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

     Laboratorio de Micología Experimental, Departamento de Ciencias Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires. Buenos Aires, Argentina

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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.

Key words: Basidiomycete, copper, lignin-modifying enzymes, Trametes trogii

	INTRODUCTION

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White rot fungi are the only organisms which efficiently degrade the complex natural polymer lignin. They produce multiple extracellular enzymes involved in lignin degradation, including lignin peroxidase, manganese peroxidase, laccase, and oxidases that generate the H₂O₂ needed for peroxidase activity. Different combinations of the enzymes are produced by lignin-degrading fungi, suggesting that there is more than one successful strategy for lignin biodegradation (Hatakka 1994 ). Ligninolytic enzymes catalyze the one-electron oxidation of lignin units, resulting in various non-enzymatic reactions that include bond cleavage. After the discovery of lignin peroxidase, it was thought that this enzyme was the main ligninolytic agent because it was able to attack the major (non-phenolic) lignin moiety. However in recent years it has been demonstrated that laccase and manganese peroxidase are able to oxidize phenolic as well as nonphenolic substrates under certain conditions (Bourbonnais and Paice 1990 , Jensen et al 1996 ). Due to the low specificity and strong oxidative abilities of their fungal lignin degradation system, white rot fungi are also capable of degrading a broad spectrum of organic chemicals containing carbon skeletons similar to those found within the lignin polymer, such as PAHs, chlorinated phenols, PCBs, dioxin, DDT, alkyl halides, nitrotoluenes, chloroanilines and dyes (Field et al 1993 ). Although the majority of the previous studies have focused on the lignin-degrading enzymes of Phanerochaete chrysosporium and Trametes versicolor, lately there has been a growing interest in studying the ligninolytic enzymes of a wider array of white rot fungi, not only from the standpoint of comparative biology but also with the expectation of finding better lignin-degrading systems for use in various biotechnological applications (D'Souza et al 1999 ). Fungi producing laccase are currently the focus of much attention (Pointing et al 2000 ), since laccase production generally exceeds that of peroxidases, and lignin degradation as well as dye decolorization (Rodriguez et al 1999 ) and PAH degradation (Johannes and Majcherczyk 2000 ) have all been demonstrated by laccase in the presence of a redox mediator. Besides playing a role in delignification, fungal laccases appear to be involved in numerous physiological functions including fruit body development, detoxification of phenolic compounds via oxidative coupling and polymerization (Thurston 1994 ), pigment production and antimicrobial activity (Eggert 1997 ).

Trametes trogii is a white rot basidiomycete of worldwide distribution. Trametes trogii strain BAFC 463, besides efficiently degrading lignin in wood (Levin and Castro 1998 ), has been tested successfully in biomechanical pulping experiments (Planes et al 1986 ), and is also a good producer of ligninases (Levin and Forchiassin 2001 ). Its ability to degrade high priority pollutants such as polychlorinated biphenyls, polyaromatic dyes and polycyclic aromatic hydrocarbons has already been shown in our laboratory (Haglund et al unpubl). The present research was undertaken to study the effect of copper on its in vitro production of extracellular ligninolytic activities. We also describe the production of laccase isoenzymes as well as some of their catalytic properties.

	MATERIALS AND METHODS

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Microorganism – Strain 463 (BAFC: Mycological Culture Collection of the Department of Biological Sciences, Faculty of Exact and Natural Sciences, University of Buenos Aires) of Trametes trogii Berk. (Polyporaceae, Aphyllophorales, Basidiomycetes) was used in these experiments. Stock cultures were maintained on malt extract agar slants at 4 C.

Basal culture medium – Glucose, 10 or 20 g; asparagine monohydrate, 3 g; MgSO₄·7H₂O, 0.5 g; H₂KPO₄, 0.5 g; HK₂PO₄, 0.6 g; CuSO₄·5H₂O, 0.4 mg; MnCl₂·4H₂O, 0.09 mg; H₃BO₃, 0.07 mg; Na₂MoO₄·2H₂O, 0.02 mg; FeCl₃, 1 mg; ZnCl₂, 3.5 mg; thiamine hydrochloride, 0.1 mg; distilled water up to 1 L. Final pH: 6.5. For the decolorization assay Poly R-478 (0.02% w/v) was added to the medium before sterilization. The effect of CuSO₄·5H₂O concentrations from 1.6 µM to 1.5 mM was tested.

Culture conditions – 250 mL Erlenmeyer flasks with 25 mL of medium were inoculated with 2 agar plugs (each of 0.25 cm²) cut out from a colony grown on Bacto-agar 2%. Incubation was carried out at 28 ± 1 C under stationary conditions. Cultures were harvested at different incubation periods, filtered through a filter paper using a Büchner funnel and dried overnight at 70 C. Dry weight of mycelia was then determined. The culture supernatants were used as enzyme sources.

Enzyme assays – Laccase activity was measured with 2,2'-azino bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) in 0.1 M sodium acetate buffer (pH 3.4) at 30 C. Oxidation of ABTS was determined by the increase in A₄₂₀ (₄₂₀ = 36/mM cm) (Collins and Dobson 1997 ). In the experiments testing the effect of temperature on laccase activity, the incubation temperature was in the range 23–70 C. In the experiments on the effect of pH on laccase activity, the ABTS was dissolved in 0.05 M citrate-phosphate buffer of different pH (2.7–7.2). For temperature stability assays, aliquots of the culture filtrate were incubated at different temperatures (23–70 C) for different periods up to 5 d. For pH stability assays aliquots of crude enzyme preparation were mixed with citrate-phosphate buffer of different pH (2.7–7.2) and incubated for different periods at 23 C. Afterwards laccase activity was measured. Manganese peroxidase activity (MnP) was measured using phenol red as the substrate in 0.1 M sodium dimethylsuccinate buffer (pH 4.5) at 30 C (₆₁₀ = 22/mM cm) (Kuwahara et al 1984 ). Glyoxal oxidase activity (GLOX) was determined by using a peroxidase-coupled assay with methylglyoxal as GLOX substrate and phenol red as the peroxidase substrate (₆₁₀ = 22/mM cm) (Kersten 1990 ). International enzymatic units (U) were used (µmol/min). Enzyme activity was expressed as U/mL of culture filtrate. The extracellular proteins were measured by the Bradford method (1976) with bovine serum albumin as the standard. Reducing sugars remaining in the medium were determined by the Somogyi-Nelson procedure (Nelson 1944 ) with glucose as standard. The decolorization of Poly R-478 polymeric dye in the liquid culture was expressed as a decrease in the absorbance ratio A₅₃₀/A₃₆₀.

Polyacrylamide gel electrophoresis (PAGE) and activity staining of gels – Electrophoresis was performed on 12% polyacrylamide gel under nondenaturing conditions. The buffer solution for the separating gel was Tris-HCl 50 mM (pH 9.5). 10 µL of supernatants from different culture days and culture conditions (water-diluted in some cases, to render a laccase activity of approx 0.5–1.5 U) were loaded onto the gel and electrophoresed with Tris-glycine buffer (pH 8.4) at 120 V and 4 C. After electrophoresis the gel was fixed for 10 min in 10% (v/v) acetic acid and 40% (v/v) methanol, and then soaked in 50 mM acetate buffer (pH 3.4) containing 2.7 mg/mL of ABTS. Protein bands exhibiting laccase activity stained green with ABTS within 5 min. For the determination of the molecular weights of the laccase isoenzymes, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS PAGE) was performed on crude filtrates as described by Laemmli (1970) . Samples were run with prestained SDS molecular weight Sigmamarkers wide range protein standards. Molecular weights of the laccase bands were calculated from relative mobility compared to standards. All chemicals were purchased from Sigma Chemical Co. The results are the average of three triplicate experiments with a standard error of less than 5%.

	RESULTS

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The influence of different C/N ratio on growth and ligninolytic enzyme production by T. trogii was studied. Not only did the fungus grow better but also the titers of laccase, MnP, and GLOX were higher in the simultaneous presence of high concentrations of nitrogen and carbon (results not shown). Figure 1 depicts the relationship between growth, glucose consumption, extracellular proteins and ligninolytic enzyme production by T. trogii in media with 20 g/L glucose, 3 g/L asparagine and different Cu-concentrations. Although the increase in Cu between 1.6 µM and 1 mM did not affect maximum growth attained, the peak of growth was registered earlier in media with increasing Cu-concentrations. Laccase and GLOX activities appeared before mycelial biomass peaked (i.e., they were present in the primary growth phase) but showed maximum activity at the beginning of secondary metabolism. MnP activity was mainly detectable in the idiophase when the glucose in the culture medium was exhausted and the mycelial dry weight was decreasing. In the basal medium two peaks of laccase activity could be detected, the first one immediately after the onset of the stationary phase, followed by a decrease in enzyme production and increasing titers in older cultures. The highest amount of extracellular proteins was recorded at 27 d simultaneously with maximal enzyme production. The titers of laccase, MnP, and GLOX were affected by the concentration of Cu in the culture medium with highest enzyme levels detected in cultures supplemented with 1mM Cu (laccase: 93.8 U/mL, MnP: 1.28 U/mL, and GLOX: 0.46 U/mL; 14–19 fold higher than those measured in the basal medium). The increase observed in enzyme production cannot be attributed to the role of Cu on fungal growth, since no major differences in mycelial yield were found, when comparing different Cu-concentrations. Taking into account that protein secretion was comparable with the different concentrations of copper tested, the specific activities of laccase, MnP, and GLOX also increased with higher Cu-concentrations, attaining in the case of laccase a specific activity of 720 U/mg protein. An increment in the percent of decolorization of the dye Poly R-478 by T. trogii with Cu supply was observed as well (33.42% after 27 d at 1.6 µM Cu, 40.18% at 0.5 mM and 64.63% at 1 mM Cu). The highest Cu-concentration assayed (1.5 mM) however, inhibited in part the growth (maximum 106 mg/25 mL) and remarkably decreased MnP production (peak at 27 d 0.37 U/mL), but did not affect laccase secretion (maximal production 90.3 U/mL at day 27).

View larger version (29K): [in this window] [in a new window]    FIG. 1. Kinetics of growth and enzyme production by T. trogii in a synthetic medium with glucose 20 g/L, asparagine 3 g/L and different Cu.SO4 concentrations

View larger version (62K): [in this window] [in a new window]    FIG. 2. (A) Laccase activity staining after SDS PAGE of T. trogii culture filtrates. Cultures grown in a synthetic medium with glucose 20 g/L, asparagine 3 g/L and different Cu.SO4 concentrations. Lane a: 1 mM Cu.SO4. Lanes b, c, and d: 1.6 µM Cu.SO4. (B) SDS PAGE of T. trogii culture grown in the same medium with 1.6 µM Cu.SO4, for 12 d. Lane 1: activity staining of gel revealed two bands corresponding to lacc 1 (mol wt 38 kDa) and lacc 2 (mol wt 60 kDa). Lane 2: prestained molecular weight protein markers

At its optimum pH for activity (pH 3.4) it retained 100% of its activity after 1 h, 60% and 20% after 6 and 24 h respectively. At pH 3 it retained 60% of its activity after 1 h but only 25% after 6 h. At lower pH values (2.7) it lost 85% of its activity within 1 h, but over pH 4.4 it was completely stable after 5 d of incubation at room temperature. Moreover, activating effects were observed at pH 4.4 and higher. Thermal stability studies showed that laccase was stable up to 5 d at 30 C, and for 24 h at 50 C. At 60 C it retained 70 and 57% of its initial activity after 1 and 2 h incubation respectively, whereas at 70 C it had 50% of its activity after 30 min and 33% after 1 h.

	DISCUSSION

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Trametes trogii produced the highest amounts of laccase, MnP, and GLOX (laccase titers being dominant) in the simultaneous presence of high concentrations of nitrogen and carbon in the medium, in agreement with the fact that its ligninolytic system is expressed constitutively. Laccase is the most common lignin-modifying enzyme produced by the white rot fungi belonging to the Polyporaceae (Pelaez et al 1995 ) and many species secrete considerable levels of this enzyme in liquid cultures. Copper has been reported to be a strong laccase inducer in several species, among them, T. versicolor (Collins and Dobson 1997 ), P. chrysosporium (Dittmer et al 1997 ) and Pleurotus eryngii (Palmieri et al 2000 ). It is known that Cu induces both laccase transcription and activity (Palmieri et al 2000 ), and the increase in activity is proportional to the amount of copper added. But in the case of T. trogii, induction of MnP and GLOX activity was observed as well. In T. trogii maximal laccase, MnP, and GLOX production were attained with 1 mM Cu. At the highest concentrations Cu appeared to be toxic, since growth was reduced. Even though ligninolytic enzyme production rose while increasing Cu (up to 1 mM), other enzyme systems may be affected, considering that no increase in extracellular proteins was detected. Heavy metals are known as inhibitors of many enzymes belonging to both primary and secondary metabolic pathways (Ramsay et al 1999 ).

Due to the participation of peroxidases in lignin breakdown, the extracellular production of H₂O₂ is essential. Glyoxal oxidase activity which is produced extracellularly and is expressed during secondary metabolism, when the ligninases are also expressed, has been suggested to be the major enzyme responsible for the production of H₂O₂ in P. chrysosporium, the best studied lignin-degrading fungus (Kersten 1990 ). High levels of GLOX activity were produced by T. trogii with Cu-addition. Only a few of the 67 strains analyzed by de Jong and coworkers (1992) tested up to 0.003 U/mL of GLOX activity. Kersten (1990) , using an optimized liquid medium, obtained 0.032 U/mL of GLOX activity in P. chrysosporium. In addition, T. trogii produced high amounts of MnP and higher laccase levels than those described up to now under similar growth conditions (maximum recorded: 30 U/mL in Pleurotus ostreatus (Palmieri et al 2000 )). Laccase has recently been purified in two other strains of T. trogii (Garzillo et al 1998 , Vares and Hatakka 1997 ), but they produced much lower titers of laccase activity (maximal production, 0.5 and 0.03 U/mL respectively) than those secreted by T. trogii BAFC 463.

As in T. trogii, laccase seems to be secreted by the basidiomycete CECT 20197 (a member of the genus Trametes) (Mansur et al 1998 ) and T. versicolor in cycles (Collins and Dobson 1997 ). The two peaks of laccase activity which were detected in culture media of T. trogii (Levin and Forchiassin 2001 , and Fig. 1D this work) are probably owing to the specific induction of both laccase isoforms secreted by the fungus. Patterns after PAGE of T. trogii laccase isoenzymes were similar in media with different C/N ratio and Cu-concentrations. Likewise, identical laccase isoforms were consistently seen in cultures of Ganoderma lucidum grown in low N synthetic medium, malt extract or wood (D'Souza et al 1999 ). The molecular masses (38 and 60 kDa) reported in this study for T. trogii laccases, are in the range observed for laccases isolated from other white rot fungi (D'Souza et al 1999 , Thurston 1994 ). Two main laccases (67 and 70 kDa) were purified from T. versicolor (Bourbonnais et al 1995 ). Two to three laccase isoforms with molecular masses 64–70 kDa were described in T. gibbosa, T. hirsuta, a different strain of T. trogii (Vares and Hatakka 1997 ) and T. hispida (Rodriguez et al 1999 ).

Laccase activity in the crude extracellular extract of T. trogii exhibited catalytic properties comparable to other laccases with respect to optimum reaction temperature and pH, and proved to be very stable even at high temperatures. Activating effects, such as those observed for laccase activity of T. trogii at pHs over 4.4, have been reported previously, although the activation was by high temperatures (Coll et al 1993 ).

The capability of Poly R-478 decolorization is indicative of peroxidase activity. Such activity includes the combined activity of H₂O₂-producing oxidases and peroxidases, and is correlated with the ability of white rot fungi to degrade the tricyclic aromatic hydrocarbon anthracene (Field et al 1993 , Rodriguez Couto et al 2000 ). The percent of decolorization of the dye Poly R-478 increased with Cu-concentration but to a lesser extent than enzyme production. The unusually high secretion of ligninolytic enzymes did not parallel the ability of the fungus to degrade this dye. This fact may be related to the pH of the media, which were not the optima for the activity of the ligninolytic enzymes. Cultures of white rot fungi grown in the presence of high nitrogen concentrations have the tendency to increase their pH, and evidence was provided indicating that this alteration indeed affects ligninolysis (Tapia and Vicuña 1995 ).

In summary, T. trogii strain BAFC 463 is a fungus which produces high amounts of ligninolytic enzymes in the presence of high nitrogen and carbon concentrations due to increased biomass yields. This characteristic makes it an outstanding candidate for large-scale fermentation in order to produce ligninolytic enzymes in bulk. Our results show that high Cu-concentrations (up to 1 mM) stimulated laccase, MnP, and GLOX production by T. trogii, resulting in higher laccase titers than those reported for other white rot fungi studied to date. Taking into account that xenobiotic compound oxidation by white rot fungi cannot be improved by overproducing peroxidases without increasing the endogenous production of H₂O₂ (Gramss et al 1999 , Kotterman et al 1996 ), the simultaneous presence of high ligninolytic and hydrogen peroxide producing activities in this fungus make it an attractive microorganism on which to base future biotechnological applications.

	ACKNOWLEDGMENTS

 
To CONICET and University of Buenos Aires for financial support.

	FOOTNOTES

 
¹ Corresponding author, lale{at}bg.fcen.uba.ar

Accepted for publication October 29, 2001.

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