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5

Biodegradation of Lignin

Prof. Dr. Annele HatakkaUniversity of Helsinki, Viikki Biocenter, Department of Applied Chemistry andMicrobiology, P.O. Box 56, 00014 University of Helsinki, Finland; Tel. : �358-9-19159314, Fax: �358-9-19159322; E-mail: [email protected]

1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130

2 Historical Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131

3 Bacteria and Microfungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1353.1 Actinomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1353.2 Other Bacteria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1363.3 Soft-Rot Fungi and Other Microfungi . . . . . . . . . . . . . . . . . . . . . . . 136

4 Brown-Rot Basidiomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

5 White-Rot Basidiomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1405.1 Mineralization of 14C-Labeled Lignins . . . . . . . . . . . . . . . . . . . . . . . 1415.2 Ligninolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1425.2.1 Lignin Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1475.2.2 Manganese Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1515.2.3 Laccase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1575.2.4 Other Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

6 Catabolism of Primary Degradation Products . . . . . . . . . . . . . . . . . . . 162

7 Outlook and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163

8 Patents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166

9 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167

129

AAO aryl alcohol oxidaseABTS 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonate)CDH cellobiose dehydrogenaseCBQ cellobiose quinone oxidoreductaseCcP cytochrome c peroxidaseCP-MAS cross polarization/magic angle spinning3-D three dimensionalDHP dehydrogenation polymerizate, synthetic ligninEDTA ethylenediaminetetraacetic acidEPR electron paramagnetic resonanceFAD flavin adenine dinucleotideG guaiacylGLOX glyoxal oxidaseGPC gel permeation chromatographyGS guaiacyl-syringylGSH glutathione3-HAA 3-hydroxyanthranilateHBT 1-hydroxybenzotriazoleHP-SEC high performance size exclusion chromatographyLiP lignin peroxidaseNAD nicotinamide adenine dinucleotideNADP nicotinamide adenine dinucleotide phosphateNMR nuclear magnetic resonanceMHQ methoxyhydroquinoneMnP manganese peroxidasePAHs polycyclic aromatic hydrocarbonsQR quinone reductaseS syringylTHB 1,2,4-trihydroxybenzeneVA veratryl alcoholVH vanillate hydroxylase

1

Introduction

Lignin is an amorphic three-dimensionalsubstance the molecular weight of which isdifficult to determine because lignins arehighly polydisperse materials (Argyropoulosand Menachem, 1997). The chemical struc-ture of lignin has also been difficult todetermine, and even very recently, newbonding patterns have been described insoftwood lignin, e.g,. dibenzodioxocin struc-

tures. Also, the isolation of native lignin iscomplicated if possible at all (Buswell andOdier, 1987). Therefore, lignin model com-pounds, e.g., dimeric b-O-4 model com-pounds and synthetic lignin (dehydrogen-ation polymerizate, DHP), are commonlyused in microbiological studies. The resultsobtained using these substrates are not easyto extrapolate to natural conditions, and thuslittle is known what happens when micro-organisms, such as white-rot fungi, degradelignin in wood.

5 Biodegradation of Lignin130

Wood-rotting basidiomycetous fungi thatcause white rot in wood are the most efficientlignin degraders in nature (Kirk and Farrell,1987; Eriksson et al., 1990), and they areperhaps nature's major agents for recyclingthe carbon of lignified tissues. No othermicroorganisms as pure culture have beendescribed to mineralize lignified tissues asefficiently (Kirk and Cullen, 1998). They are agroup of taxonomically heterogeneous high-er fungi, characterized by their unique abilityto depolymerize and mineralize lignin usinga set of extracellular ligninolytic enzymes.Lignin degradation by white-rot fungi hasbeen intensively studied during the last thirtyyears in relation to biotechnical applicationssuch as biopulping, biobleaching, treating ofpulp mill effluents, and soil bioremediation(Akhtar et al. , 1992, 1998; Lamar et al., 1992;Messner and Srebotnik, 1994). The enzymol-ogy and molecular biology of lignin degrada-tion has been mainly studied in Phanero-chaete chrysosporium (Gold and Alic, 1993;Cullen, 1997; Kirk and Cullen, 1998). How-ever, many other species of white-rot fungidegrade lignin as efficiently as P. chrysospo-rium (Hatakka, 1994). Moreover, severalfungi show better selectivity for lignin re-moval (Eriksson et al. , 1990; Blanchetteet al. , 1992; Messner and Srebotnik, 1994).

Physiological conditions for lignin degra-dation, as well as secretion patterns of theligninolytic enzymes, vary between differentfungal species (Hatakka, 1994). Variousauthors have tried to establish correlationsbetween ligninolytic enzymes and lignindegradation (Käärik, 1965; Ander and Eriks-son, 1976, 1977; Eriksson et al. , 1990; Ha-takka, 1994). However, many of the enzymesnecessary for lignin degradation were notcharacterized before the beginning of the1980s when virtually only laccase had beenknown. Since the discovery of two importantperoxidases in the beginning of the 1980s,namely lignin peroxidases (LiPs) in 1983 and

manganese peroxidases (MnPs) in 1984(Kirk and Farrell, 1987), an array of enzymeshave been isolated from fungi and charac-terized in detail. Although basidiomycetouswhite-rot fungi and related litter-decompos-ing fungi are the most efficient degraders oflignin, mixed cultures of fungi, actinomy-cetes, and bacteria in soil and compost canalso mineralize lignin (Tuomela et al., 2000).These soil-inhabiting microorganisms mayhave applications in bioremediation, andtheir enzymes may have interesting proper-ties, e.g., high thermostability and nearlyneutral pH optimum, not usually found inthe enzymes of wood-inhabiting fungi.

In this review, the focus is on the presentknowledge on lignin degradation undernatural or nearly natural conditions, i.e., onwood or straw, and in the enzymology oflignin biodegradation, including mineraliza-tion of lignin by ligninolytic enzymes in vitro.In addition, the possible involvement ofnonenzymatic agents in the fungal ligninbiodegradation process is discussed.

2

Historical Outline

Lignin degradation is in a central position inthe earth's carbon cycle, because most renew-able carbon is either in lignin or in com-pounds protected by lignin from enzymaticdegradation (cellulose and hemicellulose)(Kirk, 1983). Lignin biodegradation is alsoresponsible for much of the natural destruc-tion of wood in use, and it may have animportant role in plant pathogenesis. On theother hand, potential applications utilizinglignin-degrading organisms and their en-zymes have become attractive, because theymay provide environmentally friendly tech-nologies for the pulp and paper industry andfor the treatment of many xenobiotic com-pounds, stains, and dyes. Despite its signifi-

2 Historical Outline 131

cance, lignin biodegradation has only slowlybeen defined chemically and biochemically.One of the main reasons for that was the poorknowledge of the chemical structure of ligninuntil the late 1960s when it became betterknown (Kirk, 1971, 1983; Adler, 1977;Eriksson et al., 1990). The scheme forstructural features of conifer lignin present-ed by Adler (1977) has been commonly usedin the literature, and only recently newstructures have been discovered (Argyropou-los and Menachem, 1997; Chapter 3, thisvolume). Successful studies on the biodegra-dation of lignin require a good cooperationbetween microbiologists, biochemists, andchemists, and relatively expensive equip-ment and materials, e.g. , 14C-labeled ligninsor lignin model compounds that have notbeen commercially available. Lignin biode-gradation was also considered an unusualbiological process involving extracellularoxidations.

Prior to the 1920s, little research wasconducted on lignin biodegradation (Kirk,1983). Some findings, summarized in the1930s (for reviews, see Kirk, 1983), are stilltoday valid:

1) lignin is among the plant cell wall poly-mers the most resistant to biologicaldegradation, although it is degraded,

2) white-rot fungi degrade lignin in wood,and

3) completely selective removal of lignin(without concomitant removal of woodcarbohydrates) had not been observed.

Waksman et al. (1939) had studied lignindegradation, e.g., in compost and soil envi-ronment (reviewed by Tuomela et al. , 2000).Gottlieb and Pelczar (1951) in their reviewreported that the white-rot fungus Polyporus(syn. Trametes) versicolor used Brauns' nativeligninº as the growth substrate. Although thelignin structure is unaltered due to the mild

procedure, the preparation has a relativelylow molecular weight. This finding, indicat-ing that lignin could be used as a sole carbonand energy source for white-rot fungi, has notbeen verified. Also, many other faulty tech-niques had been used in other studies. In the1950s in addition to white-rot fungi, othergroups of fungi were found to degrade lignin,at least partially, namely basidiomycetouslitter-decomposing and brown-rot fungi aswell as soft-rot fungi (reviewed by Kirk, 1971,1983). The first lignin model compoundstudies were published in the 1960s (Russellet al. , 1961; Ishikawa et al. , 1963; Kirk et al.,1968; Fukuzumi et al. , 1969).

Biodegradation assays based on 14C-lig-nins were developed in the 1970s (Haiderand Trojanowski, 1975; Kirk et al. , 1975,1978), and using this techniques, calledradiorespirometry, it was revealed how ligninwas optimally degraded under laboratoryconditions by white-rot fungi. The white-rotfungus Phanerochaete chrysosporium wasused as the main experimental organism inUSA, while in some other laboratories, theanamorph of the same fungus, Sporotrichumpulverulentum, had been chosen for ligninbiodegradation studies (Ander and Eriksson,1976; Ander et al. , 1980). Before that,Trametes versicolor was a popular experimen-tal fungus (Cowling, 1961; Russell et al.,1961).

In the late 1970s and in the beginning ofthe 1980s many important findings in the phy-siology of lignin degradation by P. chrysospo-rium were made. The experiments werecarried out almost only in synthetic liquidmedia and using 14C-labeled synthetic lignin(DHP) (Kirk et al. , 1975, 1978). The mostimportant discoveries can be listed as fol-lows:

1) the effect of nutrient nitrogen, showingthat low nitrogen was required for lignindegradation, and indicating that the min-


eralization of lignin occurred duringsecondary metabolism,

2) the effect of atmosphere, 100% oxygengiving the highest mineralization, thusdemonstrating that lignin degradation isoxidative,

3) the detrimental effect of agitation inlignin mineralization, and

4) the concomitant production of veratrylalcohol during lignin degradation.

Some other fungi, e.g,. Phlebia radiata (Ha-takka and Uusi-Rauva, 1983; Hatakka et al.,1983, 1991) were also found to readilydegrade lignin and lignin model compoundsin a similar way.

Radical chemistry is an essential part oflignin biodegradation. Before the finding oflignin peroxidases, it was assumed that thegeneration of H2O2 and other easily diffusibleactivated oxygen species, such as hydroxylradicals (OH.), superoxide anion radical(O2

.ÿ) , and singlet oxygen (1O2) might beresponsible for fungal decay of lignin andlignocellulose (Hall, 1980). The involvementof hydroxyl radicals and singlet oxygen wasdiscounted later (Kirk and Farrell, 1987).

A breakthrough in the enzymology oflignin biodegradation occurred in 1983 ±1984 when the first extracellular enzymesinvolved in the degradation of lignin werediscovered (Tien and Kirk, 1983; Glennet al. , 1983; Kuwahara et al., 1984). Sincethat large amounts of studies on the bio-chemistry and molecular biology of theseenzymes have been published, but they wereagain strongly concentrated on the enzymesof one fungus, P. chrysosporium. The catalyticmechanism of lignin peroxidase, based oninitial one-electron oxidation of the ligninmodel compounds followed by subsequentbreakdown reactions via radical cation inter-mediates was proposed and experimentallyverified (Kersten et al., 1985; Schoemakeret al. , 1985; Kirk et al. , 1986). Numerous

publications describing the effect of lignino-lytic enzymes on lignin model compoundsalso appeared and were frequently reviewed(Higuchi, 1985; Buswell and Odier, 1987;Kirk and Farrell, 1987).

In the 1990s, in addition to detailed studieson catalytic and enzymatic properties of thelignin-modifying peroxidases as well as theirmolecular biology, major lines of researchhave dealt with the potential applications ofwhite-rot fungi and their enzymes in bio-pulping (biomechanical pulping) and pulpbleaching. The most promising fungi forbiopulping are so-called selective lignin de-graders, i.e. , fungi that degrade largeramounts of lignin relative to carbohydrates(Eriksson et al. , 1990).

Because P. chrysosporium typically pro-duces lignin peroxidases (LiPs) along withmanganese peroxidases (MnPs), LiP wasconsidered the most important lignin-de-grading enzyme. However, the optimal con-ditions for lignin degradation and expressionof LiP did not seem to be appropriate formany other fungi, and especially, the mostefficient selective lignin degraders apparent-ly did not degrade lignin under these artificialconditions. Moreover, some of these fungidid not even produce LiP at all, e.g., the mostpromising fungus for biopulping, Ceripori-opsis subvermispora, only produces MnP andlaccase. When many different fungi had beenstudied in detail, it became clear that MnP isthe most commonly occurring peroxidasewhile it was difficult to demonstrate theexpression of LiP (Hatakka, 1994).

The role of laccase, a phenol-oxidizingenzyme typically produced by almost alllignin-degrading basidiomycetous white-rotfungi (Käärik, 1965), but importantly not soby the model fungus P. chrysosporium, wasalmost totally ignored until the beginning ofthe 1990s when the efficiency of so-calledªmediatorº compounds was recognized.These compounds, e.g. , 2,2'-azinobis(3-

2 Historical Outline 133

ethylbenzthiazoline-6-sulfonate) (ABTS) or 1-hydroxybenzotriazole (HBT) (Bourbonnaisand Paice, 1990; Call and Mücke, 1997),being readily oxidizable primary substrates,extend laccase substrate range to the mostrecalcitrant nonphenolic subunits of lignin.Recombinant laccases can be relatively easilyproduced in common industrial host organ-isms, e.g., in Trichoderma reesei (Saloheimoand Niku-Paavola, 1991) or in Aspergillusoryzae (Stewart et al. , 1996), and therefore,the prospects of the use of laccase in bleach-ing of pulp, textiles, and dyes, and inbioremediation of environmental pollutantssuch as polycyclic aromatic hydrocarbons(PAHs) are realistic. Shortly after the discov-ery of these synthetic mediators, a report on anatural mediator, 3-hydroxyanthranilate (3-HAA), was published by Eggert et al.(1996a). These findings can be consideredanother breakthrough in the enzymology oflignin degradation. Due to the low appreci-ation of laccase as a lignin-degrading en-zyme, the first 3-D structure of this enzyme,although the enzyme was first described asearly as in the 1880s (Yoshida, 1883; Thurs-ton, 1994) was solved only very recently(Ducros et al., 1998).

The developments before 1983, i.e., beforeªligninaseº was found, have been summar-ized by Kirk (1983). After that some of themain events and achievements may be listedas follows:

. ligninase (lignin peroxidase, LiP) discov-ered (Tien and Kirk, 1983; Glenn et al.,1983)

. manganese peroxidase (MnP) discovered(Kuwahara et al., 1984)

. one-electron oxidation mechanism andcation radical formation by LiP discovered(Kersten et al. , 1985; Schoemaker et al.,1985)

. glyoxal oxidase (GLOX) discovered (Kers-ten and Kirk, 1987)

. the concept of ªenzymatic combustionº(Kirk and Farrell, 1987)

. molecular biological studies (earlier stud-ies reviewed by Gold and Alic, 1993)± cloning and sequencing of LiP gene

(Tien and Tu, 1987)± cloning and sequencing of MnP gene

(Pribnow et al., 1989; Mayfield et al.,1994a)

± heterologous expression of laccase(Saloheimo and Niku-Paavola, 1991)

± homologous expression of peroxidases(Mayfield et al., 1994b; Sollewijn Gelpkeet al. , 1999)

± heterologous expression of peroxidases(Doyle and Smith, 1996; Stewart et al.,1996)

± 3-D structure of LiP (Piontek et al.,1993; Poulos et al. 1993)

± 3-D structure of MnP (Sundaramoorthyet al. , 1994)

± 3-D structure of laccase (Ducros et al.,1998)

± detailed structure of MnP±LiP hybridenzyme (Ruiz-Dueæas et al., 1999a)

. different ligninolytic systems in fungirecognized (reviewed by Hatakka, 1994)

. the laccase-mediator system discovered(Bourbonnais and Paice, 1990; Call andMücke, 1997)

. a natural laccase mediator discovered(Eggert et al. , 1996a)

. the separation of LiP from lignocellulosemedium (Vares et al., 1995)

. the involvement of lipid peroxidation inlignin degradation proposed (Kapich andShishkina, 1995; Jensen et al. , 1996; Ka-pich et al. , 1999a, b)

. cell-free mineralization of 14C-labeled syn-thetic and natural lignins by MnP (Hof-richter et al., 1999a, b, c).


3

Bacteria and Microfungi

To the wood-rotting microorganisms, woodmay be considered as a series of convenientlyorientated holes surrounded by food. Theshape, size, and orientation of the holesdepend directly on the anatomy of woodspecies, and the chemical composition of thecell walls or the cell contents. These have alleffects on the particular microorganism touse them as a nutrient source (Blanchette,1995). Softwood (gymnosperms) is formedlargely by tracheids, the cell lumen beingcompletely enclosed by a cell wall. Thereforemicroorganisms must pass through the cellwall or pit membrane. Hardwood (angio-sperms) consists of vessels, fibers, andparenchyma cells as well as some tracheids,and here the vessels are formed from open-ended vessel elements. The fungal hyphae orbacteria may thus move also without passingthrough the cell wall or pit membrane. Thehemicellulose as well as lignin componentsdiffer markedly in softwood and hardwoodspecies. Softwood contains mainly gluco-mannans as hemicellulose and guaiacyl (G)type lignin, while hardwood contains glucur-onoxylans and guaiacyl±syringyl (GS) typelignin. Sapwood contains accumulated nu-trients such as starch or lipids stored by theray parenchyma cells in addition to extrac-tives, while heartwood contains more anddifferent extractives (pitch) (Sjöström,1993).

Chemically and morphologically distincttypes of decay result from attack of differentmicroorganisms. Wood decay fungi are usu-ally separated into three main groups, caus-ing white, soft, or brown rot. Bacterialdegradation of wood has also been reportedincluding erosion, tunneling, and cavityformation (Eriksson et al. , 1990; Blanchette,1995; Daniel and Nilsson, 1998).

3.1

Actinomycetes

Certain bacteria can degrade lignified cellwalls of wood. Filamentous bacteria belong-ing to the genus Streptomyces are well-knowndegraders of lignin and can mineralize up to15% of labeled lignins but usually much less(Crawford and Sutherland, 1980; Vicuæa,1988; Zimmerman, 1990; Godden et al.,1992; Berrocal et al., 1997). Typically, Strep-tomyces spp. solubilize part of lignin, and theend product is water-soluble acid-precipita-ble polymeric lignin (Crawford et al. , 1983).Berrocal et al. (1997) compared mineraliza-tion of 14C-(lignocellulose)-wheat straw by 9Streptomyces spp. Streptomyces cyaneus CECT3335 was one of the most efficient newisolates in solubilizing 45% of the 14C-(lignin) fraction and mineralizing 3% of thelabel in 21 d. However, the reference speciesS. viridosporus solubilized 59% and mineral-ized 4.5% of the same substrate. The pres-ence of phenol oxidase and peroxidase wasfound in S. cyaneus, the former activity being100 times greater as determined by theoxidation of ABTS and 2,4-dichlorophenol,respectively. S. cyaneus degraded a nonphe-nolic arylglycerol b-aryl ether by a mecha-nism indicating the cleavage of Ca±Cb bondin lignin (Zimmerman et al. , 1988).

Actinomycetes produce extracellular per-oxidases (Pasti et al., 1991; Mercer et al.,1996), e.g., lignin peroxidase-type enzyme(Ramachandra et al., 1988; Adhi et al.,1989). Streptomyces sp. EC1 produces perox-idase and cell-bound demethylase requiringH2O2 and Mn2�, both of which are producedat relatively high levels in the presence ofKraft lignin or wheat straw, while protocha-techuate 3,4-dioxygenase and b-carboxymuc-onate decarboxylase activity were less in-duced (Godden et al. , 1992). Extracellularperoxidase and catalase activity was found inall six actinomycetes strains studied (Godden

3 Bacteria and Microfungi 135

et al. , 1992). Lignin model compound stud-ies demonstrated that monomeric com-pounds, i.e., vanillic acid and protocatechuicacid were formed, indicating the cleavage ofCa±Cb bonds, as well as demethylation andoxidation of Ca in the side chain to carbonylgroup.

3.2

Other Bacteria

Nonfilamentous bacteria usually mineralizeless than 10% of lignin preparations and candegrade only the low-molecular weight partof lignin as well as degradation products oflignin (Rüttimann et al. , 1991; Vicuæa et al.,1993). Thus, they may play some role in finalmineralization of lignin. Among these eu-bacteria, Pseudomonas spp. are the mostefficient degraders (Vicuæa, 1988; Zimmer-mann, 1990). However, since these bacteriado not produce extracellular oxidoreductases,and large molecules apparently cannot betaken up into the cell, they are obviouslyunable to attack polymeric lignin.

3.3

Soft-Rot Fungi and Other Microfungi

Ascomycetes and deuteromycetes (fungiimperfecti) generally cause soft-rot decay ofwood (Blanchette, 1995; Daniel and Nilsson,1998). The decayed wood has a brown, softappearance that is cracked and checked whendry. Two forms of soft rot have been de-scribed, type I consisting of biconical or cylin-drical cavities that are formed within secon-dary walls while type II refers to an erosionform of degradation (Blanchette, 1995). Incontrast to nonselective white-rot fungi, themiddle lamella is not attacked by type II soft-rot fungi. Xylariaceous ascomycetes fromgenera such as Daldinia, Hypoxylon, andXylaria have earlier often been regarded aswhite-rot fungi, but nowadays these fungi are

grouped to soft-rot fungi since they causetypical type II soft rot. They primarily occuron hardwood, and weight losses up to 53% ofbirch wood were found within 2 months bythe most efficient fungus of this group,Daldinia concentrica (Nilsson et al. , 1989).The highest lignin loss observed was 44% atthe stage when weight loss was 77% after 4months incubation. Pine wood was degrad-ed, however, very little, only showing 2.5%weight loss (Nilsson et al. , 1989). The highconcentration of guaiacyl units in the middlelamella of coniferous wood may cause theresistance to the decay by soft-rot fungi.Ligninolytic peroxidases or laccases of soft-rot fungi may not have the oxidative potentialto attack the recalcitrant guaiacyl lignin. Onthe other hand, syringyl lignin apparently isreadily oxidized and mineralized by theenzymes of soft-rot fungi (Nilsson et al.,1989). Unfortunately, ligninolytic enzymesof xylariaceous ascomycetes are not wellknown.

Microfungi or molds, i.e., deuteromycetesand certain ascomycetes that are usuallythought to degrade mainly carbohydrates insoil, forest litter, and compost, can alsodegrade lignin in these environments (Rod-riguez et al. , 1996a; Regaldo et al. , 1997;Tuomela et al., 2000). Thus, some micro-fungi are able to mineralize grass lignins upto 27% (Haider and Trojanowski, 1980).Although actinomycetes were predominantamong 82 strains selected in a screening forligninolytic microorganisms in a forest soil,also some microfungi were identified, e.g.,Penicillium chrysogenum, Fusarium oxyspo-rum, and Fusarium solani (Rodriquez et al.,1996a). In 28 d, these fungi mineralized27.4%, 23.5%, and 22.6% of a 14C-labeledlignin prepared from milled wheat straw.However, lignin prepared from pine wasmuch less degraded, and mineralization rateof less than 3% was obtained. Another soil-inhabiting mold, Fusarium proliferatum, min-


eralized 3.5% of a 14C-(ring)-labeled DHPand 10% of 14Cb-labeled DHP in 30 d. Thedegradation was maximal during primarymetabolism (Regaldo et al. , 1997). Laccase,aryl alcohol oxidase, and superoxide radicalswere detected in liquid cultures of F. prolife-ratum, but neither MnP nor LiP were present(Regaldo et al. , 1999). Superoxide radicalsthat may generate highly reactive hydroxylradicals were found between days 3 and 7, i.e.,during the highest mineralization of lignin.In contrast, activities of extracellular laccaseand aryl alcohol oxidase reached their max-imum relatively late (Regaldo et al., 1999),and their role may be not so important in themineralization of lignin. Laccase activitieshave also been described in P. chrysogenum(Rodriguez et al. , 1996b). The fungus min-eralized 7.9% of 14Cb-DHP in 29 d.

The degradation of lignin has been studiedin the ascomycete Chrysonilia sitophila, ªredmold of breadº, which is the anamorph stage,while its teleomorph stage is Neurosporasitophila (Rodriguez et al. , 1997). Three iso-forms of LiP were purified and characterizedfrom this fungus but the production ap-peared not to be reproducible. Phenol oxidaseactivity, probably laccase, was found to bestable, and the fungus produces this activityon media containing pine wood or lignosul-fonate. MnP has not been found in thisfungus. C. sitophila caused 20% weight loss ofpine wood in 3 months, with the losses ofcarbohydrate and lignin being 18% and 25%,respectively. Analysis of the decayed ligninsuggested that oxidative Ca±Cb and b-O-arylcleavages occurred during lignin degrada-tion.

Extracellular peroxidases and oxidases,e.g., laccase (Hofrichter and Fritsche, 1996;Chefetz et al., 1998; Li et al. , 1999) areproduced also by other microfungi. Theymay not be so efficient in oxidizing lignin asthose of white-rot fungi, but they may havespecial properties. Thermoascus aurantiacus

is a thermophilic ascomycete that commonlygrows in the heated parts of wood chip pilesin Scandinavia (Bergman and Nilsson,1971). A strain of T. aurantiacus isolatedfrom eucalyptus wood in Brazil (Machucaet al. , 1995) shows unusual characteristics,i.e. , it degrades extractives of Eucalyptusgrandis and bleaches eucalyptus Kraft pulp.This strain produces high levels of phenoloxidase that has optimal pH values between2.6 and 3.0, and optimal temperature up to70±80 8C. The authors could not study highertemperatures than 80 8C, and it is possiblethat the true optimum temperature is evenhigher (Machuca et al. , 1998).

4

Brown-Rot Basidiomycetes

The largest group of fungi that degradeswood is the basidiomycetes. It has beencalculated that in North America there are1600±1700 species of wood-degrading basi-diomycetes (Gilbertson, 1980). Reproduc-tion of basidiomycetes usually occurs byhaploid basidiospores and thus the primarymycelium developed after spore germinationis also haploid. Dikaryotic secondary myce-lium arises as the result of hyphal fusionbetween different primary mycelia. Dikary-otic mycelium can be identified by thepresence of clamp connections at the septum(Alexopoulos et al. , 1996). Wood-rotting ba-sidiomycetous fungi are usually divided intowhite-rot and brown-rot fungi. They aretaxonomically closely related, and white-rotand brown-rot fungi can be found in the samegenera. Most wood rotters belong to theorders Agaricales and Aphyllophorales (phy-lum Basidiomycota) (Alexopoulos et al.,1996).

Brown-rot fungi mainly decompose thecellulose and hemicellulose components inwood, but they can also modify the lignin to a

4 Brown-Rot Basidiomycetes 137

limited extent (Eriksson et al. , 1990). Theyhave been much less investigated than white-rot fungi in spite of their enormous economicimportance in the destruction of wood.Brown-rotted wood is dark, shrink, andtypically broken into brick-shaped or cubicalfragments that easily break down into brownpowder (Blanchette, 1995). The brown colorindicates the presence of modified lignin inwood. Many brown-rot fungi such as Serpulalacrymans, Coniophora puteana, Meruliporiaincrassata, and Gloeophyllum trabeum aredestructive to wood used in buildings andother structures (Blanchette, 1995). C. pu-teana and the so-called dry-rot fungus S.lacrymans, two of the most harmful fungioccurring in wood in temperate regions,prefer softwood to hardwood as substrates.

The fungal hyphae penetrate from one cellto another through existing pores in wood cellwalls early in the decay process. The pene-tration starts from the cell lumen where thehyphae are in close connection with the S3

layer. In brown rot the decay process isthought to affect the S2 layer of the wood cellwall first (Eriksson et al. , 1990). Brown-rotfungi have a unique mechanism to breakdown wood polysaccharides. In contrast towhite-rot fungi that successively depolymer-ize cell wall carbohydrates only to the extentthat they utilize hydrolysis products in fungalmetabolism, brown-rot fungi rapidly depoly-merize cellulose and hemicellulose, anddegradation products accumulate since thefungus does not use all the products in themetabolism (Cowling, 1961).

Potential biotechnical applications ofbrown-rot fungi have been studied occasion-ally, e.g. , solid-state fermentation of pinesawdust for the production of cattle feed(Agosin et al. , 1989), or the use of brown-rotted lignin for adhesives, e.g. , to replacephenol±formaldehyde flakeboard resin (Jinet al. , 1991). Brown-rotted lignin is morereactive than native lignin due to the in-

creased content of phenolic hydroxyl groups(Jin et al. , 1990b). G. trabeum caused asignificant release of alkali-soluble ligninespecially during the first week of the growthon pine sawdust (Agosin et al. , 1989).

To some extent, brown-rot fungi havesimilar degradative capabilities and path-ways as white-rot fungi. Both wood decaymechanisms rely on radical formation, lowpH, and the production of organic acids. Theycause increased alkali solubility of lignin, andthe decay is enhanced by high oxygen tension,all of which indicate a crucial involvement ofradicals, especially in the early stages of decay(Kirk, 1975; Jin et al., 1990a). The produc-tion of lignin peroxidase and manganeseperoxidase has been described in the brown-rot fungus Polyporus ostreiformis that re-moved 18.6% of the lignin from rice strawwithin 3 weeks (Dey et al., 1994). Laccasegene-specific sequences have been detectedin brown-rot fungi (D'Souza et al. , 1996).

Usually, it has been assumed that allbrown-rot fungi use the same mechanismin wood decay, and that the decay involves aFenton-type catalytic system producing hy-droxyl radicals that attack wood components.However, analogously to different groups ofwhite-rot fungi using different mechanismsin lignin degradation, brown-rot fungi alsoseem to possess clearly different wood decaymechanisms. The first group could be Gloeo-phyllum trabeum (syn. Lenzites trabea)-typefungi, and the second group includes Co-niophora puteana and Poria (Postia) placenta-type fungi. Among brown-rot fungi, G.trabeum has been mostly studied.

The initiators of both cellulose and ligninbreakdown are suggested to be small molec-ular weight compounds that can readilydiffuse from the hyphae and penetrate intothe wood cell and start decay (Evans et al.,1994; Wood, 1994; Goodell et al. , 1997;Shimada et al. , 1997). All models suggestedto explain brown-rot decay involve hydroxyl


radical generation. However, all the proposedmodels are not fully experimentally verified.Many of these low-molecular mass agentshave been isolated from cultures of bothwhite-rot and brown-rot fungi which makes itdifficult to understand their specific role inbrown-rot decay. These compounds may be,e.g., phenolates or other types of iron-chelat-ing compounds (siderophores), oxalate, andsimple aromatic compounds (Koenigs, 1974;Fekete et al. , 1989; Espejo and Agosin, 1991;Jellison et al. , 1991; Goodell et al. , 1997;Enoki et al. , 1997; Shimada et al. , 1997).

G. trabeum showed an unusual ability torapidly degrade an aliphatic polyether viaextracellular one-electron oxidation (Keremet al. , 1998). Recently, Paszczynski et al.(1999) found that G. trabeum producessimple aromatic compounds, 4,5-dimethoxy-catechol and 2,5-dimethoxyhydroquinone.The authors suggested that these compoundsmay serve as ferric chelators, oxygen-reduc-ing agents, and redox-cycling compounds.Kerem et al. (1999) have also reported thatcultures of G. trabeum produced 2,5-di-methoxy-1,4-benzoquinone.

Certain brown-rot fungi accumulate oxalicacid causing a noticeable decrease of pH, butnot G. trabeum, probably because of oxalatedecomposing enzymes (Shimada et al.,1997). It has been suggested that thesebrown-rot fungi may use oxalic acid as aproton donor for enzymatic and nonenzy-matic hydrolysis of polysaccharides and as achelator for a Fe(II )-H2O2 system generatinghydroxyl radicals (Shimada et al. , 1997). Inthe model of Hyde and Wood (1997), whostudied Coniophora puteana, it is suggestedthat Fe(III) is reduced by cellobiose dehy-drogenase (CDH) within the fungal cells,and that then the Fe(II ) diffuses at somedistance from the hyphae, where a Fe(II )-oxalate complex is formed and again, Fentonreaction-based hydroxyl radical formationoccurs.

The radicals formed by brown-rot fungi canremove methoxyl groups from lignin andproduce methanol, and thus they leave aresidue that consists mainly of modifiedlignin (Eriksson et al., 1990; Jin et al.,1990a). Demethoxylation of methoxylgroups and aromatic hydroxylation result inincreased phenolic hydroxyl groups, whichmakes lignin more reactive. The presence ofcarboxyl and carbonyl groups is also evi-denced by an increased oxygen content (Jinet al. , 1990b). Davis et al. (1994) used 13C-CP-MAS NMR spectrometry and found that P.placenta mainly removed carbohydrate com-ponents from spruce wood, and hemicellu-loses were removed faster than cellulose,resembling thus the results obtained with G.trabeum (Agosin et al. , 1989). P. placentafurther caused demethoxylation of sprucelignin, an increase in vanillin-like structuresand a-carbonyl moieties, but there was noevidence for ring opening (Davis et al.,1994). Birch wood was degraded in a differ-ent way, and the cleavage of lignin b-O-4linkages was the most prominent reactionoccurring, rather than demethoxylation oflignin (Davis et al., 1994).

The presence of a wood environment mayhave a significant effect on the level ofdemethoxylation of lignin and lignin modelcompounds by brown-rot fungi. When G.trabeum was cultivated under optimizedconditions on pine wood flakes, the evolved14CO2 from 14C-methylated lignin was about30% in 52 d (Jin et al. , 1990a). The presenceof wood stimulated the evolution of 14CO2

from a nonphenolic (4-O14CH3)-labeled b-O-4 dimer by G. trabeum, and 30 ± 60% of theapplied radioactivity was released as 14CO2

within 8 weeks (Niemenmaa et al. , 1992). P.placenta produced 14CO2 from O14CH3-la-beled vanillic acid rather well, but poorlyfrom the b-O-4 dimer, only 3% as 14CO2

within 4 weeks under similar conditions(Niemenmaa et al., 1992).

4 Brown-Rot Basidiomycetes 139

5

White-Rot Basidiomycetes

The only organisms capable of mineralizinglignin efficiently are basidiomycetous white-rot fungi and related litter-decomposingfungi (Kirk and Cullen, 1998). White-rotfungi are a heterogeneous group of fungiclassified in the Basidiomycota. Differentwhite-rot fungi vary considerably in therelative rates at which they attack lignin andcarbohydrates in woody tissues. Some re-move lignin more readily than carbohy-drates, relative to the original amount of each(Blanchette, 1995). Many white-rot fungicolonize cell lumina and cause cell wallerosion. Eroded zones coalesce as decayprogresses and large voids filled with myce-lium are formed. This type of rot is referred toas nonselective or simultaneous rot (Blan-chette, 1995). Trametes (syn. Coriolus, Poly-porus) versicolor is a typical simultaneous-rotfungus (Cowling, 1961; Eriksson et al.,1990).

Some white-rot fungi preferentially re-move lignin without a substantial loss ofcellulose, and cause white-pocket or white-mottled type of rot, e.g., Phellinus nigrolimi-tatus (Blanchette, 1984b, 1995; Otjen andBlanchette, 1987). There are also fungi thatare able to produce both types of attack in thesame wood (Eriksson et al., 1990). Typicalexamples of such fungi are Ganodermaapplanatum and Heterobasidion annosum.Because fungi selectively degrading ligninare considered the most promising fungi forapplications in the pulp and paper industry,the search among these fungi has attained aconsiderable interest. However, the ratiolignin±hemicellulose±cellulose decayed by aselected fungus can differ enormously, andeven different strains of the same species,e.g., of Phanerochaete chrysosporium andCeriporiopsis subvermispora, may behave dif-ferently on the same kind of wood. Variations

within a substrate are also found in onefungal strain (Eriksson et al. , 1990; Blan-chette et al. , 1992). Several screening studiesto find suitable fungi for biopulping of woodor straw, have revealed fungi that, undercertain conditions, degrade lignin preferen-tially to cellulose. Such lignin-selective fungiare, e.g., P. chrysosporium, C. subvermispora(Otjen and Blanchette, 1987; Eriksson et al.,1990), Pycnoporus cinnabarinus (Ander andEriksson, 1977), Pleurotus ostreatus (Martí-nez et al. , 1994) , Pleurotus eryngii (Martínezet al. , 1994; Dorado et al., 1999), Phlebiaradiata (Ander and Eriksson, 1977), Phlebiatremellosus (syn. Merulius tremellosa) (Anderand Eriksson, 1977; Eriksson et al., 1990),Phlebia subserialis (Akhtar et al. , 1998), Phel-linus pini (Eriksson et al., 1990), and Dicho-mitus squalens (Eriksson et al., 1990). Thelignin-degrading systems of these fungi areimportant to study since they are veryefficient. C. subvermispora may be consideredas a model fungus for selective lignin degra-dation. Calcium oxalate and MnO2 accumu-late when the decay proceeds (Blanchette,1984a, 1995; Eriksson et al. , 1990). Manga-nese peroxidase (MnP) is the most importantligninolytic enzyme produced by C. subver-mispora and many other lignin-selectivefungi and will be discussed later.

White-rot fungi are more commonly foundon angiosperm than on gymnosperm woodspecies in nature (Gilbertson, 1980). Usuallysyringyl (S) units of lignin are preferentiallydegraded whereas guaiacyl (G) units aremore resistant to degradation. When grownon straw, transmission electron microscopyrevealed that C. subvermispora and P. eryngiipartially removed the middle lamella whilePhlebia radiata apparently removed ligninfrom secondary cell walls (Burlat et al.,1998). In fibers, the middle lamella containsa high concentration of G lignin whilesecondary walls contain a high proportionof S lignin.


Basic research on lignin degradation, e.g.,its mechanisms, physiology, enzymology,and molecular biology, has been mainlycarried out with the corticioid fungus P.chrysosporium (Kirk and Farrell, 1987; Eriks-son et al. , 1990; Gold and Alic, 1993). Aftermore and taxonomically different fungi hadbeen studied in more detail, it was revealedthat both the physiological conditions forlignin degradation and the enzyme systemsexpressed are fungus-specific and differ fromthose found in P. chrysosporium. Differencesmay be connected to the taxonomic positionand/or ecology of the fungi, e.g., substratespecialization (hardwood, softwood, or cer-tain wood species, heartwood or sapwood),the stage of degradation, etc. For example, theproduction of MnP in poplar wood wassignificant by Ganoderma lucidum but thefungus did not produce MnP in pine woodmedium (D'Souza et al., 1999). The fungusobviously has the genetic potential to produceLiP since lip-like sequences were found inSouthern hybridization, but LiP activitycould not be detected.

Ligninolytic activities and enzymes oflitter-decomposing basidiomycetous fungihave been only very little studied. In a studywith some fungi, e.g., from the generaStropharia and Agrocybe, the mineralizationof 14C-(ring)-labeled synthetic lignin (DHP)was about half of the level obtained withwood-inhabiting white-rot fungi (Steffenet al. , 1999). The main ligninolytic enzymesin litter-decomposing fungi such as Agaricusbisporus (Bonnen et al. , 1994) and Strophariarugosoannulata (Steffen et al., 1999) are lac-case and MnP. In a recent study, laccase wasfound to be the only ligninolytic enzyme inMarasmius quercophilus (Dedeyan et al.,2000).

5.1

Mineralization of 14C-Labeled Lignins

The determination of 14CO2 evolution from14C-DHP (dehydrogenation polymer of coni-feryl alcohol or other lignin precursors) orother 14C-(lignin)-lignocelluloses has beenthe most often adopted method to determinelignin-degrading ability (Haider and Troja-nowski, 1975; Kirk et al., 1975, 1978). Themethod has been widely used for more than20 years (Buswell and Odier, 1987; Kirk andFarrell, 1987; Eriksson et al. , 1990). Prepara-tions from different laboratories have givencomparable results (Hatakka and Uusi-Rau-va, 1983; Hatakka et al. , 1983). Buswell andOdier (1987) compared different nonradioac-tive and 14C-labeled lignin preparations formicrobiological studies and concluded thatall lignin preparations have disadvantages inreproducibility in preparation, or alteredstructure and molecular weight comparedto natural lignin. Nevertheless, the use of 14C-labeled preparations can be considered themost reliable method. In addition, methodsto study the degradation of polymeric ligninby, e.g., NMR spectroscopy have been devel-oped (Davis et al., 1994; Gamble et al.,1994), but they are not easily amenable fordetailed physiological studies with micro-organisms or biochemical studies with en-zymes. Dimeric lignin model compoundsattached to a polymer backbone, e.g., poly-styrene (Lundell et al. , 1992) or to moresoluble polymers, e.g., polyethylene glycol(Kawai et al. , 1995), have allowed to useefficient analytical tools (e.g., NMR) for moredefined structures.

Solubilization (formation of water-solublelignin fragments) and mineralization (evo-lution of 14CO2) of 14C-labeled natural andsynthetic lignins have been demonstrated forvarious white-rot fungi (Kirk et al., 1975,1978; Hatakka and Uusi-Rauva, 1983; Ha-takka et al., 1983; Boyle et al. , 1992; Hatakka,

5 White-Rot Basidiomycetes 141

1994; Vares et al., 1994; Vares and Hatakka,1997; Hofrichter et al. , 1999b). A high min-eralization of 14C-(ring)-DHP was observedin the case of Phlebia radiata that released upto 71% 14CO2 from 14C-DHP when grown in aliquid medium (Hatakka et al. , 1983). Typ-ically, only very low amounts, in the range of3% of the 14C-label from DHP, were associ-ated with the fungal biomass after 40 d ofcultivation. Similar results were obtainedusing natural 14C-labeled lignins, e.g., fromfir or oak (58 ± 61% mineralization, 12 ± 13%14C in the mycelium) (Leatham, 1986) ordifferent lignocelluloses (Hatakka et al.,1983). Nematoloma frowardii caused evenhigher mineralization (75%) of 14C-DHPduring growth on wheat straw while againonly a small percentage of the initial radio-activity (6%) was incorporated into the resid-ual straw and the fungal biomass (Hofrichteret al. , 1999b).

Usually only the end product, 14CO2, isdetermined. This may give misleading re-sults, since in some fungi or under someconditions, the rate-limiting steps may be thereactions after the initial attack on polymericlignin. Water-soluble degradation productsmay accumulate, e.g. , under conditions oflow oxygen tension (Hatakka and Uusi-Rauva, 1983). The array of different lignino-lytic enzymes may also influence the produc-tion of 14CO2.

5.2

Ligninolytic Enzymes

Enzyme systems for the degradation ofmacromolecular lignin face several challeng-es (Kirk and Cullen, 1998). The substrate is alarge heterogeneous polymer that necessi-tates attack by extracellular enzymes oragents. Lignin does not contain hydrolyzablelinkages, which means that the enzymesmust be oxidative. Lignin is stereoirregular,

which also points to more nonspecific attackcompared to many other natural polymers.

Extracellular enzymes involved in lignindegradation are lignin peroxidases (LiPs,ªligninasesº, EC 1.11.1.14) and manganeseperoxidases (MnPs, ªMn-dependent peroxi-dasesº, EC 1.11.1.13), as well as laccases(benzenediol:oxygen oxidoreductase, EC1.10.3.2). In addition, some accessory en-zymes are involved in hydrogen peroxideproduction. Glyoxal oxidase (GLOX) and arylalcohol oxidase (AAO) (EC 1.1.3.7) belong tothis group. Table 1 shows a general over-view of the main cofactors or substrates andthe principal effects or reactions of eachenzyme.

LiPs and MnPs are heme-containing gly-coproteins which require hydrogen peroxideas an oxidant. Most fungi secrete severalisoenzymes into their cultivation medium(Table 2). LiP oxidizes nonphenolic ligninsubstructures by abstracting one electronand generating cation radicals that are thendecomposed chemically (Kirk et al., 1986;Kirk and Farrell, 1987) (Figure 1). Reac-tions of LiP using a variety of lignin modelcompounds and synthetic lignin have thor-oughly been studied, the catalytic mecha-nisms elucidated, and its capability for Ca±Cb

bond cleavage, ring opening, and otherreactions has been demonstrated (Kirk andFarrell, 1987). MnP oxidizes Mn(II) toMn(III ) which then oxidizes phenolic ringsto phenoxyl radicals which lead to decom-position of compounds (Gold et al. , 1989)(Figure 2).

Studies with different white-rot fungi haveshown that MnPappears to be more commonthan LiP (Orth et al. , 1993; Hatakka, 1994;Vares and Hatakka, 1997), and during therecent years the characteristics and role ofMnP have been extensively studied. MnP hasan essential role in the depolymerization oflignin (Wariishi et al., 1991) and chloro-lignin (Lackner et al., 1991), as well as in


the demethylation of lignin and bleaching ofpulp (Paice et al., 1993). Moreover, theenzyme mediates initial steps in the degra-dation of high-molecular weight lignin (PØ-rez and Jeffries, 1992). Recently, it has beenshown that MnP in the presence of suitableorganic acids is even able to mineralize ligninand lignin model compounds to considerableamounts (Hofrichter et al. , 1999a). Table 3summarizes conditions and results of themain published in vitro experiments, whereLiPs and MnPs have been used in the mixturewith different accessory compounds.

Laccase is a copper-containing oxidase thatutilizes molecular oxygen as oxidant and alsooxidizes phenolic rings to phenoxyl radicals(Thurston, 1994) (Figure 2). It has also acapacity to oxidize nonphenolic compoundsunder certain conditions, e.g., if the reactionmixture is supplemented with ABTS (Bour-bonnais and Paice, 1990) or other mediatingmolecules (Li et al. , 1999). Most white-rotfungi typically produce laccase (Käärik, 1965;Bollag and Leonowicz, 1984).

Phylogenetic analysis of wood-rotting andother homobasidiomycetes fungi, based onribosomal DNA sequences (Hibbett et al.,1997), may give new insights into the rela-tionships of different ligninolytic systems.Production of LiP may be typical for wood-rotting fungi, phylogenetically separatedfrom ªeuagaricsº such as Pleurotus ostreatus,Agaricus bisporus, and related fungi. ªEuaga-ricº fungi typically produce MnPs and laccasebut in most cases truly lack LiP. However, thesystem of C. subvermispora, most probablybelonging to the group of wood-rotting fungi,although not included in the analysis byHibbett et al. (1997), obviously contains lip-like genes (Rajakumar et al. , 1996). Thesystem used by the white-rot fungus P.cinnabarinus is possibly unique since thisfungus does not produce any peroxidases(Eggert et al. , 1996b).

Almost all white-rot fungi produce MnPsand laccase, but only some fungi produce LiP,the only enzyme capable of attacking non-phenolic lignin substructures. Production of


Tab. 1 Enzymes involved in the degradation of lignin and their main reactions

Enzyme Activity, Abbreviation Cofactor or Substrate,ªMediatorº

Main Effect or Reaction

Lignin peroxidase, LiP H2O2, veratryl alcohol aromatic ring oxidized to cationradical

Manganese peroxidase, MnP H2O2, Mn, organic acid as chela-tor,thiols, unsaturated lipids

Mn(II) oxidized to Mn(III); che-lated Mn(III) oxidizes phenoliccompounds to phenoxyl radicals;other reactions in the presenceof additional compounds

Laccase, Lacc O2; mediators, e.g., hydroxyben-zotriazole or ABTS

phenols are oxidized to phenoxylradicals; other reactions in thepresence of mediators

Glyoxal oxidase, GLOX glyoxal, methyl glyoxal glyoxal oxidized to glyoxylic acid;H2O2 production

Aryl alcohol oxidase, AAO aromatic alcohols (anisyl, veratrylalcohol)

aromatic alcohols oxidized toaldehydes; H2O2 production

Other H2O2 producing enzymes many organic compounds O2 reduced to H2O2


Tab. 2 Molecular masses and isoelectric points of ligninolytic enzymes from selected white-rot and litter-decomposing fungi (the fungi may also produce other ligninolytic enzymes, not listed here)

Fungus Molecular Masses Mr [kDa] Isoelectric Points [pIs] Reference

Abortiporus biennis MnPs: 38±45 (many isoforms) MnPs: 3.9±6.5 (many isoforms) Vares and Hatakka (1997)Agaricus bisporus MnPs: ND

laccase: 65,66 (straw)

MnPs: 3.35, 3.25, 3.3 (strawcompost)laccase: ND

Bonnen et al. (1994), Perryet al. (1993), Leontievskyet al. (1997b)

Armillaria mellea laccase I: 60, II: 60±62 laccase I: 4,II: 3.3

Billal and Thurston (1996)

Bjerkandera sp. BOS55 LiPs: (2 isozymes) 40±42MnPs: 44, 45

NDMnPs: 3.45, 3.40

ten Have et al. (1998), Palmaet al. (2000)

Bjerkandera adusta LiPs (2 isoenzymes): 48, 48MnPs (2 isoenzymes): 44, 44

LiPs: 3.1, 3.2MnPs: 3.45, 3.35

Kimura et al. (1990),Heinfling et al. (1998a)

Ceriporiopsis subvermispora MnPs: NDlaccase: 71, 68

MnPs: 4.1-4.6 (liquid), 3.2, 3.3,3.4, 3.5 (wood)laccase: 3.4, 4.8

Lobos et al. (1994), Salaset al. (1995), Fukushima andKirk (1995)

Coprinus cinereus laccase: 58(no MnP, no LiP)

laccase: 4 Heinzkill et al. (1998),Schneider et al. (1999)

Cyathus stercoreus MnPs: NDlaccase: 70(no LiP)

MnPs: NDlaccase: 3.5, 4.8

Sethuraman et al. (1999)

Dichomitus squalens MnPs: 48, 48.9laccase: 66

MnPs: 3.9, 4.15laccase: 3.5, 3.6

PØriØ et al. (1996, 1998)

Ganoderma lucidum laccases: 40, 66 laccases: 3.0, 4.25, 4.5, 4.8, 5.1 D©Souza et al. (1999)Heterobasidion annosum MnPs: ND MnPs: 3.3, 3.5, 3.7 Maijala et al. (2000)Junghuhnia separabilima LiPs: 43±47

laccase: 58±62LiPs: 3.4±3.5laccases: 3.4±3.6

Vares et al. (1992)

Lentinula edodes MnPs: NDlaccase: 66

MnPs: 3.2laccase: ND

Forrester et al. (1990)

Marasmius quercophilus laccase: 63 (no MnP, no LiP) laccase: 3.6 Dedeyan et al. (2000)Nematoloma frowardii MnPs: 42-44 (liquid)

50 (straw)MnPs: 3.1±4.0 (liquid)3.2 (straw)

Schneegab et al. (1997),Hofrichter et al. (1999b)

Panus tigrinus MnPs: 43laccase: 64

MnPs: 2.95, 3.2laccase: 2.9±3.0

Maltseva et al. (1991)

Phanerochaete chrysosporiumBKM-F-1767(�ATCC 24725)ME446OGC101

LiPs: 38±43MnPs: 46

LiPs: 3.3±4.7MnPs: 4.2±4.9MnP: 4.9 (aspen pulp)

Farrell et al. (1989), Goldet al. (1989), Pease and Tien(1992), Datta et al. (1991)

Phanerochaete chrysosporiumME446

laccase: 100 Dittmer et al. (1997)

Phanerochaete sordida MnPs: 45(no LiP, no laccase)

MnPs: 3.3, 4.2, 5.3 Rüttimann-Johnson et al.(1994)

Phanerochaete flavido-alba LiPs: 45MnPs: 45laccase: 94

LiPs: < 3.55MnPs: 3.55±5.85laccase: < 3.55

Ben Hamman et al. (1999),PØrez et al. (1996)

Phlebia ochraceofulva LiPs: 38-46(no MnP)

ND Vares et al. (1993)

Phlebia radiata 79 (ATCC64658)

LiPs: 39±44MnPs: 44±48laccase: 64

LiPs: 3.2±4.1MnPs: 3.6, 3.85, 4.85 (liquidculture), 3.3±5.9 (straw)laccase: 3.5

Lundell and Hatakka (1994),Moilanen et al. (1996), Vareset al. (1995), Leontievskyet al. (1997b)

Phlebia tremellosa 2845 (ATCC48754)

LiPs: 35±40MnPs: activity found, not puri-fiedlaccase: 64

LiPs: 3.1±4.0MnPs: NDlaccase: ND

Vares et al. (1994), Leontiev-sky et al. (1997b)

Pleurotus eryngii laccase: 61, 65MnPs: 43 (2 isoenzymes)

laccase: 4.2, 4.1MnPs: 3.65, 3.75 (2 isoen-zymes)

Muæoz et al. (1997), Martí-nez et al. (1996)

Pleurotus ostreatus MnPs: 42-45 MnPs: 3.5, 3.7, 4.3 (liquid)3.5, 3.7 (wood)

Becker and Sinitsyn (1993),Sarkar et al. (1997)

Pycnoporus cinnabarinus laccase: 81(no MnP, no LiP)

laccase: 3.7 Eggert et al. (1996b)

Rigidoporus lignosus MnPs: 42laccase: 53

MnPs: 3.5, 3.7laccase: 3.8

Galliano et al. (1991)

Trametes hirsuta laccase: 64±68 (2±3 isoforms) laccase: 3.7±4.0 (2±3 isoforms) Vares and Hatakka (1997)Trametes trogii LiPs: 41-44 (2 isoforms) LiPs: 3.7±3.8 (2 isoforms) Vares and Hatakka (1997)

ligninolytic enzymes in many different fun-gi, belonging to the family of Corticiaceae(e.g., Phanerochaete spp., Phlebia spp.) andPolyporaceae (e.g., Trametes spp. and manyother species) and the degradation of 14C-labeled lignin by the same fungi, show great

variability (Hatakka, 1994; Vares and Hatak-ka, 1997). Based on their main expressedextracellular ligninolytic enzymes, white-rotfungi can be classified into three or fourgroups (Hatakka, 1994). Fungi from the firstgroup produce LiPs, MnPs, and laccase, and


Tab. 2 (cont.)

Fungus Molecular Masses Mr [kDa] Isoelectric Points [pIs] Reference

Trametes versicolor LiPs: 43±45MnPs: 49laccase: 64

LiPs: 3.2±3.4MnPs: 2.9±3.2laccase: ND

Johansson and Nyman(1993), Leontievsky et al.(1997b)

Trametes villosa (syn. Trametespinsitus, C. pinsitus)

laccase: dimeric, 2 subunits of63

laccase: 3.5, 6±6.5, 5±6 (3 iso-forms)

Yaver et al. (1996)

ND�not determined or reported; LiP, lignin peroxidase; MnP, manganese peroxidase

Fig. 1 Simplified reactions of lignin peroxidase and glyoxal oxidase (redrawn according to Kirk and Cullen,1998).

Fig. 2 Simplified reactions of manganese peroxidase and laccase (redrawn according to Kirk and Cullen,1998).

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