hongos enzimas

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Introduction

Lignin is an amorphic three-dimensional substance the molecular weight ofwhich is dicult to determine because lignins are highly polydispersematerials (Argyropoulos and Menachem, 199!" #he chemical structure oflignin has also been dicult to determine, and e$en $ery recently, newbonding patterns ha$e been described in softwood lignin, e"g,"diben%odio&ocin structures" Also, the isolation of nati$e lignin is complicatedif possible at all ('uswell and dier, 19)!" #herefore, lignin modelcompounds, e"g", dimeric b--* model compounds and synthetic lignin(dehydrogenation polymeri%ate, + !, are commonly used inmicrobiological studies" #he results obtained using these substrates are noteasy to e&trapolate to natural conditions, and thus little is .nown whathappens when microorganisms, such as white-rot fungi, degrade lignin inwood"

/ood-rotting basidiomycetous fungi that cause white rot in wood are the

most ecient lignin degraders in nature (0ir. and arrell, 19)2 3ri.sson etal", 1994!, and they are perhaps nature5s ma6or agents for recycling thecarbon of ligni7ed tissues" 8o other microorganisms as pure culture ha$ebeen described to minerali%e ligni7ed tissues as eciently (0ir. andullen,199)!" #hey are a group of ta&onomically heterogeneous higher fungi,characteri%ed by their uni:ue ability to depolymeri%e and minerali%e ligninusing a set of e&tracellular ligninolytic en%ymes" Lignin degradation bywhite-rot fungi has been intensi$ely studied during the last thirty years inrelation to biotechnical applications such as biopulping, biobleaching,treating of pulp mill e;uents, and soil bioremediation (A.htar et al", 199<,199)2 Lamar et al", 199<2 Messner and =rebotni., 199*!" #he en%ymology

and molecular biology of lignin degradation has been mainly studied inhanerochaete chrysosporium (>old and Alic, 199?2 ullen, 1992 0ir. andullen, 199)!" owe$er, many other species of white-rot fungi degradelignin as eciently as " chrysosporium (ata..a, 199*!" Moreo$er, se$eralfungi show better selecti$ity for lignin remo$al (3ri.sson et al", 19942'lanchette et al", 199<2 Messner and =rebotni., 199*!" hysiologicalconditions for lignin degradation, as well as secretion patterns of theligninolytic en%ymes, $ary between di@erent fungal species (ata..a, 199*!"arious authors ha$e tried to establish correlations between ligninolyticen%ymes and lignin degradation (0BBri., 19CD2 Ander and 3ri.sson, 19C,192 3ri.sson et al", 19942 ata..a, 199*!" owe$er, many of the en%ymes

necessary for lignin degradation were not characteri%ed before thebeginning of the 19)4s when $irtually only laccase had been .nown" =incethe disco$ery of two important pero&idases in the beginning of the 19)4s,namely lignin pero&idases (Lis! in 19)? and

manganese pero&idases (Mns! in 19)* (0ir. and arrell, 19)!, an array ofen%ymes ha$e been isolated from fungi and characteri%ed in detail"Although basidiomycetous white-rot fungi and related litter-decomposingfungi are the most ecient degraders of lignin, mi&ed cultures of fungi,actinomycetes, and bacteria in soil and compost can also minerali%e lignin(#uomela et al", <444!" #hese soil-inhabiting microorganisms may ha$e

applications in bioremediation, and their en%ymes may ha$e interestingproperties, e"g", high thermostability and nearly neutral p optimum, not



usually found in the en%ymes of wood-inhabiting fungi" In this re$iew, thefocus is on the present .nowledge on lignin degradation under natural ornearly natural conditions, i"e", on wood or straw, and in the en%ymology oflignin biodegradation, including minerali%ation of lignin by ligninolyticen%ymes in $itro" In addition, the possible in$ol$ement of nonen%ymatic

agents in the fungal lignin biodegradation process is discussed"istorical utline

Lignin degradation is in a central position in the earth5s carbon cycle,because most renewable carbon is either in lignin or in compoundsprotected by lignin from en%ymatic degradation (cellulose andhemicellulose! (0ir., 19)?!" Lignin biodegradation is also responsible formuch of the natural destruction of wood in use, and it may ha$e animportant role in plant pathogenesis" n the other hand, potentialapplications utili%ing lignin-degrading organisms and their en%ymes ha$ebecome attracti$e, because they may pro$ide en$ironmentally friendly

technologies for the pulp and paper industry and for the treatment of many&enobiotic compounds, stains, and dyes" +espite its signi7- < istoricalutline 1?1 cance, lignin biodegradation has only slowly been de7nedchemically and biochemically" ne of the main reasons for that was the poor.nowledge of the chemical structure of lignin until the late 19C4s when itbecame better .nown (0ir., 191, 19)?2 Adler, 192 3ri.sson et al", 1994!"

#he scheme for structural features of conifer lignin presented by Adler(19! has been commonly used in the literature, and only recently newstructures ha$e been disco$ered (Argyropoulos and Menachem, 1992hapter ?, this $olume!" =uccessful studies on the biodegradation of ligninre:uire a good cooperation between microbiologists, biochemists, and

chemists, and relati$ely e&pensi$e e:uipment and materials, e"g", 1*-labeled lignins or lignin model compounds that ha$e not been commerciallya$ailable" Lignin biodegradation was also considered an unusual biologicalprocess in$ol$ing e&tracellular o&idations" rior to the 19<4s, little researchwas conducted on lignin biodegradation (0ir., 19)?!" =ome 7ndings,summari%ed in the 19?4s ( for re$iews, see 0ir., 19)?!, are still today $alidE1! lignin is among the plant cell wall polymers the most resistant tobiological degradation, although it is degraded, <! white-rot fungi degradelignin in wood, and ?! completely selecti$e remo$al of lignin (withoutconcomitant remo$al of wood carbohydrates! had not been obser$ed"/a.sman et al" (19?9! had studied lignin degradation, e"g", in compost and

soil en$ironment (re$iewed by #uomela et al", <444!" >ottlieb and elc%ar(19D1! in their re$iew reported that the white-rot fungus olyporus (syn"

#rametes! $ersicolor used 'rauns5 nati$e ligninF as the growth substrate"Although the lignin structure is unaltered due to the mild

procedure, the preparation has a relati$ely low molecular weight" #his7nding, indicating that lignin could be used as a sole carbon and energysource for white-rot fungi, has not been $eri7ed" Also, many other faultytechni:ues had been used in other studies" In the 19D4s in addition to white-rot fungi, other groups of fungi were found to degrade lignin, at leastpartially, namely basidiomycetous litter-decomposing and brown-rot fungi as

well as soft-rot fungi (re$iewed by 0ir., 191, 19)?!" #he 7rst lignin modelcompound studies were published in the 19C4s (Gussell et al", 19C12



Ishi.awa et al", 19C?2 0ir. et al", 19C)2 u.u%umi et al", 19C9!"'iodegradation assays based on 1*-lignins were de$eloped in the 194s(aider and #ro6anows.i, 19D2 0ir. et al", 19D, 19)!, and using thistechni:ues, called radiorespirometry, it was re$ealed how lignin wasoptimally degraded under laboratory conditions by white-rot fungi" #he

white-rot fungus hanerochaete chrysosporium was used as the maine&perimental organism in H=A, while in some other laboratories, theanamorph of the same fungus, =porotrichum pul$erulentum, had beenchosen for lignin biodegradation studies (Ander and 3ri.sson, 19C2 Anderet al", 19)4!" 'efore that, #rametes $ersicolor was a popular e&perimentalfungus (owling, 19C12 Gussell et al", 19C1!" In the late 194s and in thebeginning of the 19)4s many important 7ndings in the physiology of lignindegradation by " chrysosporium were made" #he e&periments were carriedout almost only in synthetic li:uid media and using 1*-labeled syntheticlignin (+ ! (0ir. et al", 19D, 19)!" #he most important disco$eries canbe listed as followsE 1! the e@ect of nutrient nitrogen, showing that low

nitrogen was re:uired for lignin degradation, and indicating that the min-1?< D 'iodegradation of Lignin erali%ation of lignin occurred duringsecondary metabolism, <! the e@ect of atmosphere, 144 o&ygen gi$ing thehighest minerali%ation, thus demonstrating that lignin degradation iso&idati$e, ?! the detrimental e@ect of agitation in lignin minerali%ation, and*! the concomitant production of $eratryl alcohol during lignin degradation"=ome other fungi, e"g," hlebia radiata (ata..a and Husi-Gau$a, 19)?2ata..a et al", 19)?, 1991! were also found to readily degrade lignin andlignin model compounds in a similar way" Gadical chemistry is an essentialpart of lignin biodegradation" 'efore the 7nding of lignin pero&idases, it wasassumed that the generation of << and other easily di@usible acti$ated

o&ygen species, such as hydro&yl radicals (" !, supero&ide anion radical(< "J!, and singlet o&ygen (1 <! might be responsible for fungal decay oflignin and lignocellulose (all, 19)4!" #he in$ol$ement of hydro&yl radicalsand singlet o&ygen was discounted later (0ir. and arrell, 19)!" Abrea.through in the en%ymology of lignin biodegradation occurred in 19)? K19)* when the 7rst e&tracellular en%ymes in$ol$ed in the degradation oflignin were disco$ered (#ien and 0ir., 19)?2 >lenn et al", 19)?2 0uwahara etal", 19)*!" =ince that large amounts of studies on the biochemistry andmolecular biology of these en%ymes ha$e been published, but they wereagain strongly concentrated on the en%ymes of one fungus, "chrysosporium" #he catalytic mechanism of lignin pero&idase, based on

initial one-electron o&idation of the lignin model compounds followed bysubse:uent brea.down reactions $ia radical cation intermediates wasproposed and e&perimentally $eri7ed (0ersten et al", 19)D2 =choema.er etal", 19)D2 0ir. et al", 19)C!" 8umerous publications describing the e@ect ofligninolytic en%ymes on lignin model compounds also appeared and werefre:uently re$iewed (iguchi, 19)D2 'uswell and dier, 19)2 0ir. andarrell, 19)!" In the 1994s, in addition to detailed studies on catalytic anden%ymatic properties of the lignin-modifying pero&idases as well as theirmolecular biology, ma6or lines of research ha$e dealt with the potentialapplications of white-rot fungi and their en%ymes in biopulping(biomechanical pulping! and pulp bleaching" #he most promising fungi for

biopulping are so-called selecti$e lignin degraders, i"e", fungi that degradelarger amounts of lignin relati$e to carbohydrates (3ri.sson et al", 1994!"



'ecause " chrysosporium typically produces lignin pero&idases (Lis! alongwith manganese pero&idases (Mns!, Li was considered the most importantlignin-degrading en%yme" owe$er, the optimal conditions for lignindegradation and e&pression of Li did not seem to be appropriate for manyother fungi, and especially, the most ecient selecti$e lignin degraders

apparently did not degrade lignin under these arti7cial conditions" Moreo$er,some of these fungi did not e$en produce Li at all, e"g", the most promisingfungus for biopulping, eriporiopsis sub$ermispora, only produces Mn andlaccase"/hen many di@erent fungi had been studied in detail, it becameclear that Mn is the most commonly occurring pero&idase while it wasdicult to demonstrate the e&pression of Li (ata..a, 199*!" #he role oflaccase, a phenol-o&idi%ing en%yme typically produced by almost all lignin-degrading basidiomycetous white-rot fungi (0BBri., 19CD!, but importantlynot so by the model fungus " chrysosporium, was almost totally ignoreduntil the beginning of the 1994s when the eciency of so-called mediatorFcompounds was recogni%ed" #hese compounds, e"g", <,<5-a%inobis(?

ethylben%thia%oline-C-sulfonate! (A'#= ! or 1- hydro&yben%otria%ole ('# !('ourbonnais and aice, 19942 all and Mc.e, 199!, being readilyo&idi%able primary substrates, e&tend laccase substrate range to the mostrecalcitrant nonphenolic subunits of lignin" Gecombinant laccases can berelati$ely easily produced in common industrial host organisms, e"g", in

#richoderma reesei (=aloheimo and 8i.u-aa$ola, 1991! or in Aspergillusory%ae (=tewart et al", 199C!, and therefore, the prospects of the use oflaccase in bleaching of pulp, te&tiles, and dyes, and in bioremediation ofen$ironmental pollutants such as polycyclic aromatic hydrocarbons (As!are realistic" =hortly after the disco$ery of these synthetic mediators, areport on a natural mediator, ?-hydro&yanthranilate (?- AA !, was

published by 3ggert et al" (199Ca!" #hese 7ndings can be consideredanother brea.through in the en%ymology of lignin degradation" +ue to thelow appreciation of laccase as a lignin-degrading en%yme, the 7rst ?-+structure of this en%yme, although the en%yme was 7rst described as earlyas in the 1))4s (Noshida, 1))?2 #hurston, 199*! was sol$ed only $eryrecently (+ucros et al", 199)!" #he de$elopments before 19)?, i"e", beforeligninaseF was found, ha$e been summari%ed by 0ir. (19)?!" After thatsome of the main e$ents and achie$ements may be listed as followsE

" ligninase (lignin pero&idase, Li ! disco$ered (#ien and 0ir., 19)?2 >lenn etal", 19)?! " manganese pero&idase (Mn ! disco$ered (0uwahara et al",

19)*! " one-electron o&idation mechanism and cation radical formation byLi disco$ered (0ersten et al", 19)D2 =choema.er et al", 19)D! " glyo&alo&idase (>LO ! disco$ered (0ersten and 0ir., 19)! " the concept ofen%ymatic combustionF (0ir. and arrell, 19)! " molecular biologicalstudies (earlier studies re$iewed by >old and Alic, 199?! K cloning andse:uencing of Li gene (#ien and #u, 19)! K cloning and se:uencing ofMn gene (ribnow et al", 19)92 May7eld et al", 199*a! K heterologouse&pression of laccase (=aloheimo and 8i.u-aa$ola, 1991! K homologouse&pression of pero&idases (May7eld et al", 199*b2 =ollewi6n >elp.e et al",1999! K heterologous e&pression of pero&idases (+oyle and =mith, 199C2=tewart et al", 199C! K ?-+ structure of Li (ionte. et al", 199?2 oulos et al"

199?! K ?-+ structure of Mn (=undaramoorthy et al", 199*! K ?-+ structureof laccase (+ucros et al", 199)! K detailed structure of MnKLi hybrid



en%yme (Gui%-+uePas et al", 1999a! " di@erent ligninolytic systems in fungirecogni%ed (re$iewed by ata..a, 199*! " the laccase-mediator systemdisco$ered ('ourbonnais and aice, 19942 all and Mc.e, 199! " a naturallaccase mediator disco$ered (3ggert et al", 199Ca! " the separation of Lifrom lignocellulose medium (ares et al", 199D! " the in$ol$ement of lipid

pero&idation in lignin degradation proposed (0apich and =hish.ina, 199D2 Qensen et al", 199C2 0apich et al", 1999a, b! " cell-free minerali%ation of 1*-labeled synthetic and natural lignins by Mn (ofrichter et al", 1999a, b, c!"

'acteria and Microfungi #o the wood-rotting microorganisms, wood may beconsidered as a series of con$eniently orientated holes surrounded by food"

#he shape, si%e, and orientation of the holes depend directly on theanatomy of wood species, and the chemical composition of the cell walls orthe cell contents" #hese ha$e all e@ects on the particular microorganism touse them as a nutrient source ('lanchette, 199D!" =oftwood (gymnosperms!is formed largely by tracheids, the cell lumen being completely enclosed bya cell wall" #herefore microorganisms must pass through the cell wall or pitmembrane" ardwood (angiosperms! consists of $essels, 7bers, andparenchyma cells as well as some tracheids, and here the $essels areformed from openended $essel elements" #he fungal hyphae or bacteriamay thus mo$e also without passing through the cell wall or pit membrane"

#he hemicellulose as well as lignin components di@er mar.edly in softwoodand hardwood species" =oftwood contains mainly glucomannans ashemicellulose and guaiacyl (> ! type lignin, while hardwood containsglucurono&ylans and guaiacylKsyringyl (>= ! type lignin" =apwood containsaccumulated nutrients such as starch or lipids stored by the ray parenchymacells in addition to e&tracti$es, while heartwood contains more and di@erent

e&tracti$es (pitch! (=6RstrRm, 199?!" hemically and morphologically distincttypes of decay result from attac. of di@erent microorganisms" /ood decayfungi are usually separated into three main groups, causing white, soft, orbrown rot" 'acterial degradation of wood has also been reported includingerosion, tunneling, and ca$ity formation (3ri.sson et al", 19942 'lanchette,199D2 +aniel and 8ilsson, 199)!" ?"1 Actinomycetes ertain bacteria candegrade ligni7ed cell walls of wood" ilamentous bacteria belonging to thegenus =treptomyces are well-.nown degraders of lignin and can minerali%eup to 1D of labeled lignins but usually much less (rawford and=utherland, 19)42 icuPa, 19))2 Simmerman, 19942 >odden et al", 199<2'errocal et al", 199!" #ypically, =treptomyces spp" solubili%e part of lignin,

and the end product is water-soluble acid-precipitable polymeric lignin(rawford et al", 19)?!" 'errocal et al" (199! compared minerali%ation of1*-(lignocellulose!-wheat straw by 9 =treptomyces spp" =treptomycescyaneus 3# ???D was one of the most ecient new isolates in solubili%ing*D of the 1*- (lignin! fraction and minerali%ing ? of the label in <1 d"owe$er, the reference species =" $iridosporus solubili%ed D9 andminerali%ed *"D of the same substrate" #he presence of phenol o&idaseand pero&idase was found in =" cyaneus, the former acti$ity being 144 timesgreater as determined by the o&idation of A'#= and <,*-dichlorophenol,respecti$ely" =" cyaneus degraded a nonphenolic arylglycerol b-aryl ether bya mechanism indicating the clea$age of aKb bond in lignin (Simmerman

et al", 19))!" Actinomycetes produce e&tracellular pero&idases (asti et al",19912 Mercer et al", 199C!, e"g", lignin pero&idase-type en%yme



(Gamachandra et al", 19))2 Adhi et al", 19)9!" =treptomyces sp" 31produces pero&idase and cell-bound demethylase re:uiring << and Mn<T,both of which are produced at relati$ely high le$els in the presence of 0raftlignin or wheat straw, while protochatechuate ?,*-dio&ygenase and b-carbo&ymuconate decarbo&ylase acti$ity were less induced (>odden et al",

199<!" 3&tracellular pero&idase and catalase acti$ity was found in all si&actinomycetes strains studied (>odden ? 'acteria and Microfungi 1?D et al",199<!" Lignin model compound studies demonstrated that monomericcompounds, i"e", $anillic acid and protocatechuic acid were formed,indicating the clea$age of aKb bonds, as well as demethylation ando&idation of a in the side chain to carbonyl group" ?"< ther 'acteria8on7lamentous bacteria usually minerali%e less than 14 of ligninpreparations and can degrade only the low-molecular weight part of ligninas well as degradation products of lignin (Gttimann et al", 19912 icuPa etal", 199?!" #hus, they may play some role in 7nal minerali%ation of lignin"Among these eubacteria, seudomonas spp" are the most ecient

degraders (icuPa, 19))2 Simmermann, 1994!" owe$er, since thesebacteria do not produce e&tracellular o&idoreductases, and large moleculesapparently cannot be ta.en up into the cell, they are ob$iously unable toattac. polymeric lignin" ?"? =oft-Got ungi and ther MicrofungiAscomycetes and deuteromycetes ( fungi imperfecti! generally cause soft-rot decay of wood ('lanchette, 199D2 +aniel and 8ilsson, 199)!" #hedecayed wood has a brown, soft appearance that is crac.ed and chec.edwhen dry" #wo forms of soft rot ha$e been described, type I consisting ofbiconical or cylindrical ca$ities that are formed within secondary walls whiletype II refers to an erosion form of degradation ('lanchette, 199D!" Incontrast to nonselecti$e white-rot fungi, the middle lamella is not attac.ed

by type II softrot fungi" Oylariaceous ascomycetes from genera such as+aldinia, ypo&ylon, and Oylaria ha$e earlier often been regarded as white-rot fungi, but nowadays these fungi are grouped to soft-rot fungi since theycause typical type II soft rot" #hey primarily occur on hardwood, and weightlosses up to D? of birch wood were found within < months by the mostecient fungus of this group, +aldinia concentrica (8ilsson et al", 19)9!" #hehighest lignin loss obser$ed was ** at the stage when weight loss was after * months incubation" ine wood was degraded, howe$er, $erylittle, only showing <"D weight loss (8ilsson et al", 19)9!" #he highconcentration of guaiacyl units in the middle lamella of coniferous woodmay cause the resistance to the decay by soft-rot fungi" Ligninolytic

pero&idases or laccases of softrot fungi may not ha$e the o&idati$e potentialto attac. the recalcitrant guaiacyl lignin" n the other hand, syringyl ligninapparently is readily o&idi%ed and minerali%ed by the en%ymes of soft-rotfungi (8ilsson et al", 19)9!" Hnfortunately, ligninolytic en%ymes of&ylariaceous ascomycetes are not well .nown" Microfungi or molds, i"e",deuteromycetes and certain ascomycetes that are usually thought todegrade mainly carbohydrates in soil, forest litter, and compost, can alsodegrade lignin in these en$ironments (Godrigue% et al", 199Ca2 Gegaldo etal", 1992 #uomela et al", <444!" #hus, some microfungi are able tominerali%e grass lignins up to < (aider and #ro6anows.i, 19)4!" Althoughactinomycetes were predominant among )< strains selected in a screening

for ligninolytic microorganisms in a forest soil, also some microfungi wereidenti7ed, e"g", enicillium chrysogenum, usarium o&ysporum, and



usarium solani (Godri:ue% et al", 199Ca!" In <) d, these fungi minerali%ed<"*, <?"D, and <<"C of a 1*-labeled lignin prepared from milledwheat straw" owe$er, lignin prepared from pine was much less degraded,and minerali%ation rate of less than ? was obtained" Another soilinhabitingmold, usarium proliferatum, min- 1?C D 'iodegradation of Lignin erali%ed

?"D of a 1*-(ring!-labeled + and 14 of 1*b-labeled + in ?4 d" #hedegradation was ma&imal during primary metabolism (Gegaldo et al", 199!"Laccase, aryl alcohol o&idase, and supero&ide radicals were detected inli:uid cultures of " proliferatum, but neither Mn nor Li were present(Gegaldo et al", 1999!" =upero&ide radicals that may generate highlyreacti$e hydro&yl radicals were found between days ? and ,i"e", during thehighest minerali%ation of lignin" In contrast, acti$ities of e&tracellular laccaseand aryl alcohol o&idase reached their ma&imum relati$ely late (Gegaldo etal", 1999!, and their role may be not so important in the minerali%ation oflignin" Laccase acti$ities ha$e also been described in " chrysogenum(Godrigue% et al", 199Cb!" #he fungus minerali%ed "9 of 1*b-+ in <9 d"

#he degradation of lignin has been studied in the ascomycete hrysoniliasitophila, red mold of breadF, which is the anamorph stage, while itsteleomorph stage is 8eurospora sitophila (Godrigue% et al", 199!" #hreeisoforms of Li were puri7ed and characteri%ed from this fungus but theproduction appeared not to be reproducible" henol o&idase acti$ity,probably laccase, was found to be stable, and the fungus produces thisacti$ity on media containing pine wood or lignosulfonate" Mn has not beenfound in this fungus"" sitophila caused <4 weight loss of pine wood in ?months, with the losses of carbohydrate and lignin being 1) and <D,respecti$ely" Analysis of the decayed lignin suggested that o&idati$e aKband b--aryl clea$ages occurred during lignin degradation" 3&tracellular

pero&idases and o&idases, e"g", laccase (ofrichter and ritsche, 199C2hefet% et al", 199)2 Li et al", 1999! are produced also by other microfungi"

#hey may not be so ecient in o&idi%ing lignin as those of white-rot fungi,but they may ha$e special properties" #hermoascus aurantiacus is athermophilic ascomycete that commonly grows in the heated parts of woodchip piles in =candina$ia ('ergman and 8ilsson, 191!" A strain of #"aurantiacus isolated from eucalyptus wood in 'ra%il (Machuca et al", 199D!shows unusual characteristics, i"e", it degrades e&tracti$es of 3ucalyptusgrandis and bleaches eucalyptus 0raft pulp" #his strain produces high le$elsof phenol o&idase that has optimal p $alues between <"C and ?"4, andoptimal temperature up to 4K)4 )" #he authors could not study higher

temperatures than )4 ), and it is possible that the true optimumtemperature is e$en higher (Machuca et al", 199)!" * 'rown-Got'asidiomycetes #he largest group of fungi that degrades wood is thebasidiomycetes" It has been calculated that in 8orth America there are1C44K144 species of wood-degrading basidiomycetes (>ilbertson, 19)4!"Geproduction of basidiomycetes usually occurs by haploid basidiospores andthus the primary mycelium de$eloped after spore germination is alsohaploid" +i.aryotic secondary mycelium arises as the result of hyphal fusionbetween di@erent primary mycelia" +i.aryotic mycelium can be identi7ed bythe presence of clamp connections at the septum (Ale&opoulos et al", 199C!"/ood-rotting basidiomycetous fungi are usually di$ided into white-rot and

brown-rot fungi" #hey are ta&onomically closely related, and white-rot andbrown-rot fungi can be foundin the same genera" Most wood rotters belong



to the orders Agaricales and Aphyllophorales (phylum 'asidiomycota!(Ale&opoulos et al", 199C!" 'rown-rot fungi mainly decompose the celluloseand hemicellulose components in wood, but they can also modify the ligninto a * 'rown-Got 'asidiomycetes 1? limited e&tent (3ri.sson et al", 1994!"

#hey ha$e been much less in$estigated than whiterot fungi in spite of their

enormous economic importance in the destruction of wood" 'rown-rottedwood is dar., shrin., and typically bro.en into bric.-shaped or cubicalfragments that easily brea. down into brown powder ('lanchette, 199D!"

#he brown color indicates the presence of modi7ed lignin in wood" Manybrown-rot fungi such as =erpula lacrymans, oniophora puteana, Meruliporiaincrassata, and >loeophyllum trabeum are destructi$e to wood used inbuildings and other structures ('lanchette, 199D!" " puteana and the so-called dry-rot fungus =" lacrymans, two of the most harmful fungi occurringin wood in temperate regions, prefer softwood to hardwood as substrates"

#he fungal hyphae penetrate from one cell to another through e&istingporesin wood cell walls early in the decay process" #he penetration starts

from the cell lumen where the hyphae are in close connection with the =?layer" In brown rot the decay process is thought to a@ect the =< layer of thewood cell wall 7rst (3ri.sson et al", 1994!" 'rown-rot fungi ha$e a uni:uemechanism to brea. down wood polysaccharides" In contrast to white-rotfungi that successi$ely depolymeri%e cell wall carbohydrates only to thee&tent that they utili%e hydrolysis products in fungal metabolism, brown-rotfungi rapidly depolymeri%e cellulose and hemicellulose, and degradationproducts accumulate since the fungus does not use all the products in themetabolism (owling, 19C1!" otential biotechnical applications of brown-rotfungi ha$e been studied occasionally, e"g", solid-state fermentation of pinesawdust for the production of cattle feed (Agosin et al", 19)9!, or the use of

brownrotted lignin for adhesi$es, e"g", to replace phenolKformaldehydeUa.eboard resin ( Qin et al", 1991!" 'rown-rotted lignin is more reacti$e thannati$e lignin due to the increased content of phenolic hydro&yl groups ( Qinet al", 1994b!" >" trabeum caused a signi7cant release of al.ali-soluble ligninespecially during the 7rst wee. of the growth on pine sawdust (Agosin et al",19)9!" #o some e&tent, brown-rot fungi ha$e similar degradati$e capabilitiesand pathways as white-rot fungi" 'oth wood decay mechanisms rely onradical formation, low p, and the production of organic acids" #hey causeincreased al.ali solubility of lignin, and the decay is enhanced by higho&ygen tension, all of which indicate a crucial in$ol$ement of radicals,especially in the early stages of decay (0ir., 19D2 Qin et al", 1994a!" #he

production of lignin pero&idase and manganese pero&idase has beendescribed in the brownrot fungus olyporus ostreiformis that remo$ed 1)"Cof the lignin from rice straw within ? wee.s (+ey et al", 199*!" Laccase gene-speci7c se:uences ha$e been detected in brown-rot fungi (+5=ou%a et al",199C!" Hsually, it has been assumed that all brown-rot fungi use the samemechanism in wood decay, and that the decay in$ol$es a enton-typecatalytic system producing hydro&yl radicals that attac. wood components"owe$er, analogously to di@erent groups of white-rot fungi using di@erentmechanisms in lignin degradation, brown-rot fungi also seem to possessclearly di@erent wood decay mechanisms" #he 7rst group could be>loeophyllum trabeum (syn" Len%ites trabea!-type fungi, and the second

group includes oniophora puteana and oria (ostia! placentatype fungi"Among brown-rot fungi, >" trabeum has been mostly studied" #he initiators



of both cellulose and lignin brea.down are suggested to be small molecularweight compounds that can readily di@use from the hyphae and penetrateinto the wood cell and start decay (3$ans et al", 199*2 /ood, 199*2 >oodellet al", 1992 =himada et al", 199!" All models suggested to e&plain brown-rot decay in$ol$e hydro&yl 1?) D 'iodegradation of Lignin radical

generation" owe$er, all the proposed models are not fully e&perimentally$eri7ed" Many of these low-molecular mass agents ha$e been isolated fromcultures of both white-rot and brown-rot fungi whichma.esit dicult tounderstand their speci7c role in brown-rot decay" #hese compounds may be,e"g", phenolates or other types of iron-chelating compounds (siderophores!,o&alate, and simple aromatic compounds (0oenigs, 19*2 e.ete et al",19)92 3spe6o and Agosin, 19912 Qellison et al", 19912 >oodell et al", 19923no.i et al", 1992 =himada et al", 199!" >" trabeum showed an unusualability to rapidly degrade an aliphatic polyether $ia e&tracellular one-electron o&idation (0erem et al", 199)!" Gecently, as%c%yns.i et al" (1999!found that >" trabeum produces simple aromatic compounds, *,D-

dimetho&ycatechol and <,D-dimetho&yhydro:uinone" #he authors suggestedthat these compounds may ser$e as ferric chelators, o&ygen-reducingagents, and redo&-cycling compounds" 0erem et al" (1999! ha$e alsoreported that cultures of >" trabeum produced <,D-dimetho&y-1,*-ben%o:uinone" ertain brown-rot fungi accumulate o&alic acid causing anoticeable decrease of p, but not >" trabeum, probably because of o&alatedecomposing en%ymes (=himada et al", 199!" It has been suggested thatthese brown-rot fungi may use o&alic acid as a proton donor for en%ymaticand nonen%ymatic hydrolysis of polysaccharides and as a chelator for ae( II !-<< system generating hydro&yl radicals (=himada et al", 199!" Inthe model of yde and /ood (199!, who studied oniophora puteana, it is

suggested that e( III ! is reduced by cellobiose dehydrogenase (+ !within the fungal cells, and that then the e( II ! di@uses at some distancefrom the hyphae, where a e( II !- o&alate comple& is formed and again,enton reaction-based hydro&yl radical formation occurs" #he radicalsformed by brown-rot fungi can remo$e metho&yl groups from lignin andproduce methanol, and thus they lea$e a residue that consists mainly ofmodi7ed lignin (3ri.sson et al", 19942 Qin et al", 1994a!" +emetho&ylation ofmetho&yl groups and aromatic hydro&ylation result in increased phenolichydro&yl groups, which ma.es lignin more reacti$e" #he presence ofcarbo&yl and carbonyl groups is also e$idenced by an increased o&ygencontent ( Qin et al", 1994b!" +a$is et al" (199*! used 1?-MA= 8MG

spectrometry and found that " placenta mainly remo$ed carbohydratecomponents from spruce wood, and hemicelluloses were remo$ed fasterthan cellulose, resembling thus the results obtained with >" trabeum (Agosinet al", 19)9!" " placenta further caused demetho&ylation of spruce lignin, anincrease in $anillin-li.e structures and a-carbonyl moieties, but there was noe$idence for ring opening (+a$is et al", 199*!" 'irch wood was degraded in adi@erent way, and the clea$age of lignin b--* lin.ages was the mostprominent reaction occurring, rather than demetho&ylation of lignin (+a$iset al", 199*!" #he presence of a wood en$ironment may ha$e a signi7cante@ect on the le$el of demetho&ylation of lignin and lignin model compoundsby brown-rot fungi" /hen >" trabeum was culti$ated under optimi%ed

conditions on pine wood Ua.es, the e$ol$ed 1*< from 1*-methylatedlignin was about ?4 in D< d ( Qin et al", 1994a!" #he presence of wood



stimulated the e$olution of 1*< from a nonphenolic (*-1*?!-labeledb-- * dimer by >" trabeum, and ?4 K C4 of the applied radioacti$ity wasreleased as 1*< within ) wee.s (8iemenmaa et al", 199<!" " placentaproduced 1*< from 1*?-labeled $anillic acid rather well, but poorlyfrom the b--* dimer, only ? as 1*< within * wee.s under similar

conditions (8iemenmaa et al", 199<!" * 'rown-Got 'asidiomycetes 1?9 D/hite-Got 'asidiomycetes #he only organisms capable of minerali%ing lignineciently are basidiomycetous whiterot fungi and related litter-decomposing fungi (0ir. and ullen, 199)!" /hite-rot fungi are aheterogeneous group of fungi classi7ed in the 'asidiomycota" +i@erentwhite-rot fungi $ary considerably in the relati$e rates at which they attac.lignin and carbohydrates in woody tissues" =ome remo$e lignin more readilythan carbohydrates, relati$e to the original amount of each ('lanchette,199D!" Many white-rot fungi coloni%e cell lumina and cause cell wall erosion"3roded %ones coalesce as decay progresses and large $oids 7lled withmycelium are formed" #his type of rot is referred to as nonselecti$e or

simultaneous rot ('lanchette, 199D!" #rametes (syn" oriolus, olyporus!$ersicolor is a typical simultaneous-rot fungus (owling, 19C12 3ri.sson etal", 1994!" =ome white-rot fungi preferentially remo$e lignin without asubstantial loss of cellulose, and cause white-poc.et or whitemottled type of rot, e"g", hellinus nigrolimitatus ('lanchette, 19)*b, 199D2 t6en and'lanchette, 19)!" #here are also fungi that are able to produce both typesof attac. in the same wood (3ri.sson et al", 1994!" #ypical e&amples of suchfungi are >anoderma applanatum and eterobasidion annosum" 'ecausefungi selecti$ely degrading lignin are considered the most promising fungifor applications in the pulp and paper industry, the search among thesefungi has attained a considerable interest" owe$er, the ratio

ligninKhemicelluloseKcellulose decayed by a selected fungus can di@erenormously, and e$en di@erent strains of the same species, e"g", ofhanerochaete chrysosporium and eriporiopsis sub$ermispora, maybeha$e di@erently on the same .ind of wood" ariations within a substrateare also found in one fungal strain (3ri.sson et al", 19942 'lanchette et al",199<!" =e$eral screening studies to 7nd suitable fungi for biopulping ofwood or straw, ha$e re$ealed fungi that, under certain conditions, degradelignin preferentially to cellulose" =uch lignin-selecti$e fungi are, e"g", "chrysosporium, " sub$ermispora (t6en and 'lanchette, 19)2 3ri.sson etal", 1994!, ycnoporus cinnabarinus (Ander and 3ri.sson, 19!, leurotusostreatus (MartV- ne% et al", 199*!, leurotus eryngii (MartVne% et al", 199*2

+orado et al", 1999!, hlebia radiata (Ander and 3ri.sson, 19!, hlebiatremellosus (syn" Merulius tremellosa! (Ander and 3ri.sson, 192 3ri.ssonet al", 1994!, hlebia subserialis (A.htar et al", 199)!, hellinus pini (3ri.ssonet al", 1994!, and +ichomitus s:ualens (3ri.sson et al", 1994!" #he lignin-degrading systems of these fungi are important to study since they are $eryecient" " sub$ermispora may be considered as a model fungus forselecti$e lignin degradation" alcium o&alate and Mn< accumulate whenthe decay proceeds ('lanchette, 19)*a, 199D2 3ri.sson et al", 1994!"Manganese pero&idase (Mn !is themostimportant ligninolytic en%ymeproduced by " sub$ermispora and many other lignin-selecti$e fungi and willbe discussed later" /hite-rot fungi are more commonly found on angiosperm

than on gymnosperm wood species in nature (>ilbertson, 19)4!" Hsuallysyringyl (= ! units of lignin are preferentially degraded whereas guaiacyl (> !



units are more resistant to degradation" /hen grown on straw, transmissionelectron microscopy re$ealed that " sub$ermispora and " eryngii partiallyremo$ed the middle lamella while hlebia radiata apparently remo$ed ligninfrom secondary cell walls ('urlat et al", 199)!" In 7bers, the middle lamellacontains a high concentration of > lignin while secondary walls contain a

high proportion of = lignin" 1*4 D 'iodegradation of Lignin 'asic research onlignin degradation, e"g", its mechanisms, physiology, en%ymology, andmolecular biology, has been mainly carried out with the corticioid fungus "chrysosporium (0ir. and arrell, 19)2 3ri.sson et al", 19942 >old and Alic,199?!" After more and ta&onomically di@erent fungi had been studied inmore detail, it was re$ealed that both the physiological conditions for lignindegradation and the en%yme systems e&pressed are fungus-speci7c anddi@er from those found in " chrysosporium" +i@erences may be connectedto the ta&onomic position andWor ecology of the fungi, e"g", substratespeciali%ation (hardwood, softwood, or certain wood species, heartwood orsapwood!, the stage of degradation, etc" or e&ample, the production of Mn

in poplar wood was signi7cant by >anoderma lucidum but the fungus didnot produce Mn in pine wood medium (+5=ou%a et al", 1999!" #he fungusob$iously has the genetic potential to produce Li since lip-li.e se:uenceswere found in =outhern hybridi%ation, but Li acti$ity could not be detected"Ligninolytic acti$ities and en%ymes of litter-decomposing basidiomycetousfungi ha$e been only $ery little studied" In a study with some fungi, e"g",from the genera =tropharia and Agrocybe, the minerali%ation of 1*-(ring!-labeled synthetic lignin (+ ! was about half of the le$el obtained withwood-inhabiting white-rot fungi (=te@en et al", 1999!" #he main ligninolyticen%ymes in litter-decomposing fungi such as Agaricus bisporus ('onnen etal", 199*! and =tropharia rugosoannulata (=te@en et al", 1999! are laccase

and Mn" In a recent study, laccase was found to be the only ligninolyticen%yme in Marasmius :uercophilus (+edeyan et al", <444!" D"1Minerali%ation of 1*-Labeled Lignins #he determination of 1*< e$olutionfrom 1*-+ (dehydrogenation polymer of coniferyl alcohol or other ligninprecursors! or other 1*-(lignin!-lignocelluloses has been the most oftenadopted method to determine lignin-degrading ability (aider and

#ro6anows.i, 19D2 0ir. et al", 19D, 19)!" #he method has been widelyused for more than <4 years ('uswell and dier, 19)2 0ir. and arrell,19)2 3ri.sson et al", 1994!" reparations from di@erent laboratories ha$egi$en comparable results (ata..a and Husi-Gau$a, 19)?2 ata..a et al",19)?!" 'uswell and dier (19)! compared di@erent nonradioacti$e and

1*-labeled lignin preparations for microbiological studies and concludedthat all lignin preparations ha$e disad$antages in reproducibility inpreparation, or altered structure and molecular weight compared to naturallignin" 8e$ertheless, the use of 1*labeled preparations can be consideredthe most reliable method" In addition, methods to study the degradation ofpolymeric lignin by, e"g", 8MG spectroscopy ha$e been de$eloped (+a$is etal", 199*2 >amble et al", 199*!, but they are not easily amenable fordetailed physiological studies with microorganisms or biochemical studieswith en%ymes" +imeric lignin model compounds attached to a polymerbac.bone, e"g", polystyrene (Lundell et al", 199<! or to more solublepolymers, e"g", polyethylene glycol (0awai et al", 199D!, ha$e allowed to use

ecient analytical tools (e"g", 8MG ! formore de7ned structures"=olubili%ation ( formation of water-soluble lignin fragments! and



minerali%ation (e$olution of 1*<! of 1*-labeled natural and syntheticlignins ha$e been demonstrated for $arious white-rot fungi (0ir. et al", 19D,19)2 ata..a and Husi-Gau$a, 19)?2 ata..a et al", 19)?2 'oyle et al",199<2ata..a, D /hite-Got 'asidiomycetes 1*1 199*2 ares et al", 199*2ares and ata..a, 1992 ofrichter et al", 1999b!" A high minerali%ation of

1*-(ring!-+ was obser$ed in the case of hlebia radiata that released upto 1 1*< from 1*-+ when grown in a li:uid medium (ata..a etal", 19)?!" #ypically, only $ery low amounts, in the range of ? of the 1*-label from +, were associated with the fungal biomass after *4 d ofculti$ation" =imilar results were obtained using natural 1*-labeled lignins,e"g", from 7r or oa. (D) K C1 minerali%ation, 1< K 1? 1* in themycelium! (Leatham, 19)C! or di@erent lignocelluloses (ata..a et al",19)?!" 8ematoloma frowardii caused e$en higher minerali%ation (D! of1*-+ during growth on wheat straw while again only a small percentageof the initial radioacti$ity (C! was incorporated into the residual straw andthe fungal biomass (ofrichter et al", 1999b!" Hsually only the end product,

1*<, is determined" #his may gi$e misleading results, since in some fungior under some conditions, the rate-limiting steps may be the reactions afterthe initial attac. on polymeric lignin" /ater-soluble degradation productsmay accumulate, e"g", under conditions of low o&ygen tension (ata..a andHusiGau$a, 19)?!" #he array of di@erent ligninolytic en%ymes may alsoinUuence the production of 1*<" D"< Ligninolytic 3n%ymes 3n%ymesystems for the degradation of macromolecular lignin face se$eralchallenges (0ir. and ullen, 199)!" #he substrate is a large heterogeneouspolymer that necessitates attac. by e&tracellular en%ymes or agents" Lignindoes not contain hydroly%able lin.ages, which means that the en%ymesmust be o&idati$e" Lignin is stereoirregular, which also points to more

nonspeci7c attac. compared to many other natural polymers" 3&tracellularen%ymes in$ol$ed in lignin degradation are lignin pero&idases (Lis,ligninasesF, 3 1"11"1"1*! and manganese pero&idases (Mns, Mn-dependent pero&idasesF, 3 1"11"1"1?!, as well as laccases(ben%enediolEo&ygen o&idoreductase, 3 1"14"?"<!" In addition, someaccessory en%ymes are in$ol$ed in hydrogen pero&ide production" >lyo&alo&idase (>LO ! and aryl alcohol o&idase (AA ! (3 1"1"?"! belong to thisgroup" #able 1 shows a general o$er$iew of the main cofactors or substratesand the principal e@ects or reactions of each en%yme" Lis and Mns areheme-containing glycoproteins which re:uire hydrogen pero&ide as ano&idant" Most fungi secrete se$eral isoen%ymes into their culti$ation

medium (#able <!" Li o&idi%es nonphenolic lignin substructures byabstracting one electron and generating cation radicals that are thendecomposed chemically (0ir. et al", 19)C2 0ir. and arrell, 19)! (igure 1!"Geactions of Li using a $ariety of lignin model compounds and syntheticlignin ha$e thoroughly been studied, the catalytic mechanisms elucidated,and its capability for aKb bond clea$age, ring opening, and otherreactions has been demonstrated (0ir. and arrell, 19)!" Mn o&idi%esMn( II ! to Mn( III ! which then o&idi%es phenolic rings to pheno&yl radicalswhich lead to decomposition of compounds (>old et al", 19)9! (igure <!"=tudies with di@erent white-rot fungi ha$e shown thatMnappears to bemorecommon than Li (rth et al", 199?2 ata..a, 199*2 ares and ata..a,

199!, and during the recent years the characteristics and role of Mn ha$ebeen e&tensi$ely studied" Mn has an essential role in the depolymeri%ation



of lignin (/ariishi et al", 1991! and chlorolignin (Lac.ner et al", 1991!, as wellas in 1*< D 'iodegradation of Lignin the demethylation of lignin andbleaching of pulp (aice et al", 199?!" Moreo$er, the en%yme mediates initialsteps in the degradation of high-molecular weight lignin (X- re% and

Qe@ries, 199<!" Gecently, it has been shown that Mn in the presence of

suitable organic acids is e$en able tominerali%e lignin and ligninmodelcompounds to considerable amounts (ofrichter et al", 1999a!" #able ?summari%es conditions and results of the main published in $itroe&periments, where Lis andMns ha$e been usedin themi&ture withdi@erent accessory compounds" Laccase is a copper-containing o&idase thatutili%es molecular o&ygen as o&idant and also o&idi%es phenolic rings topheno&yl radicals (#hurston, 199*! (igure <!" It has also a capacity too&idi%e nonphenolic compounds under certain conditions, e"g", if thereaction mi&ture is supplemented with A'#= ('ourbonnais and aice, 1994!or other mediating molecules (Li et al", 1999!" Most white-rot fungi typicallyproducelaccase (0BBri., 19CD2 'ollag and Leonowic%, 19)*!" hylogenetic

analysis of wood-rotting and other homobasidiomycetes fungi, based onribosomal +8A se:uences (ibbett et al", 199!, may gi$e new insights intothe relationships of di@erent ligninolytic systems" roduction of Li may betypical for woodrotting fungi, phylogenetically separated from euagaricsFsuch as leurotus ostreatus, Agaricus bisporus, and related fungi" 3uagaricFfungi typically produce Mns and laccase but in most cases truly lac. Li"owe$er, the system of " sub$ermispora, most probably belonging to thegroup of wood-rotting fungi, although not included in the analysis by ibbettet al" (199!, ob$iously contains lipli.e genes (Ga6a.umar et al", 199C!" #hesystem used by the white-rot fungus " cinnabarinus is possibly uni:ue sincethis fungus does not produce any pero&idases (3ggert et al", 199Cb!" Almost

all white-rot fungi produce Mns andlaccase, but only some fungi produceLi, the only en%yme capable of attac.ing nonphenolic lignin substructures"roduction of D /hite-Got 'asidiomycetes 1*? #ab" 1 3n%ymes in$ol$ed inthe degradation of lignin and their main reactions 3n%yme Acti$ity,Abbre$iation ofactor or =ubstrate, MediatorF Main 3@ect or GeactionLignin pero&idase, Li <<, $eratryl alcohol aromatic ring o&idi%ed to cationradical Manganese pero&idase, Mn <<, Mn, organic acid as chelator,thiols, unsaturated lipids Mn( II ! o&idi%ed to Mn( III !2 chelated Mn( III !o&idi%es phenolic compounds to pheno&yl radicals2 other reactions in thepresence of additional compounds Laccase, Lacc < 2 mediators, e"g",hydro&yben%otria%ole or A'#= phenols are o&idi%ed to pheno&yl radicals2

other reactions in the presence of mediators >lyo&al o&idase, >LO glyo&al,methyl glyo&al glyo&al o&idi%ed to glyo&ylic acid2 << production Arylalcohol o&idase, AA aromatic alcohols (anisyl, $eratryl alcohol! aromaticalcohols o&idi%ed to aldehydes2 << production ther << producingen%ymes many organic compounds < reduced to << 1** D'iodegradation of Lignin #ab" < Molecular masses and isoelectric points ofligninolytic en%ymes from selected white-rot and litterdecomposing fungi(the fungi may also produce other ligninolytic en%ymes, not listed here!ungus Molecular Masses Mr Y.+aZ Isoelectric oints YpIsZ GeferenceAbortiporus biennis MnsE ?)K*D (many isoforms! MnsE ?"9KC"D (manyisoforms! ares and ata..a (199! Agaricus bisporus MnsE 8+ laccaseE CD,

CC (straw! MnsE ?"?D, ?"<D, ?"? (straw compost! laccaseE 8+ 'onnen et al"(199*!, erry et al" (199?!, Leontie$s.y et al" (199b! Armillaria mellea



laccase IE C4, IIE C4KC< laccase IE *, IIE ?"? 'illal and #hurston (199C!'6er.andera sp" '=DD LisE (< iso%ymes! *4K*< MnsE **, *D 8+ MnsE?"*D, ?"*4 ten a$e et al" (199)!, alma et al" (<444! '6er.andera adustaLis (< isoen%ymes!E *), *) Mns (< isoen%ymes!E **, ** LisE ?"1, ?"< MnsE?"*D, ?"?D 0imura et al" (1994!, einUing et al" (199)a! eriporiopsis

sub$ermispora MnsE 8+ laccaseE 1, C) MnsE *"1-*"C (li:uid!, ?"<, ?"?, ?"*,?"D (wood! laccaseE ?"*, *") Lobos et al" (199*!, =alas et al" (199D!,u.ushima and 0ir. (199D! oprinus cinereus laccaseE D) (no Mn, no Li !laccaseE * ein%.ill et al" (199)!, =chneider et al" (1999! yathus stercoreusMnsE 8+ laccaseE 4 (no Li ! MnsE 8+ laccaseE ?"D, *") =ethuraman et al"(1999! +ichomitus s:ualens MnsE *), *)"9 laccaseE CC MnsE ?"9, *"1DlaccaseE ?"D, ?"C XriX et al" (199C, 199)! >anoderma lucidum laccasesE *4,CC laccasesE ?"4, *"<D, *"D, *"), D"1 +[=ou%a et al" (1999! eterobasidionannosum MnsE 8+ MnsE ?"?, ?"D, ?" Mai6ala et al" (<444! Qunghuhniaseparabilima LisE *?K* laccaseE D)KC< LisE ?"*K?"D laccasesE ?"*K?"Cares et al" (199<! Lentinula edodes MnsE 8+ laccaseE CC MnsE ?"<

laccaseE 8+ orrester et al" (1994! Marasmius :uercophilus laccaseE C? (noMn, no Li ! laccaseE ?"C +edeyan et al" (<444! 8ematoloma frowardiiMnsE *<-** (li:uid! D4 (straw! MnsE ?"1K*"4 (li:uid! ?"< (straw! =chneegabet al" (199!, ofrichter et al" (1999b! anus tigrinus MnsE *? laccaseE C*MnsE <"9D, ?"< laccaseE <"9K?"4 Maltse$a et al" (1991! hanerochaetechrysosporium '0M--1C (TA# <*<D! M3**C >141 LisE ?)K*?MnsE *C LisE ?"?K*" MnsE *"<K*"9 MnE *"9 (aspen pulp! arrell et al"(19)9!, >old et al" (19)9!, ease and #ien (199<!, +atta et al" (1991!hanerochaete chrysosporium M3**C laccaseE 144 +ittmer et al" (199!hanerochaete sordida MnsE *D (no Li, no laccase! MnsE ?"?, *"<, D"?Gttimann-Qohnson et al" (199*! hanerochaete Ua$ido-alba LisE *D MnsE

*D laccaseE 9* LisE \ ?"DD MnsE ?"DDKD")D laccaseE \ ?"DD 'en ammanet al" (1999!, Xre% et al" (199C! hlebia ochraceoful$a LisE ?)-*C (no Mn !8+ ares et al" (199?! hlebia radiata 9 (A# C*CD)! LisE ?9K** MnsE**K*) laccaseE C* LisE ?"<K*"1 MnsE ?"C, ?")D, *")D (li:uid culture!,?"?KD"9 (straw! laccaseE ?"D Lundell and ata..a (199*!, Moilanen et al"(199C!, ares et al" (199D!, Leontie$s.y et al" (199b! hlebia tremellosa<)*D (A# *)D*! LisE ?DK*4 MnsE acti$ity found, not puri7ed laccaseEC* LisE ?"1K*"4 MnsE 8+ laccaseE 8+ ares et al" (199*!, Leontie$s.y et al"(199b! leurotus eryngii laccaseE C1, CD MnsE *? (< isoen%ymes! laccaseE*"<, *"1 MnsE ?"CD, ?"D (< isoen%ymes! MuPo% et al" (199!, MartV- ne% etal" (199C! leurotus ostreatus MnsE *<-*D MnsE ?"D, ?", *"? (li:uid! ?"D,

?" (wood! 'ec.er and =initsyn (199?!, =ar.ar et al" (199! ycnoporuscinnabarinus laccaseE )1 (no Mn, no Li ! laccaseE ?" 3ggert et al" (199Cb!Gigidoporus lignosus MnsE *< laccaseE D? MnsE ?"D, ?" laccaseE ?")>alliano et al" (1991! #rametes hirsuta laccaseE C*KC) (<K? isoforms!laccaseE ?"K*"4 (<K? isoforms! ares and ata..a (199! #rametes trogiiLisE *1-** (< isoforms! LisE ?"K?") (< isoforms! ares and ata..a (199!ligninolytic en%ymes in many di@erent fungi, belonging to the family oforticiaceae (e"g", hanerochaete spp", hlebia spp" ! and olyporaceae(e"g", #rametes spp" and many other species! and the degradation of1*labeled lignin by the same fungi, show great $ariability (ata..a, 199*2ares and ata..a, 199!" 'ased on their main e&pressed e&tracellular

ligninolytic en%ymes, white-rot fungi can be classi7ed into three or four



groups (ata..a, 199*!" ungi from the 7rst group produce Lis, Mns, andlaccase, and

hongos enzimas

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