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

Glossary
basidiomycetes A large taxon of the filamentous fungi that produce club-shaped spores. Many members of this 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 of these 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.

Abbreviations
3-HAA ABTS CBQ CDH EPR 3-hydroxyanthranilic acid 2,29-azino-big(3-ethylbenzthiazoline-6sulfonic acid cellobiose:quinone oxidoreductase cellobiose dehydrogenase Electron Paramagnetic Resonance

GSH HBT HO HPLC LiP MnP

glutathione hydroxybenzotriazol hydroxyl radical high performance liquid chromatography lignin peroxidase 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 enzymatic degradation of lignocellulosic materials is one
y

of the natures most important biological reactions. Understanding these mechanisms is therefore of fundamental interest due to their environmental and technological implications.

Introduction
The energy crisis during the early 1970s turned interest toward the utilization of renewable resources lignocellulosic materials in particular instead of fossil fuels, for

Deceased.

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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 particularly 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 lignocellulosic materials. The enzyme mechanisms involved in the degradation of cellulose and the hemicelluloses are investigated 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, particularly 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 mechanical 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 producing 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, xylanases have been used at one stage in the bleaching of wood 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 production 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, hemicelluloses, and lignins in various proportions. The most important lignocellulosic materials are wood and agricultural 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 cellulose 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 galactoglucomannan. 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 polymers are formed by the oxidation of the phenolic monomers to their corresponding phenoxy radicals by the enzymes peroxidases and laccases. These radicals polymerize spontaneously and, as far as known, without the aid of any enzyme. It appears that the process of lignin deposition within the cell wall during xylem formation is highly controlled; 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 lignification process also remain controversial. However, there is a consensus that lignins are synthesized by free-radical polymerization of the three different phenylpropanoid structures. Softwood lignins are made up almost entirely

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of guaiacyl-type (coniferyl alcohol) structures, while hardwood 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 substructures present in softwood lignins are guaiacylglycerol-aryl ether linkages (4060%), phenylcoumaran (10%), dibenzodioxin (10%), diarylpropane (<5%), pinoresinol (<5%), biphenyl (510%), and diphenyl ether (5%). Hardwood lignins, mainly composed of guaiacyl and syringyl subunits, are also connected by similar linkages; yet, the ratio of the syringyl unit to the guaiacyl unit varies from 1:1 to 1:3 in different species. Grass lignins, composed of guaiacyl, syringyl, and p-hydroxyphenyl (p-coumaryl) units also contain similar structures. p-Coumaric acid makes up 510% of the lignin and is mostly esterified at the -position of its propyl group. The highest concentration of lignin is in the middle lamella, but it is also distributed throughout the secondary wall. Since the secondary wall is the largest portion of the total cell wall area, most of the lignin, 6080%, is located here. The three-dimensional structure of lignins is unknown, although they are second only to cellulose in natural abundance and, thus, occupy an important position in the global carbon cycle. In the hemicellulosic polysaccharides, side chains are present at more or less regular intervals, whose conformational preference determines how the lignin and the hemicellulose polymers interact. Figure 1 shows a detailed composition of a softwood tracheid. Lignin is the last component to be synthesized in

Layering of a mature cell wall

S3

S2

Secondary wall

S1
Primary wall (P)

Figure 1 Diagrammatic representation of a softwood tracheid. te Reproduced from Co WA Jr. (1967) Wood Ultrastructure An Atlas of Electron Micrographs. Seattle: University of Washington Press.

the fiber cell wall. It initially appears at the cell corners of the middle lamella. When the deposition of cellulose microfibrils in the S3 layer of the secondary wall is completed, lignification proceeds extensively. The lignin monomers are polymerized and deposited within a preformed cell wall matrix. Accordingly, the space available for lignin deposition is defined from the outset. Recent molecular biological research, utilizing largescale generation of the expressed sequence tags from xylem tissues of various species, revealed a great deal of information about lignin biosynthesis at the transcriptional level. Moreover, sophisticated methodologies such as lignin transcriptional profiling using microarray technology helped identifying genes encoding the enzymes responsible for lignocellulose biosynthesis in different developmental stages of wood-forming tissues. Another high-tech method, infrared microspectroscopy, particularly in combination with synchrotron radiation, facilitated greatly the in vivo studies of the cell wall architecture and the major cell wall components: lignin, cellulose, and the hemicelluloses. Multi-angle laser light scattering, atomic force microscopy, Raman imaging and near-infrared spectroscopy also found significant use in physical characterization of lignins. However, the results of these studies are not the focus of this article, and thus, are not presented here. Industrial use of wood and their components would greatly benefit from wood biotechnology in terms of both quality and quantity. A top-down approach of lignin-free or lignin-reduced tree production using biotechnology is an important goal. These goals may be possible to achieve by using genetic engineering methods allowing for the downregulation or knocking-out of the genes encoding for enzymes involved in the synthesis of lignin monomers. For example, downregulation of 4-hydroxycinnamate:CoA ligase in aspen exhibits a 45% reduction of lignin content and a 15% increase in cellulose content. The growth rate of aspen plants transformed with the horseradish peroxidase gene increases significantly. The average stem length increases by 25%, and elevated peroxidase levels are detected. Another example is the tobacco plants transformed with transcription factor Ntlim1. The transformed plants show considerably lower activities of some enzymes in the lignin biosynthetic pathway, and consequently the lignin content decreases by 27%. As mentioned earlier, lignin serves several important structural, protective, and supportive functions in the cell wall matrix of plants. Unfortunately, in consideration of utilization of lignocellulosic material, these same positive traits of lignins for plants and trees turn into a negative impact on mankind and the environment in several different ways. In production of wood pulp, lignin is removed from wood chips by cooking and bleaching. When chlorine and chlorine derivatives were used to a higher degree than is the case now, it resulted in the generation of toxic

376 Applied Microbiology: Industrial | Lignin, Lignocellulose, Ligninase

compounds constituting environmental hazards. Lignins are also an obstacle for efficient bioconversion of cell wall polysaccharides in biomass to useful sugars for fermentation 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 revolution, 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 nanofibrillar structure, are capable of becoming multifunctional, and can self-assemble into well-defined architectures.

Microorganisms Involved in the Degradation of Lignocelluloses

Lignocellulosic materials are decomposed in nature by microorganisms. However, the conditions must be conducive 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 decomposers 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 degradation will be discussed through the rest of this article. Lignin catabolism does not resemble that of other polymeric biomolecules. First of all, most natural polymers such as proteins, carbohydrates, and nucleic acids can be synthesized and decomposed by the same organism, 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 degradation of lignin is accomplished only by enzyme catalyzed oxidation reactions, usually accompanied by nonenzymatic rearrangements. The phenomena of wood decay and wood decomposition 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 platforms, such as within the secondary cell wall. They slowly degrade cell wall polysaccharides in the immediate vicinity of their hyphae. The hyphae may be observed in channels within the secondary wall. It is easy to distinguish 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 mycelia 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 2 Simultaneous attack on all the wood components by the white-rot fungus Phanerochaete chrysosporium. Courtesy of T Nilsson.

Figure 3 Selective attack on the lignin by the white-rot fungus Phellinus pini. Courtesy of RA Blanchette.

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white rot was used mostly for fungi that preferentially attacked lignin. Later, the white rotters were characterized as white-pocket, white-mottled, white-stringy rotters, and so on, depending on the microscopic characteristics 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 delignification always. (Figure 3). When the middle lamellae are extensively attacked, it causes a separation of the cells. In a specific attack, lignin is then also degraded in the secondary 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 not produce 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 questioned 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 carbon 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 organism 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 triggering 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 systems 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 available 14 C-ring-labeled carbon from synthetic lignin as 14CO2 after 35 days of incubation. However, cultures maintained at 21 and 100% oxygen, respectively, generated approximately 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 of P. chrysosporium cultures completely repressed LiP production and lignin metabolism (14C-lignin ! 14CO2). However, later conflicting 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 from 14 C-ring-labeled synthetic lignin. 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 H2O2generating 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 elements to induce starvation. Moreover, expression of some isozymes is differentially regulated under starvation conditions. Meanwhile, laccase production is not repressed 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 of P. 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 different 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 different aryl alcohol oxidases, four multicopper oxidases (which are not conventional laccases) and a ferroxidaselike 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 carbohydrate esterases, and 57 glycosyltransferases. Among these, 40 putative endoglucanases, 7 exo-cellobiohydrolyses, and at least 9 beta-glucosidases are also identified. These data are sufficient to emphasize the great complexity of the machinery employed, thus, a tremendous capability of the white-rot fungi for decomposing biopolymers as well as various other related bioorganic compounds.

been demonstrated to produce veratryl alcohol de novo. Veratryl alcohol is synthesized from glucose using the phenyl-alanine pathway. Its production appears to be parallel to that of LiP production regulated by N starvation. Addition of veratryl alcohol to cultures of P. 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 production by P. chrysosporium of both LiP and MnP was clear evidence that these two enzymes were necessary for lignin degradation. However, it has later been shown (Table 1) that only about 40% of all studied white-rot fungi produce LiP. LiP catalyzes a large variety of reactions, 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 lignin subunits. The importance of LiP in the degradation of lignin has been demonstrated in several studies. The enzyme 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 repolymerize. LiP also oxidizes and degrades in vitro a 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

Low Molecular Weight Compounds Play Role in Expression of Ligninases and/or Lignin 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 particularly difficult barrier for the ligninolytic enzymes to penetrate. Therefore, various low molecular weight mediators 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 transcription is also regulated by Mn(II). Surprisingly, it is also observed that LiP levels decrease in the presence of the same 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

Applied Microbiology: Industrial | Lignin, Lignocellulose, Ligninase

Table 1 Distribution of ligninolytic peroxidases in white-rot fungi Organisms Coriolopsis occidentalis 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

379

Fe3+ 3 A AH Fe2+ 2 O2 Excess H2O2 H2O2 H2O AH AH2 6 Compound lll

Fe3+ O2

LiP

MnP ? ? ? ?

Lac ?a ? ? ?

O 4 Compound ll Fe lV

O 5 Compound l Fe lV (P)+

Fe2+ O2

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. Biochemistry 25: 16261631.

?, Information not given.

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 reduction 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 formation 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 reaction 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 understood why P. chrysosporium, and also other white-rot fungi, produce 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 modifications 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 molecular 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 structure of the lignin-degrading enzyme system produced by P. chrysosporium and 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 breakthrough in the understanding of lignin degradation. To

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make matters even more confusing, an increasing number of studies have indicated that the value of P. 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 peroxidase. It was first identified in ligninolytic cultures of P. chrysosporium as an Mn(II)- and H2O2-dependent oxidase. 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 compounds, 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 1.6fold 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 be listed as important examples. Most of these enzymes contain flavin cofactors or copper sites. The crystal structure of Mn(II)-bound MnP has been resolution. The active site contains a elucidated at 1.45 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 shown that 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 substructures 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 is that 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 compared to that under aerobic conditions. It has also been demonstrated that, in the presence of long-chain unsaturated 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 information, radical formation is a very important concept in MnP-catalyzed substrate degradation. Phenoxyl and aminoxyl 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 cultures of 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

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facilitates MnP production but also provides extra Mn(II) for MnP catalysis. (3) Quinones are reduced to their corresponding 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 peroxidases

Except for the MnP and LiP described above, some whiterot fungi are reported to produce hybrid (versatile) peroxidases that exhibit the characteristics of both MnPs and LiPs, such as oxidation of both phenolic and nonphenolic lignin structures. Some Pleurotus and Bjerkandera species are such examples. The enzyme appears to act like a LiP, yet it contains an Mn-binding site near an internal heme propionate and binds by the carboxylates of three acidic residues to directly transfer electrons to one of the heme propionates. However, nonphenolic substrates are oxidized at the protein surface via a long range of electron transfers utilizing 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 different 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 6070 kDa 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 substrates 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 of substrates has been investigated. 2,6-Dimethoxyphenol, ABTS, guaiacol, syringaldazine, vanillic acid, hydroquinone, sinapic acid, syringic acid, polycyclic aromatic hydrocarbons, 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-containing oxidases such as cyanide, sodium azide, or fluoride. Laccases are members of the blue copper oxidase enzyme family. They are monomeric, dimeric, or tetrameric 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 also been 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 resoluCu-depleted laccase from Coprinus cinereus at 2.2 A tion has been reported.

382 Applied Microbiology: Industrial | Lignin, Lignocellulose, Ligninase

(a)

His 452 Cu His 400 H2O

His 111

His 395 Cu His 396 His 64 His 454 Cu His 66 T2 T3 His 109
T1

OH His 458

Cys 453
Cu

Phe 463

(b)

4 Sub

4 Sub

CuII CuII CuII CuII CuI

CuI CuI CuI

Fully oxidized copper cluster

Fully reduced copper cluster

2H2O

O2

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, substrate molecule; Sub?, oxidized substrate radicals. Reproduced from Riva S (2006) Laccases: Active site structure and catalytic cycle. Trends in Biotechnology 24(5): 219226.

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 purposes. 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 lignin degradation 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 nonphenolic 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 nonphenolic 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 alcohols. 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 demethylated by laccase from T. versicolor in 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-hydroxybenzotriazol (l-HBT). Laccases have gained interest in various industrial applications. Examples are bioremediation of industrial wastewaters; 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 applications 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 identify 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 degradation. This fungus produces only one isoelectric form of laccase, small amounts of an as yet unidentified peroxidase, 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. cinnabarinus laccase 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 characterization with Electron Paramagnetic Resonance (EPR) technique showed a typical laccase spectrum both in the UV and in the visible regions. These studies also confirmed 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.

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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 demonstrated 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. cinnabarinus laccase. 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 surmounted 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 system. 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 lignin structure, such as the ketone II, is easily degraded by hydrogen 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

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.

3 O2 H2O Laccase LaccaseOX 1-HBTOX 1-HBT dimer l dimer ll

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 Biochemistry 27: 239243. Portland Press on behalf of the IUBMB.

were studied for the redox-mediated oxidation of the nonphenolic lignin dimer I in Scheme I. In the presence of the redox mediators l-HBT or violuric acid, the oxidation rates of dimer I by different laccases were found to vary considerably. In the oxidation of dimer I, both lHBT 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 this inactivation. 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) versicolor was grown on lignin agar plates supplemented with cellobiose or cellulose,

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Table 2 Summary of oxidative lignin modification reactions Enzyme system LiP H2O2 Laccase O2 mediators MnP H2O2 Mn(II) mediators Laccase O2 MnP H2O2 Mn(II) Non-enzymatic reactions Phenolic lignin substructures 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 Phenoxy radicals Substrate(s) Phenolic lignin substructures Non-phenolic lignin substructures Products Phenoxy radicals Aryl cation radicals or cation radicals

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

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 different from CBQ since it was found to be a flavocytochrome 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 radicals, 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 of whether or not it should be characterized as a lignin degrading 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 formation 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 complete degradation 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) Microbial and Enzymatic Degradation of Wood and Wood Components. Berlin: Springer Verlag. Glenn JK, Morgan MA, 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/lHBT redox system. Biotechnology and Applied Biochemistry 27: 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. Biochemistry 25: 16261631. Riva S (2006) Laccases: Blue enzymes for green chemistry. Trends in Biotechnology 24(5): 219226. Sethuraman A, Akin DE, and Eriksson K-EL (1998) Plant-cell-walldegrading enzymes produced by the white-rot fungus Ceriporiopsis subvermispora. Biotechnology and Applied Biochemistry 27: 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 sustainable technologies. 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.