Inhibition of the proteasome strongly affects cadmium stimulated laccase activity in Trametes versicolor

Most proteins in eukaryotic cells are degraded by a highly selective non-lysosomal pathway that requires ATP and a large multicatalytic proteinase complex known as the 26S proteasome. In the present study, we evaluated the possibility that the proteasome-mediated pathway is involved in the regulation of laccase production by the efficient lignin-degrading basidiomycete Trametes versicolor in response to cadmium. These studies were performed using MG132 and lactacystin beta-lactone as specific proteasome inhibitors separately added to the culture medium of 7-day-old mycelia of T. versicolor at the start of incubation with 10-200 muM CdCl(2). We found that Cd(2+) stimulated laccase activity at all concentrations tested. The highest increase was observed at 100 muM Cd(2+), where laccase activity was three to fivefold higher than in Cd(2+)-free cultures. Blocking of proteasome function in Cd(2+)-supplemented cultures resulted in the considerably lower laccase activity in comparison to controls with no proteasomal inhibitor added. The decline of extracellular laccase activity triggered by the proteasome inhibitors was especially significant in the case of cultures with 100 muM Cd(2+), where around seven and threefold lost of laccase activity was observed for MG132 and lactacystin beta-lactone, respectively. Similar findings were obtained for intracellular laccase. In contrast to Cd(2+)-supplemented cultures, no significant change in laccase activity could be detected for Cd(2+)-free cultures after exposure to the proteasome inhibitors. Effects observed with chloroquine, the inhibitor of lysosomal proteolysis, added to T. versicolor cultures were markedly different from those found in the case of the proteasome inhibitors. We also showed that addition of Cd(2+) to growing cultures of T. versicolor did not significantly affect proteasome activities detected in high molecular (above 500 kDa) fractions of mycelial extracts. Our results strongly support the interpretation that the proteasome-mediated proteolytic pathway plays an important role in the regulation of T. versicolor laccase activity in response to Cd(2+).     

Biodegradation of endocrine-disrupting bisphenol A by white rot fungus Irpex lacteus

Biodegradation of endocrine-disrupting bisphenol A was investigated with several white rot fungi (Irpex lacteus, Trametes versicolor, Ganoderma lucidum, Polyporellus brumalis, Pleurotus eryngii, Schizophyllum commune) isolated in Korea and two transformants of T versicolor (strains MrP 1 and MrP 13). I. lacteus degraded 99.4% of 50 mg/l bisphenol A in 3 h incubation and 100% in 12 h incubation. which was the highest degradation rate among the fungal strains tested. T. versicolor degraded 98.2% of 50 mg/l bisphenol A in 12 h incubation. Unexpectedly, the transformant of the Mn-repressed peroxidase gene of T. versicolor, strain MrP 1, degraded 76.5% of 50 mg/l bisphenol A in 12 h incubation, which was a lower degradation rate than wild-type T. versicolor. The removal of bisphenol A by I. lacteus occurred mainly by biodegradation rather than adsorption. Optimum carbon sources for biodegradation of bisphenol A by I. lacteus were glucose and starch, and optimum nitrogen sources were yeast extract and tryptone in a minimal salts medium; however, bisphenol A degradation was higher in nutrient-rich YMG medium than that in a minimal salts medium. The initial degradation of endocrine disruptors was accompanied by the activities of manganese peroxidase and laccase in the culture     

Effect of phenolic compounds on cellulose degradation by some white rot basidiomycetes

Abstract Cellulose degradation by several white rot fungi was investigated. In most fungi cellulase production was stimulated by lignin-related phenolics. Detailed investigation of Tremetes versicolor showed that this stimulation was not directly effected by phenols but was due to an indirect induction. The phenol was oxidized by laccase to quinone. The quinone was then reduced by the enzyme cellobiose: quinone-oxidoreductase while cellobiono-lactone was formed from cellobiose. The cellobiono-lactone was responsible for the increased cellulase production in submerged cultures with cellulose as the sole carbon source.     

Degradation of phenanthrene by Trametes versicolor and its laccase

Phenanthrene is a three-ring polycyclic aromatic hydrocarbon and commonly found as a pollutant in various environments. Degradation of phenanthrene by white rot fungus Trametes versicolor 951022 and its laccase, isolated in Korea, was investigated. After 36 h of incubation, about 46% and 65% of 100 mg/l of phenanthrene added in shaken and static fungal cultures were removed, respectively. Phenanthrene degradation was maximal at pH 6 and the optimal temperature for phenanthrene removal was 30 degrees C. Although the removal percentage of phenanthrene was highest (76.7%) at 10 mg/l of phenanthrene concentration, the transformation rate was maximal (0.82 mg/h) at 100 mg/L of phenanthrene concentration in the fungal culture. When the purified laccase of T versicolor 951022 reacted with phenanthrene, phenanthrene was not transformed. The addition of redox mediator, 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) or 1-hydroxybenzotriazole (HBT) to the reaction mixture increased oxidation of phenanthrene by laccase about 40% and 30%, respectively.     

Degradation of 2,4-dichlorophenol and pentachlorophenol by two brown rot fungi

Wheat straw cultures of the brown rot fungi Gloeophyllum striatum and G. trabeum degraded 2,4-dichlorophenol and pentachorophenol. Up to 54% and 27% 14CO2, respectively, were liberated from uniformly 14C-labeled substrates within 6 weeks. Under identical conditions Trametes versicolor, a typical white rot species employed as reference, evolved up to 42% and 43% 14CO2 and expressed high activities of laccase, manganese peroxidase, and manganese-independent peroxidase. No such activity could be detected in straw or liquid cultures of Gloeophyllum. Moreover, G. striatum degraded both chlorophenols most efficiently under non-cometabolic conditions, i.e. on a defined mineral medium lacking sources of carbon, nitrogen and phosphate.     

Extracellular oxidative enzyme production and PAH removal in soil by exploratory mycelium of white rot fungi

Selected strains of three species of white rot fungi, Pleurotus ostreatus, Phanerochaete chrysosporium and Trametes versicolor, were grown in sterilized soil from straw inocula. The respective colonization rates and mycelium density values decreased in the above mentioned order. Three- and four-ringed PAHs at 50 ppm inhibited growth of fungi in soil to some extent. The activities of fungal MnP and laccase (units per g dry weight of straw or soil), extracted with 50 mM succinate-lactate buffer (pH 4.5), were 5 to 20-fold higher in straw compared to soil. The enzyme activities per g dry soil in P. ostreatus and T. versicolor were similar, in contrast to P. chrysosporium, where they were extremely low. Compared to the aerated controls, P. ostreatus strains reduced the levels of anthracene, pyrene and phenanthrene by 81–87%, 84–93% and 41–64% within 2 months, respectively. During degradation of anthracene, all P. ostreatus strains accumulated anthraquinone. PAH removal rates in P. chrysosporium and T. versicolor soil cultures were much lower.     

Enzymatic activity, osmotic stress and degradation of pesticide mixtures in soil extract liquid broth inoculated with Phanerochaete chrysosporium and Trametes versicolor

In this study we examined the extracellular enzymatic activity of two white rot fungi (Phanerochaete chrysosporium and Trametes versicolor) in a soil extract broth in relation to differential degradation of a mixture of different concentrations (0-30 p.p.m.) of simazine, dieldrin and trifluralin under different osmotic stress (-0.7 and -2.8 MPa) and quantified enzyme production, relevant to P and N release (phosphomonoesterase, protease), carbon cycling (beta-glucosidase, cellulase) and laccase activity, involved in lignin degradation. Our results suggest that T. versicolor and P. chrysosporium have the ability to degrade different groups of pesticides, supported by the capacity for expression of a range of extracellular enzymes at both -0.7 and -2.8 MPa water potential. Phanerochaete chrysosporium was able to degrade this mixture of pesticides independently of laccase activity. In soil extract, T. versicolor was able to produce the same range of enzymes as P. chrysoporium plus laccase, even in the presence of 30 p.p.m. of the pesticide mixture. Complete degradation of dieldrin and trifluralin was observed, while about 80% of the simazine was degraded regardless of osmotic stress treatment in a nutritionally poor soil extract broth. The capacity of tolerance and degradation of high concentrations of mixtures of pesticides and production of a range of enzymes, even under osmotic stress, suggest potential bioremediation applications.     

Purification and characterization of laccase from the white rot fungus Trametes versicolor

Laccase is one of the ligninolytic enzymes of white rot fungus Trametes versicolor 951022, a strain first isolated in Korea. This laccase was purified 209-fold from culture fluid with a yield of 6.2% using ethanol precipitation, DEAE-Sepharose, Phenyl-Sepharose, and Sephadex G-100 chromatography. T. versicolor 951022 excretes a single monomeric laccase showing a high specific activity of 91,443 U/mg for 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as a substrate. The enzyme has a molecular mass of approximately 97 kDa as determined by SDS-PAGE, which is larger than those of other laccases reported. It exhibits high enzyme activity over broad pH and temperature ranges with optimum activity at pH 3.0 and a temperature of 50 degrees C. The Km value of the enzyme for substrate ABTS is 12.8 micrometer and its corresponding Vmax value is 8125.4 U/mg. The specific activity and substrate affinity of this laccase are higher than those of other white rot fungi, therefore, it may be potentially useful for industrial purposes.     

Degradation of anthracene by selected white rot fungi

Abstract Approximately 60% of the originally supplied anthracene (AC) was degraded in ligninolytic stationary cultures of selected white rot fungi within 21 days. All the white rot fungi tested oxidized AC to anthraquinone (AQ). Unlike Phanerochaete chrysosporium and strain Px, with Pleurotus ostreatus, Coriolopsis polyzona and Trametes versicolor , AQ did not accumulate in the cultures, indicating that AQ was degraded further and its degradation did not appear to be a rate-limiting step. However, P. ostreatus and C. polyzona failed to degrade AQ in the absence of AC. P. ostreatus, T. versicolor and strain Px did not produce lignin peroxidase (ligninase) (LIP) under the test conditions but oxidized AC to AQ suggesting that white rot fungi produce enzyme(s) other than LIP capable of oxidizing compounds with high ionization potential like AC. Moreover, in the case of Ph. chrysosporium and C. polyzona , AC degradation started earlier than the production of LIP. Veratryl alcohol (VA) seemed to be playing a role in AC oxidation catalyzed by LIP in Ph. chrysosporium .     

Novel enzymatic oxidation of Mn2+ to Mn3+ catalyzed by a fungal laccase.

Fungal laccases are extracellular multinuclear copper-containing oxidases that have been proposed to be involved in ligninolysis and degradation of xenobiotics. Here, we show that an electrophoretically homogenous laccase preparation from the white rot fungus Trametes versicolor oxidized Mn2+ to Mn3+ in the presence of Na-pyrophosphate, with a Km value of 186 microM and a Vmax value of 0.11 micromol/min/mg protein at the optimal pH (5.0) and a Na-pyrophosphate concentration of 100 mM. The oxidation of Mn2+ involved concomitant reduction of the laccase type 1 copper site as usual for laccase reactions, thus providing the first evidence that laccase may directly utilize Mn2+ as a substrate.     

Biodegradation of tetrabromobisphenol A by oxidases in basidiomycetous fungi and estrogenic activity of the biotransformation products

Tetrabromobisphenol A (TBBPA) degradation was investigated using white rot fungi and their oxidative enzymes. Strains of the Trametes, Pleurotus, Bjerkandera and Dichomitus genera eliminated almost 1 mM TBBPA within 4 days. Laccase, whose role in TBBPA degradation was demonstrated in fungal cultures, was applied to TBBPA degradation alone and in combination with cellobiose dehydrogenase from Sclerotium rolfsii. Purified laccase from Trametes versicolor degraded approximately 2 mM TBBPA within 5 h, while the addition of cellobiose dehydrogenase increased the degradation rate to almost 2.5 mM within 3 h. Laccase was used to prepare TBBPA metabolites 2,6-dibromo-4-(2-hydroxypropane-2-yl) phenol (1), 2,6-dibromo-4-(2-methoxypropane-2-yl) phenol (2) and 1-(3,5-dibromo-4-hydroxyphen-1-yl)-2,2',6,6'-tetrabromo-4,4'-isopropylidene diphenol (3). As compounds 1 and 3 were identical to the TBBPA metabolites prepared by using rat and human liver fractions (Zalko et al., 2006), laccase can provide a simple means of preparing these metabolites for toxicity studies. Products 1 and 2 exhibited estrogenic effects, unlike TBBPA, but lower cell toxicity.     

杂色云芝漆酶基因(Lcc1)的克隆及在甲醇毕赤酵母中的表达

以白腐菌杂色云芝Coriolus versicolor RNA为模板,通过RT-PCR获得漆酶Leel基因的cDNA片段。构建了甲醇酵母表达质粒pMETA-Lccl载体,并将其线性化后用电穿孔法导入Pichia methabolica PMAD16,部分阳性克隆的PCR结果表明Lccl基因已经整合到甲醇毕赤酵母染色体上,经摇瓶培养筛选出表达水平较高的酵母工程菌株。漆酶酶活力达53U/L     

Growth, dye degradation and ligninolytic activity studies on Zimbabwean white rot fungi

White rot fungi were collected from Chirinda and Chimanimani hardwood forests in Zimbabwe and studied with respect to growth temperature optima and dye decolorization. Temperature optima were found to vary (between 25-37 degrees C) amongst the isolates. The isolates were screened for their ability to degrade the polymeric dyes; blue dextran and Poly R478 and the triphenylmethane dyes; cresol red, crystal violet and bromophenol blue. Semi-quantitative determination of the hydrolytic enzyme activities possessed by the white rot fungi was determined using the API ZYM system. Lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase activities in the fungi were also determined. No LiP was detected in any of the isolates but all isolates showed manganese peroxidase and laccase activities. Time related decolorization studies and optimum pH determinations for Poly R478 degradation by the isolates were carried out in liquid cultures. The most significant rates of Poly R478 decolorization in liquid cultures were found with the following isolates: Trametes cingulata, Trametes versicolor, Trametes pocas, DSPM95 (a species to be identified), Datronia concentrica and Pycnoporus sanguineus.     

Laccase induction in fungi and laccase/N-OH mediator systems applied in paper mill effluent

There has been increasing interest in extracellular enzymes from white rot fungi, such as lignin and manganese peroxidases, and laccases, due to their potential to degrade both highly toxic phenolic compounds and lignin. The optimum cultivation conditions for laccase production in semi-solid and liquid medium by Trametes versicolor, Trametes villosa, Lentinula edodes and Botrytis cinerea and the effects of laccase mediator system in E1 effluent were studied. The higher laccase activity (12756 U) was obtained in a liquid culture of T. versicolor in the presence of 1 mM of 2,5-xylidine and 0.4 mM copper salt as inducers. The effluent biotreatments were not efficient in decolorization with any fungal laccases studied. Maximum phenol reduction was approximately 23% in the absence of mediators from T. versicolor. The presence of 1-hydroxybenzotriazole did not increase phenol reduction. However, acetohydroxamic acid, which was not degraded by laccase, acted very efficiently on E1 effluent, reducing 70% and 73% of the total phenol and total organic carbon, respectively. Therefore, acetohydroxamic acid could be applied as a mediator for laccase bioremediation in E1 effluent.     

Biodegradation of DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] by the white rot fungus Phanerochaete chrysosporium.

Extensive biodegradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by the white rot fungus Phanerochaete chrysosporium was demonstrated by disappearance and mineralization of [14C]DDT in nutrient nitrogen-deficient cultures. Mass balance studies demonstrated the formation of polar and water-soluble metabolites during degradation. Hexane-extractable metabolites identified by gas chromatography-mass spectrometry included 1,1,-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol (dicofol), 2,2-dichloro-1,1-bis(4-chlorophenyl)ethanol (FW-152), and 4,4'-dichlorobenzophenone (DBP). DDD was the first metabolite observed; it appeared after 3 days of incubation and disappeared from culture upon continued incubation. This, as well as the fact that [14C]dicofol was mineralized, demonstrates that intermediates formed during DDT degradation are also metabolized. These results demonstrate that the pathway for DDT degradation in P. chrysosporium is clearly different from the major pathway proposed for microbial or environmental degradation of DDT. Like P. chrysosporium ME-446 and BKM-F-1767, the white rot fungi Pleurotus ostreatus, Phellinus weirii, and Polyporus versicolor also mineralized DDT.     

Biodegradation of DDT [1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane] by the white rot fungus Phanerochaete chrysosporium

Extensive biodegradation of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by the white rot fungus Phanerochaete chrysosporium was demonstrated by disappearance and mineralization of [14C]DDT in nutrient nitrogen-deficient cultures. Mass balance studies demonstrated the formation of polar and water-soluble metabolites during degradation. Hexane-extractable metabolites identified by gas chromatography-mass spectrometry included 1,1,-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD), 2,2,2-trichloro-1,1-bis(4-chlorophenyl)ethanol (dicofol), 2,2-dichloro-1,1-bis(4-chlorophenyl)ethanol (FW-152), and 4,4'-dichlorobenzophenone (DBP). DDD was the first metabolite observed; it appeared after 3 days of incubation and disappeared from culture upon continued incubation. This, as well as the fact that [14C]dicofol was mineralized, demonstrates that intermediates formed during DDT degradation are also metabolized. These results demonstrate that the pathway for DDT degradation in P. chrysosporium is clearly different from the major pathway proposed for microbial or environmental degradation of DDT. Like P. chrysosporium ME-446 and BKM-F-1767, the white rot fungi Pleurotus ostreatus, Phellinus weirii, and Polyporus versicolor also mineralized DDT.     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).     

Biodegradation of pentachlorophenol by the white rot fungus Phanerochaete chrysosporium.

Extensive biodegradation of pentachlorophenol (PCP) by the white rot fungus Phanerochaete chrysosporium was demonstrated by the disappearance and mineralization of [14C]PCP in nutrient nitrogen-limited culture. Mass balance analyses demonstrated the formation of water-soluble metabolites of [14C]PCP during degradation. Involvement of the lignin-degrading system of this fungus was suggested by the fact the time of onset, time course, and eventual decline in the rate of PCP mineralization were similar to those observed for [14C]lignin degradation. Also, a purified ligninase was shown to be able to catalyze the initial oxidation of PCP. Although biodegradation of PCP was decreased in nutrient nitrogen-sufficient (i.e., nonligninolytic) cultures of P. chrysosporium, substantial biodegradation of PCP did occur, suggesting that in addition to the lignin-degrading system, another degradation system may also be responsible for some of the PCP degradation observed. Toxicity studies showed that PCP concentrations above 4 mg/liter (15 microM) prevented growth when fungal cultures were initiated by inoculation with spores. The lethal effects of PCP could, however, be circumvented by allowing the fungus to establish a mycelial mat before adding PCP. With this procedure, the fungus was able to grow and mineralize [14C]PCP at concentrations as high as 500 mg/liter (1.9 mM).