Lignin, Lignocellulose, Ligninase

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Lignin, Lignocellulose, Ligninase

K-E L Eriksson, University of Georgia, Athens, GA, USA

H Bermek, Istanbul Technical University, Istanbul, Turkey

2009 Elsevier Inc. All rights reserved.

Defining Statement

Introduction

Lignocellulose

Microorganisms Involved in the Degradation of

Lignocelluloses

Expression of Ligninolytic Enzymes Physiological

Demands

Low Molecular Weight Compounds Play Role in

Expression of Ligninases and/or Lignin Degradation

by White-Rot Fungi

Ligninases

Further Reading

Glossarybasidiomycetes A large taxon of the filamentous fungi

that produce club-shaped spores. Many members ofthis taxon are industrially important.

lignin(s) Highly stable polymers of mostly

methoxylated phenyl-propanoic residues, synthesized

as part of the cell wall of vascular plants, constituting the

second most abundant organic polymer on earth after

cellulose. The monomeric components in lignins are

p-coumaryl alcohol (p-hydroxyphenyl unit), coniferyl

alcohol (guaiacyl unit), and sinapyl alcohol (syringyl unit).

ligninases Oxidoreductases (phenol oxidases),

produced mainly by white-rot fungi, that are capable of

depolymerization and modification of lignins. The most

studied of these enzymes are lignin peroxidase (LiP),

manganese peroxidase (MnP), and laccase. Each ofthese three enzymes participates in various ways in the

degradation of lignins.

lignocellulose The woody material in plants (trees) in

which the main components are cellulose, lignins, and

hemicelluloses. The proportions among these three

main components vary considerably in different plants

(trees).

mediator Low molecular weight organic compounds

facilitating lignin oxidation reactions catalyzed by

ligninases.

Abbreviations3-HAA 3-hydroxyanthranilic acid

ABTS 2,29-azino-big(3-ethylbenzthiazoline-6-

sulfonic acid

CBQ cellobiose:quinone oxidoreductase

CDH cellobiose dehydrogenase

EPR Electron Paramagnetic Resonance

GSH glutathione

HBT hydroxybenzotriazol

HO hydroxyl radical

HPLC high performance liquid chromatography

LiP lignin peroxidase

MnP manganese peroxidase

Defining Statement

The main structural components of wood are the

polysaccharides, cellulose and the hemicelluloses, and

lignins. These substructures are efficiently decomposed

by wood-rotting fungi employing various enzyme

systems. The mechanisms of these systems are extremely

complex and highly interactive. Microbial and enzy-

matic degradation of lignocellulosic materials is one

of the natures most important biological reactions.Understanding these mechanisms is therefore of funda-

mental interest due to their environmental and

technological implications.

Introduction

The energy crisis during the early 1970s turned interest

toward the utilization of renewable resources lignocellu-

losic materials in particular instead of fossil fuels, foryDeceased.

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energy production. To release the solar energy stored in

various lignocellulosic materials has been a prime target for

research in laboratories around the world. The tremendous

efforts devoted to understanding the mechanisms involved

in the degradation of wood and plant materials particu-

larly by fungi and their enzymes which are essential for

the successful utilization of these resources, contribute a

vast literature.

It is now known in considerable detail how the three

main groups of wood-rotting fungi, that is, white-rot,

brown-rot, and soft-rot fungi attack and degrade lignocel-

lulosic materials. The enzyme mechanisms involved in the

degradation of cellulose and the hemicelluloses are inves-

tigated and known in depth, and also the complex

mechanisms of lignin degradation are known in some detail.

Substantial efforts for a technical utilization of this new

knowledge have been made. Some white-rot fungi, parti-

cularly those that more or less specifically attack and

degrade lignin, have been tried for delignification of

wood chips to save energy in the production of mechan-ical and chemical pulp and also to upgrade straw and

sugar cane bagasse for feed. However, a full-scale use of

these possibilities has not yet been realized.

White-rot fungi were also used in pilot plant scale to

remove chlorinated aromatic and aliphatic components in

waste bleach waters. However, before this technology was

used in full scale, bleaching with molecular chlorine,

which gives rise to the formation of the highly toxic

dioxins, was discontinued in most pulp and paper produ-

cing countries.

Cellulose degrading enzymes are now produced in

large scale by several producers worldwide and at very

low prices. These enzymes have found industrial use in

food and beverage industries, and huge amounts are used

in the pulp and paper industry, particularly for deinking

of recycled paper. When ethanol production from wood

and other lignocellulosic materials comes into technical

use, an enormous demand for these enzymes can be

expected. However, acidic hydrolysis is also an option for

lignocellulosic material decomposition, and it remains to be

seen which technique will be used for this purpose.

Cellulases have also found use in the textile industry,

particularly for softening of denim in blue jeans production.

Among the hemicellulose-degrading enzymes, xyla-

nases have been used at one stage in the bleaching ofwood pulp. The treatment of kraft pulp with xylanases

cuts down the use of chlorine dioxide, which could be a

limiting factor in many mills.

Of the ligninases, only laccase has found a technical

use, mainly in the textile industry. Laccase treatment

changes the blue and indigo colors in a desirable way.

The focus of this article is mainly on the white-rot

fungi, the only microorganisms that, to any extent, can

degrade all of the lignocellulosic components. The pro-

duction and characteristics of the three essential

extracellular enzymes employed for this purpose are

described here in some detail.

Lignocellulose

Lignocellulose is made up mainly of cellulose, hemicel-

luloses, and lignins in various proportions. The most

important lignocellulosic materials are wood and agricul-

tural wastes, such as straw of various kinds and sugar cane

bagasse.

The lignin content of angiosperms (hardwoods) and

gymnosperms (softwoods) varies between 2025% and

2832%, respectively. Lignins are usually distributed

together with hemicelluloses in the space between cellu-

lose microfibrils in both primary and secondary cell walls,

and in the middle lamellae for cell adhesion as well as

for reinforcing the cell walls of the xylem tissues. In the

absence of lignin, the plant does not have the strength to

stand up, as in the case of the mosses (phylum Bryophyta),where the plant is only millimeters tall. Water-dwelling

plants float and therefore do not need the reinforcement of

lignins. Other well-known functions of the lignins are to

help sap conduction through vascular elements and to

defend the plant from attackers such as microorganisms

and insects.

There are major differences in the structure of the

hemicelluloses present in hardwoods and softwoods. The

content of glucuronoxylan is high in hardwoods, while the

dominating hemicellulose in softwood is galactoglucoman-

nan. However, there is a great deal of variation among

different woods, in their chemical composition and also in

the composition of different types of cells in a tree.

Lignin monomer biosynthesis is accomplished via a

complex biochemical reaction pathway, called the

cinnamate pathway, by utilizing glucose, shikimic acid,

L-phenylalanine, and cinnamic acid. This pathway appears

to be very costly in terms of energy demand and has been

elucidated using 14C-labeled precursors. The lignin poly-

mers are formed by the oxidation of the phenolic

monomers to their corresponding phenoxy radicals by

the enzymes peroxidases and laccases. These radicals poly-

merize spontaneously and, as far as known, without the aid

of any enzyme. It appears that the process of lignin deposi-

tion within the cell wall during xylem formation is highlycontrolled; it requires initiation sites and a complement of

cell wall localized enzymes. The process is believed to be

an example of template polymerization. The nature and

the role of the initiating sites as well as the roles of the

enzymes are not totally clear. Besides, the relative roles

played by the peroxidases and the laccases in the lignifica-

tion process also remain controversial. However, there is a

consensus that lignins are synthesized by free-radical poly-

merization of the three different phenylpropanoid

structures. Softwood lignins are made up almost entirely

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of guaiacyl-type (coniferyl alcohol) structures, while hard-

wood lignins are made of equal amounts of guaiacyl and

syringyl (sinapyl alcohol) type structures. Softwood lignin

is a three-dimensional heterogeneous polymer where more

than 90% of the guaiacyl monomers are connected by

ether and carboncarbon linkages. The most frequent sub-

structures present in softwood lignins are guaiacylglycerol-

-aryl ether linkages (4060%), phenylcoumaran (10%),

dibenzodioxin (10%), diarylpropane (


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compounds constituting environmental hazards. Lignins

are also an obstacle for efficient bioconversion of cell wall

polysaccharides in biomass to useful sugars for fermenta-

tion to liquid fuels. Lignins also limit the digestibility of

straw and other lignocellulosic materials by cattle and

other ruminants. On the contrary, degraded lignocellulosic

materials, lignins in particular, form the bulk of soil humus,

without which life on earth cannot be sustained.

Since it is now considered as the next industrial revolu-

tion, a great deal of research is invested in nanotechnology

nanobiotechnology. In search of new nanobiomaterials,

cellulose and lignocellulose appear to have great potential

since they are ubiquitous and renewable, have nanofibril-

lar structure, are capable of becoming multifunctional,

and can self-assemble into well-defined architectures.

Microorganisms Involved in theDegradation of Lignocelluloses

Lignocellulosic materials are decomposed in nature by

microorganisms. However, the conditions must be condu-

cive to microbial activity, or the degradation process will

not start or will be interrupted. Fungi, which by their

hyphae effectively penetrate wood, are also major decom-

posers of wood. A great majority of the wood-rotting fungi

that have been identified are white rotters. Most of the white

rotters colonize hardwood trees with lower lignin content

and higher hemicellulose content, but many also degrade

softwoods. Since white-rot fungi are so dominating in wood

degradation and also are the only ones that to any extent can

degrade lignin, the focus here will be on this particular type

of wood-rotting fungi. Their mechanisms of lignin degrada-

tion will be discussed through the rest of this article.

Lignin catabolism does not resemble that of other

polymeric biomolecules. First of all, most natural poly-

mers such as proteins, carbohydrates, and nucleic acids

can be synthesized and decomposed by the same organ-

ism, while lignin cannot be degraded by its producer, the

plant. Moreover, while most polymeric biomolecules are

degraded via simple hydrolytic reactions, lignin is

resistant to this type of degradation. The biological degra-

dation of lignin is accomplished only by enzyme

catalyzed oxidation reactions, usually accompanied by

nonenzymatic rearrangements.The phenomena of wood decay and wood decomposi-

tion have been studied since the mid-nineteenth century.

The three main types of wood-rotting fungi are white

rotters, brown rotters, and soft rotters. The latter two

mainly degrade wood polysaccharides. In comparison

with the white-rot fungi, brown rotters seem to be better

equipped with efficient mechanisms for depolymerization

of wood polysaccharides and get access to their sugars

without wasting energy on lignin degradation. They can

methylate lignin, but do not depolymerize it. Soft-rot

fungi, however, prefer to grow on more localized plat-

forms, such as within the secondary cell wall. They slowly

degrade cell wall polysaccharides in the immediate vici-

nity of their hyphae. The hyphae may be observed in

channels within the secondary wall. It is easy to distin-

guish between white and brown rotters by the color of the

rotted wood. The ability of white rotters to degrade lignin

and the difference in color of advanced decay suggest that

different enzymes are employed by these two types of

wood-rotting fungi. Color formation around fungal myce-

lia when phenols and tannins are added to a growth

medium is one way to distinguish between white-rot

and brown-rot fungi (Bavendamms test). Only the white

rotters excrete phenoloxidases and, therefore, convert the

added phenols to the more strongly colored quinones.

White-rot fungi commonly decay wood by attacking

all the cell wall components simultaneously (Figure 2).

However, there are also others that preferentially degrade

the lignin component (Figure 3). Originally, the term

Figure 3 Selective attack on the lignin by the white-rot fungus

Phellinus pini. Courtesy of RA Blanchette.

Figure 2 Simultaneous attack on all the wood components by

the white-rot fungus Phanerochaete chrysosporium. Courtesy of

T Nilsson.



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white rot was used mostly for fungi that preferentially

attacked lignin. Later, the white rotters were charac-

terized as white-pocket, white-mottled, white-stringy

rotters, and so on, depending on the microscopic charac-

teristics of the attack.

In the most interesting type of white rot, lignins are

preferentially degraded within all cell wall layers. Some

species, such as Phellinus pini, Ganoderma tsugae, and

Ceriporiopsis subvermispora, seem to cause selective deligni-

fication always. (Figure 3). When the middle lamellae areextensively attacked, it causes a separation of the cells. In

a specific attack, lignin is then also degraded in the sec-

ondary wall, but there are no visible lysis zones, erosion

troughs, or thinned areas. In a selective attack of the

lignin, the crystalline nature of cellulose is not destroyed.

It is well known that degradation of crystalline cellulase

takes place essentially only when there is a concerted

action of both endo- and exoglucanases. In-depth studies

of the plant cell wall degrading enzymes produced by

C. subvermispora demonstrated that this fungus did notproduce an exoglucanase (cellobiohydrolase).

In fungal wood degradation, reactive oxygen species

play important roles. The fungi produce fair amounts of

H2O2 in well-aerated environments. Since wood contains

Fe(II), a very active hydroxyl radical (HO_) is formed from

H2O2 via the Fenton reaction. This radical can virtually

attack any organic molecule and is also capable of

depolymerizing lignins. However, since lignin peroxidase

(LiP), a H2O2-dependent enzyme, was first evidenced in

1983, the importance of these radicals has been ques-

tioned and reevaluated from time to time by various

research groups.

Expression of Ligninolytic Enzymes Physiological Demands

White-rot basidiomycetes degrade lignin more rapidly and

extensively than other groups of microorganisms.

However, for lignin degradation to take place, white-rot

fungi require an additional, more easily metabolizable car-

bon source. It has not been possible to demonstrate that

lignin can serve as the sole carbon and energy source for

any known microorganism. However, degradation of lignin

enables fungi to gain access to cellulose and hemicellulose.Phanerochaete chrysosporium has been the model organ-

ism for studies of lignin degradation by white-rot fungi.

The ligninolytic system of P. chrysosporium is triggered

mainly by nitrogen starvation, but it can also be triggered

by carbon or sulfur starvation. The system operates only

under secondary metabolism. These phenomena for trig-

gering secondary metabolism are probably true for most

white-rot fungi, although there are also examples of fungi

that are not so strongly regulated by nitrogen starvation.

Such fungi may be found in nitrogen-rich environment,

such as in cattle dung piles, whereas in fungi growing on

wood, where they encounter low nitrogen concentrations,

lignin degradation would be repressed by a high nitrogen

concentration. In studies with certain fungi, addition of

organic ammonia or L-amino acids did not appear to

repress ligninolytic activity. Therefore, ligninolytic sys-

tems of all fungi are not necessarily nitrogen regulated.

As mentioned earlier, lignin degradation is an almost

entirely oxidative process, which is why increased oxygen

levels enhance lignin degradation considerably in various

white-rot fungi. Cultures of P. chrysosporium, kept at an

atmosphere of 5% O2, released only 1% of totally available14C-ring-labeled carbon from synthetic lignin as 14CO2after 35 days of incubation. However, cultures maintained

at 21 and 100% oxygen, respectively, generated approxi-

mately 47 and 57% of total 14C-label as 14CO2. The

maximum rate of 14CO2 evolution is approximately three

times higher in 100% O2 atmosphere compared to that in

air. This beneficial effect of O2 on lignin biodegradation is

probably applicable to white-rot fungi in general.It was originally reported that agitation ofP. chrysosporium

cultures completely repressed LiP production and lignin

metabolism (14C-lignin ! 14CO2). However, later conflict-

ing results concerning agitation and lignin degradation

appeared in the literature, and it was reported that agitated

cultures of P. chrysosporium, in which the mycelium had

formed a single large pellet, readily produced 14CO2 from14C-ring-labeled syntheticlignin.Production of LiP and also

a complete oxidation of labeled lignin to 14CO2 have later

been demonstrated in agitated cultures of both wild-type

and mutant strains of P. chrysosporium. Effects on enzyme

production of other chemical and physical parameters such

as pH, temperature, buffers used, or ionic strength vary

among the studied fungi.

LiP-, manganese peroxidase (MnP)-, and the H2O2-

generating systems seem to be the major components of

the extracellular lignin degradation system in P. chrysosporium.

Both LiP and MnP are regulated at the gene transcription

level, for example, by the depletion of nutrient nitrogen or by

the presence of Mn(II). The promoter regions of MnP and

LiP genes in most organisms contain cAMP response ele-

ments to induce starvation. Moreover, expression of some

isozymes is differentially regulated under starvation condi-

tions.Meanwhile, laccase production is notrepressed by high

nitrogen content; in contrast, it can even be stimulated.Laccases can be divided as constitutive and inducible

on the basis of gene expression. The inducible ones are

stimulated by copper, ferulic acid, veratric acid, xylidine,

and so on.

Following the recent completion of the whole genome

sequencing ofP. chrysosporium, using a pure whole genome

shotgun approach, the enzymatic processes of fungal

wood degradation was demonstrated to be possibly even

more complicated than anticipated. The genome was

shown to contain an impressive array of oxidative

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enzymes such as copper radical oxidases, FAD-dependent

oxidases, peroxidases, and hydrolytic enzymes that play a

role in wood decay. The organism utilizes for wood decay

ten LiP-type and five MnP-type genes encoding for dif-

ferent isozymes, a gene encoding a different hybrid

peroxidase that resembles the characteristics of both

LiPs and MnPs, and a cellobiose dehydrogenase (CDH,

see CDH) gene. Moreover, at least six genes for copper

radical oxidases and a glyoxal oxidase, at least four dif-

ferent aryl alcohol oxidases, four multicopper oxidases

(which are not conventional laccases) and a ferroxidase-

like protein are also included in the genome. The position

of these copper radical oxidase genes is suggested to be an

indication of a functional dependency between LiPs and

copper radical oxidases. What is even more astonishing is

the number of the putative carbohydrate-active enzymes.

The organism contains totally 240 genes, which are divided

into 69 distinct families: 166 glycoside hydrolases, 14 car-

bohydrate esterases, and 57 glycosyltransferases. Among

these, 40 putative endoglucanases, 7 exo-cellobiohydro-lyses, and at least 9 beta-glucosidases are also identified.

These data are sufficient to emphasize the great complex-

ity of the machinery employed, thus, a tremendous

capability of the white-rot fungi for decomposing biopoly-

mers as well as various other related bioorganic

compounds.

Low Molecular Weight Compounds PlayRole in Expression of Ligninases and/orLignin Degradation by White-Rot Fungi

Wood and other plant cell walls are made up of bulky

polymers that are difficult to penetrate. Lignins, with

their complex three-dimensional structures, form a parti-

cularly difficult barrier for the ligninolytic enzymes to

penetrate. Therefore, various low molecular weight med-

iators seem to be important both for triggering the fungal

production of these enzymes and for facilitating the

enzyme attack on the polymer itself.

Mn(II) is usually found in wood in high concentrations.

MnP is dependent on Mn(II) for its activity, and so is the

production of this enzyme since the mnp gene transcrip-

tion is also regulated by Mn(II). Surprisingly, it is also

observed that LiP levels decrease in the presence of thesame cation. Also, Mn(II) might, in various ways, influence

the expression of various MnP isozymes. The promoter

regions of most MnP and LiP genes contain xenobiotic

response elements and also heat shock elements.

Therefore, under stress conditions such as in the presence

of elevated concentrations of H2O2, arsenite, ethanol, and

other components, MnP production can be stimulated.

Veratryl alcohol is a secondary metabolite in some

white-rot fungi associated with their ligninolytic system,

particularly in those producing LiP. P. chrysosporium has

been demonstrated to produce veratryl alcohol de novo.

Veratryl alcohol is synthesized from glucose using the

phenyl-alanine pathway. Its production appears to be par-

allel to that of LiP production regulated by N starvation.

Addition of veratryl alcohol to cultures ofP. chrysosporium

has been demonstrated to induce LiP, and so does addition

of lignin. Surprisingly, the increased LiP activity does not

seem to give rise to a significant increase in the conversion

of 14C-labeled synthetic lignin to 14CO2 in this fungus,

which indicates that LiP is not the sole rate-limiting

component in lignin metabolism.

Oxalate, an important metabolite and a major aliphatic

organic acid, is produced and decomposed by white-rot

fungi. Upon its decomposition, reactive oxygen species that

are able to facilitate ligninolysis are formed. Oxalate has

been shown to reduce the veratryl alcohol cation radical as

well as Mn(III) and is therefore capable of inhibiting

ligninolysis.

Ligninases

LiP

LiP (ligninase, EC 1.11.14) was first discovered in 1983 in

ligninolytic cultures of P. chrysosporium where it seems to

constitute one of the major components of the ligninolytic

system. It was, for a long time, believed that the produc-

tion by P. chrysosporium of both LiP and MnP was clear

evidence that these two enzymes were necessary for lig-

nin degradation. However, it has later been shown

(Table 1) that only about 40% of all studied white-rotfungi produce LiP. LiP catalyzes a large variety of reac-

tions, such as the cleavage of -0-4 ether bonds and of

CC linkages in lignins. Cleavage of these bonds is

essential for the depolymerization of lignins. The enzyme

also catalyzes oxidation of aromatic C alcohols to

C-oxo compounds, hydroxylation, quinone formation,

and aromatic ring cleavage. LiP oxidizes its substrates

by two consecutive one-electron oxidation steps. Cation

radicals are intermediates in these reactions. LiP has,

compared to other phenol oxidases and peroxidases, an

unusually high redox potential and can oxidize not only

phenolic but also nonphenolic, methoxy-substituted lig-

nin subunits. The importance of LiP in the degradation of

lignin has been demonstrated in several studies. Theenzyme can depolymerize dilute solutions of lignins.

However, the net depolymerization is not that great,

since phenoxy radicals are generated in the oxidation of

phenolic substrates, as well as in demethoxylation and in

ether cleavage reactions. These radicals readily repoly-

merize. LiP also oxidizes and degrades in vitroa variety of

dimers and oligomers structurally related to lignins and

catalyzes the production of activated oxygen species.

LiP has a catalytic cycle similar to that of horseradish

peroxidase (Figure 4). The native Fe(III) enzyme is first



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oxidized by H2O2 to compound I. One-electron reduction

of compound I with veratryl alcohol then takes place, or

H2O2 oxidation results in compound II. Electron reduc-

tion of compound II by veratryl alcohol returns the

enzyme to its native form, thus maintaining the catalytic

cycle. However, in competition with a reducing substrate,

compound II can react with H2O2 and result in the for-mation of the catalytically less active compound III,

which is stable but inactivated in the presence of H2O2.

Compound III can transform back to the native enzyme,

and the cycle can get restarted.

It seems likely that the cation radicals of veratryl

alcohol, the products of LiP catalysis, may mediate in the

oxidation of lignin. These radicals may also assist in the

reaction of LiP, compound II, with the reductant

and, thereby, maintain the active peroxidase cycle.

Therefore, veratryl alcohol appears to have three separate

functions for the action of LiP: acting as a mediator in

electron transfer, completing the catalytic cycle by acting

as a substrate for compound II, and finally, restoring the

enzymes activity from the inactive compound III, a reac-

tion accomplished by the veratryl alcohol cation radical.

Since their discovery, LiPs from various white-rot

fungi have been thoroughly studied. The LiP family

contains multiple isozymes with a molecular weight

range of 38 00043 000 and isoelectric points range of

3.34.7. LiPs are glycoproteins of the oligomannose type

with a number of possible O-glycosylation sites and one

or more N-glycosylation sites. It is not well understoodwhy P. chrysosporium, and also other white-rot fungi, pro-

duce so many LiP isozymes. One question has, therefore,

been, are there specific roles, if any, for the individual

isozymes in lignin degradation? Also, do the different

enzymes represent different posttranslational modifica-

tions of the product of a single gene, or are the isozymes

encoded by different genes? While there is no answer to

the first question, all evidence indicates that each enzyme

is encoded by a different gene. In addition to the mole-

cular genetic studies of these LiPs, the X-ray crystal

structure of LiP is known. These studies have, no doubt,

led to a better understanding of the regulation and struc-ture of the lignin-degrading enzyme system produced by

P. chrysosporiumand other white-rot fungi. Yet, with all of

these advances, it has proven surprisingly difficult to

demonstrate extensive ligninolytic activity using either

isolated LiP or MnP. In fact, several investigators have

reported polymerization, rather than depolymerization,

of lignin interaction with LiP in vitro. So far, there has

not been any application for this particular enzyme,

which originally was thought to be an important break-

through in the understanding of lignin degradation. To

Table 1 Distribution of ligninolytic peroxidases in white-rot

fungi

Organisms LiP MnP Lac

Coriolopsis occidentalis ?a

Phlebia brevispora

Phlebia radiata

Pleurotus ostreatus

Pleurotus sapidus

Trametes gibbosa

Trametes hirsuta

Trametes versicolor

Phanerochaete chrysosporium

Perenniporia medulla-panis

Trametes cingulata

Phanerochaete sordida

Bjerkandera sp.

Ceriporiopsis subvermispora

Cyathus stercoreus

Daedaleopsis confragosa

(Coriolus pruinosum) ?

Dichomitus squalens

Ganoderma valesiacum Ganoderma colossum

Ganoderma lucidum

Grifola frondosa

Lentinus (Lentinula) edodes

Panus tigrinus

Pleurotus eryngii ?

Pleurotus pulmonarius ?

Rigidoporus lignosus

Stereum hirsutum

Stereum spp.

Trametes villosa

Pycnoporus cinnabarinus

Junghuhnia separabilima

Phlebia tremellosa (Merulius tremellosus) ?

Bjerkandera adusta (Polyporus adustus)

?

Coriolus consors ? ?

a?, Information not given.

3

4 Compound ll Fe lV

O

A

Fe3+

O2

AH2

ExcessH

2O

2

2

56

Fe2+

Fe3+ O2

AH

H2O2

H2O

Compound lCompound lll Fe lV (P)+

O

Fe2+ O2

AH

Figure 4 The five redox states of lignin peroxidase.

Reproduced from Renganathan V and Gold MH (1986) Spectral

characterization of the oxidized states of lignin peroxidase, an

extracellular heme enzyme from the white rot basidiomycete

Phanerochaete chrysosporium. Biochemistry25: 16261631.



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make matters even more confusing, an increasing number

of studies have indicated that the value ofP. chrysosporium

as a model organism for lignin degradation might be

limited, since the majority of species within the group of

white-rot fungi do not produce LiP.

MnP

MnP is another heme-containing extracellular fungal per-

oxidase. It was first identified in ligninolytic cultures of

P. chrysosporium as an Mn(II)- and H2O2-dependent oxi-

dase. It has then been purified and characterized from

many other white-rot fungi. The mechanisms of MnP

catalysis have been studied in detail. The catalytic cycle

of the enzyme resembles very much that of LiP (Figure 4)with the only difference being that MnP can accept Mn(II)

for transformation to compound II from compound I

instead of being dependent on another reducing substrate.

Finally, to revert to the native ferric form, the enzyme has

to oxidize another Mn(II). Thus, in a mixture with Mn(II)and H2O2, MnP oxidizes Mn(II) to Mn(III), which in turn,

oxidizes lignins, phenols, phenolic lignin model com-

pounds, and high molecular weight chlorolignins. For

Mn(II) to diffuse away and oxidize MnP substrates, it

must be sufficiently stable and able to disassociate from

the active site of the enzyme. Organic acids, metabolic

products of white-rot fungi, form complexes with Mn(II).

These complexes are stable entities and allow for

dissociation from the active site of the enzyme. Most of

these chelators are carboxylic acids such as malonate,

oxalate, and lactate. H2O2 may function to induce MnP

gene transcription. In P. chrysosporium, MnP is induced 1.6-

fold upon the addition of Mn(II) and H2O2 compared to

that in their absence. However, induction of MnP by

Mn(II) does not seem to be a general trait in white-rot

fungi. In Phlebia radiata, another white-rot fungus, high

concentrations of Mn(II) had no influence on the induced

levels of MnP, LiP, or laccase. It was also demonstrated

that high Mn-containing cultures exhibited less efficient

mineralization of synthetic lignin.

As mentioned earlier, H2O2 required for the activity of

both MnP and LiP can be provided by fungal systems.

The enzymes responsible of producing H2O2 are fungal

oxidases. Among them, glucose oxidases, glyoxal oxidases,

aryl alcohol oxidases, and methanol oxidase could belisted as important examples. Most of these enzymes

contain flavin cofactors or copper sites.

The crystal structure of Mn(II)-bound MnP has been

elucidated at 1.45 A resolution. The active site contains a

His-ligand hydrogen bonded to an Asn residue and a distal

side peroxide binding pocket formed by a catalytic His and

Arg. The Mn(II)-binding site is at the propionate end of the

heme, Mn(II) being hexacoordinated by an Asp, two Glu

residues, a heme propionate, and two water molecules.

Trivalent cations, such as lanthanides were shown to

mimic Mn(III), thus inhibiting Mn(II) oxidation. Besides,

Cd(II) exhibited a ligation geometry similar to that of

Mn(II), however, acting as an inhibitor.

Each MnP molecule was also found to contain five

disulfide bridging elements and two Ca(II) ions, which are

believed to have a structural role. It has also been shownthat

the thermal stability of the enzyme depends on the presence

of these Ca(II) ions. Thermal inactivation appears to be a

two-step process, and loss of Ca(II) decreases the enzyme

stability. If excess Ca(II) is added to the medium, the enzyme

can be reactivated. However, inactivation, caused by the loss

of the heme component, cannot be reversed.

Both MnP and laccase can oxidize phenolic lignin sub-

structures but not nonphenolic lignin structures. However,

both enzymes can attack nonphenolic lignin substructures

in the presence of certain low molecular weight organic

compounds, which act as mediators. It has, thus, been

found that MnP, in the presence of glutathione (GSH),

could efficiently oxidize veratryl alcohol, anisyl alcohol,

and benzyl alcohol. The mechanism for this oxidation isthat the formed Mn(II) oxidizes thiol to a thiyl radical,

which abstracts a hydrogen from the substrate to form the

corresponding aldehyde. This substrate oxidation was at

least twofold higher under anaerobic conditions com-

pared to that under aerobic conditions. It has also been

demonstrated that, in the presence of long-chain unsatu-

rated fatty acids, Tween 80 or other lipids, MnP Mn(II)

could oxidize a -0-4 lignin model compound without

the need for H2O2. This mechanism, by which lipid

peroxy radicals are generated, is called MnP-mediated

lipid peroxidation. The peroxy radicals easily abstract

hydrogen from MnP substrates. Therefore, the biogenic

peroxyl radicals may be considered as agents in lignin

biodegradation. As can be seen from the above informa-

tion, radical formation is a very important concept in

MnP-catalyzed substrate degradation. Phenoxyl and ami-

noxyl radicals are also formed by basic hydrogen

abstraction, and aryl cation radicals are produced from

nonphenolic substrates. The spontaneous interaction of

O2 and alkyl radicals, formed by the reaction of chelates

of Mn(III) with carboxylic acids, give rise to new reactive

oxygen species.

Several other extracellular fungal enzymes are produced

simultaneously with MnP and appear to work in accord

with this enzyme. Laccase, which coexists in culturesof various fungi, was shown to work in concert with MnP

in the degradation of lignosulfonates and solubilization of

lignins. The highest degradation rates were obtained when

the enzymes were working together. Similarly, an

interaction between MnP and CDH, both produced by

Trametes versicolor, was also proposed. It is obvious that

CDH can support MnP in different ways. (1) CDH oxidizes

cellobiose to cellobionic acid, an efficient Mn(II) chelator.

(2) CDH returns insoluble MnO2 to the soluble Mn pool in

the form of Mn(II) and Mn(III). This reaction not only



9/12

facilitates MnP production but also provides extra Mn(II)

for MnP catalysis. (3) Quinones are reduced to their corre-

sponding phenols by CDH. These phenols are substrates

for MnP as explained in CDH below.

Several white-rot fungi, including P. chrysosporium and

T. versicolor, are able to degrade lignin and to bleach kraft

pulp. There is a strong correlation between these abilities

and the MnP activity in the culture solutions. The ability

of MnP to increase brightness and decrease pulp kappa

numbers has been well established. MnP purified from

cultures of T. versicolor was found to cause most of the

demethylation and delignification of kraft pulp when

compared to the effect of the complete, cell-free culture

solution. It was also demonstrated that MnP bleached

kraft pulp brown stock, thereby releasing methanol.

Maximal bleaching effect was observed in cultures of

T. versicolor when MnP production and activity were at

their peak values.

Hybrid peroxidasesExcept for the MnP and LiP described above, some white-

rot fungi are reported to produce hybrid (versatile) perox-

idases that exhibit the characteristics of both MnPs and

LiPs, such as oxidation of both phenolic and nonphenolic

lignin structures. Some Pleurotusand Bjerkanderaspecies are

such examples. The enzyme appears to act like a LiP, yet it

contains an Mn-binding site near an internal heme propio-

nate and binds by the carboxylates of three acidic residues

to directly transfer electrons to one of the heme propio-

nates. However, nonphenolic substrates are oxidized at the

protein surface via a long range of electron transfers utiliz-

ing a surface tryptophan residue. Thus, while the enzyme

can oxidize nonphenolic substrates via aromatic radicals, it

also oxidizes Mn(II) to Mn(III). The oxidation occurs at the

binding site near the heme cofactor. These hybrid enzymes

seem to work on substrates that neither MnP nor LiP can

efficiently oxidize.

Laccase

Laccase was first identified in the 1880s as a proteinaceous

substance that catalyzed the lacquer curing process. With

one of the defining reactions catalyzed by the enzyme,

that is, the ability to oxidize hydroquinone, the name

laccase was implemented in the 1890s.Laccases are ubiquitous in the fungi. Laccases have

also been found in a large variety of plant species, in

insects, and, also in a bacterium, Azospirillum lipoferum.

The combination, in white-rot fungi, of laccase with LiP

and/or MnP seems to be a more common combination

of phenoloxidases than the LiP/MnP pattern found in

P. chrysosporium (Table 1). Laccases can function in dif-ferent ways, such as participation in lignin biosynthesis,

degradation of plant cell walls, plant pathogenicity, and

insect sclerotization. In contrast to LiP and MnP, laccases

are not strictly extracellular; high intracellular levels have

also been demonstrated in certain fungi. In T. versicolor

and P. ostreatus, for example, laccases were found to be

associated with the cell wall.

Laccases (benzenediol: oxygen oxidoreductase; EC

1.10.3.2.) are 6070kDa glycoproteins with an average pI

of 4.0. They catalyze the oxidation of a variety of phenols,

simultaneously reducing dioxygen to water. Not only

p-diphenols, but also o-diphenols, polyphenols, polyamines,

aryldiamines, aminophenols, and hydroxyindols as well as

some inorganic ions may be oxidized by laccases.

Differentiation between laccase and other phenol oxidases

is not a trivial matter, due to the relative nonspecificity of

laccases in terms of their substrates. Therefore, to distinguish

between laccases and other oxidases such as tyrosinases or

catechol oxidases, it is best to study the pure enzymes and to

calculate kinetic parameters using high-affinity laccase sub-

strates like 2,29-azino-bis(3-ethylbenzthiazoline-6-sulfonic

acid) (ABTS), syringaldazine, and low affinity substrates

like tyrosine. Specificity of laccases for a wide variety ofsubstrates has been investigated. 2,6-Dimethoxyphenol,

ABTS, guaiacol, syringaldazine, vanillic acid, hydroquinone,

sinapic acid, syringic acid, polycyclic aromatic hydrocar-

bons, pentachlorophenol, dihydroxyphenylalanine, gallic

acid, pyrogallol, protocathechuic acid, orcinol, resorcinol,

and so on are only a few of such substrates studied.

Laccases can also act like MnP, since, in the presence of

organic acids such as pyrophosphate, malonate, or oxalate,

they can oxidize Mn(II) to produce Mn(III)organic acid

complexes. The organic acids possibly facilitate this catalysis

by decreasing the high redox potential of the Mn( II)/Mn(III)

couple. Substrate specificity of laccases is often quite broad

and also varies with the source of the enzyme. The enzyme

is inhibited by a variety of general inhibitors of metal-con-

taining oxidases such as cyanide, sodium azide, or fluoride.

Laccases are members of the blue copper oxidase

enzyme family. They are monomeric, dimeric, or tetra-

meric glycoproteins. Members of this family are

characterized by having four cupric (Cu(II)) ions distributed

in three different redox centers, where each of the known

magnetic species (type 1, type 2, and type 3) is associated

with a single polypeptide chain. The Cu(II) domain is

highly conserved in the blue oxidases, and gives the enzyme

its characteristic deep blue color. Although white and

yellow laccases lacking type 1 copper center have alsobeen reported to exist, it is controversial to call them

laccases. It would be more appropriate if the definition of

laccase is limited to blue copper oxidases (Figure 5).While the crystallographic structure of a laccase is yet

to be published, the crystallographic structure of ascorbate

oxidase, another member of the blue copper oxidases, has

been a valuable model for the structure of the laccase

active site. Recently, the crystal structure of the type 2

Cu-depleted laccase from Coprinus cinereusat 2.2 A resolu-

tion has been reported.



10/12

Laccases produced by white-rot fungi are believed to

participate mainly in the degradation of lignin, while

laccases from other fungal species can serve different pur-

poses. Fungal laccases can have different characteristics,

such as carbohydrate content, redox potential, substrate

specificity, and thermal stability, depending on the fungal

species. The problem in assigning a role for laccase to

substitute for the roles of either LiP or MnP in lignindegradation has been its low redox potential. The redox

potentials, around 450700 mV, of laccases studied so far,

have not been high enough to extract electrons from non-

phenolic aromatic substrates. Temperature optima of

laccases usually range between 50 and 70 C. However,

lower optima have also been observed for these enzymes

from some organisms.

Laccases alone cannot oxidize the predominantly non-

phenolic structures of lignin, which make up for 90% of

lignin structures. However, it was demonstrated in the

1980s that laccase could oxidize a nonphenolic aromatic

compound, rotenone, in the presence of chlorpromazine.

It was further demonstrated that laccase in the presence of

syringaldehyde could oxidize methoxylated benzyl alco-

hols. However, it was only when researchers at the

Canadian Pulp and Paper Research Institute showed

that two artificial laccase substrates, ABTS and remazol

blue could act as redox mediators, which enable laccase to

oxidize nonphenolic lignin model compounds also, that

the possible importance of laccase in lignin degradation

was realized. When the same laboratory later demonstrated

that kraft pulp could be partially delignified and demethy-

lated by laccase from T. versicolorin the presence of ABTS,

the importance of these findings became obvious. German

researchers were then, in the mid-1990s, the first to apply

the laccase mediator concept to pulp bleaching in pilot

plant scale. The redox mediator, they used was l-hydro-

xybenzotriazol (l-HBT).

Laccases have gained interest in various industrial

applications. Examples are bioremediation of industrialwastewaters; in food and beverage industries, for removal

of phenols from alcoholic and nonalcoholic beverages; in

textile industry, for decolorization or synthesis of textile

dyes and bleaching of denims; for utilization in a wide

variety of organic syntheses; nanobiotechnologic applica-

tions such as biosensors to detect phenolics, catecholamines,

morphine, codeine for use in electroimmunoassays; for

cosmetic and dermatologic preparations. All these examples

are only a few headings of the many applications.

With the rapidly developing interest for laccase-based

bleaching of wood pulp, investigations regarding the role

played by laccases in lignin degradation by white-rot

fungi were started at the University of Georgia. To iden-

tify white-rot fungi producing large amounts of laccase,

extensive screening was undertaken. Pycnoporus cinnabarinus,

a white-rot fungus isolated from decaying pine wood in

Queensland, Australia, was identified in this screening. It

turned out to be an ideal candidate for in-depth studies to

investigate the importance of laccase in lignin degrada-

tion. This fungus produces only one isoelectric form of

laccase, small amounts of an as yet unidentified peroxi-

dase, and neither LiP nor MnP. The rate of lignin

degradation by P. cinnabarinus is comparable to that of

P. chrysosporium, despite the lack of both LiP and MnP.

Contrary to what was initially thought, P. cinnabarinuslaccase had the same traits as practically all other laccases

from white-rot fungi. The redox potential was not any

higher, the molecular mass, 76 500 Da, was comparable to

that of other fungal laccases, and spectroscopic character-

ization with Electron Paramagnetic Resonance (EPR)

technique showed a typical laccase spectrum both in the

UV and in the visible regions. These studies also con-

firmed the presence of four Cu ions typical for an intact

active center of a laccase. Glycosylation of this laccase

was about 9%, just about average for fungal laccases.

2H2O

H2O

His 396His 64

His 454

His 66 His 109

His 458 Phe 463

His 395

His 400

His 111His 452

OH

O2

CuII

CuII

CuII

CuII

CuI

T1

Cu

CuI

CuI

CuI

4 Sub

T2 T3

Cu

Cu

Cu

4 Sub

Cys 453

(b)

Fully oxidizedcopper cluster

Fully reducedcopper cluster

(a)

Figure 5 (a) Model of the catalytic center of the laccase from

Trametes versicolor. Type 1 (T1) copper is the site of substrate

oxidation, while type 2 (T2) and type 3 (T3) copper form a

trinuclear cluster, where reduction of molecular oxygen and

release of water takes place. (b) A representation of a laccase

catalytic cycle producing two molecules of water from the

reduction of one molecule of molecular oxygen and the

concomitant oxidation (at the T1 copper site) of four substrate

molecules to the corresponding radicals. Sub, substratemolecule; Sub?, oxidized substrate radicals. Reproduced from

Riva S (2006) Laccases: Active site structure and catalytic cycle.

Trends in Biotechnology24(5): 219226.



11/12

Comparison of the N-terminal amino acid sequences of

this laccase with those of other fungal laccases showed the

closest similarity to a laccase from T. versicolor (86%).

High similarity was also found with laccases from other

white-rot fungi, while, in contrast, the N-terminal

sequences of laccases isolated from nonwood-rotting

fungi were significantly different. These results seem to

demonstrate that the lack of LiP and MnP does not

exclude lignin degradation by a fungus producing only a

laccase. These results were also taken as support for the

possible production by the fungus P. cinnabarinus of its

own laccase redox mediator system, allowing for the

oxidation of nonphenolic lignin structures. Such a redox

mediator system was also found, and it turned out to be

3-hydroxyanthranilic acid (3-HAA). It was demonstrated

that P. cinnabarinus laccase, in the presence of 3-HAA,

could oxidize also a nonphenolic lignin model dimer.

This laccase redox mediator system was also able to

depolymerize synthetic lignin into low molecular weight

oligomers.The importance of laccase for lignin degradation by

the white-rot fungus P. cinnabarinus was further demon-

strated by production of laccaseless mutants of the fungus.

It was shown that these laccaseless mutants were greatly

reduced in their ability to metabolize 14C-ring-labeled

synthetic lignin. However, 14CO2 evolution could be

restored in cultures of these mutants, to levels comparable

to those of the wild-type cultures, by the addition of

purified P. cinnabarinuslaccase. This clearly demonstrates

that laccase is absolutely essential for lignin degradation

by this fungus.

Although a laccase mediator system could be both an

interesting and a promising method for environmentally

benign pulp bleaching, there are certain hurdles to be sur-

mounted for such a system to be applied in pulp mills. The

laccase mediators found so far are still too expensive; the

effect of laccase mediator systems in pulp bleaching is still

not satisfactory; and the mechanism for lignin degradation

by the laccase mediator system is yet unclear. To screen for

more efficient laccase mediators, researchers at the

University of Georgia have developed a fast screening sys-

tem. Monitoring the oxidation of compound I to compound

II, Figure 6, by high performance liquid chromatography(HPLC) was found to be useful for a fast screening of

potentially effective laccase mediators (Scheme I). A ligninstructure, such as the ketone II, is easily degraded by hydro-

gen peroxide under alkaline conditions. This would

depolymerize lignin macromolecules in pulp treated with

an efficient laccase mediator system, followed by treatment

with an alkaline solution of hydrogen peroxide. This was

also demonstrated to be true, and substantial efforts to find

effective laccase mediators have been made in many

laboratories.

To investigate the importance, not only of laccase

mediators, but also of laccases per se, several laccases

were studied for the redox-mediated oxidation of the

nonphenolic lignin dimer I in Scheme I. In the presenceof the redox mediators l-HBT or violuric acid, the oxida-

tion rates of dimer I by different laccases were found to

vary considerably. In the oxidation of dimer I, both l-

HBT and violuric acid were consumed, to some extent.

The redox mediators were simply converted to inactive

components, such as benzoltriazol in the case of l-HBT.

Also, both l-HBT and violuric acid inactivate the laccases.

However, the presence of dimer I, or any other lignin

model compound in the reaction mixture, slows down thisinactivation. The inactivation seems to be due mainly to

the reaction of the redox mediator free radicals, created

by the laccases, with certain amino acids in the laccase

molecule. With the present state of the art, it seems

unlikely that laccase plus a redox mediator could evolve

as an efficient pulp bleaching stage.

CDH

When the fungus Coriolus (Trametes) versicolorwas grown on

lignin agar plates supplemented with cellobiose or cellulose,

Figure 6 Structures of mediator and lignin model compounds.

Reproduced from Li K, Helm RF, and Eriksson K-EL (1998)

Mechanistic studies of the oxidation of a non-phenolic lignin

model compound by the laccase/l-HBT redox system.

Biotechnology and Applied Biochemistry 27: 239243.

Portland Press on behalf of the IUBMB.

LaccaseO2

H2O LaccaseOX

1-HBTOX

1-HBT dimer ll

dimer l

3

Scheme 1 Proposed mechanism for the laccase mediator

oxidation of nonphenolic lignins. The number 3 in the scheme

refers to compound 3 in Figure 6. Reproduced from Li K, Helm

RF, and Eriksson K-EL (1998) Mechanistic studies of the

oxidation of a non-phenolic lignin model compound by the

laccase/l-HBT redox system. Biotechnology and Applied

Biochemistry27: 239243. Portland Press on behalf of the

IUBMB.



12/12

but not with glucose, quinone formation was suppressed.

This phenomenon led to the discovery of a new FAD

enzyme, cellobiose:quinone oxidoreductase (CBQ, EC

1.1.5.1). Another enzyme, cellobiose dehydrogenase

(CDH, EC 1.1.99.18), with similar reaction patterns, was

later isolated from P. chrysosporium. However, CDH is dif-

ferent from CBQ since it was found to be aflavocytochrome enzyme containing both FAD and heme

as prosthetic groups.

Despite their direct and indirect important functions in

lignin degradation, CBQ and CDH are not considered as

ligninases. The well-understood function of CDH is

to withdraw two electrons from certain oligomeric sugars

to convert these substrates to their corresponding lactones.

The electrons are transported to quinones, phenoxy radi-

cals, dioxygen, and chelated Fe(III) and Cu(II).

The joint effects of CDH and MnP were explained

above in MnP. In addition to these joint effects, CDH

can also directly modify lignins, raising the question ofwhether or not it should be characterized as a lignin degrad-

ing or modifying enzyme. Studies with the nonphenolic

lignin model compound 3,4-dimethoxyphenyl glycol

showed that CDH can modify lignins by (1) breaking -

ether bonds, (2) demethoxylating aromatic structures, and

(3) introducing hydroxyl groups in nonphenolic lignins

through hydroxyl radicals produced by CDH. MnP does

not normally oxidize nonphenolic substrates as mentioned

above. However, in the presence of cellobiose and H2O2,

when the substrates are pretreated with CDH, the forma-

tion of hydroxyl radicals may enable MnP and laccases to

further oxidize the modified lignin substrate

(Table 2)However, it is not yet known whether a completedegradation of lignin is possible with a CDHMnP or a

CDHlaccase system. If it is found to be possible , then

CDH may, in addition to its other functions, be considered

as a ligninase.

See also: Cellulases; Enzymes, Industrial (overview);

Wastewater Treatment (not infectious hazards);

Xylanases

Further Reading

Ayers AR, Ayers SB, and Eriksson K-EL (1978) Cellobiose oxidase,

purification and partial characterization of a heme protein from

Sporotrichum pulverulentum. European Journal of Biochemistry

90: 171181.

Dean JFD and Eriksson K-EL (1994) Laccase and the deposition of

lignin in vascular plants. Holzforschung 48: 2124.

Eggert C, Temp U, and Eriksson K-EL (1997) Laccase is essential for

lignin degradation by the white-rot fungus Pycnoporus cinnabarinus.

FEBS Letters 407: 8992.

Eriksson K-EL, Blanchette RA,and Ander P (1990) Microbialand Enzymatic

Degradation of Wood and Wood Components. Berlin: Springer Verlag.

Glenn JK, MorganMA, Mayfield MB, Kuwahara M, and Gold MH (1983) An

extracellular H2O2-requiring enzyme preparation involved in lignin

biodegradation by the white rot basidiomycete Phanerochaete

chrysosporium. Biochemical and Biophysical Research

Communications 114: 10771083.

Higuchi T (2006) Look back over the studies of lignin biochemistry.

Journal of Wood Science 52: 28.

Kirk TM and Farrell R (1987) Enzymatic combustion: The microbial

degradation of lignin. Annual Review of Microbiology 41: 465505.

Li K, Helm RF, and Eriksson K-EL (1998) Mechanistic studies of the

oxidation of a non-phenolic lignin model compound by the laccase/l-

HBT redox system. Biotechnology and Applied Biochemistry27: 239243.

Martinez D, Larrondo LF, Putnam N, et al. (2004) Genome sequence of

the lignocellulose degrading fungus Phanerochaete chrysosporium

strain RP78. Nature Biotechnology 22(6): 695700.

Renganathan V and Gold MH (1986) Spectral characterization of the

oxidized states of lignin peroxidase, an extracellular heme enzyme

from the white rot basidiomycete Phanerochaete chrysosporium.

Biochemistry25: 16261631.

Riva S (2006) Laccases: Blue enzymes for green chemistry. Trends in

Biotechnology24(5): 219226.

Sethuraman A, Akin DE, and Eriksson K-EL (1998) Plant-cell-wall-

degrading enzymes produced by the white-rot fungus Ceriporiopsis

subvermispora. Biotechnology and Applied Biochemistry27: 3747.

Tien M and Kirk TK (1983) Lignin-degrading enzyme from the

hymenomycete Phanerochaete chrysosporium burds. Science

221: 661663.

Viikari L (2003) Lignocellulose modifying enzymes for sustainabletechnologies. In: Mansfield SD and Saddler JN (eds.) Applications of

Enzymes to Lignocellulosics. Washington, DC: American Chemical

Society ACS symposium series, vol. 855, pp. 3044.

Westermark U and Eriksson K-E (1974) Cellobiose: Quinone

oxidoreductase, a new wood degrading enzyme from white-rot

fungi. Acta Chemica Scandinavica B28: 209214.

Table 2 Summary of oxidative lignin modification reactions

Enzyme system Substrate(s) Products

LiP H2O2Laccase O2 mediators

Phenolic lignin substructures Phenoxy radicals

MnP H2O2 Mn(II) mediatorsNon-phenolic lignin substructures Aryl cation radicals or

cation radicals

Laccase

O2 Phenolic lignin substructures Phenoxy radicalsMnP H2O2 Mn(II)

Non-enzymatic reactions

Homolytic or heterolytic cleavage of side chains and aromatic rings

Products of O2 attack on carbon-centered radical intermediates

Products of nucleophilic attack by H2O or ROH on aryl or C cations

Reproduced from Higuchi T (2006) Look back over the studies of lignin biochemistry. Journal of Wood Science 52: 28.


Documents

Lignin, Lignocellulose, Ligninase