Trametes Trogii Research Articles

Modeling the 3-D Structure of a Recombinant Laccase from Trametes trogii Active at a pH Close to Neutrality

A cDNA encoding a novel laccase from the white-rot fungus Trametes trogii was cloned and expressed in Pichia pastoris. The recombinant protein (Lcc2) exhibited kinetic parameters for both phenolic and non phenolic substrates that were different from the previously described Lcc1, the main laccase isoform expressed by T. trogii; in addition, the pH/activity profiles for phenolic substrates of Lcc2 were shifted upward by 1-1.5 pH units towards neutrality as compared to Lcc1. Comparative modeling of the two laccases (69.2% identity) showed that the overall fold of Lcc2 is very similar to Lcc1 and other laccases. The substrate cavity of Lcc2 contains the Asp residue which is thought to mediate the laccase activity at acidic pHs, whereas two hydrophobic residues (Phe, Ile) on the cavity orifice of Lcc2 replace the two polar residues (Thr, Ser) of Lcc1. These structural differences may be responsible for the unique kinetic performances of Lcc2.

Effect of high ammonia concentrations on fungal treatment of Tunisian landfill leachates

Landfill leachates (LFL) collected from Djebel Chekir are highly loaded with COD ranging from 4.5 to 45 g l–1, ammonia ranging from 0.64 to 4.99 g l–1 and salts (14–32 mScm–1). They also contain high concentrations of phenols and hydrocarbons. Consequently, LFL exhibit high toxicity since they completely inhibit the bioluminescence of Vibrio fischeri and the germination of Lepidium sativum seeds. Assays of a biological treatment of LFL, in order to detoxify the effluent by selected strains of white rot fungi (Trametes trogii and Phanerochaete chrysosporium), were carried out. Since the effluent contained an important amount of ammonia which is problematic for microorganisms, we first studied the effect of different concentrations of ammonia on mycelia growth and enzymes secretion. Results obtained during 14 days of fungal culture showed that ammonium chloride was not problematic at concentrations up to 2 g l–1 added to the synthetic medium. However, at a concentration of 5 g l–1 added, it exerted inhibitor effects on mycelia growth and enzymes secretion. Experiments with real LFL using T. trogii and P. chrysosporium were carried out at 50% of diluted effluent. COD removal efficiencies for T. trogii and P. chrysosporium were 79% and 68%, respectively. This was accompanied by an important enzyme secretion for each strain and a reduction in phenols and hydrocarbons concentrations. Consequently, an important reduction in the toxicity of the 50% diluted LFL was achieved.

Crystal structure of the blue multicopper oxidase from the white-rot fungus Trametes trogii complexed with p-toluate

A multicopper oxidase, the fungal laccase glycoenzyme from the white-rot basidiomycete fungus Trametes (Funalia) trogii, was crystallized and its crystal structure was solved at 1.58 Å using molecular replacement techniques. Model refinement resulted in R-factor and R-free values of 17.4% and 19.0%, respectively. The T. trogii laccase structural model reveals the presence of a ligand bound to the T1 active site which resembles a p-toluate molecule, such bound compound is most probably a fungal metabolite. The p-toluate is bound into the T1 active site of the laccase forming, with one of the carboxylate oxygens, a H-bond with His455, one of the T1 copper ion ligands, whereas the methyl group presents hydrophobic interactions within a pocket composed by Phe331, Phe336, Pro390 and Val162. The coordination geometries, the bond distances and the oxidation states of the T1 and T2/T3 copper active sites are analyzed and discussed in terms of the enzymatic mechanism and catalytic functionality.

Preliminary studies on the biobleaching of loblolly pine Kraft pulp with Trametes trogii crude extracts

Trametes trogii (BAFC 463), Coriolus versicolor var. antarcticus (BAFC 266) and Trametes villosa (BAFC 2755) are Argentinean white-rot fungi that proved to be efficient ligninolytic enzyme producers in previous studies. The aim of the present work was investigating the ability of their crude extracellular extracts for the biobleaching of loblolly pine Kraft pulp. The production of laccase, manganese peroxidase, endoxylanase and endoglucanase was also evaluated. Out of the three fungi studied, the best biobleaching results were obtained with culture supernatants from T. trogii grown for 21 days in a synthetic medium with glucose and asparagine as carbon and nitrogen sources, respectively, with the addition of 1 mM copper sulfate. Such medium rendered high level of ligninolytic enzymes: 0.25 U ml −1 of manganese peroxidase and 44 U ml −1 of laccase. A strong brightness increase was attained after the enzymatic treatment followed by H 2O 2. Bleaching up to 82% ISO brightness (compared with 37% in the peroxide-bleached control) was obtained. Moreover, after the enzymatic treatment, hydrogen peroxide requirements decreased (as reflected by the higher residual peroxide), and the pulp luminance reached a value of 92.5. Addition of known laccase mediators such as HBT, ABTS, and HBA did not improve the process. The supernatant did not significantly lose its bleaching capacity even after successive recovery and reapplication to unbleached pulp. Correspondingly, laccase and manganese peroxidase activities in the recovered supernatant, exhibited near 100% of their initial values.

Optimization of lignocellulolytic enzyme production by the white-rot fungus Trametes trogii in solid-state fermentation using response surface methodology

The white-rot basidiomicete Trametes trogii (MYA 28-11) is an outstanding producer of laccase. A Doehlert experimental design was applied to optimize its lignocellulolytic enzyme production in solid-state fermentation. The impact on enzyme production of three quantitative variables, namely pH, copper and nitrogen concentrations, was investigated by using a wood-based solid medium supplemented with malt extract. Optimization was aimed at simultaneously minimizing cellulase activity and maximizing ligninolytic enzyme production. Such conditions were: pH 4.5, peptone 12.5 g l −1, and CuSO 4 11 mM. Highest activities achieved were: laccase 901 and Mn-peroxidase 20 U g −1. There was not a direct correlation between the levels of enzymatic activities and the extent of losses of wood weight or components. The factorial approach also allowed us to quantify the enzyme activity in any part of the experimental domain, consequently, it was also possible to determine a culture medium to obtain crude extracellular extracts with high laccase (510 U g −1) and endoxylanase (780 U g −1) yields. These enzymes have gained renewed interest mainly due to their applications in paper industries for pulp treatment, improving the effectiveness of conventional bleaching. Considering the results obtained, T. trogii could be an attractive source of both enzymes.

Laccase purification and characterization from Trametes trogii isolated in Tunisia: decolorization of textile dyes by the purified enzyme

A white-rot basidiomycete, isolated from decayed acacia wood (from Northwest of Tunisia) and identified as Trametes trogii, was selected in a broad plate screening because of its ability to degrade commercial dyes. In liquid cultures using a glucose–peptone medium, the sole ligninolytic activity detected was laccase. The highest laccase levels were obtained in presence of CuSO 4 as inducer (around 20000 U/l). Two isoenzymes, were purified using anion-exchange and size-exclusion chromatographies. Both isoenzymes are monomeric proteins, with M w around 62 kDa and isoelectric points of 4.3 and 4.5, showing similar stability at pH and temperature, optimum pH and substrate specificity. The highest oxidation rate was obtained at pH 2 and 2.5 for ABTS and DMP, respectively. They were stable up to 50 °C for 24 h and the stability was higher at alkaline pH. Activity increased by the addition of 10 mM Ni, Mo or Mn but it was not affected by Cd, Al, Li and Ca. Identical N-terminal sequences were determined in both laccases. The crude enzyme, as well as the purified laccase, was able to decolorize dyes from the textile industry.

Optimization of a culture medium for ligninolytic enzyme production and synthetic dye decolorization using response surface methodology.

A Box-Wilson central composite design was applied to optimize copper, veratryl alcohol and l-asparagine concentrations for Trametes trogii (BAFC 212) ligninolytic enzyme production in submerged fermentation. Decolorization of different dyes (xylidine, malachite green, and anthraquinone blue) by the ligninolytic fluids from the cultures was compared. The addition of copper stimulated laccase and glyoxal oxidase production, but this response was influenced by the medium N-concentration, with improvement higher at low N-levels. The medium that supported the highest ligninolytic production (22.75 U/ml laccase, 0.34 U/ml manganese peroxidase, and 0.20 U/ml glyoxal oxidase) also showed the greatest ability to decolorize the dyes. Only glyoxal oxidase activity limited biodecoloration efficiency, suggesting the involvement of peroxidases in the process. The addition of 1-hydroxybenzotriazole (a known laccase mediator) to the ligninolytic fluids increased both their range and rate of decolorization. The cell-free supernatant did not decolorize xylidine, poly R-478, azure B, and malachite green as efficiently as the whole broth, but results were similar in the case of indigo carmine and remazol brilliant blue R. This indicates that the mycelial biomass may supply other intracellular or mycelial-bound enzymes, or factors necessary for the catalytic cycle of the enzymes. It also implies that this fungus implements different strategies to degrade dyes with diverse chemical structures.

Degradation of organic pollutants by the white rot basidiomycete Trametes trogii

The ability of the white rot basidiomycete Trametes trogii (strain BAFC 463) to degrade in vitro concentrations of 250 –500 ppm of nitrobenzene and anthracene was analyzed. Within 12 – 24 days, more than 90% of the organic pollutants added to the fungal cultures were removed, as demonstrated by gas–liquid chromatography. Enzyme estimations indicated a high and relatively stable activity of laccase, as well as a lower production of manganese peroxidase. Laccase activity could be implicated in the degradation of these xenobiotic compounds by T. trogii. Earlier work showed that this fungus almost completely removed anthraquinone dyes and polychlorinated biphenyls. In view of the results obtained, this strain seems promising for detoxi5cation. ? 2002 Elsevier Science Ltd. All rights reserved.

Copper induction of lignin-modifying enzymes in the white-rot fungus Trametes trogii

Trametes trogii, a white rot basidiomycete involved in wood decay worldwide, produces several ligninolytic enzymes, laccase being the dominant one, with higher titers than those reported for most other white rot fungi studied up to date. The effect of copper on in vitro production of extracellular ligninolytic activities was studied. CuSO4·5H2O concentrations from 1.6 μM to 1.5 mM were tested in a synthetic medium with glucose 20 g/L and asparagine 3 g/L. The addition of copper (up to 1 mM) did not affect growth but strongly stimulated ligninolytic enzyme production; faster decolorization of the polymeric dye Poly R-478 was observed as well. Maximal production of manganese peroxidase, laccase, and glyoxal oxidase [1.28 U/mL, 93.8 U/mL (with a specific activity of 720 U/mg protein), and 0.46 U/mL respectively] was attained with 1 mM CuSO4·5H2O. However, higher copper concentrations inhibited growth and notably decreased manganese peroxidase production, although they did not affect laccase secretion. Laccase activity in the culture filtrate was maximal at 50 C and pH 3.4, and the enzyme was completely stable at pH 4.4 and above, and at 30 C for up to 5 d. Denaturing polyacrylamide gel electrophoresis of extracellular culture fluids showed two laccase activity bands (mol wt 38 and 60 kDa respectively). The pattern of isoenzyme production was not affected by medium composition but differed with culture age.

Relationship between production of 3-indoleacetic acid and peroxidase-laccase activities depending on the culture periods in Funalia trogii (Trametes trogii).

The relationship between production of 3-indoleacetic acid (IAA) and peroxidase and laccase activity was investigated in white-rot fungus Funalia trogii (Trametes trogii). F. trogii produced IAA and peroxidase and laccase as both primary metabolite and secondary metabolite; the levels of IAA may be influenced by peroxidase and laccase. A correlation exists between the levels of IAA and peroxidase-laccase activity.

Lignin-degrading activity and ligninolytic enzymes of different white-rot fungi: effects of manganese and malonate

Ten species of white-rot fungi, mainly belonging to the family Polyporaceae (Basidiomycotina), were studied in terms of their ability to degrade14C-ring labelled synthetic lignin and secrete ligninolytic enzymes in liquid cultures under varying growth conditions. Lignin mineralization by the fungi in an air atmosphere did not exceed 14% within 29 days. Different responses to the elevated Mn2+concentration and the addition of a manganese chelator (sodium malonate) were observed among various fungal species. This could be related with the utilization of either lignin peroxidase (LiP) or manganese peroxidase (MnP) for lignin depolymerization, i.e., some fungi apparently had an LiP-dominating ligninolytic system and others an MnP-dominating ligninolytic system. The LiP isoforms were purified from Trametes gibbosa and Trametes trogii. Isoelectric focusing of purified ligninolytic enzymes revealed the expression of numerous MnP isoforms in Trametes gibbosa, Trametes hirsuta, Trametes trogii, and Abortiporus biennis grown under a high (50-fold) Mn2+level (120 μM) with the addition of the chelator. In addition, two to three laccase isoforms were detected. Key words: white-rot fungi, lignin degradation, lignin peroxidase, manganese peroxidase, manganese, malonate.