Manganese dependent peroxidase (MnP) is the most ubiquitous enzyme produced by white-rot fungi, MnP is known to be involved in lignin degradation, biobleaching and oxidation of hazardous organopollutants. Bjerkandera fumosa is a nitrogen-unregulated white-rot fungus, which produces high amounts of MnP in the excess of N-nutrients due to increased biomass yield. The objective of this study was to optimize the MnP production in N-sufficient cultures by varying different physiological factors such as Mn concentration, culture pH, and incubation temperature. The growth of fungus was optimal in pH 4.5 at , -unregulated white-rot fungus produces high amounts of MnP in the excess N-nutrients. The fungus produced the highest level of MnP (up to ) with asparagine and as N source at 1.5 mM concentration, pH value of 4.5 at . Purification of MnP revealed the existence of two isoforms: MnPl and MnP2. The molecular masses of the purified MnPl and MnP2 were in the same range of 42~45 kDa. These isoforms of B. fumosa strictly require Mn to oxidize phenolic substrates. Concerned to kinetic constants of B. fumosa MnPs, B. fumosa has similar Km value and Vmax compared to the other white-rot fungi.

Effects of paraquat dichloride (PQ) on the laccase (LAC) activity and some biochemical parameters of Trametes versicolor and Abortiporus biennis strains belonging to white rot Basidicunycetes fungi were examined. PQ water solution was added to 10-day-old stationary cultures cultivated on a liquid medium. Having measured the activity of extracellular laccase during the first 120h, we found that the addition of 25 M paraquat to T versicolor and 20 M paraquat to A. biennis cultures significantly stimulated the LAC activity in comparison to the control value (without PQ). Native PAGE gel analysis demonstrated that no new isoforms of laccase appeared in the presence of PQ stress. The increase of LAC activity was connected with dry weight loss. Enhanced activity of extracellular superoxide dismutase was observed during the first 48 h after PQ application in both investigated strains. The PQ-treatment also caused an evident increase of catalase activity, formaldehyde level and depletion of glutathione in T versicolor as well as in A. biennis mycelia. (c) 2006 Elsevier Inc. All rights reserved.

The influence of aqueous extracts of alfalfa (Medicago saliva) tops, roots, seeds and isolated total saponins (SAP) from alfalfa roots on the growth and biological activity of Trametes versicolor was examined. Results indicate that saponins from roots of a Medicago saliva strain are very good inductors of extracellular laccase (LAC). This strain was cultivated on liquid, mineral medium for 30 days. A 10-fold increase in the LAC activity was observed after the addition of saponins. Both crude saponin extracts and highly refined saponins were examined. The kinetic data were calculated for crude LAC prepared from the liquid growth medium of T versicolor. These data were obtained with three different substrates: 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), 2,6-dimethoxyphenol (DMP) and o-methoxyphenol (o-MP) in the presence of SAP. Using ABTS as substrate, the quantity of SAP did not make any difference for the Km and Vmax. The same results were obtained using DMP as substrate in the presence of 0.1 and 1 mg of SAP and the lowest results were obtained with o-MP as substrate. The results of the present study suggest that saponins may be used as natural inductors of fungal LAC and this would obviate the need to use toxic xenobiotics.

Results are reported for biosensors based on two different preparations of the same enzyme, laccase from Cerrena unicolor, one lyophilized and one stored frozen at - 18degreesC, for monitoring phenolic compounds. The enzyme was adsorbed on graphite electrodes and these were used in a flow through wall jet cell connected to a flow injection set-up. The electrodes were used at - 50 mV vs. Ag\AgCl. The effect of PH, flow rate of the carrier buffer was investigated as well as the operational and storage stability. Biosensors based on the frozen enzyme preparation were shown to be superior for biosensor construction.

The effect of cadmium ions on ligninolytic and decolourizing activities in cultures of two white-rot fungi, Cerrena unicolor and Trametes versicolor, were examined. Cadmium was added to the shallow stationary cultures growing on a liquid mineral medium. Both examined strains sorbed Cd ions in the first 24 hr of incubation. An appreciable stimulation of the activity of extracellular laccase (LAC) and inhibition of the extracellular manganese-dependent peroxidase (MnP) were simultaneously observed when 25 and 50 of cadmium ions were added to the cultures. On the other hand, the addition of cadmium ions also resulted in stimulating the decolorization activity of C. unicolor to decolorize Remazol Brilliant Blue R (RBBR) in the cultures, but decreasing it in the culture of T. versicolor, which is compared to the inhibition of MnP activity in this fungus. Our data indicate that the presence of Cd(II) ions can affect the ligninolytic activity of white-rot fungi. It was found that C. unicolor is a strain resistant to the presence of Cd ions in the liquid culture media, and has a potential to use this strain for bioremediation of sites contaminated with both heavy metals and aromatic pollutants.

The effects of cadmium Cd (II) ions on the physiology and biological activity of Trametes versicolor, a strain belonging to white‐rotting Basidiomycetes, were examined. Cd (II) ions were added to 10‐day‐old cultures grown on a liquid medium, or at the time of inoculation. Our experiments showed that T. versicolor is a good cadmium biosorbent from aqueous solution, this strain removing almost all the Cd (ll) ions over the first 2h of incubation by what appears to be a rapid, energy‐independent surface binding phenomenon, at the rate of ∼2mg Cd per g mycelial dry weight. An additional slower and energy‐dependent transport mechanism was also present, taking in ∼0.3mg Cd (II) perg dry weight. It is also shown that these Cd (II) ions significantly stimulate the activity of extracellular laccase when added to 10‐day‐old cultures.

A cycle of four enzymatic reactions with repression or allosteric inhibition of the enzymes has been proposed by analogy to a neural oscillator. The system is analysed in a situation remote from full symmetry. Asymmetry has been introduced by treating one of the reagents as a reservoir substance with constant concentration and having essentially different rate constants for forward and backward reactions. It is demonstrated that, for certain values of parameters, the system can work as a strength of stimulus to frequency transducer. For other values of the parameters, it acquires the features of an excitable system.

The antioxidant activity of the liquid media from laboratory cultures of four basidiomycete strains Trametes versicolor, Pleurotus cystidiosus, Cerrena unicolor and Bjerkandera fumosa were tested. The proteins less media were concentrated, and the water fractions after ethyl acetate extractions were precipitated with acetone and divided on TLC plates. The active fractions were rich in organic acids and their antioxidant activity was between 0.73 and 1.47 of Trolox waits. The antistress activity of fungal samples were tested on the leukocyte cultures which were move out in vivo from normal blood of carcinoma larynges patients. In these cultures the oxygen burst was initiated with the aim of phorbol ester, which force granulocytes to production of superoxide free radicals. The antioxidant effectiveness of fungal samples was revealed there. The mentioned results could be information that the protein less media after fungal cultivation can be useful remedy for natural scavenger of superoxide free radicals during anticancer therapy.

The influence of excess arginine and ammonium chloride on the demethylation of veratric acid by Rhodococcus erythropolis was examined. The reactions were compared with regards to changes of oxygen uptake, free radicals, hydrogen peroxide, superoxide dismutase - like activity, formaldehyde and phenolic acids levels. The addition of ammonium chloride caused the lengthening of the period of oxygen uptake contrary to the action of arginine. The presence of nitrogen sources modified the production of demethylation products (vanillic, isovanillic and protocatechuic acids). In the presence of arginine in the incubation medium the first products od demethylation - vanillic or isovanillic acid - were not produced, although protocatechuic acid appeared at the same time. These processes were correlated with free radical transformations and the generation of formaldehyde.

One hundred and fifteen fungi from different physio-ecological groups were compared for their capacity to decolourize two structurally different dyes in agar plates. We found that the azo dye, Acid Red 183, was much more resistant to decolourization by the examined strains in both solid and liquid cultures. Among the tested fungi, 69 strains showed decolourization of the anthraquinonic dye, Basic Blue 22, within 5–14 days, and only 16 strains were able to decolourize the azo dye, Acid Red 183, within 21 days. Furthermore, the potential of selected strains for decolourization of dyes was examined with regard to their extracellular oxidative factors both enzymatic and non-enzymatic. In static aqueous culture, the three selected fungi (Bjerkandera fumosa, Kuehneromyces mutabilis, and Stropharia rugoso-annulata) formed fungal mats, which did not decolourize any dye beyond some mycelial sorption. In comparison to the static cultures, the agitated cultures (180 rpm) removed 75 to 100% of the colour of Basic Blue 22 and 20 to 100% of Acid Red 183 colour.

Peroxidase and laccase activities increased rapidly up to the formation of primordia and then declined throughout the entire stage of fruiting. In the case of Pleurotus ostreatus, the level of Mn-dependent peroxidase was very low in primordia and fruiting stages but gradually increased with the growth of the fruit-body, whereas no activity was detected in Pleurotus sajor-caju during all growth stages. Superoxide dismutase activity was observed mainly at the fruiting stages. These results show that changes in concentration of lignin-related enzymes are associated with the fruiting process.

Highly purified Cerrena unicolor laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) caused the demethoxylation of milled wood lignin and several lignin related substances. The constitutive form of the enzyme produced extracellularly by C. unicolor fermenter culture was isolated and purified by ion-exchange chromatography on the DEAE-Toyopearl column and by affinity chromatography on a ConA-Sepharose and Syringyl-AH-Sepharose 4B columns. The enzyme was further immobilized on functionalized porous glass (CPG) and keratin coated CPG. The demethylating activity was monitored both by estimation of released methanol and by detection of the level of methoxyl groups (also in some water miscible solvents) after incubation of lignin materials with laccase preparations (free and immobilized). The effects of the incubation time and temperature on the demethoxylating activity of immobilized laccase preparations were also studied.

Although Rhodococcus spp. strains are able to degrade methoxyphenols by enzymatic means, the contact with veratric acid (3,4-dimethoxybenzoic acid, hereafter called veratrate) is very stressful for the cells ofRhodococcus erythropolis DSM 1069 (Rh). Within 5min of contact veratrate in phosphate buffer, the emergence of many vacuoles was observed in the cell body and respiratory bursts, with violent endogenous oxygen uptake, took place several times during the 24h incubation. During these peaks (where the cells were in their MAX states), increased activity of NADH oxidase was noted, accompanied by maximal accumulation of vanillic and isovanillic acids (3-methoxy-4-hydroxybenzoic acid and 4-hydroxy-3-methoxybenzoic acid respectively, hereafter called vanillates) in the incubation medium, which appeared to be products of veratrate demethylation. At the troughs (cell in their MIN state), the vacuoles disappeared from the cell body, oxygen uptake was normal, and the pool of vanillates decreased while the veratrate level in the medium increased. The cells from MAX and MIN states reacted in opposite ways in the presence of either formaldehyde and GSH, or paraquate and cAMP. The NADH oxidase activity, measured as oxygen uptake against NADH in the membrane pellets of MAX and MIN stage cells, differed in their response to the exogenous presence of FAD, ATP, cAMP, catalase, GSH, H2O2and methoxyphenolic substrates. The periodic character of these events is described here. Co-operation between two multiprotein membrane complexes (NAD(P)H oxidase and 3-O/4-O-demethylases) in Rhodococcus erythropolis cells and their competition for two common substrates—NAD(P)H and O2—is proposed as an explanation for rhythmical nature of these reactions.

The ability of several wood rotting fungi for decolorization of two anthracene derivatives, Carminic acid (CA) and Remazol brilliant blue R (RBBR), and hardwood KP bleaching liquor (BL) as well as laccase activities in these fungi were studied. The enzyme activity appeared exclusively in fungi destaining RBBR and CA, but in the case of BL, such relationship was not observed. The laccase enzyme was released into the decolorization media and its inducible (but not constitutive) forms shown destaining activity. The purified inducible forms of Kuehneromyces mutabilis and Pleurotus ostreatus laccase destained CA. Thus the possible differentiation between specificity of particular LAC forms was confirmed. In addition the nitrogen starvation induced both laccase and CA destaining activities, but the increase was higher for decolorization of CA than LAC activity. Probably LAC would be only partly responsible for decolorization of this dye. This results suggested that purified LACs decolorize CA, however its destaining activities were considerably lower than the activities on syringaldazine.

SummaryA screening of wood-rotting basidiomycete fungi was conducted for glucose 1-oxidase (GOD) and laccase (LAC) production as well as for ligninolytic activity measured by a Rhemazol reaction. The results showed that genera rich in GOD are lignin degraders as well as effective producers of extracellular LAC. The fungi poor in GOD neither showed LAC, nor ligninolytic activity. The Björkman lignin and 3 phenolic compounds, hydroquinone and syringic and vanillic acids, were tested on the sequential activity of LAC and GOD. In the presence of LAC, quinoid intermediates formed from Björkman lignin and phenolic compounds were observed. The addition of GOD caused a diminution of the quinone level. During incubation of Björkman lignin with LAC and GOD depolymerization occurred, and in the experiments omitting GOD the quantities of low molecular products were markedly lower. Consequently, the consecutive ping-pong activity of LAC and GOD reduced the polymerization and improved the efficiency of depolymerization processes.

Selected strains of basidiomycetes (Abortiporus biennis, Trametes versicolor and Cerrena unicolor) were shown to produce enhanced extracellular peroxidase (EP), superoxide dismutase (SOD) and laccase activities following the exposure of 10-day-old fungal cultures to separate high and low temperature stress. The stressful conditions also caused an increase in the concentrations of phenol compounds and superoxide anion radicals in these cultures. At first, peroxidase activity was observed at 12 hours from the moment of temperature stress application. Laccase activity appeared at 96 hours after the maximum levels of superoxide anion radicals (48 h) and SOD activity (36-72 h). The concentration of phenolic substances grew steadily during the period of cultivation. These relations between laccase, SOD and EP as well as superoxide radicals and phenol levels in the environment of Ligninolytic fungi seems to be important in the course of the biosynthesis or biodegradation of lignin, as the consequence of adaptation of these basidiomycetes to environmental temperature conditions.

The laccase activity in the medium of Cerrena unicolor cultivated under high temperature and cadmium stress conditions were determined. After five and fifteen days fromthe stress beginning, the changes in the PAGE patern of laccase isoforms were observed. Among six possible forms of laccase, three lighter forms characterised the initial control state and five heavier forms were stimulated in stress conditions.The simultaneous action of two stressors in the fungus cultures strongly decreased the laccase level while the condition of mycelium improved.