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th day of the cultivation. The evolution of carbon dioxide from aliphatic chain cleavage cinnamate showed very similar trend which demonstrated simultaneous process with the decarboxylation ones on all tested media. About 30% of carbon dioxide evolved from carboxyl-labeled cinnamate. The radioactivity in the mycelium was also above 10% in all cases and can be read as partially degraded or metabolized to the other components. The cellulose slightly repressed the decarboxylation of cinnamate in opposition to wheat straw and spruce wood, which doubled degradation. The maximum rate of decarboxylation was ca. 1.2% of the applied activity evolved as 14 CO2 per hour. According to identified metabolites, at first the substrates after decarboxylation, and aliphatic chain cleavage were demethylated in the position 4 following the demethylation in the position 3 and finally aromatic ring cleavage were observed in air and oxygen aeration on the media with glucose, cellu- lose, wheat straw and spruce wood.

14 C either at carboxyl, methoxyl, or aromatic ring was studied during the 10-day cultivation of Phlebia radiata. Seventy percents of carbon dioxide evolved from carboxyl-labeled vanillate and about 40% from ring carbon-labeled vanillate and car- boxyl-labeled benzylvanillate. The degradation of methoxyl group from vanillic and benzylvanillic acids reached a similar level, yielding about 25% after one week growth of P. radiata in these conditions. The radioactivity inside mycelium reached two maximas, the first on the 2 nd -3 rd day after the induction and the second on the 7 th day after the induction. The first peak could be derived from high polymerization which can accompany high laccase activity. These polymers of decarboxylated quinones probably attach them- selves to the mycelium and can be subsequently degraded by the P. radiata extracellular enzymatic sys- tem. These enzymes can demethylate monomeric and polymeric structures as well as cleave the aromatic ring. The late second peak inside the mycelium can be attributed to the cleavage of monomers originating from the degradation of polyquinones or from the secondary metabolites.

Suggested applications of bio–ligninolytic systems in pulp and paper industry are usually based on selective removal or in situ modification of lignin. For example, it is possible using lignin–degrading white– rot fungi to decrease the amount of energy needed for mechanical pulping, i.e. biopulping, or to facilitate pulp bleaching (Kirk and Chang, 1990; Messner and Srebotnik, 1994; Ragauskas, 2002, Hatakka et al., 2002). Besides wood, annual plants, e.g. cereal straw and grass species contain substantial amounts of cellulose, which could be used as feed for ruminants, as a growth substrate for microorganisms and also as raw material for pulp and paper industry. However, due to the close association between cellulose and other plant cell wall polymers, i.e. lignin and hemicellulose (Eriksson et al., 1990; Daniel, 1994) cellulose is not easily available as a carbon or fiber source unless the structure of lignin is chemically or biologically modified or lignin is partially removed. Microbial delignification may be considered as a noteworthy alternative (Kirk and Farrell, 1987; Hatakka et al., 1989; Lewis and Yamamoto, 1990; Sarikaya and Ladish, 1997; Scott et al., 2002). In these cases a key question is how to break down the connection between lignin and carbohydrates, especially hemicellulose. The knowledge of the critical factors that are involved in the biodegradation of lignin carbohydrate complex (LCC) would help to understand and regulate selective delignification. The white–rot fungi that belong to Basidiomycetes are the most efficient of all known lignin degraders (Eriksson et al., 1990, Hatakka, 2001) but they also degrade cellulose and hemicellulose. Among these fungi Phlebia radiata is a very efficient degrader of lignin (Hatakka and Uusi–Rauva, 1983), it preferentially degrades lignin (Ander and Eriksson, 1977; Hatakka, 1983, 1994) but also was characterized as cellulase (Rogalski et al., 1993; Longa, 1997) and hemicellulase producer (Rogalski et al., 1993a; Tokarzewska– Zadora, 2001). Phlebia. radiata produces directly lignin–modifying enzymes, including lignin peroxidase (ligninase, LiP; EC 1.11.1.14), manganese peroxidase (MnP; EC 1.11.1.13), laccase (EC 1.10.3.2) and dioxygenase (EC 1.13.11), which have been characterized (Niku–Paavola et al., 1988; 1990; Hatakka et al., 1989; Karhunen et al., 1990; Lundell, 1993; Vares et al., 1995; Rogalski et al., 1996). The second group of enzymes involved in lignin degradation does not attack wood components directly. They include aryl alcohol oxidase and glyoxal oxidase providing H2O2 for the peroxidases, and also the feed–back type enzymes as glucose oxidase and cellobiose:quinone oxidoreductase (cellobiose dehydrogenase) that play an important role in joining the metabolic chains during the bioconversion of high– molecular mass wood constituents. All these enzymes Directional Degradation of Lignocellulose by Phlebia radiata

The ability of Bjerkandera adusta R59 strain to degrade anthraquinonic antibiotic (daunomycin) points on its possible aptitudes for decomposing of other anthraquinonic derivatives, e.g. lignocellulose sub- units or metabolically related lipids, present in wood. This study was performed to investigate the possibil- ity of B. adusta R59 to synthesize enzymes participating in decay of wood compounds (including lignin, celluloses, hemicelluloses and lipids). Geotrichum-like strain, anamorphic stadium of B. adusta, white-rot fungus, was isolated from soil. It was found to completely decolorize and degrade 10% daunomycin post- production effluent during 10 days of incubation at 26 °C. R59 strain produces only small activities of ligno- lytic enzymes when grown on wheat straw or beech sawdust-containing media but in the presence of humic acids derived from brown coal synthesizes significant activities of laccase and lipase. This phenomenon was coupled with entering the idiophase by this fungus and appearance of aerial mycelium. The ability of B. adusta R59 strain to degrade humic acids from brown coal could be useful in constructing of new genera- tion of biologically active filters for purification of humic acids-contaminated comestible waters.

It has recently been established that most short- and long-lived cellular proteins (80-90%) are degrad- ed by a highly selective non-lysosomal pathway that requires ATP and a large (~2.5 MDa) multisubunit, multicatalytic proteinase complex known as the 26S proteasome. It degrades many important proteins involved in signaling pathway, in cell cycle control, and in general metabolism, including transcription fac- tors and key metabolic enzymes. Here, we demonstrated all distinct proteasome activities: chymotrypsin- like, trypsin-like, and caspase-like (peptidylglutamyl-peptide hydrolyzing) in mycelial extracts of the white-rot fungi Trametes versicolor and Phlebia radiata by monitoring cleavage of three different fluoro- genic peptide substrates: Suc-LLVY-MCA, Z-GGR-MCA, Z-LLE-βNA, respectively. We also found that this cleavage was ATP-dependent. Reagents that inhibit proteasome-mediated protein degradation in intact cells have recently become available, including substrate-related peptide aldehydes. These inhibitors are useful tools to demonstrate that a process exhibits proteasome-dependent biochemical regulation. In the present study, we report that in vivo Cbz-LLLal treatment strongly inhibited all tested proteasome activi- ties and affected ligninolytic activities in nutrient deprived cultures of both fungi.

Several wood rotting fungi decolourized Remazol brilliant blue R (RBBR) and carminic acid (CA). Parallel activity of laccase in these fungi was studied. The addition of acetovanillone (AV) or ace- tosyringone (AS) intensified these processes: decolourization was more extensive than in the experiment omitting these compounds. At the presence of AS the decourization was more extensive than AV. However the level of destaining was relatively low in comparison to laccase activity on syringaldazine. The highly purified constitutive form of Cerrena unicolor and inducible form of Trametes versicolor laccases also decolourized both dyes. The fungus, Lentinus lepideus, which has no laccase activity did not decolourize at all. Nitrogen starvation induced the laccase and decolourization activity in both organisms, irrespective of nitrogen availability. This fact indicates laccase not solely responsible for decolourization, and probably decolourization of dyes involves more than one mechanism.

This study was performed in order to investigate the effect of selected xenobiotic compounds, such as different stress factors, Cd(II), Zn(II), Cu(II) and Mn(II) ions or some pro-oxidants: menadione, paraquat and hydrogen peroxide, on laccase activity with some white-rot fungi. Changes in laccase activity in the presence of natural fungicidals, the saponins from Medicago sativa roots, were also examined. The intro- duction of both metal ions and some pro-oxidants into the medium enhanced a multiple increase in extra- cellular laccase activity for the two fungal strains, Trametes versicolor and Abortiporus biennis. An nota- ble increase in laccase level had already appeared in the 36 hour by the treatment with oxidative stress fac- tors (menadion, paraquat and hydrogen peroxide), anti-fungal substance saponines as well as metal ions. The enhanced activity of laccase under heavy metal ions presence also suggests its important role in the fun- gal adaptation processes, what could be utilized to heavy metals containing waste materials. These strains of white-rot fungi could also be used to remove heavy metals from aqueous solutions by adsorbing the met- als on mycelium and to biodegrade various organic xenobiotics by extracellular laccase. The presence of harmful compounds in the medium which might appear lethal for other organisms arouses detoxification abilities of this fungal group by increasing extracellular discharge of ligninolytic enzymes, in particular lac- case. This offers extremely large possibilities for biotechnological use of white-rot fungi.