Gonzalez2008 Res Microb

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Gonzalez2008 Res Microb

  1. 1. Research in Microbiology 159 (2008) 103e109 www.elsevier.com/locate/resmic Melanoidin-containing wastewaters induce selective laccase gene expression in the white-rot fungus Trametes sp. I-62 Tania Gonzalez1, Marıa Carmen Terron, Susana Yague, Howard Junca2, Jose Marıa Carbajo3, ´ ´ ´ ¨ ´ ´ Ernesto Javier Zapico , Ricardo Silva , Ainhoa Arana-Cuenca , Alejandro Tellez6, 4 5 6 ´ Aldo Enrique Gonzalez* ´ Department of Molecular Microbiology, Centro de Investigaciones Biologicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain ´ Received 24 July 2007; accepted 23 October 2007 Available online 21 November 2007 Abstract Wastewaters generated from the production of ethanol from sugar cane molasses may have detrimental effects on the environment due to their high chemical oxygen demand and dark brown color. The color is mainly associated with the presence of melanoidins, which are highly recal- citrant to biodegradation. We report here the induction of laccases by molasses wastewaters and molasses melanoidins in the basidiomycetous fungus Trametes sp. I-62. The time course of effluent decolorization and laccase activity in the culture supernatant of the fungus were correlated. The expression of laccase genes lcc1 and lcc2 increased as a result of the addition of complete molasses wastewater and its high molecular weight fraction to fungal cultures. This is the first time differential laccase gene expression has been reported to occur upon exposure of fungal cultures to molasses wastewaters and their melanoidins. Ó 2007 Elsevier Masson SAS. All rights reserved. Keywords: Melanoidin; Molasses wastewaters; Laccase; Decolorization; Fungi; Ligninolytic enzymes; Basidiomycete 1. Introduction Final effluents produced from alcoholic fermentation of molasses are among the most environmentally harmful * Corresponding author. Tel.: þ34 9 1837 3112x4413/4414; fax: þ34 9 1536 wastewaters generated by the sugar and byproduct industries. 0432. These effluents, also known as vinasses, contain persistent toxic ´ E-mail address: aldo@cib.csic.es (A.E. Gonzalez). 1 ´ Present address: Departamento de Microbiologıa, Instituto Cubano de los chemicals which have a harmful impact on aquatic ecosystems, ´ ´ Derivados de la Ca~a de Azucar (ICIDCA), Vıa Blanca 804 y Carretera Cen- n not only by increasing the chemical oxygen demand (COD) but ´ tral, San Miguel del Padron, Havana, Cuba. also as a result of their dark brown color. This leads to a reduc- 2 Present address: Biodegradation Research Group e GBF, Environmental tion in penetration of sunlight in rivers, lakes and lagoons, Microbiology, Mascheroder Weg 1 D-38124, Braunschweig, Germany. which in turn decreases oxygenation by photosynthesis and 3 Present address: CIFOR-INIA, Crta. de La Coru~a Km. 7, 28040 Madrid, n Spain. causes multiple damaging effects to aquatic life. The organic 4 Present address: Biotechnology Department, Technische Universitat ¨ matter can be degraded by conventional anaerobic-aerobic Hamburg-Harburg, Biotechnology II, Denickestrasse, 15, 21073 Hamburg, treatments, but the colored compounds of molasses effluents ap- Germany. pear to be recalcitrant to biodegradation [31]. The characteristic 5 ´ Present address: Fac. Ciencias Forestales, Dpto. de Ingenierıa de la very dark color is mainly due to the presence of melanoidins. Madera, Universidad de Chile, Santa Rosa, 11315 Santiago, Chile. 6 ´ ´ Present address: Departamento Biotecnologıa, Universidad Politecnic de These brown polymers, which are formed by amino-carbonyl ´ Pachuca, Ex-Hacienda de Santa Barbara Km 20, Carretera Pachuca-Ciudad reactions, are widely distributed in nature and are not readily Sahagun, Zempoala, CP. 43830, Estado de Hidalgo, Mexico. susceptible to microbial degradation. 0923-2508/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.resmic.2007.10.005
  2. 2. 104 T. Gonzalez et al. / Research in Microbiology 159 (2008) 103e109 ´ Thus, there is an urgent need to develop alternative bio- 2.4. Culture conditions technological processes to effectively remove light-absorptive compounds from molasses effluents. White-rot basidiomyce- Submerged cultures were prepared from 7-day-old cultures tous fungi can degrade lignin and a broad range of environ- of the fungus grown on agar plates with Kirk medium [17]. mentally persistent xenobiotics, organopollutants and Eight plugs (1 cm2) were cut and inoculated under sterile industrial wastewaters [18,37]. Some of these fungi have conditions in 500 ml culture flasks containing 300 ml of the also been shown to be effective in the decolorization of natu- same growth medium and four 1.5 cm diameter glass beads. ral and synthesized melanoidins and molasses wastewaters They were incubated for 24 h at 28 C in an orbital shaker (MWWs) [16]. A complex non-specific enzyme system (100 rpm). A 1:10 (v/v) inoculum was transferred into 250 ml secreted by these organisms has been shown to be associated flasks containing 75 ml (total volume) of Kirk medium supple- with their degradative capacities. The ligninolytic system mented with 20% (v/v final concentration) of MWW. Controls consists of two main groups of enzymes: peroxidases (lignin were prepared in the same way, except that Kirk medium was peroxidases and manganese peroxidases) and laccases not supplemented with the wastewater. Abiotic controls con- [1,2,11,18]. Although the enzymatic system associated with tained Kirk medium and the effluent, but were not inoculated decolorization of melanoidins appears to be related to the with fungus. presence and activity of fungal ligninolytic mechanisms, this Fungal cultures were incubated at 28 C in an orbital shaker relation is as yet not completely understood [12]. (100 rpm) for 16 days. COD, decolorization, lignin peroxidase Trametes sp. I-62 (CECT 20197) is a white-rot fungus (LiP), manganese peroxidase (MnP) and laccase activity strain with a high detoxification capacity towards molasses ef- measurements were monitored on a daily basis, in triplicate. fluents. It also represents a model strain for studying the diver- To study the effect of both MWW and molasses melanoi- sity [1,13,14,20], transcriptional [21,34] and postranslational dins on lcc gene transcription, they were added to 8-day-old regulation of laccases in a single organism. Four laccase genes fungal cultures in Kirk medium (28 C, 100 rpm) at a final have been reported thus far in this basidiomycete [14,20]. concentration of 0.37 mM total phenols in the culture medium. In the present work, we analyzed the relationship between Laccase activity was monitored over a 43 h period and fresh the production of ligninolytic enzymes and decolorization of 10 mg mycelium samples were harvested at different time MWW by Trametes sp. I-62. The effect of molasses effluents points (7, 19, 31 and 43 h) following the addition of either and molasses melanoidins on laccase gene transcription was effluent or melanoidins to the cultures. also evaluated. 2.5. Analytical methods 2. Materials and methods Color units and COD were determined according to CPPA 2.1. Wastewater [8] and ‘‘Standard methods for the examination of water and wastewater’’ [33], respectively. The concentration of total phe- The final effluent from the distillation of ethanol produced nols was determined by the Folin-Ciocalteu method [30] with from sugar-cane molasses was provided by a distillery in minor modifications [4] using gallic acid (Sigma Chemicals) Havana, Cuba. MWW are complex organic mixtures including as a reference standard. melanoidins, which results in acid dark-brown solutions. The effluent used in the present study has the following physical 2.6. Enzyme assays characteristics: pH 4.0, color units 60,923 Æ 100, and a COD of 55.5 Æ 1.2 g/l. Laccase activity in the culture supernatant was determined by the method of Mansur and coworkers [20] using ABTS 2.2. Separation of molasses melanoidins (2,20 -azinobis-3-ethylbenzthiazoline-6-sulfonate) as the sub- strate. Lignin and manganese peroxidases were determined The MWW was centrifuged at 12,000 rpm for 15 min to as previously described by Tien and Kirk [35], and Pick and eliminate suspended solids. The resultant supernatant was Keisare [27], using veratryl alcohol and phenol red, respec- dialyzed against running tap water through a 10 kD membrane tively, as substrates. Enzyme activities were expressed in units (Pierce) at room temperature for 2 days, and then against de- defined as 1 mmol product formed per min. ionized water for another two days. The resulting solution of non-dialyzable compounds was used as a solution of molasses 2.7. Total RNA preparation and cDNA synthesis melanoidins. RNA extraction was performed using the Fast RNA kit-Red, 2.3. Organism following the manufacturer’s instructions (BIO 101). In order to remove contaminating DNA, 1 unit per mg of RNA of RQ1 Basidiomycete Trametes sp. I-62 (CECT 20197) was iso- DNase enzyme (Promega) was added to each RNA sample ´ lated from decayed wood in Pinar del Rıo, Cuba [21]. The fun- and subsequently incubated for 30 min at 37 C. First-strand gal culture was maintained on agar plates with Medium-7 [20]. cDNA synthesis was carried out using 2 mg of total RNA as Plates were grown for 7 days at 28 C and stored at 4 C. template and the cDNA synthesis kit from Roche.
  3. 3. T. Gonzalez et al. / Research in Microbiology 159 (2008) 103e109 ´ 105 2.8. Multiplex PCR reaction cultures with effluent nor in the controls during the 16 days of the experiment, under the assayed conditions. Laccase Design and optimization of this method have been previ- was the only detectable ligninolytic activity produced under ously described [13]. Briefly, a PCR mix was prepared by add- these conditions. Levels of this enzyme in the medium supple- ing together the three pairs of primers used to amplify lcc1, mented with effluent were always significantly higher than lcc2 and lcc3 (GenBank accession numbers AF548032, those of the controls (Fig. 1A). Maximal differences of 13- AF548034, AF548035, respectively) gene fragments. A total and 19-fold were achieved in the 8- and 16-day cultures, of 100 ml PCR mixtures also contained 5 ml of reverse tran- respectively. In contrast, decolorization of 76.9% and a COD scription product, 2.5 U Taq polymerase (Perkin Elmer) and reduction of 71% with respect to the initial values were all the rest of the standard components of a PCR DNA ampli- achieved at the end of the experiment (Fig. 1 and data not fication reaction [29]. PCR reactions were performed in shown). The time course of effluent decolorization and laccase a Rapidcycler (Idaho Technology) thermocycler. The basic activity detected in the culture supernatant of Trametes sp. I- program comprised an initial denaturizing step at 95 C for 62 showed a similar trend. Statistical analysis revealed a signif- 1 min followed by 30 cycles of 95 C for 45 s, 30 s at the icant correlation of 96% between the two variables. annealing temperature (59 C) and 72 C for 2 min, one final extension step at 72 C for 7 min and incubation at 4 C until 3.2. Effect of MWW and of molasses melanoidins on further storage of reactions at À20 C. The same procedure laccase gene transcription was performed to amplify a fragment of the constitutively expressed glyceraldehyde-3-phosphate dehydrogenase gene The effect of complete MWW and of the high molecular ( gpd1, GenBank acc. No. AF297874) from the RT reaction, weight fraction (corresponding to melanoidins) upon induction which was used as a control to normalize differences in the to- of laccase gene expression was compared. The time course tal RNA input or in the reverse transcription reaction efficien- analysis of laccase activity (Fig. 1B) indicated that 30 min cies. The only changes were that amplification was performed after their addition, both the complete effluent and the isolated for 25 cycles and the annealing temperature was set at 55 C. molasses melanoidins caused an increase in extracellular lac- case activity. Although the time course was very similar, the 2.9. Quantitative and statistical analysis maximal level of activity was slightly higher and more rapidly achieved (after 11 h) in the presence of the melanoidin fraction Three independent amplification reactions were performed with respect to those media amended with complete MWW, in for each condition assayed. PCR products (10 ml of each reac- which maximal levels were detected after 24 h. Laccase activ- tion) were separated by 1.5% agarose gel electrophoresis and ity was minimal in controls, and no significant changes were visualized after staining for 10 min in a 1 mg/ml ethidium produced in these samples during the assay. bromide solution. Densitometric analysis of Polaroid film gel Both complete molasses effluents and melanoidins selec- images was performed using ‘‘Image Quant 3.3’’ software tively induced lcc1 and lcc2 laccase gene transcription (Figs. (Molecular Dynamics). Levels of lcc mRNAs were expressed 1C and 2A,B). However, a higher increase in lcc transcript in arbitrary units, as the rate between lcc transcript levels levels was observed in the presence of the complete molasses (previously normalized according to size differences) and effluent, with induction being detected from the first sampling those of gpd1 calculated by the following equation: laccase/ time (30 min) after the addition of the effluent. In contrast, at ( gpd1sample/gpd1average). For all experiments and determi- that time no lcc gene expression could be detected in the media nations, variability coefficients between triplicate samples supplemented only with the molasses melanoidins. Maximal were calculated. Statistical differences were determined by induction of lcc1 transcripts was detected 7 h after supplemen- the ‘‘t test’’ for mean comparison (with P 0.001). tation with melanoidins and at 19 h following addition of the complete molasses effluent. In terms of lcc2 transcript levels, 3. Results maximal expression of lcc2 was observed 7 h after the addition of both molasses effluent and melanoidins. However, no lcc3 3.1. Ligninolytic enzyme profile and gene expression could be detected in any of the samples. MWW decolorization When the time course of total transcripts was analyzed (Fig. 2C), it could be noted that the highest lcc levels were ob- In a previous study we reported the optimization of differ- served 7 h after the addition of both MWW and melanoidins. ent parameters in submerged cultures of Trametes sp. I-62 in However, overall induction of lcc gene expression was initially order to attain the maximum reduction in color and COD of produced in the presence of the complete MWW and was the MWW [12]. slightly higher than induction observed with the isolated mel- These optimal parameters were applied here to analyze the anoidins. Nevertheless, the later decrease, after 19 h, was more relationship between the production of ligninolytic enzymes pronounced in the presence of the complete MWW; indeed, and effluent decolorization. We monitored culture supernatant after 31 h, laccase levels were lower than those corresponding for the presence of both lignin peroxidase (LiP) and manga- to addition of the melanoidins. No lcc gene expression were nese peroxidase (MnP) activity as well as for laccase activity. detected in any of the control samples throughout the course Neither LiP nor MnP activities could be detected in the of the experiment.
  4. 4. 106 T. Gonzalez et al. / Research in Microbiology 159 (2008) 103e109 ´ 1 100 [12]. Those studies were reviewed by Coulibaly and coworkers A vinasse lac. act. [7] and by Pant and coworkers [26]. The results in our work control lac. act. 0,8 80 compare with some of the best results reported in relation to decol. the extent of decolorization and COD removal. Paradoxically, Lac. Act. (U/ml) the first studies on the enzymatic system involved in these Decol. (%) 0,6 60 processes did not focus on ligninolytic enzymes. In fact, intra- cellular sugar oxidase enzymes were considered as having the 0,4 40 most important role in decolorization [36]. Miyata and coworkers [23] subsequently proposed the participation of lig- 0,2 20 ninolytic enzymes, particularly peroxidases, in the degradation of melanoidins. 0 In the present work, MnP and LiP do not appear to be in- 0 2 4 6 8 10 12 14 16 volved in decolorization of MWW by Trametes sp. I-62, since days neither of these enzymatic activities could be detected under conditions that resulted in maximal color reduction. Laccase was the only enzyme which could be detected at high levels B in culture supernatants. Previous studies to detect ligninolytic 0,036 enzymes in this fungus showed that the major ligninolytic ac- tivity in culture supernatants of Trametes I-62 was laccase, in 0,030 conjunction with small amounts of manganese peroxidase Lac. Act. (U/ml) 0,024 [20]. No lignin peroxidase has been detected thus far in this strain, even in culture conditions that permit the expression 0,018 of this enzyme in a Trametes versicolor strain used as a control melanoidin fraction (data not published). A study on decolorization of colored ef- complete MWW fluents from textile, paper and pulp mill and distillery waste 0,012 control with a marine basidiomicetous fungus has been recently pub- 0,006 lished by D’Souza and coworkers [10]. The authors also report laccase as being the dominant lignin-degrading enzyme, with very low activities of manganese-dependent peroxidase and no 0 6 12 18 24 30 36 42 lignin peroxidase activity. h Although induction of laccase activity in various basidio- mycete grown on MWW has previously been reported gpd1 C lcc [12,15] no clear correlation with effluent decolorization was 0 0,5 7 19 31 43 0 0,5 7 19 31 43 h ´ observed. More recently, Rodrıguez and coworkers [28] sug- gested an important role for laccases from Pleurotus ostreatus Control lcc1 – lcc2 – in MWW decolorization, as well as the involvement of other lcc3 – enzymes or mechanisms when nutrient levels become restric- complete tive. On the other hand, D’Souza and co-workers [10] de- MWW melanoidin lcc1 – lcc2 – scribed induction of laccase activity by MWW and lcc3 – decolorization of the effluent with a partially purified laccase preparation. Results presented here strengthen the role of lac- lcc1 – lcc2 – cases in MWW color reduction, since decolorization corre- lcc3 – lated directly with laccase activity throughout the experiment. Fig. 1. Effect of MWW and molasses melanoidins on Trametes sp. I-62 laccase Recent works have focused on the use of different natural activity and lcc transcript levels. (A) Biotreatment with the fungus grown in or synthetic compounds to induce laccases and to improve Kirk medium with 20% vinasses for 16 days. Laccase activity in cultures their secretion by white-rot fungi [3,15,22], but fewer have with effluent and corresponding decolorization rate values are represented. determined the inductive effect of these compounds on the Controls were grown on Kirk medium without effluent. (B) Changes in laccase activity after the addition of the complete MWW or the melanoidin fraction to expression of laccase genes [5,6,32]. Previous work by our 8-day cultures of Trametes sp. I-62 in Kirk medium. (C) Effect on lcc tran- group has shown that lcc gene expression in Trametes sp. I- script levels. Amplification of a fragment from the gpd1 gene was used as 62 is induced by veratryl alcohol, by two of its isomers and an internal control for each sample. by different aromatic monomers [13,21,34]. We now report for the first time that lcc gene expression in this fungus can 4. Discussion also be induced by MWW and by molasses melanoidins. Maximal levels of lcc transcripts were detected in the The application of basidiomycetous fungi to melanoidin de- presence of the complete and the high molecular weight frac- colorization has been studied for more than 30 years. Various tion of the effluent, and they were similar to those observed strains can degrade these polymers and decolorize MWW following induction of the best aromatic monomers tested
  5. 5. T. Gonzalez et al. / Research in Microbiology 159 (2008) 103e109 ´ 107 thus far ( p-coumaric acid, guaiacol and p-methoxyphenol) under identical culture conditions in Trametes sp. I-62 [34]. Nevertheless, both the complete MWW and the dialyzed frac- A 30 tion resulted in a more rapid increase in laccase activity than those obtained with aromatic monomers in the previous study. lcc1 25 lcc2 On the other hand, it would seem contradictory that maximal lcc3 extracellular laccase levels occurred first in the presence of the isolated melanoidins, even when lcc genes were previously arbitrary units 20 induced by the complete MWW. Various factors would explain these phenomena, considering that the effect of the effluent on 15 laccase activity can occur at a number of different levels to produce more active protein. For instance, it has been reported 10 that humic acids possess surfactant properties, which have been proposed to favor the liberation of enzymes in Trametes 5 versicolor and Phanerochaete chrysosporium [9]. Thus, due to the structural similarity between humic acids and melanoidins, 0 it seems reasonable to suggest that melanoidins play a similar 0 6 12 18 24 30 36 42 role, resulting in increased release of enzymes from fungus Time (h) grown on cultures amended by these compounds. In addition, melanoidins, as a result of the presence of phenolic groups in their structure, may also act as laccase stabilizers. Indeed, Mai B 30 and coworkers [19] reported enhancement of laccase stability lcc1 in the presence of phenolics compounds. Prior to the addition 25 lcc2 of the effluent to Trametes sp. I-62 cultures, low levels of ex- lcc3 tracellular laccase were measured. If both the afore mentioned arbitrary units 20 factors are considered, namely stimulation of laccase secretion from the fungus coupled with potential stabilization of the 15 enzyme in culture media, then the more marked increment in laccase activity observed in media with isolated melanoi- dins, which occurs even before detection of lcc gene induction, 10 can be explained. The rapid induction of lcc genes in media with complete 5 molasses effluent may be associated with the presence of low molecular weight compounds that can be easily trans- 0 ported through fungal membranes. The inductive effect of 0 6 12 18 24 30 36 42 melanoidins at the genetic level may, in fact, be mediated by Time (h) the action of lower molecular weight compounds derived 60 from their degradation. Another factor to consider is that mel- C anoidins are potent copper chelators. It has been shown that complete MWW their chromophore groups are related to this property, since 50 melanoidin liberation of chelated copper was detected when melanoidin degradation occurs as part of the decolorization process [24]. 40 If we consider that laccase genes can be induced by copper arbitrary units [25,32], then degradation of melanoidins could result in induc- 30 tion of lcc gene expression as a consequence of the release of copper into the culture media. These are tentative explanations which require additional studies for confirmation. 20 Fig. 2. Changes in the relative transcript levels of lcc1, lcc2 and lcc3 at differ- 10 ent times following addition of the complete MWW (A), and of the melanoidin fraction (B) to 8-day old cultures in Kirk medium analyzed by multiplex RT- PCR. Each data point represents the mean PCR product yield from two inde- 0 pendent amplifications. Arbitrary units express the ratio between lcc transcript 0 6 12 18 24 30 36 42 levels (normalized according to PCR product size) and those of glyceralde- Time (h) hyde 3-phosphate dehydrogenase ( gpd1). This means: laccase/( gpd1sample/ gpd1average). (C) Total lcc transcript levels calculated from the addition of the relative levels of lcc1, lcc2 and lcc3 mRNA in each sample, at different times, following addition of the complete MWW or the melanoidin fraction.
  6. 6. 108 T. Gonzalez et al. / Research in Microbiology 159 (2008) 103e109 ´ In conclusion, results presented in this work indicate a close role in biological systems Vol. 37 (pp. 559e586). New York: Marcel relationship between decolorization of MWW and selective Dekker. [12] ´ ´ Gonzalez, T., Terron, M.C., Yague, S., Zapico, E., Galletti, G.C., ¨ induction of laccase activity in Trametes sp. I-62. Two laccase ´ Gonzalez, A.E. (2000) Pyrolysis/gas chromatography/mass spectrometry genes, lcc1 and lcc2, are overexpressed during the decoloriza- monitoring of fungal-biotreated distillery wastewater using Trametes sp. tion process. Differential laccase gene expression occurs upon I-62 (CECT 20197). Rapid Comm. Mass Spectrom 14, 1417e1424. exposure of fungal cultures to both molasses wastewaters and [13] ´ ´ ´ Gonzalez, T., Terron, M.C., Zapico, E., Tellez, A., Yague, S., ¨ their melanoidins, the high molecular weight fraction of these Carbajo, J.M., et al. (2003) Use of multiplex reverse transcription-pcr to study the expression of a laccase gene familiy in a basidiomycetous effluents. These findings could have important implications for fungus. Appl. Environ. Microbiol. 69, 7083e7090. a better understanding of molecular processes involved in [14] ´ ´ ´ Gonzalez, T., Terron, M.C., Zapico, E., Yague, S., Tellez, A., Junca, H., ¨ depollution of distillery industrial effluents. et al. (2003) Identification of a new laccase gene and confirmation of genomics predictions by cDNA sequences of Trametes sp. I-62 laccase family. Mycol. Res. 107, 727e735. Acknowledgments [15] Kahraman, S., Gurdal, I. (2002) Effect of synthetic and natural culture media on laccase production by white rot fungi. Biores. Technol. 82, 215e217. We are grateful to G. del Solar, M. Espinosa and A. Dobson [16] Kahraman, S., Yesilada, O. (2003) Decolorization and bioremediation of for their critical reading of the manuscript. We also molasses wastewater by white-rot fungi in semi-solid-state condition. ´ acknowledge the valuable help of L. Rodon and F.J. Carbajo Folia Microbiol. 48, 525e528. with some of the figures. [17] Kirk, T.K., Croan, K.S., Tien, M., Murtagh, K.E., Farrell, R.L. (1986) This work was supported by projects BIO95-2065-E and Production of multiple ligninases by Phanerochaete chrysosporium: ´ effect of selected growth conditions and use of a mutant strain. Enz. BIO97-0655 from the Comision Interministerial de Ciencia Microbiol. Technol. 8, 27e32. ´ ´ y Tecnologıa (CICYT, Madrid, Spain). T. Gonzalez acknowl- [18] Leonowicz, A., Matuszewska, A., Luterek, J., Ziegenhagen, D., Wojta- s edges support from a Mutis Program doctoral grant from Wasilewska, M., Cho, N.-S., et al. (1999) Biodegradation of Lignin by AECI (Spain) as well as from The International Foundation White Rot Fungi. Fungal Genet. Biol. 27, 175e185. ´ for Science (grant F/3899-1). M.C. Terron acknowledges [19] Mai, C., Schormann, W., Milstein, O., Huttermann, A. (2000) Enhanced ¨ ´ ´ stability of laccase in the presence of phenolic compounds. Appl. a post-doctoral grant from the Consejerıa de Educacion y Microbiol. Biotechnol. 54, 510e514. ´ Cultura de la Comunidad Autonoma de Madrid (Spain). [20] ´ ´ Mansur, M., Suarez, T., Fernandez-Larrea, J.B., Brizuela, M.A., ´ Gonzalez, A.E. (1997) Identification of a laccase gene family in the new lignin-degrading basidiomycete CECT 20197. Appl. Environ. References Microbiol. 63, 2637e2646. [21] ´ ´ Mansur, M., Suarez, T., Gonzalez, A.E. (1998) Differential gene ´ ´ [1] Arana, A., Roda, A., Tellez, A., Loera, O., Carbajo, J.M., Terron, M.C., expression in the laccase gene family from Basidiomycete I-62 (CECT et al. (2004) Comparative analysis of laccase-isozymes patterns of 20197). Appl. Environ. Microbiol. 64, 771e774. several related Polyporaceae species under different culture conditions. [22] Marques De Souza, C.G., Tychanowicz, G.K., De Souza, D.F., J. Basic Microbiol. 44, 79e87. Peralta, R.M. (2004) Production of laccase isoforms by Pleurotus [2] Baldrian, P. (2006) Fungal laccases-occurrence and properties. FEMS pulmonarius in response to presence of phenolic and aromatic Microbiol. Rev. 30, 215e242. compounds. J. Basic Microbiol. 2, 129e136. [3] Birhanli, E., Yesilada, O. (2006) Increased production of laccase [23] Miyata, N., Iwahori, K., Fujita, M. (2000) Microbial decolorization of by pellets of Funalia trogii ATCC 200800 and Trametes versicolor melanoidin-containing waste waters: combined use of activated sludge ATCC 200801 in repeated-batch mode. Enzyme Microb. Technol. 39, and the fungus Coriolus hirsutus. J. Biosci. Bioeng. 89, 145e150. 1286e1293. [24] Murata, M., Terazawa, N., Homma, S. 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