Decomposition Of Biopolymers

2.1 Decomposition of Cellulose 2.1.1 Enzyme-Catalyzed Decomposition

Cellulose is the main polymeric component of the plant cell wall and is the most abundant polysaccharide on earth. The chemical composition is simple: it consists of D-glucose residues linked by ¿6-1,4-glycosidic bonds to form linear polymeric chains of over 10,000 glucose residues. Cellulose contains both highly crystalline regions where individual chains are linked to each other, and less-ordered amorphous regions (Hon, 1994). Although chemically simple, the extensive intermolecular bonding pattern of cellulose generates a crystalline structure that can result in a very complex morphology.

For the efficient decomposition of cellulose, an enzymatic system must include endo and exotype hydrolases and ¿S-glucosidases, the complementary activities acting synergistically. Endo-1,4-S-glucanases (EG, EC 3.2.1.4) belong to endotype enzymes that hydrolyse cellulose molecules preferentially in the amorphous parts of the fibril. Endoglucanase activity probably exists in all wood-degrading fungi including brown-rot fungi (Highley, 1988). Cellobiohydrolases (CBH, EC 3.2.1.91) are exotype enzymes that attack cellulose fibres from both reducing and nonreducing ends. No CBH activity has been detected in most brown-rot fungi, which makes their ability to degrade pure crystalline cellulose questionable. Exceptions can be found in a few of the Coniophoraceae (Nilsson and Ginns, 1979; Schmidhalter and Canevascini, 1993a, 1993b). The product of CBH action, cellobiose, is hydrolyzed by 1,4-S-glucosidases (EC 3.2.1.21) to two glucose units. Cellobiose and glucose can be taken up and assimilated by the hyphae. The cellobiose is probably hydrolyzed to glucose by cell wall bound or intracellular ¿S-glucosidases. All cellulolytic enzymes share the same chemical specificity for ¿-1,4-glycosidic bonds, which they cleave by a general acid-catalyzed hydrolysis. Most cellulolytic hydrolases are proteins of 30-50 kDa molecular mass with acidic pH optima between 2.5 and 4.5.

Oxidative enzymes participating in cellulose hydrolysis were first detected in Phanerochaete chrysosporium in the 1970s (Westermark and Eriksson, 1975). Biochemically the enzyme is currently described as cellobiose dehydrogenase (CDH, EC 1.1.3.25). CDH will oxidize cellobiose, lactose and mannobiose that all have ¿S-1,4-bonds, whereas monosaccharides and a-1,4-glycosidic bond containing maltose are not oxidized (Henriksson et al., 2000). As electron acceptors CDH reduces quinones, phenoxy radicals, cytochrome c, complexed Fe3+, manganese and molecular oxygen, which leads to the production of hydrogen peroxide; production of the latter having been demonstrated in P. chrysosporium CDH (Kremer and Wood, 1992). These properties enable this enzyme to combine cellulose and lignin decomposition. CDHs have been characterized from several white-rot fungi, e.g. Heterobasidion annosum, P. chrysosporium, Pycnoporus cinna-barinus, Schizophyllum commune, and Trametes versicolor (Henriksson et al., 2000), and are also known from the brown-rot fungus Coniophora puteana (Schmidhalter and Canevascini, 1992) and several ascomycetes.

Cellulolytic enzyme systems in white-rot basidiomycetes have received less attention than in asco- and deuteromycetes, but P. chrysosporium or its anamorph, Sporotrichum pulverulentum (Eriksson and Hamp, 1978) and Ischnoderma resinosum (Sutherland, 1986) have been studied. The work with P. chrysospor-ium has shown that in this fungus the cellulase systems are analogous to systems well known in the ascomycete Trichoderma reesei (Lynd et al., 2002). P. chrysosporium produces a cellulase system with seven CBH, of which CBHI-4 is the major cellobiohydrolase (van den Wymelenberg et al., 1993). Until now, only a 28-kDa endoglucanase (EG28) lacking cellulose-binding motif (CBM) has been isolated from P. chrysosporium and synergism between the EG28 and the CBH was demonstrated. Birch et al. (1995) reported differential splicing in the CBM-encoding region of the cbh1.2 gene, depending on whether micro-crystalline or amorphous cellulose was used as the substrate. They proposed that differential splicing of the cbhI-like genes could yield cellobiohydrolase and endoglucanase activity. Apart from CBH and endoglucanase activities, P. chrysosporium also produces ^-glucosidase and CDH (Lynd et al., 2002).

Amongst brown-rot fungi, cellulolysis by Gloeophyllum trabeum is best understood. It has a hydroquinone-driven system for the production of extracellular reactive oxygen species, a yS-glucosidase, a xylanase, and three endo-glucanases, one of which is a processive enzyme that was reported to degrade crystalline cellulose (Cohen et al., 2005). The relative role of the enzymatic and nonenzymatic decomposition of cellulose in this species is unclear.

2.1.2 Nonenzymatic Decomposition of Cellulose

Recently, several systems of nonenzymatic decomposition of cellulose by brown-rot fungi have been proposed that generate free reactive radicals in the modifications of the Fenton reaction—the decomposition of hydrogen peroxide in the presence of ferrous ions (Goodell, 2003). The two systems with the best experimental support are based on the studies with G. trabeum and C. puteana.

G. trabeum exhibits the rapid ability to degrade an aliphatic polyether via extracellular one-electron oxidation (Kerem et al., 1998), it produces simple aromatic compounds, 4,5-dimethoxycatechol and 2,5-dimethoxyhydroquinone, these compounds may serve as ferric chelators, oxygen-reducing agents and re-dox-cycling compounds (Kerem et al., 1999). It also produces the necessary NADH-dependent quinone reductases (Jensen et al., 2002). The above phenolic compounds participate in the endocleavage of cellulose by the quinone cycling mechanism generating reactive hydroxyl radicals.

Certain brown-rot fungi accumulate oxalic acid causing a noticeable decrease of pH probably because of the presence of oxalate decomposing enzymes (Shimada et al., 1997). It has been suggested that these brown-rot fungi may use oxalic acid as a proton donor for enzymatic and nonenzymatic hydrolysis of polysaccharides and as a chelator for a Fe2+-H2O2 system generating hydroxyl radicals (Shimada et al., 1997). Hyde and Wood (1997), who studied C. puteana, suggested that Fe3+ is reduced by CDH within fungal cells, and that the Fe2+ diffuses at some distance from the hyphae, where a Fe2+-oxalate complex is formed and a Fenton reaction-based hydroxyl radical formation occurs.

Another brown-rot fungus, Piptoporus betulinus, produces three endoglu-canases and two ^-glucosidases (Valaskova and Baldrian, 2006a). Nonenzymatic decomposition of cellulose has not been P. betulinus, and it is questionable how this species decomposes crystalline cellulose since the enzymatic system is unable to attack it (Valaskova and Baldrian, 2006a).

2.2 Hemicellulose Degrading Enzymes

Hemicellulose is a low molecular weight linear or branched polymer usually containing several different sugar units and substituted side chains. Xylans, consisting of D-xylose units, and glucomannans, consisting of D-glucose and D-mannose units, are the main hemicelluloses of angiosperm and conifer trees, respectively, while other lignocellulosic materials may additionally contain considerable amounts of arabinogalactans and galactans (Dekker, 1985). Branched polymers contain neutral and/or acidic side groups that render hemi-celluloses noncrystalline or poorly crystalline. Hemicelluloses thus usually form a matrix together with pectins and proteins in primary plant cell walls and with lignin in secondary cell walls.

Enzymatic decomposition of hemicelluloses requires a complex set of different enzymes reflecting structural variability. Hemicellulose hydrolysis proceeds through the action of endotype enzymes that liberate shorter fragments of substituted oligosaccharides, which are further degraded by side-group cleaving enzymes and exotype enzymes. Cleavage results in liberation of monomeric sugars and acetic acid. Similarly to cellulose hydrolysis, the hydrolases act synergistically to convert hemicellulose polymer into soluble units.

Xylanases have been widely studied due to their biotechnological importance (Subramaniyan and Prema, 2002). Endo-1,4-^-xylanases (EC 3.2.1.8) catalyse the random hydrolysis of ¿-1,4-glycosidic bonds in xylans. A range of different end-oxylanases with different specificities have been found in both white-rot and brown-rot fungi. They show the highest activity against polymeric xylan, and the rate of the hydrolysis decreases with decreasing chain length (Coughlan and Hazlewood, 1993). 1,4-^-xylosidase (EC 3.2.1.37), which is needed for the release of xylose monomers, has also been reported from several white-rot and brown-rot basidiomycetes. The effective native xylan decomposition seems to involve another three enzyme types: 1,4-^-glucuronidases (EC 3.2.1.131), ¿-arab-inofuranosidases (EC 3.2.1.55) and acetyl xylan esterases (EC 3.2.1.72). They all differ in specificity with respect to the neighbouring substituents and chain length. For example, in conifers, where the xylan has arabinose as a substituent, xylan decomposition requires a ¿-arabinofuranosidase but not the esterase. ¿-Glucuronidases have been characterized from P. chrysosporium (Castanares et al., 1995) and S. commune (Johnson et al., 1989), and ¿-arabinofuranosidase from P. chrysosporium (Coughlan and Hazlewood, 1993).

Complete hydrolysis of glucomannans also requires a wide set of enzymes. Endo-1,4-p-mannanases (EC 3.2.1.78) hydrolyse randomly the 1,4-6-mannopyra-nosyl linkages of gluco- and galactoglucomannans, releasing oligomeric fragments. Acetyl(gluco)mannan esterase removes the acetyl groups and 1,4-6-galactosidase (EC 3.2.1.22) removes galactose. 1,4-6-mannosidase (EC 3.2.1.25) and ¿S-glucosidase cleave the ¿-1,4 linkages between oligomeric fragments. However, few studies have been conducted on these enzymes from wood-rotting basidiomycetes, although they obviously effectively remove mannan from the cell walls of wood (Eriksson et al., 1992). Apparently, many species produce a wide set of hemicel-lulose-degrading enzymes. For example, endomannanase, endoxylanase, ¿-mannosidase and ¿S-xylosidase were produced by the white-rot fungi Pleurotus ostreatus, T. versicolor and the brown-rotter P. betulinus while the latter also produced ¿-galactosidase activity (Valaskova and Baldrian, 2006b). Hemicellulase production does not appear to be as strictly controlled by substrate induction as cellulase production (Valaskova and Baldrian, 2006a, 2006b).

Hemicelluloses are bound to lignin by three types of covalent linkages (Spanikova and Biely, 2006). The first involves p-coumaric or ferulic acid, linked by ether bonds to lignin, and esterically to hemicellulose sugars. This linkage could be cleaved by feruloyl esterase (EC 3.1.1.73) that is typical of filamentous fungi and has been demonstrated in the ligninolytic basidiomycetous yeast Aureobasidium pullulans (Rumbold et al., 2003) but not yet in other ligninolytic species. The second is represented by ether linkages between OH-groups of saccharides and lignin alcohols. The third are ester linkages between 4-O-methyl-D-glucuronic acid or D-glucuronic acid residues of glucuronoxylans and hydroxyl groups of lignin alcohols, which can potentially be cleaved by glucuronoyl esterase reported from S. commune (Spanikova and Biely, 2006). The physiological role of the recently found Pleurotus sapidus carboxylesterase has not yet been assigned (Zorn et al, 2005).

The nonenzymatic systems described above for the cleavage of cellulose, work equally well for hemicellulose decomposition. Although the above definitions of enzymes with respect to the reactions they catalyze seem to be clear, in reality the substrate specificity often overlaps. Since some endoglucanases, recently denominated ''processive endoglucanases'' (Gilad et al., 2003) show oligosaccharide-releasing activity, they can be classified as both endoglucanase and cellobiohydrolase. Also the cellulolytic and hemicellulolytic enzymatic systems cannot be separated completely since the substrates are chemically analogous and individual enzyme molecules frequently exhibit activities with more than one substrate (Copa-Patino and Broda, 1994; Cohen et al., 2005). In the case of P. betulinus purified ''¿S-glucosidase'' also exhibited ¿S-galactosidase, ¿S-man-nosidase and ¿S-xylosidase activities and a weak cellobiohydrolase activity (Valaskova and Baldrian, 2006a). The classification according to the major activity can thus be misleading with respect to the actual physiological role, and even the purified enzymes described so far probably exhibit additional activities that have not been searched for.

2.3 Decomposition of Lignin

Lignin is a branched polymer of substituted phenylpropane units joined by carbon-carbon and ether linkages. The monolignol precursors p-coumaryl, coniferyl and sinapyl alcohol form p-hydroxyphenyl-, guaiacyl-, and syringyl type units in lignin. The major linkage in lignin, the arylglycerol-p-aryl ether substructure, comprises about half of the total interunit linkages. Lignin of gym-nosperms contains mainly guaiacyl type units with some p-coumaryl units, whereas angiosperm lignin consists of both guaiacyl and syringyl type lignin with few p-coumaryl residues (Sjostrom, 1993).

The ligninolytic systems consist of oxidases, peroxidases and hydrogen peroxide producing enzymes. Ligninolytic oxidase—laccase—oxidizes its substrates using molecular oxygen, while the peroxidases need the supply of extracellular hydrogen peroxide which is formed by the oxidation of different extracellular metabolites.

Laccases (EC 1.10.3.2) have been known for many years in plants, fungi, and insects and have recently been found in bacteria. Although they exhibit low redox potentials they can oxidize a wide variety of substrates and were thus proposed to play a variety of roles, including synthesis of pigments, fruit-body morphogenesis and detoxification (Thurston, 1994; Mayer and Staples, 2002; Baldrian, 2006). Their role in lignin decomposition has recently been reviewed (Leonowicz et al., 2001). Laccases are typically proteins of 50-70 kDa with acidic pH optima (3.0-5.5 for 2,6-dimethoxyphenol and 4.0-6.0 for guaiacol). They are produced by white-rot and litter-decomposing basidiomycetes and by a range of ascomycetes (Baldrian, 2006). Although there are some records of laccases in my-corrhizal basidiomycetes, and in the brown-rotter C. puteana (Lee et al., 2004), it seems that their significance for these functional groups is limited (Baldrian, 2006). Recently it was proposed that several compounds produced during lignin transformation can act as redox mediators and thus make it possible to mediate the oxidation of compounds with high redox potential (Camarero et al., 2005; Baldrian, 2006), which is important for the understanding of lignin decomposition.

Lignin peroxidase and manganese peroxidase were discovered in the mid-1980s in P. chrysosporium and described as true ligninases because of their high redox potential (Gold et al., 2000; Martinez, 2002). Lignin peroxidase degrades nonphenolic lignin units (up to 90% of the polymer), whereas Mn-peroxidase generates Mn3+, which acts as a diffusible oxidizer on phenolic or nonphenolic lignin units (Jensen et al., 1996; Hofrichter, 2002).

Lignin peroxidases (LiP, EC 1.11.1.14) have molecular masses of approximately 40 kDa and very low pH optima, approximately 2.5-3.0. Fungi commonly produce several enzyme isoforms or isoenzymes of LiP but the significance of such multiplicity is not known (Hatakka, 2001; Hilden et al., 2006). LiP preferentially catalyzes the cleavage of the Ca-CS bond in the propyl side chain of lignin (Kirk and Farrell, 1987). Veratryl alcohol, an aromatic compound produced by some white-rot fungi during secondary metabolism, is necessary for LiP catalysis. It acts as a cation radical redox mediator of remote substrates, protects LiP from inac-tivation by H2O2 and completes the catalytic cycle of the enzyme (Hatakka, 2001). LiP is produced by far fewer white-rot fungi than Mn-peroxidase and laccase, e.g. P. chrysosporium, Bjerkandera spp., Trametes spp. and Phlebia spp.

Mn-peroxidases (MnP, EC 1.11.1.13) are somewhat larger heme proteins than LiPs with molecular masses of 47-60 kDa, glycosylated, and have usually acidic pH optima. Although MnP is able to oxidize phenolic substrates, it most frequently oxidizes Mn2+ to Mn3+ that is stabilized by organic acids such as oxalate, malate, lactate or malonate. The chelated Mn3+ is diffusible and can oxidize a wide range of substrates including phenols, nonphenolic aromatic compounds, carboxylic acids, thiols and unsaturated aliphatic compounds (e.g. fatty acids). The initial oxidation can be followed by a sequence of radical-based or oxidative reactions leading to lignin decomposition and mineralization (Hatakka, 2001; Hofrichter, 2002). The prerequisite of MnP action is a massive production of organic acids by fungi that can be as high as 45 mM malate, 3.5 mM fumarate and 10 mM oxalate (Hofrichter et al., 1999). Production of MnP is widespread among white-rot basidiomycetes and it is usually produced in several isoforms (Hofrichter, 2002).

Versatile peroxidase (VP, EC 1.11.1.7) has been described in Pleurotus spp. and other basidiomycetes as a third type of ligninolytic peroxidase that combines the catalytic properties of LiP, MnP and plant/microbial peroxidases oxidizing phenolic compounds (Martinez et al., 1996; Heinfling et al., 1998; Ruiz-Duenas et al., 1999). The role of heme-thiolate haloperoxidases, newly detected in a range of litter-decomposing basidiomycetes in lignin decomposition, is still questionable. The enzyme is able to catalyze several reactions including oxidations and halogenations of aromatic compounds, but exhibits an unusually high pH optimum around 7 (Hofrichter and Ullrich, 2006).

Other extracellular enzymes involved in wood lignin decomposition are oxidases generating H2O2, and mycelium-associated dehydrogenases that reduce lignin-derived compounds. The former include the extracellular aryl-alcohol oxidase (AAO, EC 1.1.3.7) described in Bjerkandera adusta (Muheim et al., 1990) and other fungi, and P. chrysosporium glyoxal oxidase (Kersten, 1990). Pyranose oxidases (EC 1.1.3.10) produce hydrogen peroxide in the hyphal periplasmic space of several white-rot fungi, e.g. P. chrysosporium, T. versicolor or Phlebiopsis gigantea, during oxidation of glucose or xylose, the major sugars derived from wood (Giffhorn, 2000). Fungal aryl-alcohol dehydrogenases (AAD, EC 1.1.1.91) and quinone reductases (QR) are also involved in lignin decomposition (Gutieirrez et al., 1994; Guillen et al., 1997).

Decomposition of lignin by saprotrophic basidiomycetes is a complex process including several enzymatic and nonenzymatic reactions (Figure 1). Laccases or ligninolytic peroxidases produced by white-rot fungi oxidize the lignin polymer, thereby generating aromatic radicals (1) (Eriksson et al., 1992). These are involved in different nonenzymatic reactions that include demethoxylation (2), C4-ether breakdown (3), aromatic ring cleavage (4) and Ca-Cb breakdown (5) (Hatakka, 2001; Martinez et al., 2005). The aromatic aldehydes released from Ca-Cb breakdown of lignin, or synthesized de novo by fungi are the substrate for AAO that generate H2O2 in cyclic redox reactions (6, 7). AAD are also involved (Guillen et al., 1994; Gutierrez et al., 1994). Phenoxy radicals from C4-ether breakdown (3) can be reduced by oxidases to phenolic compounds (8), as reported for AAO (Marzullo et al., 1995) or repolymerize back with the lignin polymer (9). The phenolic compounds can be reoxidized by laccases or peroxidases (10). Phenoxy radicals can also be subjected to Ca-Cb breakdown (11), yielding p-quinones. Quinones from the reactions (7) and (11) contribute to oxygen activation in redox cycling reactions involving laccases, peroxidases and QR (12, 13) (Guillen et al., 1997). This results in reduction of Fe2+ present in wood (14), either by superoxide cation radical or directly by the semiquinone radicals, and its reoxidation with concomitant reduction of H2O2 to hydroxyl free radicals (OH) (15) (Guillein et al., 2000; Hammel et al., 2002). The hydroxyl radical is a strong oxidizer that can attack lignin (16) and probably participates in the initial phases of wood decay when the cell wall is intact and pore size prevents the penetration of ligninolytic enzymes (Flournoy et al., 1991,1993; Evans et al., 1994). In the final steps, simple products from lignin decomposition enter hyphae and are incorporated into intracellular catabolic routes (Martinez et al., 2005).

The above mechanism, however, refers only to white-rot fungi capable of production of ligninolytic enzymes. Brown-rot fungi can only affect lignin by the formation of hydroxyl radicals (see Section 2.1.2.) that can remove methoxyl

HOCH2 Hi HC.

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Figure 1 Schematic representation of processes involved in the decomposition of lignin by white-rot basidiomycetes (see text for explanations). Source: Based on Hatakka (2001) and Martinez et al. (2005).

groups from lignin and produce methanol, and thus they leave a residue that consists mainly of modified lignin. Demethoxylation of methoxyl groups and aromatic hydroxylation makes the modified lignin more reactive (Hatakka, 2001).

White-rot fungi can be classified into four groups based on the extracellular lignolytic enzymes produced (Hatakka, 1994): (1) LiPs, MnPs and laccase; (2) MnPs and laccase; (3) LiPs and laccase; (4) no peroxidases, only laccases. Recently, it was confirmed that P. chrysosporium does not produce true laccase, but a functionally related enzyme, perhaps belonging to a very specific fifth group (Baldrian, 2006).

2.4 Enzymes Degrading Pectic Compounds, Starch and Chitin

2.4.1 Pectic Compounds

Pectic substances are complex polysaccharides being major components of middle lamellae, in the form of calcium pectate and magnesium pectate. Pectic substances account for 0.5-4.0% of the fresh weight of plants. Their relative molecular masses range from 25 to 360 kDa. Pectic substances mainly consist of galacturonans and rhamnogalacturonans — in which the C-6 carbon of galactate is oxidized to a carboxyl group, the arabinans and the arabinogalactans. Based on the increasing methoxylation of galacturonate groups, pectic substances can be divided into pectic acids, pectinic acids and pectin (Jayani et al., 2005). The pec-tinolytic enzymes may all be divided into three groups: (1) protopectinases degrade insoluble protopectin, resulting in highly polymerized soluble pectin; (2) esterases catalyze the de-esterification of pectin by the removal of methoxy esters; and (3) depolymerases catalyze the hydrolytic cleavage of the a-1,4-glycosidic bonds in the D-galacturonic acid moieties of the pectic substances. Depolymerases act on pectic substances by two different mechanisms: hydrolysis, in which they catalyze the hydrolytic cleavage with the introduction of water across the oxygen bridge, and trans-elimination lysis, in which they break the glycosidic bond by a trans-elimination reaction (Conner, 2001).

Pectinolytic enzymes are produced by phytopathogenic basidiomycetes that have to penetrate pectin-rich plant tissues (Jayani et al., 2005) and are also present in wood-rotting saprotrophs. Wood can contain up to 4% pectin and its decomposition enables penetration of bordered and pinoid pits and accelerates colonization. Endopolygalacturonase (EC 3.2.1.15) found in several brown-rot and white-rot basidiomycetes has a pH optimum between 3.0 and 4.5 (Green and Clausen, 1999; Xavier-Santos et al., 2004). The purified enzymes from Postia placenta, P. chrysosporium and Chondrostereum purpureum have molecular masses of 34-42 kDa and are produced as multiple isoenzymes (Miyairi et al., 1985; Shanley et al., 1993; Clausen and Green, 1996). In addition to endopolygalacturonase, pectate lyase (EC 4.2.2.2) was also detected in C. purpureum (Miyairi et al., 2002) and pectin lyase (EC 4.2.2.10) but not pectate lyase in Trametes. trogii (Levin and Forchiassin, 1998). Although only a few species have been tested, pectinolytic enzymes seem to be widespread among wood-rotting saprotrophs.

2.4.2 Starch

Starch serves as a storage polysaccharide contained in wood, roots and leaves, mainly in parenchyma cells (Willfor et al., 2005). The main enzyme responsible for its decomposition is amylase, 1,4-a-glucosidase (EC 3.2.1.3) which has been identified in several wood-rotting basidiomycetes including Ceriporiopsis sub-vermispora (Sethuraman et al., 1998), P. chrysosporium (Dey et al., 1991) and

S. commune (Shimazaki et al., 1984). It is also produced by soil-inhabiting sap-rotrophs, e.g. Coprinus sp. (Stephens et al., 1991). Production can be induced, e.g. by the interaction of saprotrophic fungi and invertebrates (Dyer et al., 1992), but the ecological significance of the enzyme is not yet fully clarified.

2.4.3 Chitin

Chitin, an unbranched homopolymer of 1,4-S-linked N-acetyl-D-glucosamine, is widely distributed in nature, present mainly as a cell wall component in fungi (including basidiomycetes) and in the exoskeletons of insects and other invertebrates. It is believed to be the second most abundant polysaccharide on earth, next to cellulose (Duo-Chan, 2006). The main difference from all the previously mentioned biopolymers is the presence of 7% nitrogen in the molecule. The chitin content of fungal mycelium harvested from wood has been estimated to be around 2%. Assuming an N content of 0.25-3% in the mycelium of wood-degrading basidiomycetes, 5-50% of the total N in wood-degrading mycelium would be found in chitin (Lindahl and Finlay, 2006). Chitin in fungal mycelium is therefore an important nitrogen source.

The decomposition of chitin is initiated by endochitinases (EC 3.2.1.14) that hydrolyse the bonds between N-acetylglucosamine residues at random locations within the chitin macromolecule, disrupting the structural integrity of chitin and producing oligosaccharides of varying length. ¿S-N-acetylhexosaminidases (EC 3.2.1.52) further degrade the oligosaccharides, releasing monosaccharides from the nonreducing ends. A third type of enzyme — chitobiosidase, cleaves disaccharides of N-acetylglucosamine from the ends of chitin chains (Lorito, 1998; Lindahl and Finlay, 2006).

Compared to phytopathogenic fungi, the occurrence of chitinase in sap-rotrophic basidiomycetes has not yet attracted much attention. Endochitinase and ¿-N-acetylhexosaminidase have been purified from the white-rotter P. cinnabarinus. The enzymes had molecular masses of 60 and 36 kDa and acidic pH optima at 2.5-4.5 (Ohtakara, 1988). Production of extracellular chit-inolytic enzymes has also been documented for the root-infecting bas-idiomycetes H. annosum and Armillaria ostoyae and several ectomycorrhizal (ECM) fungi (Hodge et al., 1995). All reports about the presence of chitinolytic enzymes however do not necessarily mean that the fungus uses them for saprotrophic purposes. In some cases the enzymes can be merely involved in the reconfiguration of the chitin-containing cell wall. Recently, Lindahl and Finlay (2006) studied the production of chitinolytic enzymes by three secondary wood colonizers Coniophora arida, Hypholoma capnoides and Resinicium bicolor. All of the three tested fungi produced endochitinases, chitobiosidases and N-acetylhexosaminidases during colonization of wood. N-acetylhexosami-nidase activity, and in some cases also chitobiosidase and endochitinase activities, were higher during secondary overgrowth of another fungus than during primary colonization of noncolonized wood. The results suggest that wood-degrading fungi degrade their own cell walls as well as the hyphae of earlier colonizers.

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