substrate derived radicals in Fe(II) oxalate/ hydrogen peroxide reaction. The importance of the reduction of Fe(III) oxalate by oxygen-conjugated radicals to regenerate Fe(II) in reaction of carbohydrates and model compounds. J. Chem. Soc., Perkin Trans. 2 1999. 923-931. Gierer, J. Formation and involvement of superoxide (O2•-/HO2•) and hydroxyl (OH•) radicals in TCF bleaching processes: A review. Holzforschung 1997, 51, 34-46. Halliwell, B.; Gutteridge, J. M. C. Free radicals in biology and medicine, 3rd ed.; Oxford University Press: Oxford, 1998. Vandegaer, J.; Chaberek, S.; Frost, A. E. Iron chelates of diethylenetriaminepentaacetic acid. J. Inorg. Nucl. Chem. 1959, 11, 210221. Sandstrom, B. E.; Svoboda, P.; Granstrom, M.; Harms-Ringdahl, M.; Candeias, L. P. H2O2-driven reduction of the Fe3+-quin2 chelate and the subsequent formation of oxidizing species. Free Radical Biol. Med. 1997, 23, 744-753.

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