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                Tannic acid induces transcription of laccase gene
                cglcc1 in the white-rot fungus Co...
1042                                                                                               Can. J. Microbiol. Vol....
Carbajo et al.                                                                                                            ...
1044                                                                                                 Can. J. Microbiol. Vo...
Carbajo et al.                                                                                                            ...
1046                                                                                                   Can. J. Microbiol. ...
Carbajo et al.                                                                                                            ...
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  1. 1. 1041 Tannic acid induces transcription of laccase gene cglcc1 in the white-rot fungus Coriolopsis gallica José M. Carbajo, Howard Junca, María C. Terrón, Tania González, Susana Yagüe, Ernesto Zapico, and Aldo E. González Abstract: Laccase, a phenoloxidase enzyme secreted by white-rot fungi, has a significant role in the degradation of lignin and environmental pollutants. Coriolopsis gallica is a ligninolytic basidiomycete that produces high levels of this extracellular enzyme. A laccase gene cglcc1 from this fungus has been cloned and sequenced. The capacity of C. gallica to efficiently degrade polyphenols has been successfully applied in our laboratory to the biotreatment and decolorization of several industrial wastewaters. This study focused on the effect of tannic acid, a natural compound widely distributed in plants, on the production of laccase activity by C. gallica. Our results showed an evident increase of extracellular laccase levels when C. gallica was grown in the presence of tannic acid. Concentrations of 50 and 100 µM of this compound increased laccase activity when compared with control samples grown without tannic acid. In addition, we found an increase in laccase transcript levels in C. gallica grown in culture media supplemented with tannic acid. The role of tannic acid was shown to be an inductor of laccase activity in this fungus, due to the enhance- ment of expression of the laccase gene at the transcriptional level. Key words: laccase, tannic acid, Coriolopsis gallica, induction, gene transcription. Résumé : La laccase est une enzyme phénoloxydase secrétée par les champignons responsables al. la pourriture Carbajo et de blanche et qui joue un rôle significatif dans la dégradation de la lignine et de divers polluants environnementaux. Le Coriolopsis gallica est un basidiomycète qui produit des quantités élevées de cette enzyme extracellulaire. Un gène cglcc1 de la laccase de ce champignon a été cloné et séquencé. La capacité de C. gallica à dégrader efficacement les polyphénols a été appliquée avec succès dans notre laboratoire pour le biotraitement et la décoloration de quelques eaux usées industrielles. La présente étude a vérifié l’effet de l’acide tannique, un produit naturel largement répandu chez les plantes, sur la production de l’activité laccase par C. gallica. Les résultats obtenus ont démontré une nette augmentation des niveaux de laccase extracellulaire lorsque C. gallica était cultivé en présence d’acide tannique. Des concentrations de 50 et 100 µM de ce produit ont fortement augmenté l’activité laccase comparativement à des échan- tillons de contrôle cultivés en absence d’acide tannique. Nous avons de plus constaté une augmentation des niveaux de transcription de la laccase chez C. gallica cultivé dans des milieux enrichis d’acide tannique. Nous démontrons ainsi le rôle de l’acide tannique comme inducteur de la laccase chez ce champignon à cause d’une augmentation de l’expression du gène et de la transcription de la laccase. Mots clés : laccase, acide tannique, Coriolopsis gallica, induction, transcription d’un gène. [Traduit par la Rédaction] 1047 Introduction (Pointing 2001). A highly nonspecific ligninolytic enzymatic system secreted by white-rot fungi is known to be involved in White-rot basidiomycetous fungi are gaining interest be- this biodegradation (Thurston 1994; Leonowicz et al. 1999). cause of their capability to degrade a wide variety of natural Research on this topic has been undertaken recently in many and synthetic materials and environmentally persistent laboratories because of its great potential in several biotech- organopollutants, such as chlorophenols, polycyclic aromatic nological applications, such as bioremediation (Roy-Arcand hydrocarbons, polychlorinated biphenyls, and industrial dyes and Archibald 1991; Pointing 2001), animal feed improve- Received 8 July 2002. Revision received 20 November 2002. Accepted 28 November 2002. Published on the NRC Research Press Web site at on 7 January 2003. J.M. Carbajo, H. Junca,1 M.C. Terrón, T. González,2 S. Yagüe, E. Zapico,3 and A.E. González.4 Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas (CIB), Consejo Superior de Investigaciones Científicas (CSIC), Velázquez 144, E-28006, Madrid, Spain. 1 Present address: Department of Environmental Microbiology, Gesellschaft für Biotechnologische Forchung (GBF)-National Research Centre for Biotechnology, D-38124 Braunschweig, Germany. 2 Present address: Instituto Cubano de Derivados de la Caña de Azúcar, Havana, C.P. 11000 Cuba. 3 Present address: Biotechnology Department, University of Hamburg, Hamburg, D-21073 Germany. 4 Corresponding author (e-mail: Can. J. Microbiol. 48: 1041–1047 (2002) DOI: 10.1139/W02-107 © 2002 NRC Canada
  2. 2. 1042 Can. J. Microbiol. Vol. 48, 2002 ment (Akin et al. 1993), pulp and paper production (Messner Materials and methods and Srebotnik 1994), and wastewater treatment (Terrón et al. 1993; Garg and Modi 1999). Chemicals Studies on lignin-degrading enzymes have been mainly fo- TA (tannic acid powder pure, United States Pharmaco- cused on peroxidases (lignin and manganese peroxidases) and poeia (USP) empirical formula C 76H 52O 46), was obtained laccases (benzenediol:oxygen oxidoreductase, EC from Merck (Darmstadt, Germany). 2,2′-Azino-bis(3- Although biochemical, genetic, and regulatory aspects of ethylbenzthiazoline-6-sulfonate) (ABTS) was purchased peroxidases have been relatively well studied (Cullen 1997), from Boehringer Mannheim (Mannheim, Germany) and information on laccases is still scarce. Nevertheless, the im- 2,6-dimethoxyphenol from Fluka (Buch, Switzerland). portant role of laccases in degradation of lignin and other All other chemicals were reagent grade obtained from phenolic molecules has been confirmed (Eggert et al. 1997). Merck, Boehringer Mannheim, or Sigma–Aldrich Corp., To date, there are few reports focused on the regulatory St. Louis, Mo. mechanisms involved in laccase production (Collins and Dobson 1997; Mansur et al. 1998; Palmieri et al. 2000). Organism and maintenance Still, these studies are of interest given that large quantities Coriolopsis gallica A-241 was obtained from the IJFM of the enzyme will be required for further practical applica- (Instituto Jaime Ferrán de Microbiología) collection. The tions in bioremediation and biotechnology (Cullen 1997). fungal culture was maintained on malt agar slants (2% malt There are several studies demonstrating increased laccase extract, 2% Bacto agar, Difco, Detroit, Mich.), grown for activity levels in basidiomycetes caused by natural and 10 days at 28°C and stored at 4°C. synthetic aromatic substances (Pickard and Westlake 1970; Arora and Sandhu 1984). In contrast, there are few reports Culture conditions on the effect of phenolics and other substances on the The fungus was grown on agar plates with modified enhancement of laccase gene transcription (Linden et al. Czapek’s medium (Guillén et al. 1990) for 7 days at 28°C. 1991; Collins and Dobson 1997; Mansur et al. 1998). Ten plugs (1 cm2) were cut and inoculated under sterile con- Some of these inducers are synthetic molecules, such as 1- ditions into 500-mL culture flasks containing 300 mL of hydroxybenzotriazole, cycloheximide, and 2,5-xylidine, Kirk growth medium (nitrogen-limited defined medium) which show a significant degree of toxicity to animal and (Kirk et al. 1986). After incubation for 48 h at 28°C in an human health. Given the toxicity associated with these sub- orbital shaker (200 rpm), 10 mL of the culture medium con- stances, the search for less harmful compounds that are able taining little fragments of fungal mycelia was used to inocu- to increase laccase levels is of environmental importance. late 250-mL Erlenmeyer flasks containing 90 mL of Kirk The ligninolytic basidiomycete Coriolopsis gallica has medium. Samples were incubated at 28°C and 125 rpm for been selected in our laboratory, based on the results of a 7 days. After this time, a filter-sterilized (0.22 µm) solution previous screening performed among more than 90 fungal of 50 mM TA in distilled water was added to the culture me- species to select the most efficient decolorizer of a lignin- dium to reach a final concentration of 50, 100, or 200 µM. containing paper-industry effluent; a laccase enzyme is sug- Controls without TA were also grown. In all cases, the final gested to be involved in this process (Calvo et al. 1998). pH of the culture media was 4.6 ± 0.1, which is optimum for Moreover, another strain of C. gallica has recently been re- C. gallica growth (Calvo et al. 1998). Laccase activity was ported to be an effective pollutant degrader (Pickard et al. measured in the extracellular fluid throughout the following 1999). To date, we have evidence of only one genomic se- 9 days of incubation. quence for a laccase gene in C. gallica, even though there are reports of gene families for laccase in some white-rot Enzymatic activities assays fungi (Yaver and Golightly 1996; Mansur et al. 1997; Laccase activity was determined in the extracellular fluid Palmieri et al. 2000), and in spite of our numerous attempts of fungal cultures by the method of Wolfenden and Willson to find more laccase sequences in this fungus (Zapico 1999). (1982), using ABTS as the substrate. Lignin and manganese This laccase gene (cglcc1) has been cloned and sequenced peroxidases were measured as described by Tien and Kirk (GenBank accession No.: AY017340). (1984) and Paszczy½ski et al. (1988), respectively. One unit Different types of industrial effluents (paper industry, dis- of enzymatic activity is defined as the formation of 1 µmol tillery, beer factory, and oil mill wastewaters) have been of product per min. shown to increase laccase activity in various white-rot fungi (Mansur et al. 1997; Calvo et al. 1998; Pérez et al. 1998; Zymograms of fungal laccase activity González et al. 2000; Tsioulpas et al. 2002). All of these Polyacrylamide gel electrophoresis was performed at alka- effluents derive from industries that use plants as a raw ma- line pH under nondenaturating conditions using a Mini- terial, and the presence of tannic compounds has been re- Protean (Bio-Rad Laboratories, Hercules, Calif.) electropho- ported in some of them (Maestro-Durán et al. 1993). After resis cell. The separating gel contained 12% acrylamide, and lignin, tannins are the most abundant group of plant the buffer solution was 375 mM Tris–HCl (pH 8.8). The polyphenols and share with lignin a series of common struc- stacking gel contained 5% acrylamide, and the buffer solu- tural polyphenolic features (William et al. 1986). Tannic tion was 125 mM Tris–HCl (pH 6.8). The electrode buffer acid (TA) is the tannin most widely distributed in nature. solution contained 25 mM Tris–HCl and 122 mM glycine The possible role of TA as a laccase inductor in C. gallica (pH 8.8). Prestained molecular weight standards (Bio-Rad was studied from a physiological and molecular point of Laboratories) were used. All attempts to determine total pro- view, and the results are presented in this work. teins in the extracellular medium were inaccurate because of © 2002 NRC Canada
  3. 3. Carbajo et al. 1043 the interference caused by TA. This problem was overcome Fig. 1. Time course for laccase activity monitored in the by loading the same amount (25 µL) of extracellular fluid extracellular medium of Coriolopsis gallica grown in the absence from induced and control cultures. To detect laccase activ- () and in the presence of 50 µM ( ) and 100 µM (Ž) tannic ity, the gels were previously equilibrated in 100 mM acetate acid. Time is expressed in days after the addition of tannic acid. buffer at pH 5.0 and stained using 10 mM 2,6-dimethoxy- Experimental data are the means of three experiments, and the phenol as the substrate. experimental error was never greater than 5%. RNA preparations Total RNA was prepared using the Fast RNA Kit following the manufacturer’s instructions (BIO 101 Inc., San Diego, Ca- lif., U.S.A.) from fresh C. gallica mycelium collected at day 5 of incubation with different concentrations of TA and from controls without TA. PCR PCR was performed to obtain the hybridization probes for cglcc1 and gpd1 (glyceraldehyde-3-phosphate-dehydrogenase gene from C gallica). Taq DNA polymerase (Perkin Elmer Corp., Norwalk, Conn.) was used as recommended by the manufacturer, using a Rapidcycler (Idaho Technology, Idaho Falls, Idaho, U.S.A.) thermocycler with a PCR temperature program of 95°C for 1 min, followed by 30 cycles of 95°C for 40 s, 55°C for 40 s, 72°C for 1 min, and a final extension staining, and the RNA was then blotted by capillary transfer at 72°C for 8 min. All the primers were used at a final con- to a Hybond-N membrane (Amersham Biosciences Corp., centration of 0.4 µM. Piscataway, N.J.) in 20× SSPE (0.36 M NaCl, 20 mM The hybridization probe for cglcc1 was obtained using as a NaH2PO4, 2 mM EDTA, pH 7.7). The membrane was then template the plasmid pYES1, which contains the full length hybridized with the homologous DIG-labeled probes under cDNA of the C. gallica laccase gene (GenBank acc. No. high stringency conditions, following the manufacturer’s in- AF263467). The primers 5RT (5′-GCGATTGGCCCCAAGA- structions (Boehringer Mannheim). Hybridization signals CTG-3′) and 3RT (5′-CAGTGGCTGCGTGTTCACAC-3′) and RNA bands were quantified by densitometry using were designed to anneal at specific sites of this gene, ampli- Imagequant Software (Molecular Dynamics, Sunnyvale, Ca- fying an intragenic region of 690 bp. lif.). The transcript levels of the cglcc1 gene were calculated The probe to detect gpd1 transcripts, used as a signal of by dividing their hybridization signal percentages by the constitutive expression, was obtained using primers DIM (5′- corresponding gpd1 constitutive signals. TCAACGGTTTCGGTCGTATT-3′) and RM2 (5′-GTGG- ACGGTGGTCATGAGAC-3′) that amplify a highly con- Experimental reproducibility served fragment of 515 bp of the gpd1 gene from C. gallica All the experiments were performed three times. The stan- (GenBank acc. No. AF297874). The template DNA was the dard deviation in all the analytical assays was always less plasmid pHJC5 that included a partial gpd1 cDNA, synthe- than 5%. sized previously by RT-PCR and cloned on pGEM-T (Promega, Madison, Wisc.). Labeling of probes was performed using the PCR- Results amplified fragments of cglcc1 and gpd1 that were run on a 1% agarose gel and eluted with the UltraClean-15 DNA pu- Influence of different concentrations of TA on laccase rification kit (MO BIO, Solana Beach, California). One mi- production: spectrophotometric time course crogram of each purified fragment was diluted in H2O to a A time course experiment for laccase activity in the total volume of 16 µL, then the DNA samples were extracellular fluid of C. gallica showed that it was clearly in- denatured in a boiling water bath for 10 min and chilled on fluenced by the addition of different concentrations of TA ice. After that, 4 mL of digoxigenin (DIG) High Prime (Fig. 1). At day 2 of fungal growth in the presence of 50 µM (Boehringer Mannheim) were added, mixed, and spinned TA, laccase values were higher than those of the controls down briefly. The reaction was then incubated at 37°C over- and reached a maximum after 4 days. From day 4 to 9 of in- night and stopped with 2 µL of 0.2 M EDTA. cubation with 50 µM TA, the activity decreased, although at day 9, it was still higher than controls. At a final concen- Northern analysis tration of 100 µM TA, the increase of laccase activity was The expression of cglcc1 and gpd1 genes was estimated much higher than that detected at 50 µM but occurred later, using the hybridization probes described above. Approxi- reaching a maximum after 7 days following TA addition. mately 10 µg of total RNA were electrophoresed overnight The highest TA concentration assayed (200 µM) caused a at 1.2 V/cm in a 1.2% agarose–formaldehyde gel with partial inhibition of C. gallica growth. The same observa- 40 mM MOPS (morpholinepropanesulfonic acid) – 10 mM tion regarding the negative effect of tannic compounds on sodium acetate (pH 7.0) – 1 mM EDTA. RNA band intensi- the growth of several filamentous fungi has been reported ties were densitometrically measured after ethidium bromide (Davidson et al. 1938; Nobles 1948; Scalbert 1991) and for © 2002 NRC Canada
  4. 4. 1044 Can. J. Microbiol. Vol. 48, 2002 Fig. 2. Zymograms of laccase activity monitored in the Transcription analysis of cglcc1 extracellular fluid of Coriolopsis gallica grown in the absence The possible correlation between the increase in laccase (lanes b) and in the presence of 50 µM (lanes c) and 100 µM activity observed in culture media supplemented with TA (lanes d) tannic acid at different days of incubation after the ad- and the levels of cglcc1 transcripts was analyzed by North- dition of tannic acid. Molecular weight standards are represented ern blotting. RNA was prepared from C. gallica mycelium by lanes a. harvested on day 5 after the addition of 50, 100, and 200 µM of TA and from controls. Good quality RNA was obtained from controls and from samples with 50 and 100 µM, but all the attempts to extract RNA from mycelium incubated in the presence of 200 µM TA were unsuccessful. The interference of tannic compounds in the extraction of RNA has previ- ously been reported in the case of polyphenolic-rich materi- als (John 1992). In the same way, TA also causes an inaccurate spectrophotometric quantification of RNA; this problem was overcome by using the hybridization signal of gpd1 from C. gallica as a loading control. The effect of TA on cglcc1 expression is shown in Fig. 3, which also shows the ratio between the hybridization signals of cglcc1 and gpd1 (Fig. 3D). In the range of TA concentra- tions tested, the results showed the existence of a clear cor- relation between TA concentration and induction of cglcc1 expression. Our results showed that relative levels of cglcc1 transcripts in cultures containing 50 and 100 µM TA were higher than controls without TA, in agreement with the laccase activity determined spectrophotometrically and the this reason, the results of laccase activity at this TA concen- zymogram data obtained on day 5. This observation indi- tration were considered not to be comparable. cates that TA can have an important effect on the induction In all cases, except in controls without TA, an increase of of cglcc1 gene transcription, suggesting that these transcripts brown color in the extracellular fluid was observed during are translated to an active laccase protein. the first days after the addition of TA (data not shown), at which time laccase activity also started to be detected (Fig. 1). Moreover, the color intensity was directly corre- Discussion lated with TA concentration in the cultures containing 50 and 100 µM of TA. This color became less intense as the Until now, little work has been done regarding the regula- length of incubation increased, along with increasing values tion of laccase gene expression in white-rot fungi (Eggert et of laccase activity in the extracellular fluid. al. 1996; Collins and Dobson 1997; Mansur et al. 1998; Lignin peroxidase and manganese peroxidase were not de- Palmieri et al. 2000). The addition of inducers is one of the tected in the extracellular fluid of C. gallica grown with dif- simplest methods to increase the yield of enzyme produc- ferent concentrations of TA nor in the controls without TA. tion. It has been shown that tannic compounds are able to in- crease laccase activity in several basidiomycetes (Pickard and Westlake 1970; Arora and Sandhu 1984) and ascomy- Influence of different concentrations of TA on laccase cetes (Kim et al. 1995), although, as far as we know, the mo- production: zymograms lecular basis of such induction has not been reported. The The results of zymograms of laccase activity from day 1 results presented here demonstrate that the expression of the to 9 after the addition of various concentrations of TA are laccase gene cglcc1 of C. gallica is transcriptionally induci- shown in Fig. 2. The increase in the laccase activity detected ble by TA, giving rise to an enhancement of laccase activ- in the fungal culture containing 50 and 100 µM TA (Fig. 1) ity. The induction of laccase in several fungi by a number was also observed in the zymograms (Fig. 2). As in the of low molecular weight phenolic compounds (Koroljova- spectrophotometric assays, the effect of TA on increased Skorobogat’ko et al. 1998), aromatic acids (Farnet et al. laccase activity was observed earlier in the medium contain- 1999), a variety of flavonoids (Pickard and Westlake 1970), ing 50 µM TA than in the culture with 100 µM TA (Fig. 2). and different lignin preparations (Arora and Sandhu 1984) In the last 5 days of incubation, the induction of laccase pro- has been demonstrated at the physiological level. Our results duction became stronger in the cultures containing 100 µM showed that TA can be considered as an efficient inducer of TA, whereas a decrease in laccase signals were observed in laccase production by C. gallica, as revealed by the increase the presence of 50 µM TA, although levels were still higher of enzymatic activity detected and, as will be discussed later, than controls at day 9. In addition, the density of the induced by the enhancement of the laccase gene at the transcription enzymes bands was greater than those of controls without level. TA. Moreover, the bands of laccase activity in the controls An increased level of laccase activity in C. gallica and showed a sharp pattern during the first 4 days assayed, be- other white-rot fungi growing in the presence of diverse coming more diffuse towards the latter part of the experi- types of industrial wastewaters has been frequently detected ment (Fig. 2, lanes b). (Ardon et al. 1998; Pérez et al. 1998; Calvo et al. 1998; © 2002 NRC Canada
  5. 5. Carbajo et al. 1045 Fig. 3. (A) Total RNA samples from mycelium of Coriolopsis gallica harvested on day 5 after the addition of 0, 50, and 100 mM of TA (lanes 1, 2, and 3, respectively). Northern blot analysis of cglcc1 gene expression (B) and gpd1 gene transcripts (C). (D) cglcc1 transcript levels represented as the ratio between the hybridization signals of cglcc1 and gpd1. González et al. 2000; Yagüe et al. 2000; Tsioulpas et al. compounds present in distillery vinasses in Trametes sp. I- 2002), and the presence of tannins has been described in 62 (Mansur et al. 1997). some of them (Maestro-Durán et al. 1993; Yagüe et al. At the transcriptional level, induction of laccase in the 2000). Our results suggest that laccase induction produced ascomycete Neurospora crassa has been well characterized by this type of industrial effluents could be ascribed, in part, (Linden et al. 1991; Tamaru et al. 1994). Moreover, to TA or to structurally related molecules. Schouten and coworkers (2002) have recently described the A detoxification of toxic phenols by means of polymeriza- induction of transcripts for the laccase gene bclcc2 from the tion reactions has been suggested as one of the physiological ascomycete Botrytis cinerea when a solution of tannic acid roles of fungal laccases (Bollag et al. 1988; Thurston 1994). was added to the culture medium. In contrast, in basidio- Our observation that an increase of color in extracellular fluid mycetous fungi, studies regarding induction of laccase tran- of cultures containing TA coincided with detection of laccase scription are still scarce (Eggert et al. 1996; Collins and supports the proposed role of the enzyme in the detoxification Dobson 1997; Mansur et al. 1998; Palmieri et al. 2000). Our of phenolic compounds by polymerization reactions. In addi- results showed a direct relation between an increase in tion, the fact that this color became less intense with time, cglcc1 transcript levels and the enhancement of laccase ac- along with increasing values of laccase activity, suggests the tivity values produced by C. gallica in the presence of TA. involvement of laccase in the further degradation of these The presence of various laccase isozymes in the extra- compounds (Bollag et al. 1988) without disregarding the pos- cellular fluid of C. gallica growing in TA is suggested by the sible contribution of other enzymes. Studies using different thick shape of the bands attained in the zymograms pre- basidiomycetous strains are being carried out in our labora- sented in this study. Laccase isozymes can be ascribed to a tory to obtain further information on this subject. laccase gene family or to postranslational modifications of a Laccase induction by TA could be indicative of an envi- single protein. The latter could be the case for cglcc1, given ronmental switching response developed by fungi to oxidize that several potential places of glycosylation can be found in and, therefore, to decrease the potentially toxic effect of TA the putative amino acidic sequence deduced from this gene. and related compounds. Recently, some of our results re- Glycosylation is a common feature that has been demon- garding the decolorization of tannin-rich wastewaters also strated in many other fungal laccase proteins from basidio- suggested a toxic effect of polyphenols on the growth of C. mycetes (Perry et al. 1993; Giardina et al. 1996). The gallica and a further adaptation of the fungus along with a increase of laccase activity could be, among others, the re- polymerization of phenol compounds (Yagüe et al. 2000). sult of several aspects: (i) an increased production of laccase Similarly, an induction of laccase by other potential toxic mRNA, (ii) an increased stability of laccase mRNA tran- compounds in connection with an adaptative fungal response scripts, (iii) increased production of the active protein, and to diminish their toxic effect has also been suggested for (iv) an increased half-life of laccase protein. Work is cur- 2,5-xylidine in Pycnoporus cinnabarinus (Eggert et al. rently underway to examine some of these possibilities. 1996), 2,5-xylidine and 1-hydroxybenzotriazole in Trametes The induction of laccase production in different filamen- versicolor (Collins and Dobson 1997), and for undefined tous fungi seems to be specific for certain aromatic © 2002 NRC Canada
  6. 6. 1046 Can. J. Microbiol. Vol. 48, 2002 compounds; therefore, some precise mechanisms of transcrip- Coll, P.M., Tabernero, C., Santamaría, R., and Pérez, P. 1993. tional activation are likely to be involved. We have found a Characterization and structural analysis of the laccase I gene putative xenobiotic response element (XRE) in the promoter from the newly isolated ligninolytic basidiomycete PM1 (CECT of cglcc1 of C. gallica (GenBank acc. No. AY017340). The 2971). Appl. Environ. Microbiol. 59: 4129–4135. sequence GTGCCAT, reported by Fujisawa-Sehara et al. Collins, P.J., and Dobson, A.D.W. 1997. Regulation of laccase (1988) (coincidences with the XRE consensus sequence are gene transcription in Trametes versicolor. Appl. Environ. underlined), is present 115 bp upstream of the TATA box. Microbiol. 63: 3444–3450. The presence of these putative XREs has been reported in Cullen, D. 1997. Recent advances on the molecular genetics of several white-rot fungal lcc promoters (Coll et al. 1993; ligninolytic fungi. J. Biotechnol. 53: 273–289. Giardina et al. 1995; Collins and Dobson 1997; Mansur et Davidson, R.W., Cambell, W.A., and Baisdell, D.J. 1938. Differenti- al. 1997), suggesting that transcription of laccase genes may ation of wood-decaying fungi by their reactions on gallic or tan- be activated by stressing compounds, such as TA. nic acid medium. J. Agric. Res. (Washington, D.C.), 57: 683–695. The high degree of toxicity for human and animal health Eggert, C., Temp, U., and Eriksson, K.-E.L. 1996. The ligninolytic shown by some synthetic laccase inducers makes their use system of the white rot fungus Pycnoporus cinnabarinus: purifi- cation and characterization of the laccase. Appl. Environ. rather limited as potential enhancers of laccase production Microbiol. 62: 1151–1158. on an industrial scale in the future. TA is one of the first re- Eggert, C., Temp, U., and Eriksson. K.-E.L. 1997. Laccase is es- ported molecules of natural origin that is able to increase sential for lignin degradation by the white-rot fungus laccase levels, making its use, in principle, more suitable Pycnoporus cinnabarinus. FEBS Lett. 407: 89–92. from an industrial standpoint. In addition, the synthetic ori- Farnet, A.M., Tagger, S., and Le Petit, J. 1999. Effects of copper gin of some of the inducers studied to date make it difficult and aromatic inducers on the laccases of the white-rot fungus to elucidate their role in nature. In contrast, tannic com- Marasmius quercophilus. C. R. Acad. Sci. Ser. III, 322: 499– pounds are natural molecules widely distributed in the envi- 503. ronment, mainly as components of the external surfaces of Fujisawa-Sehara, A., Yamane, M., and Fujii-Kuriyama, Y. 1988. A plants, which represent the first defense mechanism against DNA-binding factor specific for xenobiotic responsive elements external aggressions. The results presented here permit us to of P-450c gene exists as a cryptic form in cytoplasm: its possi- propose an essential role of tannins as inducers of fungal ble translocation to nucleus. Proc. Natl. Acad. Sci. U.S.A. 85: laccases in the initial steps of wood degradation in natural 5859–5863. systems. Garg, S.K., and Modi, D.R. 1999. Decolorization of pulp-paper mill effluents by white-rot fungi. Crit. Rev. Biotechnol. 19: 85– Acknowledgements 112. Giardina, P., Cannio, R., Martirani, L., Marzullo, L., Palmieri, G., We are grateful to G. del Solar, M. Espinosa, and A.D.W. and Sannia, G. 1995. Cloning and sequencing of a laccase gene Dobson for their critical reading of the manuscript. Research from the lignin-degrading basidiomycete Pleurotus ostreatus. was financed by Comisión Interministerial de Ciencia y Appl. Environ. Microbiol. 61: 2408–2413. Tecnología (CICYT, Madrid, Spain) BIO 97-0655-E. H. Giardina, P., Aurilia, V., Cannio, R., Marzullo, L., Amoresano, A., Junca acknowledges financial support from G. Díaz de Siciliano, R., Pucci, P., and Sannia, G. 1996. The gene, protein Junca. T. González and E. Zapico acknowledge support from and glycan structures of laccase from Pleurotus ostreatus. Eur. a Mutis Programme doctoral grant from Agencia Española J. Biochem. 235: 508–515. de Cooperación Internacional (AECI) (Spain). S. Yagüe ac- González, T., Terrón, M.C., Yagüe, S., Zapico, E., Galletti, G.C., knowledges a grant from Ministerio de Ciencia y Tecno- and González, A.E. 2000. Pyrolysis/gas chromatography/mass logia. M.C. Terrón acknowledges a postdoctoral grant from spectrometry monitoring of fungal-biotreated distillery waste- Conserjería de Educación y Cultura de la Comunidad water using Trametes sp. I-62 (CECT 20197). Rapid Commun. Autónoma de Madrid (Spain). Mass Spectrom. 14: 1417–1424. 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