Influence of aromatic compounds on biodegradation of [14C]-labeled xylan and mannan by the white-rot fungus Phlebia radiata.

Radiolabeled [14C]arabinoxylan from wheat meal and [14C]galactoglucomannan from red clover meal were prepared by using 14CO2 as a precursor. Twice as much mannan was mineralized than xylan after 14 days of incubation with Phlebia radiata. Low-molecular-weight phenolic compounds structurally related to lignin increased during mineralization of both hemicellulose fractions. Veratryl alcohol increased degradation of arabinoxylan by approximately 28.5%, whereas veratric acid increased it by only 9.0%. Vanillic acid and ferulic acid also stimulated degradation by 16.6% and 34.7%, respectively. Veratryl alcohol and ferulic acid increased degradation of galactoglucomannan by approximately 75%. Veratraldehyde in both cases repressed the degradation process (23.6% arabinoxylan, 43.8% galactoglucomannan). These results indicate that the degradation of hemicelluloses, e.g., xylan and mannan, by P. radiata is enhanced by addition of aromatic compounds.

Fungal laccase: properties and activity on lignin.

The sources of ligninocellulose that occur in various forms in nature are so vast that they can only be compared to those of water. The results of several, more recent experiments showed that laccase probably possesses the big ability for "lignin-barrier" breakdown of ligninocellulose. The degradation of this compound is currently understood as an enzymatic process mediated by small molecules, therefore, this review will focus on the role of these mediators and radicals working in concert with enzymes. The fungi having a versatile machinery of enzymes are able to attack directly the "lignin-barrier" or can use a multienzyme system including "feed-back" type enzymes allowing for simultaneous transformation of lignin and carbohydrate compounds.

Production of lignolytic and feed-back type enzymes by Phlebia radiata on different media.

Low molecular-weight compounds, structurally related to lignin, increase the production of laccase, lignin peroxidase, manganese dependent peroxidase, and feed-back type enzymes such as glucose oxidase, cellobioso-quinone oxidoreductase, and glyoxal oxidase in the culture of the white rot fungus Phlebia radiata growing on different carbon sources.

Studies on the role of proteases in the white-rot fungus Trametes versicolor: effect of PMSF and chloroquine on ligninolytic enzymes activity.

In the present study we investigated the possibility of proteinases, intracellular and extracellular, being involved in the regulation of ligninolytic activities in cultures of Trametes versicolor during the shift from primary growth (i.e. trophophase) to idiophase triggered by nitrogen or carbon starvation. These studies were performed using specific inhibitors added to the cultures of T. versicolor. Addition of PMSF (irreversible inhibitor of serine proteinases) or chloroquine (the lysosomotropic agent inhibiting intralysosomal degradation of proteins) revealed distinct differences in the activity of ligninolytic enzymes between nutrient-deprived and non-starved cultures. The addition of PMSF during the transfer of mycelia to the nutrient limited media significantly enhanced the activities of laccase (2-7-fold) and of unspecified peroxidases (2-4-fold). The activity of lignin peroxidase decreased with PMSF, both in tropho- and in idiophasic cultures. The enhanced activities of laccase and general peroxidases (horseradish peroxidase-like, HRP-like) were accompanied by markedly altered patterns of both intracellular and extracellular proteolytic activities revealed by electrophoretic analysis with polyacrylamide gels containing the copolymerized substrate (haemoglobin or gelatin, respectively). The experiments with chloroquine added to nutrient-deprived cultures showed that inhibition of vacuolar proteolysis resulted in lowered activities of laccase and peroxidase. Electrophoretic analysis revealed altered patterns of intracellular proteinases upon chloroquine addition to nutrient-starved cultures. Moreover, chloroquine was found to enhance the activity of proteases secreted in carbon-starved cultures. From the results it is concluded that both intracellular (including vacuolar) and extracellular proteases are involved in the regulation of laccase and peroxidase activity in cultures of T. versicolor under nutrient limitation.

Demethylation of [14C]-labelled veratric acid and oxidation of methanol and formaldehyde by the white-rot fungus Phlebia radiata.

Veratric acids 14C-labelled in carboxyl group, 3-OCH3, 4-OCH3, or aromatic ring together with unlabelled veratric acid were supplemented in the cultures of the white-rot fungus Phlebia radiata. The effect of various carbon sources on the release of 14CO2 was studied. Veratric acid was readily decarboxylated, maximally already on day 1 from the addition of [14COOH]-veratric acid. High amounts (4%) of glucose slightly repressed the decarboxylation. In medium supplemented with cellulose the methoxyl group in position 4 was much more readily mineralized to CO2 than the group in position 3. The maximum evolution was achieved on day 5, two days from the addition. Cellulose did not repress methanol oxidation but repression of methanol oxidation by glucose was detected in media supplemented with [O14CH3]-veratric acids and 14CH3OH. However, glucose did not repress oxidation of H14CHO. The apparent uptake of 14C by fungal mycelium, especially from methoxyl groups, but also from the aromatic ring, may partially be due to the strong slime formation observed in cellobiose medium. Also in cellobiose medium apparent uptake of 14C from 14C-labelled methoxyl groups was observed.

Biodegradation of lignin by white rot fungi.

A review is presented related to the biochemistry of lignocellulose transformation. The biodegradation of wood constituents is currently understood as a multienzymatic process with the mediation of small molecules; therefore, this review will focus on the roles of these small molecular compounds and radicals working in concert with enzymes. Wood rotting basidiomycetous fungi penetrate wood and lead to more easily metabolized, carbohydrate constituents of the complex. Having a versatile machinery of enzymes, the white rot fungi are able to attack directly the "lignin barrier." They also use a multienzyme system including so-called "feed back" type enzymes, allowing for simultaneous transformation of both lignin and cellulose. These enzymes may function separately or cooperatively.

Formaldehyde as a proof and response to various kind of stress in some Basidiomycetes.

The influence of cadmium chloride and/or high temperature on the level of selected parameters were examined in both medium and mycelium of some Basidiomycetes belonging to white-rot fungi: Abortiporus biennis, Trametes versicolor and Cerrena unicolor. We investigated changes in the formaldehyde (FA) level and in the level of superoxide radical anions (SR). Accordingly, the capacity of three enzymes was also studied: two enzymes of the cellular antioxidative system - superoxide dismutase (SOD; EC 1.15.1.1), and catalase (CAT; EC 1.11.1.6), and laccase (LAC; EC 1.10.3.2) the main lignin-modifying enzyme, which is produced by white-rot fungi. During the first 24 hours after application of separate stressors, or jointly with two stressors to 10-day-old cultivation, changes in all selected parameters were observed. Moreover, we found significant changes in the levels of extracellular SR, FA and extra- and intracellular activity of LAC. Simultaneous action of two stressors in the fungal cultures decreased the extracellular LAC level, intracellular CAT activity and the level of SR in the medium compared to the control values, while using the two stress factors separately would strongly make these values increase. Stressful conditions brought a rapid increase in the level of FA in all fungal species. The results of our study, carried out on selected strains of Basidiomycetes, seem to have shown that: (I) LAC, the lignin-modifying enzyme, may have an important application in fungal stress response, and take part in the cross-protection between responses to heat shock and cadmium resistance; (2) the oxidative burst as a result of cadmium and high temperature treatment is an additional factor to damage fungal cells; (3) FA may be a determining factor in the phases of fungal stress syndrome.

Affinity chromatography as a rapid and convenient method for purification of fungal laccases.

A rapid and convenient method for graduation, isolation, and purification of laccase from Trametes versicolor and Fomes fomentarius culture fluids was developed. For purification affinity chromatography on syringyl- and vanillyl-controlled porosity glass (CPG) columns was applied. The purified laccase of F. fomentarius was immobilized on porous glass. Some properties of the immobilized enzyme in comparison to the free one are discussed.

Formation and Action of Lignin-Modifying Enzymes in Cultures of Phlebia radiata Supplemented with Veratric Acid.

Transformation of veratric (3,4-dimethoxybenzoic) acid by the white rot fungus Phlebia radiata was studied to elucidate the role of ligninolytic, reductive, and demeth(ox)ylating enzymes. Under both air and a 100% O(2) atmosphere, with nitrogen limitation and glucose as a carbon source, reducing activity resulted in the accumulation of veratryl alcohol in the medium. When the fungus was cultivated under air, veratric acid caused a rapid increase in laccase (benzenediol:oxygen oxidoreductase; EC 1.10.3.2) production, which indicated that veratric acid was first demethylated, thus providing phenolic compounds for laccase. After a rapid decline in laccase activity, elevated lignin peroxidase (ligninase) activity and manganese-dependent peroxidase production were detected simultaneously with extracellular release of methanol. This indicated apparent demethoxylation. When the fungus was cultivated under a continuous 100% O(2) flow and in the presence of veratric acid, laccase production was markedly repressed, whereas production of lignin peroxidase and degradation of veratryl compounds were clearly enhanced. In all cultures, the increases in lignin peroxidase titers were directly related to veratryl alcohol accumulation. Evolution of CO(2) from 3-OCH(3)-and 4-OCH(3)-labeled veratric acids showed that the position of the methoxyl substituent in the aromatic ring only slightly affected demeth(ox)ylation activity. In both cases, more than 60% of the total C was converted to CO(2) under air in 4 weeks, and oxygen flux increased the degradation rate of the C-labeled veratric acids just as it did with unlabeled cultures.

Dearomatization of lignin derivatives by fungal protocatechuate 3,4-dioxygenase immobilized on porosity glass.

Protocatechuate 3,4-dioxygenase (see protocatechuate: oxygen 3,4-oxidoreductase, EC 1.13.11.3) was isolated from the mycelium of Pleurotus ostreatus (induced with p-hydroxybenzoic acid) and immobilized on controlled porosity glass beads. Four fractions of Na-lignosulfonates (varying in M(r), after chromatography on Sephadex G-50) were treated with the immobilized enzyme. The products after incubation showed the same M(r) as the untreated fractions, but their light absorption at 280 nm considerably decreased. These studies indicate that dioxygenase causes partial dearomatization of lignin macromolecule.

Intensification of oak sawdust enzymatic hydrolysis by chemical or hydrothermal pretreatment.

The activities of cellulases and xylanase were determined in laboratory cultures of Aspergillus terreus F-413 performed on natural and chemically or hydrothermally pretreated oak sawdust. The best stimulation effects were obtained in the cultures containing sawdust treated with dioxane, sodium hydroxide, or phosphoric acid. Moreover, the sawdust pretreatment distinctly affected its enzymatic hydrolysis, especially when the preparation of hydrolase complex was isolated from the culture of A. terreus F-413 growing on the modified sawdust as a sole carbon source. The highest saccharification effect was observed when the sawdust was treated with dioxane, sodium hydroxide, or phosphoric acid. Glucose was the main product of sawdust decomposition found in the hydrolyzates.

Comparative studies of extracellular fungal laccases.

Various basidiomycetes, ascomycetes, and deuteromycetes, grown in a sugar-rich liquid medium, were compared for laccase-producing ability and for the inducing effect of 2,5-xylidine on laccase production. Clear stimulation of the extracellular enzyme formation by xylidine was obtained in the cultures of Fomes annosus, Pholiota mutabilis, Pleurotus ostreatus, and Trametes versicolor, whereas Rhizoctonia praticola and Botrytis cinerea were not affected by the xylidine, and in the case of Podospora anserina a decrease in laccase activity was observed. The laccases were purified, and electrophoresis on polyacrylamide gels indicated a particular pattern for each laccase. The bands of the induced forms appeared only with basidiomycetes. The optimal pH of R. praticola laccase was in the neutral region, whereas the optima of all the other exolaccases were significantly lower (between pH 3.0 and 5.7). All laccases oxidized the methoxyphenolic acids under investigation, but there existed quantitative differences in oxidation efficiencies which depended on pH and on the nature (noninduced or induced) of the enzyme. The sensitivity of all enzymes to inhibitors did not differ considerably.

Oxidative and demethylating activity of multiple forms of laccase from Pholiota mutabilis.

1. The inducible and constitutive forms of laccase from the fungus Pholiota mutabilis show both the oxidative and demethylating activity, which proves the bifunctional character of the enzyme. 2. The oxidative/demethylating activity ratio of the forms induced either with ferulic or syringic acid is different from that shown by the constitutive form. 3. Splitting of one methoxyl group from the methoxyphenol substrate is associated with the release of one molecule of methanol.

Induction of laccase in Basidiomycetes: the laccase-coding messenger.

Formation of the mRNA specific for the inducible forms of laccase was evidenced in Coriolus versicolor, Pleurotus ostreatus and Pholiota mutabilis. The half-life time of these mRNAs in the fungi species studied were, respectively, 30, 37 and 24 min. Molecular weight of the newly synthesized mRNA in Pleurotus ostreatus was about 4.5X10(5), consistently with the size of the inducible laccase protein. The polysome obtained from the ferulic acid-treated mycelium, synthesized in vitro a polypeptide with the electrophoretic mobility similar to that of laccase.

Induction of laccase in Basidiomycetes: apparent activity of the inducible and constitutive forms of the enzyme with phenolic substrates.

The purified preparations of the inducible and constitutive forms of laccase (EC 1.14.18.1) have been obtained from mycelia of Trametes versicolor, Pleurotus ostreatus and Pholiota mutabilis. The activities of the inducible forms of laccase with ferulic acid and other phenolic hydrogen donors were found to be several-fold higher as compared with the constitutive forms.