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Degradation of cellulose and hemicelluloses by the brown rot fungus Piptoporus betulinus – production of extracellular enzymes and characterization of the major cellulases

Vendula Valáková

Laboratory of Biochemistry of Wood-Rotting Fungi, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeská 1083, 14220, Prague 4, Czech Republic

Correspondence
Petr Baldrian
baldrian{at}biomed.cas.cz

	ABSTRACT

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Piptoporus betulinus is a common wood-rotting fungus parasitic for birch (Betula species). It is able to cause fast mass loss of birch wood or other lignocellulose substrates. When grown on wheat straw, P. betulinus caused 65 % loss of dry mass within 98 days, and it produced endo-1,4--glucanase (EG), endo-1,4--xylanase, endo-1,4--mannanase, 1,4--glucosidase (BG), 1,4--xylosidase, 1,4--mannosidase and cellobiohydrolase activities. The fungus was not able to efficiently degrade crystalline cellulose. The major glycosyl hydrolases, endoglucanase EG1 and -glucosidase BG1, were purified. EG1 was a protein of 62 kDa with a pI of 2.6–2.8. It cleaved cellulose internally, produced cellobiose and glucose from cellulose and cellooligosaccharides, and also showed -xylosidase and endoxylanase activities. The K_m for carboxymethylcellulose was 3.5 g l^–1, with the highest activity at pH 3.5 and 70 °C. BG1 was a protein of 36 kDa with a pI around 2.6. It was able to produce glucose from cellobiose and cellooligosaccharides, but also produced galactose, mannose and xylose from the respective oligosaccharides and showed some cellobiohydrolase activity. The K_m for p-nitrophenyl-1,4--glucoside was 1.8 mM, with the highest activity at pH 4 and 60 °C, and the enzyme was competitively inhibited by glucose (K_i=5.8 mM). The fungus produced mainly -glucosidase and -mannosidase activity in its fruit bodies, while higher activities of endoglucanase, endoxylanase and -xylosidase were found in fungus-colonized wood.

	INTRODUCTION

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Brown rot basidiomycetes cause the most destructive form of wood decay by rapidly depolymerizing the polysaccharidic component (Eriksson et al., 1990). They also belong to the principal recyclers of lignocellulose in forest ecosystems (Gilbertson & Ryvarden, 1986). Although brown rot fungi can cause extensive lignin modification, little lignin is removed from the wood, probably due to the lack of ligninolytic peroxidases or oxidases. However, brown rot fungi rapidly cleave the cellulose and hemicelluloses in wood, and subsequently remove it so completely that extensively brown-rotted wood consists almost entirely of modified lignin (Eriksson et al., 1990).

The enzymic cellulolytic system of brown rot fungi did not attract as much attention as that of other well-characterized groups of cellulose-degrading micro-organisms. The white rot fungus Phanerochaete chrysosporium effectively degrades crystalline cellulose via a synergistic mechanism between endoglucanases and cellobiohydrolases (Eriksson et al., 1990). Endoglucanases cleave within cellulose molecules, generating non-reducing ends. Cellobiohydrolases (exoglucanases) act processively from these free ends, remaining attached to the cellulose and releasing soluble cellobiose molecules, which are subsequently hydrolysed to assimilable glucose by -glucosidases (Beguin & Aubert, 1994).

Endoglucanases have been isolated from several brown rot fungi, including Gloeophyllum sepiarium (Mansfield et al., 1998), Gloeophyllum trabeum (Herr et al., 1978a; Mansfield et al., 1998), Polyporus schweinitzii (Bailey et al., 1969), Serpula incrassata (Kleman-Leyer & Kirk, 1994) and Tyromyces palustris (Ishihara & Shimizu, 1984). In general, it appears that most endoglucanases of brown rot fungi are constitutively expressed and are not catabolically repressed by glucose (Cotoras & Agosin, 1992; Highley, 1973). Purified glucosidases have so far been reported only from G. trabeum (Herr et al., 1978b) and Poria vailantii (Sison et al., 1958).

While brown rot fungi do not produce cellobiohydrolase, with the exception of Coniophora puteana (Sethuraman et al., 1998), and most of them have not been reported to degrade crystalline cellulose, there is an open question how they produce the substrate for their -glucosidases if cellobiohydrolases are not involved. It was recently reported that the brown rot fungus G. trabeum produces a processive endoglucanase, an enzyme with the ability to cleave cellulose internally but also release soluble oligosaccharides (Cohen et al., 2005). However, there is currently no information about similar activity in other brown rot fungi.

Radical-generating reactions have been confirmed to participate in cellulose degradation by several brown rot fungi (Goodell, 2003; Hammel et al., 2002). These include the involvement of quinone cycling (Jensen et al., 2001) and cellobiose dehydrogenase-catalysed reactions (Hyde & Wood, 1997). However, production of reactive radicals has not been found in all brown rot species tested (Kleman-Leyer & Kirk, 1994). The enzymic system of these fungi is thus of primary importance for the utilization of cellulose and hemicelluloses. Only scant information is available about the enzymic activity during wood decay, i.e. in colonized wood and fungal fruit bodies.

The aim of this study was to estimate the activity of glycosylhydrolases of the brown rot fungus Piptoporus betulinus, detect these activities in the natural substrate and characterize the main cellulolytic enzymes. P. betulinus is a hardwood-specific parasite of birch (Betula species) trees in northern temperate forests and causes a very fast wood decay. It is also one of the most common brown rot species in central Europe (Baldrian & Gabriel, 2002), and has been reported to depolymerize cellulose (Bell & Burnett, 1966). The fungus exhibits a high rate of wheat straw degradation accompanied by a high production of hydrolytic enzymes, which makes it interesting for potential biotechnological use.

	METHODS

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Reagents.
Chemicals were obtained from Sigma-Aldrich/Fluka. Phosphoric acid swollen cellulose (PASC) was prepared from Avicel, as described previously (Wood, 1988).

Organism and maintenance.
The wood-rotting basidiomycete P. betulinus CCBAS585 was obtained from the Culture Collection of Basidiomycetes (CCBAS, Institute of Microbiology Academy of Sciences of the Czech Republic). For the preparation of inocula, the fungus was grown on malt extract (ME) agar plates (15 g malt extract l^–1, 20 g agar l^–1) at 28 °C for 7 days. Mycelium agar plugs 7 mm in diameter (cut along the edge of an actively growing colony) were used as inocula.

Degradation of lignocellulose.
For enzyme activity estimations, the cultivation of P. betulinus proceeded in 100 ml Erlenmeyer flasks containing 5 g air-dried milled wheat straw. The straw was moistened with 15 ml distilled water (final water content 75 %). The flasks were stoppered, autoclaved (2x25 min at 121 °C) and inoculated with two agar plugs with mycelium. The cultures were incubated at 28 °C in the dark (Baldrian & Gabriel, 2003). Three flasks were collected on each sampling day (days 14, 28, 42, 56, 70 and 98).

Enzyme activity in fruit bodies and colonized wood.
Fresh fruit bodies of P. betulinus were collected from dead birch trees (Betula pendula) from several sites in central Bohemia, Czech Republic. Samples of wood immediately adjacent to the fruit bodies with apparent fungal growth were also collected. Natural samples were stored at 4 °C until analysed (less than 48 h). pH was determined in water extracts of all samples.

Enzyme extraction.
Buffers of different composition (pH 5–7, 100 mM phosphate buffer, 160 mM acetate buffer) and distilled water were compared for the maximum enzyme activity recovery from straw, fruit bodies and wood. The highest recovery from wheat straw was achieved with 160 mM sodium acetate buffer (pH 5.0), while distilled water was the most effective extractant for both fruit bodies and wood. Each sampling day, straw cultures in Erlenmeyer flasks were cut into small pieces and soaked with 40 ml sodium acetate buffer (160 mM, pH 5.0), while the fresh fruit bodies and wood were homogenized using a laboratory blender and soaked with 40 ml distilled water. The homogenized substrates were extracted at 4 °C for 2 h on a shaker. Extracts were filtered and the filtrates were kept at 4 °C until analysed (less than 24 h). Low molecular mass (LMM) and high molecular mass (HMM) fractions of culture extract from day 56 were prepared by ultrafiltration using a 10 kDa-cutoff membrane with an Amicon ultrafiltration cell (Millipore) and desalting on PD10 columns (Amersham).

The solid residues of the straw substrate, fruit bodies or wood were collected after filtration, dried at 105 °C to constant mass and weighed. The loss of dry mass in the straw cultures was calculated as the difference against the dry mass at day 0.

Assays.
Activities of endo-1,4--glucanase (EG), endo-1,4--xylanase and endo-1,4--mannanase were routinely measured with azo-dyed carbohydrate substrates [carboxymethyl cellulose (CMC), birchwood xylan and galactomannan, respectively], using the protocol of the supplier (Megazyme). The reaction mixture contained 0.2 ml 2 % dyed substrate in 200 mM sodium acetate buffer (pH 5.0), and 0.2 ml sample. The reaction mixture was incubated at 40 °C for 20–60 min and the reaction was stopped by adding 1 ml ethanol followed by 10 s vortexing and 10 min centrifugation (Baldrian et al., 2005). The amount of released dye was measured spectrophotometrically at 595 nm and the enzyme activity was calculated according to standard curves that correlated the dye release with the release of reducing sugars.

Endoglucanase activity was also assayed in 10–30 min reactions, using a 1 % (w/v) solution of CMC in 50 mM citrate/phosphate buffer (pH 5.0) at 40 °C. Xylanase and mannanase were assayed likewise with 1 % (w/v) birchwood xylan or galactomannan as the substrates. Activities against Avicel and PASC were determined in 250 µl reactions that contained 1 % (w/v) substrate in 50 mM citrate/phosphate (pH 5.0) and 0.02 % (w/v) NaN₃. The capped mixtures were shaken at 40 °C for 1–24 h. Reducing sugars were determined as glucose equivalents by the ferricyanide method (Sethuraman et al., 1998). For all glycosylhydrolase assays, one unit of activity was defined as the amount of enzyme that converted one nanomole of substrate per minute.

The activity of 1,4--glucosidase (BG) was determined using p-nitrophenyl--D-glucoside (pNPG) in 50 mM sodium acetate buffer (pH 5.0) in 20–60 min reactions at 40 °C. The release of p-nitrophenol from the substrate was determined by the increase in absorbance at 400 nm (=18.3 mM^–1 cm^–1) after addition of Na₂CO₃ to the reaction mixtures (Sadana & Patil, 1988). Activities of cellobiohydrolase, 1,4--xylosidase and 1,4--mannosidase were assayed using p-nitrophenyl--D-cellobioside (pNPC), p-nitrophenyl--D-xyloside (pnpx) and p-nitrophenyl--D-mannoside (pNPM), respectively, using the same method.

Cellobiose dehydrogenase was assayed by monitoring 2,6-dichlorophenolindophenol-reducing activity in 50 mM sodium acetate buffer (pH 5.0) (Temp & Eggert, 1999).

Laccase activity was measured using 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid in citrate/phosphate (100 mM citrate, 200 mM phosphate) buffer (pH 5.0). Activities of peroxidases, including Mn-peroxidase were assayed using 3,3-dimethylaminobenzoic acid and 3-methyl-2-benzothiazolinone hydrazone in succinate/lactate buffer (100 mM, pH 4.5), as described previously (Ngo & Lenhoff, 1980; Bourbonnais & Paice, 1990; Baldrian et al., 2000). All spectrophotometric measurements were done in a microplate reader (Sunrise, Tecan) or a UV-VIS spectrophotometer (Lambda 11, Perkin-Elmer).

Statistical analyses were performed by the Statistica 7.0 software package (StatSoft). Differences at P <0.05 were regarded as statistically significant unless otherwise stated. For better comparison of values obtained in a system with changing water content and dry mass, enzyme activities during lignocellulose degradation were expressed per gram of original dry mass of straw.

Purification of EG.
The water extract of a 42-day culture grown on milled wheat straw was filtered and concentrated by ultrafiltration using a 10 kDa-cutoff membrane with an Amicon ultrafiltration cell (Millipore). The concentrate was loaded onto a DEAE-Sepharose HR 10/10 column (Pharmacia LKB) equilibrated with 20 mM phosphate buffer, pH 6.0. Proteins were eluted with a gradient of 0–0.5 M NaCl in 20 min at a flow rate of 0.5 ml min^–1. The EG fractions were pooled, concentrated and applied to a Superdex 200 HR 10/30 column (Pharmacia LKB). Elution was performed with 20 mM phosphate buffer, pH 6.0, containing 0.15 M NaCl, at a flow rate of 0.5 ml min^–1. Desalted EG fractions were applied to MonoQ HR 5/5 anion-exchange column (Pharmacia LKB) equilibrated with 20 mM phosphate buffer, pH 6.0, and eluted with a gradient of 0–0.5 M NaCl in 20 min at a flow rate of 0.5 ml min^–1. In the next step, EG fractions were desalted and chromatographed again on MonoQ equilibrated with 20 mM phosphate buffer, pH 7.5, under the conditions described above. The purity of the enzyme was checked using SDS-PAGE. The concentrated EG fractions were desalted to deionized water and stored frozen (–18 °C).

Purification of BG.
The water extract of a 42-day culture grown on milled wheat straw was filtered and concentrated by ultrafiltration using a 10 kDa-cutoff membrane with an Amicon ultrafiltration cell (Millipore). The concentrate was loaded onto a DEAE-Sepharose HR 10/10 column (Pharmacia LKB) equilibrated with 20 mM phosphate buffer, pH 6.0. Proteins were eluted with a gradient of 0–0.5 M NaCl in 20 min at a flow rate of 0.5 ml min^–1. Desalted BG fractions were applied to a MonoQ HR 5/5 anion-exchange column (Pharmacia LKB) equilibrated with 20 mM phosphate buffer, pH 6.0, and eluted with a gradient from 0 to 0.5 M NaCl in 20 min at a flow rate of 0.5 ml min^–1. The BG fractions were pooled, concentrated and applied to a Superdex 200 HR 10/30 column (Pharmacia LKB). Elution was performed with 20 mM phosphate buffer, pH 6.0, containing 0.15 M NaCl, at a flow rate of 0.5 ml min^–1. In the next step, BG fractions were again desalted and chromatographed, employing MonoQ equilibrated with 20 mM phosphate buffer, first at pH 6.0 and then at pH 7.5, and 50 mM sodium acetate buffer, pH 4.5, respectively. Purity of the enzyme was checked using SDS-PAGE. The concentrated BG fractions were desalted to deionized water and stored frozen (–18 °C).

Characterization of purified enzymes.
SDS-PAGE was performed using a 10 % polyacrylamide gel. Analytical IEF was carried out with a Multiphor II electrophoresis system (Pharmacia LKB). The IEF gel (7.5 %) was prepared using ampholines of pI 2.5–5.0 and pI 3.5–10.0 (Pharmacia LKB). A Low pI protein calibration kit, pI 2.5–6.5 (Pharmacia LKB), was used for the estimation of isoelectric point. Gels were stained using a Silver Stain Plus kit (Bio-Rad) and BG gels were activity-stained with pNPG. Protein concentrations were determined with the Bio-Rad Protein Assay kit by the method of Bradford, with BSA as the standard (Bradford, 1976). The molecular mass of the enzymes was estimated by gel filtration on a Superdex 200 HR 10/30 column (Pharmacia LKB) with gel filtration standards (Pharmacia LKB), and by SDS-PAGE with Sigma CK molecular mass markers.

The effect of pH on enzyme activity was examined in the pH range 2.25–8.0 in 50 mM citrate/phosphate buffer. CMC was used as substrate for EG, and pNPG, pNPX, pNPM and p-nitrophenyl--D-galactoside (pNPGa) were used as substrates for BG. Microcal Origin Professional 7.0 software was used for curve fitting. The effect of temperature on the enzyme activity and stability was determined in the range 10–80 °C. Substrate specificity for EG and BG was tested in 50 mM citrate/phosphate buffer, pH 5.0, with a range of substrates (Table 3). For EG, the K_m and k_cat were determined for CMC in 50 mM citrate/phosphate buffer (pH 2.5), and for pNPC, pNPG and pNPX in 50 mM citrate/phosphate buffer (pH 5.0); for BG, the K_m and k_cat were determined for pNPG, pNPM and pNPX in 50 mM citrate/phosphate buffer at the respective optimal pHs. The inhibitory effect on pNPG cleavage by BG was tested for glucose, cellobiose, mannose, galactose and xylose in 50 mM citrate/phosphate buffer (optimal pH) and respective K_i values were calculated. The kinetic parameters K_m, k_cat and K_i were obtained by a non-linear least-square fitting procedure using the ordinary Michaelis–Menten equation (Lineweaver–Burk plots) and using a non-linear regression with Microcal Origin Professional 7.0 software.

	RESULTS

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Degradation of lignocellulose
P. betulinus colonized the whole volume of wheat straw substrate in the first 2 weeks after inoculation. The initial rate of substrate degradation was relatively low and reached 11.4 % after 21 days of cultivation. The highest rate of dry mass loss was observed during the subsequent weeks (52.1 % on day 56). At the end of the experiment on day 98, the loss of the straw substrate dry mass reached 65.3 %.

P. betulinus cultures exhibited all seven tested cellulose and hemicellulose-degrading activities: EG, endo-1,4--xylanase, endo-1,4--mannanase, cellobiohydrolase, BG, 1,4--xylosidase and 1,4--mannosidase. The activity of ligninolytic enzymes, laccase and peroxidases, was not detected, nor was the activity of cellobiose dehydrogenase. EG was the major endo-cleaving polysaccharide hydrolase, accounting for more than 85 % of the total activity, with a mean activity of 6400 U g^–1 compared to 720 U g^–1 for endo-1,4--xylanase and 120 U g^–1 for endo-1,4--mannanase (Fig. 1). Peak activity was reached on day 56 for EG (11 300 U g^–1), while it was earlier for endo-1,4--xylanase (day 42, 1450 U g^–1) and endo-1,4--mannanase (day 21, 345 U g^–1). BG was the major exo-cleaving hydrolase, with a mean activity of 810 000 U g^–1, accounting for approximately 67 % of the total activity of this group of enzymes. The activity of 1,4--mannosidase was higher than that of 1,4--xylosidase (199 000 and 106 000 U g^–1, respectively), and a lower yet significant cellobiohydrolase activity was detected (88 000 U g^–1). The activity of BG increased rapidly during the first 4 weeks of the experiment, reaching a peak value of 1.4x10⁶ U g^–1 on day 28 (Fig. 1). The activity of 1,4--mannosidase peaked on day 42 at 380 000 U g^–1, and both 1,4--xylosidase and cellobiohydrolase activities were relatively stable during the whole cultivation period. The hydrolytic activity towards all glycosylhydrolase substrates was associated exclusively with the HMM fraction of P. betulinus culture extract, and no activity was detected with the LMM fraction or the LMM fraction plus 0.1 mM FeCl₃ and H₂O₂.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Time-course of enzyme activities during the growth of P. betulinus on wheat straw. (a) Endo-cleaving hydrolases: EG (), endo-1,4--xylanase () and endo-1,4--mannanase (). (b) Exo-cleaving hydrolases: BG (), 1,4--xylosidase (), 1,4--mannosidase () and cellobiohydrolase (). Means of three replicates and standard errors are shown.

View larger version (73K): [in this window] [in a new window]   Fig. 2. SDS-PAGE (a) and IEF (b) of purified BG1 and EG1. The gels were stained by silver staining; lanes M contain molecular mass markers (a) and pH markers (b). Numbers indicate molecular mass in kDa (SDS-PAGE) and pH (IEF).

View larger version (19K): [in this window] [in a new window]   Fig. 3. Effect of pH on the activity of EG and BG of P. betulinus. Open symbols, EG; filled symbols, BG. Substrates: pNPG (squares), pNPM (circles), pNPX (triangles), pNPGa (inverted triangles). Data points represent means of three replicate estimations, and are expressed as a percentage of the highest activity; relative standard errors did not exceed 5 %.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Effect of temperature on the activity of EG1 and BG1 of P. betulinus. , EG; , BG. The data are expressed as a percentage of the highest activity; relative standard errors did not exceed 5 %.

Oligoglucosides produced from cellulase substrates
Analysis of oligoglucoside cleavage products confirmed that no detectable amounts of reducing oligosaccharides were produced by P. betulinus culture extract from Avicel. Both glucose and cellobiose were produced from PASC, and glucose was the only product of oligosaccharide cleavage (Fig. 5). EG1 released cellobiose and glucose from PASC and cellotriose, and cellobiose alone from cellotetraose. BG1 released glucose as the reaction product from cellobiose, cellotriose and cellotetraose, and also from PASC.

View larger version (59K): [in this window] [in a new window]   Fig. 5. TLC showing products released from Avicel, PASC and oligoglucosides. The standard lanes (Stds) include glucose (G1), cellobiose (G2), cellotriose (G3) and cellotetraose (G4). The other lanes show, from left to right, products released by P. betulinus culture extract, purified EG1 and purified BG1 from Avicel (Avic.), PASC and oligoglucosides G2–G4.

Exo-cleaving glycosylhydrolases were the dominant enzymes in fungal fruit bodies. Among them, BG activity was the highest, with tens to hundreds of thousands of units per gram dry mass, while the activities of 1,4--mannosidase and cellobiohydrolase ranged from below ten to tens of thousands of units per gram dry mass, and 1,4--xylosidase activity was even lower (Table 4). The activity of endo-cleaving hydrolases was often not detected in the fruit bodies at all, and ranged from below ten to a few tens of units per gram dry mass. Endo-1,4--mannanase activity was much lower than that of the other two enzymes (Table 4). Within a fruit body, enzyme activities of monosaccharide hydrolases were usually higher in the hymenium compared to the trama, but the differences were not statistically significant. The size (mass) of the fruit bodies as an approximate indication of their developmental stage did not show any correlation with enzyme activities.

The activities of individual enzymes in wood and fruit bodies were significantly different, both when the whole sets of data or the pairs of fruit body/adjacent wood were compared. The difference was significant at a probability level of 0.01 in all cases except endo-1,4--mannosidase, the enzyme with low activity in both fruit bodies and wood. Also, the pH values of fruit bodies and wood were significantly different, with more acidic values of around 3.0–3.5 in wood and higher values of 4.5–5.5 in the fruit bodies (Table 4).

	DISCUSSION

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The brown rot basidiomycete P. betulinus exhibited activities of all tested glycosylhydrolases and caused a rapid degradation of wheat straw during its growth. The mass loss after 98 days was comparable to that of the white rot fungus Pleurotus ostreatus, while the activities of glycosylhydrolases were higher by up to two orders of magnitude (Baldrian et al., 2005).

The participation of reactive oxygen species and reactive radicals in brown rot fungi has repeatedly been demonstrated (Hyde & Wood, 1997; Jensen et al., 2001; Hammel et al., 2002; Goodell, 2003). P. betulinus did not produce cellobiose dehydrogenase, nor was there a detectable hydrolytic activity of the LMM (<10 kDa) fraction of the culture extracts after the addition of Fe(III), and 2,5-dimethoxybenzoquinone was not found in the cultures (data not shown). However, the involvement of quinone cycling in the radical reactions (Jensen et al., 2001) cannot be ruled out.

Like several other brown rot fungi (Ishihara & Shimizu, 1984; Kleman-Leyer & Kirk, 1994; Mansfield et al., 1998; Cohen et al., 2005), P. betulinus produced more enzymes with endoglucanase activity. The molecular mass of the major endoglucanase EG1 (62 kDa) was higher than that of the endoglucanases of most other brown rot fungi. With the exception of the LMM enzymes Cel 25 from S. incrassata (Kleman-Leyer & Kirk, 1994) and Cel 12a from G. trabeum (Herr et al., 1978a; Cohen et al., 2005), the molecular mass is typically between 35 and 50 kDa (Keilich et al., 1969; Ishihara & Shimizu, 1984; Kleman-Leyer & Kirk, 1994; Clausen, 1995; Mansfield et al., 1998; Cohen et al., 2005). Larger enzymes (Cel 57) have been found only in fungi that produce a greater number of isoenzymes: Gl. sepiarium (Bhattacharjee et al., 1993) and S. incrassata (Kleman-Leyer & Kirk, 1994). The isoelectric point of EG1 was more acidic than the 3.1–4.9 reported for other enzymes (Kleman-Leyer & Kirk, 1994; Mansfield et al., 1998; Cohen et al., 2005), and the pH optimum was also more acidic: 3.5 compared to the 4.0–4.5 for Gl. sepiarium, G. trabeum and P. schweinitzii endoglucanases. However, all of these enzymes are highly active at pH <4.0 (Keilich et al., 1969; Herr et al., 1978a; Mansfield et al., 1998). The low pH optimum of endoglucanase corresponds with the low pH of wood colonized by P. betulinus (Table 4).

EG1 exhibited high affinity towards its best substrate: the K_m for CMC was 3.5 g l^–1. EGS endoglucanase from Gl. sepiarium, EGT from G. trabeum and a 29 kDa endoglucanase from G. trabeum had K_m values of 7.6, 6.3 and 13.1 g l^–1, respectively. EG1 also exhibited detectable activity towards a range of other substrates. Interestingly, it also cleaved pNPX with high activity, but showed little activity with galactomannan and pNPG. The activity with Avicel was extremely low. With respect to the oligosaccharide hydrolysis, EG1 showed a combination of EGT and Cel5A activities from G. trabeum: like EGT it yielded glucose and cellobiose from PASC, but unlike EGT, EG1 also cleaved cellotriose (Mansfield et al., 1998; Cohen et al., 2005). Although a low activity with pNPG was detected, EG1 was unable to release glucose from cellobiose. Like both EGT and Cel5A, EG1 is also a processive enzyme able to cleave oligosaccharides from HMM cellulose (although not crystalline cellulose).

Although all brown rot fungi probably produce -glucosidase activity (Goodell, 2003), only the enzymes from Por. vailantii and G. trabeum have been purified, and the latter is the only well-characterized -glucosidase (Sison et al., 1958; Herr et al., 1978b). The G. trabeum -glucosidase is a large enzyme with a molecular mass of 320 000 Da, active with glucose and cellooligosaccharides, but inactive with CMC or crystalline cellulose. Its K_m values for pNPG and pNPX are 0.4 and 3.3 mmol l^–1, respectively (Herr et al., 1978b). BG1, the enzyme responsible for most of the P. betulinus -glucosidase activity, is much smaller, with a molecular mass even smaller than that of -glucosidases from white rot fungi (Deshpande et al., 1978; Smith & Gold, 1979; Copa-Patino & Broda, 1994; Cai et al., 1998). The K_m value of P. betulinus BG1 for pNPG is lower than that of G. trabeum, but is well within the relatively broad range (0.1–5.3 mmol l^–1) found in wood-rotting fungi (Smith & Gold, 1979; Copa-Patino & Broda, 1994; Lymar et al., 1995; Wei et al., 1996; Morais et al., 2002). In addition to its activity against cellobiose and cellooligosaccharides, BG1 also exhibits low activity against PASC. The wide range of substrates hydrolysed by BG1, with no preference for one principal substrate, makes this enzyme a unique molecule among cellulases of wood-rotting basidiomycetes (Deshpande et al., 1978; Herr et al., 1978b; Smith & Gold, 1979; Copa-Patino & Broda, 1994; Cai et al., 1998).

From the overlapping substrate specificities of P. betulinus enzymes it seems obvious that the classification of glycosylhydrolases into endo- and exo-cleaving enzymes cannot be strict while ‘processive’ endoglucanases show oligosaccharide-releasing activity. Also, the cellulolytic and hemicellulolytic enzymic systems cannot be separated, since several enzymes show activity against more than one substrate (Copa-Patino & Broda, 1994; Cohen et al., 2005). The classification according to the major activity can thus be misleading with respect to the actual physiological role.

Although growth in wood and the production of fruit bodies are the most ecologically relevant modes of the existence of wood-rotting fungi, relatively little attention has been paid to the production of enzymes under these conditions, and there are no reports about cellulolytic enzymes in the fruit bodies of brown rot fungi growing under natural conditions. Poria placenta produces several endo-cleaving glycosylhydrolases and 1,4--glycosidases during growth in Liquidambar styraciflua (sweetgum) wood, with xylanase as the main enzyme (Highley, 1973). Production of xylanase and BG has also been detected in wood colonized by the white rot fungus Ceriporiopsis subvermispora. All of the hydrolytic activities detected in P. betulinus-colonized straw were also present in the colonized wood, despite the great range of detected activities. Activities of endo-1,4--xylanase, EG and 1,4--xylosidase were detected in all samples of wood colonized by P. betulinus, while not all samples exhibited detectable endo-1,4--mannanase, BG or 1,4--mannosidase activity. The pH in the wood was low, probably due to the production of organic acids (Takao, 1965) that formed a favourable environment for endoglucanase. This enzyme was possibly also responsible for at least some 1,4--xylosidase activity detected in wood. Interestingly, fruit bodies of P. betulinus with pH values more favourable for BG activity frequently contained no activity of endo-cleaving hydrolases. It seems that the endo-cleavage of polysaccharides by P. betulinus, along with the processive production of cellobiose, is preferentially performed in wood, while the fruit bodies contain mostly disaccharide and oligosaccharide hydrolases.

This work shows that P. betulinus is able to perform fast lignocellulose hydrolysis due to its hydrolytic enzymes with relatively wide substrate specificities. The enzymes are produced both during fungal colonization of wood and on wheat straw, the latter fact being of possible use for the fast production of cellulolytic enzymes for biotechnology purposes.

	ACKNOWLEDGEMENTS

 
This work was supported by the Grant Agency of the Czech Republic (204/02/P100) and by the Institutional Research Concept no. AV0Z50200510 of the Institute of Microbiology, Academy of Sciences of the Czech Republic.

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Received 19 May 2006; revised 27 July 2006; accepted 18 August 2006.

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