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HONDA, Yoichi

KUWAHARA, Masaaki

TERANISHI, Hiroko

WATANABE, Takashi

Kyoto University Research Information Repository

TitleSuppression of the Fenton Reaction by Ceriporic AcidsProduced by a Selective Lignin-Degrading Fungus,Ceriporiopsis subvermisporaAuthor(s)TERANISHI, Hiroko; HONDA, Yoichi; KUWAHARA,Masaaki; WATANABE, TakashiCitationWood research : bulletin of the Wood Research Institute KyotoUniversity (2003), 90: 13-14Issue Date2003-09-30URL http://hdl.handle.net/2433/53107RightType Departmental Bulletin PaperTextversionpublisherKyoto UniversityPreliminarySuppression of the Fenton Reaction by Ceriporic Acids Produced bya Selective Lignin-Degrading Fungus, Ceriporiopsis subvermisporaHiroko TERANISHI*l, Yoichi HONDA*l, Masaaki KUWAHARA*2and Takashi WATANABE*1,3(Received May 31, 2003)Keywords: ceriporic acid, Ceriporiopsis subvermispora, white rot fungi, ligninBiodegradation of lignin by white rot fungi proceeds inconcert with the activation of molecular oxygen and redoxcycling of transition metals, with the aid of oxidative andreductive enzymes. When wood degrading fungi arecolonized in woods, their extracellular enzymes are notable to diffuse into the intact wood cell walls because theenzymes are too large to penetrate the pores of the woodcell walls1). A cellulolytic active oxygen species, hydroxylradical (' OH) is a principal low molecular mass oxidantthat erodes wood cell walls to enhance the accessibility ofthe extracellular enzymes of wood rot fungi to wood cellwall components2). Hydroxyl radicals are produced bythe reaction of Fe2+ with H 20 2 (Fenton reaction; Fe2+ +H 20 2- F:e3+ + OH- + 'OH), although involvement ofother oxidizing species such as (Fe=0)2+ have often beensuggested. It is known that Fe3+ also reacts with H 20 2 togenerate Fe2+ and 02' . This reaction is slower thanthe reaction of H 20 2 with Fe2+ at physiological pH andmuch depends on the ligand to the iron. In the Fentonsystem, catalysts for the reductive half cycle (Fe3+ - Fe2+)accelerate the hydroxyl radical formation. Wood rotfungi have versatile enzymatic and non-enzymatic systemsto accelerate the reductive half cycle. For instance,reduction ofFe3+ is catalyzed by O 2' - which is producedby the reduction of molecular oxygen with reductiveradicals such as CO2"- and semiquinone radicals fromlignin fragments. Fe3+ is directly reduced by lignin-derived phenols such as guaiacol and catechol. Since thebiodegradation oflignin produces free radicals and phenolsin the presence of molecular oxygen and iron, oxidativedegradation of cellulose by hydroxyl radicals is inevitable ifsome inhibition systems for the iron redox reactions are notinvolved during wood decay processes.In contrast to brown rot and non-selective white rotfungi, selective lignin-degrading fungi like Ceriporiopsissubvermispora are able to decompose lignin in wood cellwalls without the intensive damage of cellulose 1) . Thisindicates that the selective white rot fungus possessesunknown extracellular systems that attenuate theproduction of hydroxyl radicals. We herein report thatceriporic acid B, an alkylitaconate produced from C.subvermispora strongly suppressed the Fenton reactions evenin the presence of the Fe3+ reductants such as*1 Wood Research Institute, Kyoto University.*2 Institute of Wood Technology, Akita Prefectural University.*3 Corresponding author, twatanab@kuwri.Kyoto-u.ac.jp- 13hydroquinone.Effect of ceriporic acid B on reduction of ferric ionsI-Nonadecene-2,3-dicarboxylic acid (ceriporic acid B)was synthesized using the Grignard reaction. The NMRand MS spectra of the synthetic compound were identicalto those isolated from the cultures of C. subvermispora FP90031 3). Inhibitory effects of ceriporic acids on reductionofFe3+ were examined by pre-mixing a solution (1,600 pI)of 0.02 mM FeCls and 0.3% Tween 20 in 20 mM sodiumsuccinate buffe~ (pH 4.0) for 0.5 h at 25°C, with or without0.02 mM ceriporic acid B. Next, 200 pI of 3.0 mMdisodium bathophenanthroline disulfonate (BPS) and 100pI of 40 mM reductant were added and absorption at 533nm was monitored on a Hitachi U-2000A spectro-photometer (Tokyo Japan). As a reductant, freshlyprepared solutions of ascorbic acid were used. Theresults obtained showed that reduction of Fe3+ ions byascorbic acid was inhibited by the presence of ceriporicacid B in the same succinate buffer. The extent ofinhibition for the reduction by ascorbic acid during the first3 min reached 96 % .Suppression of the Fenton reactionsby ceriporic acid BHydroxyl radicals produced by the reaction of iron andH202 were determined by the spin trapping of thesecondary radicals from ethano14 ). ESR spectralrecordings were made in a flat cell on a JEOL FR-30 x-band ESR spectrometer operating at room temperaturewith a modulation amplitude of 0.079 mT, a time constantof 0.10 sec, a scanning time of 2 min and a microwavepower of4 mW. Recently, we reported inhibition of ,OHproduction by ceriporic acid B in aqueous mediacontaining no buffer salts. In the present study, theeffects of ceriporic acid B on the 'OH production wasquantified in sodium succinate buffer (pH 4.0). WhenH 20 2 (0.5 mM) was added to a solution (200 pI)containing Fe3+ (0.1 mM), ethanol (3.42 M), hydro-quinone (0.05 mM) and 4-POBN (100 mM), ~ spin adductof the a-hydroxylethyl radical was produced. However,in the presence of l-nonadecene-2,3-dicarboxylic acid(ceriporic acid B,. 3 mM), the production of 'OH wasstrongly inhibited as observed in the reactions without thesuccinate buffer5). In reactions of Fe3+ with H 20 2 withand without hydroquinone, inhibition of 'OH production-------------WOOD RESEARCH No. 90 (2003)x· +Depolymerization of cell wallpolysaccharides, cellulose andhemcelluloses(+ degradation of lignin)'1IIOH-,Fenton reaction"" " "Superoxide "-""Fe{lI) ~ ~ H202J Hydroxyl radicalFe{lII) " ~ HO- -...+IIIIInhibition "IRadicals from ligninand fungal metabolites'--- ,," --..J",----------------------~IIIIIIIIII1--'"Erosion of wood cell wallsCOOHH2C1)"J/""V/"'v/'''v/',,//''v//'''-//'~/COOH ceriporic acidFig. 1. Acceleration and inhibition of the production of hydroxyl radicals by wood rot fungi. In selective whiterot, lignin is degraded without penetration of the extracellular enzymes into wood cell wall regions.Ceriporic acids, metabolites of C. subvermispora inhibit the iron redox reactions to suppress the formation of·OH.by ceriporic acid B after 15 min in the buffer reached 92and 94%, respectively. In direct reactions of Fe2+ withH 20 2, inhibition of the· OH production by ceriporic acidB during the first 1 min was 56%. However, ·OH wasnot produced after 1 min. This is in accordance with theexperiments without the bufferS). The marked differencesin the inhibition rate before and after 1 min suggest thatinhibitory effects of ceriporic acid B on the •ORproduction is ascribed to the reductive half cycle from Fe3 +to Fe2 + The inhibition was not observed when dimethylester of the alkylitaconic acid was used instead of the freedicarboxylic acid. Thus, ceriporic acids suppressed theFenton reaction by ion-redox interactions at weak acidicmedia suitable for the growth of this fungus on wood mealcultures. Differences in reactivity with and withoutsuccinate buffer were found in the reactions of itaconicacid. Without succinate buffer, itaconic acid promotedthe hydroxyl radical production. However, in succinatebuffer, the rate of . OR production in. the presence ofitaconate was the same as that of the control experimentswithout the chelator compound.In summary, ceriporic acids strongly attenuated theFenton reaction by suppressing iron redox reactions in aweak acidic medium. Studies on ceriporic acids will leadto further understandings of the selective white rot by C.subvermispora.References1) K. MESSNER and E. SREBOTNIK: FEMS Microbiol. Rev., 13,351-364 (1994).2) P.M. WOOD: FEMS Microbiol Rev., 13, 313-320 (1994).3) M. ENOKI, Y. HONDA, M.KuWAHARA and T. WATANABE:Chem Phys. Lipids, 120, 9-20 (2002).4) C.M. ROSEN, S. POU, RE. BRITIGAN and M.S. COHEN: InOxygen Radicals in Biological Systems Part C, Methods inEn;:,ymology, 233, 105-111 (1994).5) T. WATANABE, H. TERANISHI, Y. HONDA and M.KUWAHARA: Biochem. Biophys. Res. Commun., 297, 918-923(2002).-14-

Suppression of the Fenton Reaction by Ceriporic Acids Produced by a Selective Lignin-Degrading Fungus, Ceriporiopsis subvermispora

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Suppression of the Fenton Reaction by Ceriporic Acids Produced by a Selective Lignin-Degrading Fungus, Ceriporiopsis subvermispora

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