Received 2 January 2002/Accepted 10 April 2002

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 2002, p Vol. 68, No /02/$ DOI: /AEM Copyright 2002, American Society for Microbiology. All Rights Reserved. Laccase-Catalyzed Oxidation of Mn 2 in the Presence of Natural Mn 3 Chelators as a Novel Source of Extracellular H 2 O 2 Production and Its Impact on Manganese Peroxidase Dietmar Schlosser 1 * and Christine Höfer 2 UFZ Centre for Environmental Research Leipzig-Halle, D Halle, 1 and Biotype Gesellschaft für Molekularbiologische Analytik AG, D Dresden, 2 Germany Received 2 January 2002/Accepted 10 April 2002 A purified and electrophoretically homogeneous blue laccase from the litter-decaying basidiomycete Stropharia rugosoannulata with a molecular mass of approximately 66 kda oxidized Mn 2 to Mn 3, as assessed in the presence of the Mn chelators oxalate, malonate, and pyrophosphate. At rate-saturating concentrations (100 mm) of these chelators and at ph 5.0, Mn 3 complexes were produced at 0.15, 0.05, and 0.10 mol/min/mg of protein, respectively. Concomitantly, application of oxalate and malonate, but not pyrophosphate, led to H 2 O 2 formation and tetranitromethane (TNM) reduction indicative for the presence of superoxide anion radical. Employing oxalate, H 2 O 2 production, and TNM reduction significantly exceeded those found for malonate. Evidence is provided that, in the presence of oxalate or malonate, laccase reactions involve enzyme-catalyzed Mn 2 oxidation and abiotic decomposition of these organic chelators by the resulting Mn 3, which leads to formation of superoxide and its subsequent reduction to H 2 O 2. A partially purified manganese peroxidase (MnP) from the same organism did not produce Mn 3 complexes in assays containing 1 mm Mn 2 and 100 mm oxalate or malonate, but omitting an additional H 2 O 2 source. However, addition of laccase initiated MnP reactions. The results are in support of a physiological role of laccase-catalyzed Mn 2 oxidation in providing H 2 O 2 for extracellular oxidation reactions and demonstrate a novel type of laccase-mnp cooperation relevant to biodegradation of lignin and xenobiotics. Laccases (EC ) are extracellular multicopper oxidases produced by different kinds of fungi (39), which oxidize lignin and many organic xenobiotics (7, 23, 27, 45). These enzymes couple four one-electron substrate oxidations to the four-electron reduction of dioxygen to water, without formation of free reduced oxygen species (7, 45). Manganese peroxidases (MnP; EC ) are part of the ligninolytic system of white rot and litter-decaying basidiomycetes. During the catalytic cycle, the active center is oxidized by H 2 O 2. Reduction to the resting enzyme is achieved by two successive one-electron transfers, thereby oxidizing Mn 2 to Mn 3, respectively. This is facilitated by fungal organic acids such as oxalate or malonate upon chelation of the highly reactive Mn 3 state (4, 20 22, 40, 43). MnP catalyzes the oxidation of lignin, humic substances, and many organopollutants (16, 20, 29). Extracellular H 2 O 2 is required as a substrate for ligninolytic peroxidases. Extracellular enzymes like aryl alcohol oxidase (31) and glyoxal oxidase (19) produce H 2 O 2. This compound is also formed upon oxidation of hydroquinones by ligninolytic enzymes and autoxidation of the resulting semiquinones concomitantly reducing O 2 to superoxide anion radical (13, 27). Mn 2 reduces superoxide to H 2 O 2 and is thereby oxidized to * Corresponding author. Mailing address: UFZ Centre for Environmental Research Leipzig-Halle, Microbiology of Subterrestrial Aquatic Systems Group, Theodor-Lieser-Strasse 4, D Halle, Germany. Phone: Fax: Mn 3 (2, 27). Furthermore, superoxide may dismutate to H 2 O 2 and O 2. Oxidation of oxalate, glyoxylate, and malonate by Mn 3 was also considered to be a source of H 2 O 2 (16, 42). For oxalate, the following reactions are well established (41, 42): Mn 3 COOH-COOH 3 Mn 2 CO 2 CO 2 2H (1) CO 2 O 2 3 O 2 CO 2 (2) O 2 Mn 2 2H 3 Mn 3 H 2 O 2 (3) O 2 H 3 1 2H 2 O 2 1 2O 2 (4) For abiotic decomposition of malonate, the following reactions were proposed (16): Mn 3 COOH-CH 2 -COOH 3 Mn 2 COOH-CH 2 (5) CO 2 H COOH-CH 2 O 2 3 COOH-CH 2 OO (6) COOH-CH 2 OO O 2 3 O 2 COOH-COOH H (7) COOH-CH 2 OO Mn 2 H 3 Mn 3 COOH-CH 2 OOH (8) Superoxide and oxalate derived from reaction 7 subsequently can contribute to reactions 1 to 4. Autocatalytic generation of traces of Mn 3, which leads to H 2 O 2 (16) and the release of small H 2 O 2 concentrations from fungal mycelia (42), was considered to initiate MnP reactions, thereby enhancing the Mn 3 concentration and facilitating H 2 O 2 production in the aforementioned follow-up reactions. In a previous paper, we have demonstrated that a purified 3514

2 VOL. 68, 2002 H 2 O 2 PRODUCTION THROUGH Mn 2 OXIDATION BY LACCASE 3515 laccase from the white rot fungus Trametes versicolor oxidized Mn 2 to Mn 3 in the presence of the Mn 3 chelator pyrophosphate (15). Mn 2 oxidation involved concomitant reduction of laccase type 1 copper, thus providing evidence that Mn 2 oxidation occurs via one-electron transfer to type 1 copper as usual for substrate oxidation by blue laccases (7, 45). A Phellinus ribis laccase devoid of type 1 copper did not oxidize Mn 2 in the presence of pyrophosphate (25). The litter-decaying basidiomycete Stropharia rugosoannulata degrades chlorophenols (36), the fluoroquinolone antibacterial drug ciprofloxacin (44), 2,4,6-trinitrotoluene (33), and synthetic lignin (38) to CO 2 and H 2 O. Here, we show that a catalytic system consisting of purified laccase from S. rugosoannulata, Mn 2, and organic Mn chelators such as oxalate and malonate generates H 2 O 2. We further assessed key aspects of the catalytic mechanism underlying this new reaction and the impact on MnP produced along with laccase by S. rugosoannulata under certain culture conditions. This study was attempted to provide evidence for a physiological role of laccase-catalyzed Mn 2 oxidation and establishes a novel type of laccase-mnp cooperation with potential significance for lignocellulose breakdown and degradation of xenobiotics. MATERIALS AND METHODS Organism. S. rugosoannulata DSM was an isolate of the Institute of Microbiology, University of Jena, Jena, Germany, and was maintained on malt agar plates (35). Culture conditions and production of ligninolytic enzymes. For laccase production, S. rugosoannulata was pregrown on liquid malt medium (44) and then transferred into defined medium (35) containing 56 mm glucose and 30 M Mn 2 as MnSO 4, which was modified as follows. Diammonium tartrate was employed at 12 mm. 2,5-Xylidine and CuSO 4 were included at 200 and 50 M, respectively. Culture flasks were incubated on a Multitron rotary shaker (IN- FORS, Bottmingen, Switzerland) at 24 C under agitation (60 rpm). For MnP production, defined medium (35) containing 56 mm glucose and 1.2 mm diammonium tartrate was supplemented with additional MnSO 4 (final concentration, 100 M) and directly inoculated with S. rugosoannulata pregrown on malt agar plates (35). Incubation was carried out at 24 C without agitation. Enzyme isolation and purification. Cell-free culture filtrates were concentrated as described in reference 15. Proteins in concentrates were separated on a Mono Q HR 5/5 anion-exchange column (Amersham Pharmacia Biotech, Freiburg, Germany) under the conditions described before (15). MnP elutions were monitored at 405 nm (heme). For further MnP purification, (NH 4 ) 2 SO 4 at 35% saturation was added to enzyme pools derived from Mono Q separations. Precipitated proteins were removed by centrifugation (model 5415C centrifuge; Eppendorf, Hamburg, Germany) (14,000 rpm, 15 min), and MnP-containing supernatant was applied to a Phenyl Superose HR 5/5 hydrophobic interaction chromatography column (Amersham Pharmacia Biotech), preequilibrated with 10 mm Na-acetate buffer (ph 5.5) containing (NH 4 ) 2 SO 4 at 60% saturation. Proteins were eluted at 1 ml/min with 10 mm Na-acetate buffer (ph 5.5) without (NH 4 ) 2 SO 4. Enzyme-containing fractions (1 ml) were pooled, reconcentrated, and stored at 20 C (15). Protein concentrations were determined according to the method of Bradford (5). Gel electrophoresis and staining. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE were performed as described in reference 15. Gels were stained with Coomassie brilliant blue R-250 (Serva Feinbiochemica, Heidelberg, Germany) and 2,2 -azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) for protein and laccase activities, respectively (15). Molecular weights of proteins were determined with commercial molecular weight markers (Bio-Rad, Munich, Germany). Spectrophotometric determinations. Routine determinations of ligninolytic enzyme activities are described in reference 15. Laccase was assessed with ABTS at ph 4.5. MnP, lignin peroxidase (LiP; EC ), and manganese-independent peroxidase (MiP; EC ) activities were monitored upon formation of Mn 3 -malonate, oxidation of veratryl alcohol, and ABTS oxidation, respectively. Where indicated, 2,6-dimethoxyphenol (2,6-DMP) was additionally employed (15). Enzyme activities were expressed as units, where 1 U 1 mol of product formed per min. Mn 3 -oxalate (ε mm 1 cm 1 ) (22) and Mn 3 -malonate (ε mm 1 cm 1 ) (43) concentrations were determined at 270 nm, and Mn 3 -pyrophosphate (ε mm 1 cm 1 ) (18) concentrations were monitored at 258 nm. Chelators were employed as sodium salts at concentrations and ph values (adjusted with phosphoric acid) indicated in the text. Mn 2 and Mn 3 were always applied as MnSO 4 and Mn 3 -acetate, respectively. H 2 O 2 concentrations were determined with the horseradish peroxidase (HRP)-catalyzed oxidation of 4-hydroxyphenylacetic acid (PHPA) to the fluorescent product 2,2 -dihydroxyphenyl-5,5 -diacetic acid (17). Assays (final volume, 400 l) contained up to 300 l of enzyme-free sample, 15 U of HRP (type II; Sigma-Aldrich Chemie GmbH, Steinheim, Germany), and PHPA at 1.3 mm in 100 mm phosphate buffer (ph 7.4). Enzyme-free samples were obtained by ultrafiltration with 10-kDa-cutoff centrifuge filters (Sartorius, Göttingen, Germany). The samples from abiotic experiments were treated as the enzymatic ones. After incubation for 5 min and addition of NaOH (final concentration, 200 mm), assay mixtures were applied to a spectrofluorophotometer (34) operated at a 323-nm excitation wavelength. The fluorescence signal was integrated over an emission range from 335 to 550 nm. Calibration curves were established for each of the chelators employed with known concentrations of H 2 O 2. Assays omitting HRP served as blanks. The reduction of tetranitromethane (TNM) used to indicate superoxide anion radical was monitored at 350 nm (ε mm 1 cm 1 ) (26). For this, 270 l of ultrafiltrated sample was mixed with 30 l of 10 mm TNM in methanol, and the initial linear increase in absorbance was used to calculate TNM-reducing activity. All spectrophotometric determinations were carried out in air-saturated solutions at 35 C by using a double-beam spectrophotometer (15). Absence of low-molecular-weight compounds in the purified enzyme fractions. Since Mn 3 may also be formed during enzymatic oxidation of organic compounds (1, 27), the absence of hypothetical fungal redox mediators was ensured by directly applying purified enzyme fractions to high-performance liquid chromatography (HPLC) (15). Chemicals. All reagents were of analytical grade and were purchased from either Sigma-Aldrich Chemie GmbH, Merck, Darmstadt, Germany, or Fluka Chemie, Neu Ulm, Germany. Superoxide dismutase (SOD) (specific activity, 5,180 U/mg of protein) was obtained from Fluka. RESULTS Production and purification of laccase and MnP. Culture filtrates of agitated S. rugosoannulata cultures expressed a laccase activity of 3,191 U/liter at the time point of harvest (culture day 7). Neither MnP, MiP, nor LiP was detectable. Culture filtrate (235 ml) was 23.5-fold concentrated and applied to a Mono Q HR 5/5 column. Laccase activity was recovered as a major peak in fractions eluted at 2 to 3 ml and two minor peaks at 8 to 11 and 11 to 15 ml of elution volume, respectively. Pooled major activity fractions were used for further experiments, corresponding to a specific activity of 158 U/mg of protein, a 3.7-fold purification, and a yield of 14%. This pool showed the typical absorbance maximum at nearly 610 nm indicative of type 1 copper (7) (an A 280 /A 610 ratio of approximately 19) and did not contain any activity of MnP, MiP, or LiP. SDS-PAGE revealed one protein band with a molecular mass of approximately 66 kda (Fig. 1A). Native PAGE and activity staining with ABTS also visualized a single band (not shown in Fig. 1). HPLC analysis of the preparation did not lead to any indication of a hypothetical natural redox mediator. Time courses of extracellular MnP and laccase activities in nonagitated S. rugosoannulata cultures are depicted in Fig. 2, where MnP was the predominant enzyme. No attempt was made to purify the laccase because of its low activity. After harvesting on culture day 19, culture filtrate (3,830 ml) was 106-fold concentrated and applied to a Mono Q HR 5/5 column. MnP activity was recovered as a single peak in the frac-

3 3516 SCHLOSSER AND HÖFER APPL. ENVIRON. MICROBIOL. FIG. 1. Coomassie brilliant blue R-250-stained gels (SDS-PAGE) containing Mono Q-separated laccase (L) (A) and phenyl Superoseseparated (B) MnP. M, molecular mass markers. tions between 15 and 20 ml of elution volume, together with a tailed 405-nm heme absorbance peak. Laccase and LiP were not detectable in any fraction. Pooled MnP fractions corresponding to an elution volume of 17 to 18 ml, which additionally contained 7% (as related to the MnP activity) MiP, were applied to a phenyl Superose HR 5/5 column. Again, MnP activity eluted as a single peak between 21 and 24 ml, together with a distinct main absorbance peak at 405 nm. Pooled fractions eluted at 20 to 23 ml were used for further experiments, achieving a specific activity of 187 U/mg of protein, a 5.5-fold purification, and a yield of 2.4%. This pool oxidized ABTS, 2,6-DMP, and veratryl alcohol neither with nor without H 2 O 2, as proven over a ph range from 2.5 to 7.0. Traces of MiP (5% of the MnP pool activity) eluted at 18 to 19 ml, concomitant with a small A 405 peak. SDS-PAGE of the reconcentrated MnP pool revealed two protein bands with molecular masses of approximately 40 and 41 kda (Fig. 1B). The absence of hypothetical fungal redox mediators was ensured as described above. FIG. 2. Time course of extracellular laccase ( ) and MnP ( ) activities in nonagitated S. rugosoannulata cultures on defined medium containing 56 mm glucose, 1.2 mm diammonium tartrate, and 100 M Mn 2. Symbols represent means standard deviations for triplicate cultures. FIG. 3. Typical time courses of Mn 3 -oxalate (trace 1), -pyrophosphate (trace 2), and -malonate (trace 3) complex formation catalyzed by laccase. Assay mixtures (ph 5.0) contained 0.2 U of laccase/ml, 1 mm Mn 2, and 100 mm chelator, respectively. Laccase-catalyzed oxidation of Mn 2 to Mn 3 and formation of reduced oxygen species. Representative kinetics of Mn 3 -oxalate, -malonate, and -pyrophosphate formation catalyzed by S. rugosoannulata laccase are shown in Fig. 3. Corresponding UV-visible spectra revealed specific absorbance maxima at nearly 270 (22, 41) and 500 (43) nm for Mn 3 - oxalate, 270 nm for Mn 3 -malonate (43), and 258 (18) and 478 (1) nm for Mn 3 -pyrophosphate. The spectra of synthetic Mn 3 complexes, prepared by dissolving 100 M Mn 3 -acetate in either 100 mm Na-oxalate, -malonate, or -pyrophosphate (ph 5.0) prior to use, were identical to those of enzymatically generated Mn 3 complexes. No Mn 3 complex formation was observed in assays containing heat-inactivated enzyme. Formation of Mn 3 complexes was dependent on both the kind and concentration of the respective chelator (Fig. 4). Saturation of Mn 2 oxidation was observed at 100 mm for both organic acids, since oxalate and malonate concentrations of 200 mm did not further enhance the Mn 3 -oxalate and -malonate concentrations, respectively (not shown in Fig. 4). At 100 mm chelator and ph 5.0, formal enzyme activities of 0.15, 0.10, and 0.05 U/mg of protein were obtained for Mn 3 - oxalate, -pyrophosphate, and -malonate production, respectively. Mn 2 oxidation was optimal at ph 5.0 (oxalate and pyrophosphate) and 4.5 (malonate) (Fig. 5A). The highest laccase activity was obtained with ABTS at ph 2.5, followed by 2,6-DMP at ph 3.5 (Fig. 5B). In enzyme assays containing oxalate or malonate, Mn 2 oxidation was unequivocally accompanied by formation of H 2 O 2, whereas only insignificant levels were monitored in the absence of Mn 2 (Table 1). No remarkable H 2 O 2 formation could be detected upon application of pyrophosphate. Employing oxalate, the H 2 O 2 concentration was more than three times higher than in the presence of malonate. The ph dependency of H 2 O 2 formation upon oxalate application was assessed and revealed an optimum at ph 5.0 (Fig. 5C), thus fitting the ph

4 VOL. 68, 2002 H 2 O 2 PRODUCTION THROUGH Mn 2 OXIDATION BY LACCASE 3517 FIG. 4. Effect of oxalate, malonate, and pyrophosphate concentrations on laccase-catalyzed formation of Mn 3 -oxalate ( ), -malonate ( ), and -pyrophosphate ( ). Air-saturated reaction mixtures (ph 5.0) contained 0.2 U of laccase/ml, 1 mm Mn 2, and chelators as indicated. At chelator concentrations of 0, 25, and 50 mm, respectively, 100, 75, and 50 mm Na 2 HPO 4 were additionally employed. Concentrations of Mn 3 complexes were determined after2hofincubation at 35 C in the dark. Symbols represent means standard deviations for triplicate experiments. optimum determined for Mn 3 -oxalate production (Fig. 5A). Both the Mn 3 -oxalate and H 2 O 2 concentration nearly linearly increased over a tested range of up to 0.5 U of laccase/ml (Fig. 6). TNM reduction used to indicate superoxide anion radical (26) was significantly higher in enzymatic assays containing the organic chelators and Mn 2 than in those omitting Mn (Table 1), which was not observed during application of pyrophosphate. Oxalate employment led to an approximately sevenfoldhigher TNM-reducing activity than malonate application. These results are indicative of abiotic cleavage of oxalate and malonate by enzymatically formed Mn 3 according to reactions 1 to 7. In addition, H 2 O 2 may have been decomposed to a certain extent upon reduction of Mn 3 in a reaction not generating free superoxide (2, 43). Certain amounts of reduced oxygen species further may have been produced during sample preparation and in Mn-omitting reaction mixtures upon autoxidation of contaminating Mn. Experiments omitting laccase confirmed that Mn 3 is the species responsible for H 2 O 2 production and TNM reduction under conditions mimicking enzymatic reactions with respect to chelator and ph (Table 1). Addition of Mn 3 caused a significant higher H 2 O 2 concentration and TNM-reducing activity in the presence of oxalate compared to those with malonate. This is qualitatively consistent with the results of the laccase experiments. Only very low levels of reduced oxygen species were detected upon addition of Mn 2 as well as in the absence of Mn. In confirmation, application of pyrophosphate led to similarly low values under any condition. Interaction of laccase and MnP. In previous studies, essentially no MnP reactions were observed in enzymatic systems containing purified MnP, Mn 2, and 50 mm malonate (16) or 20 mm oxalate (41), but no additional source of H 2 O 2. Lower oxalate concentrations ranging from 0.5 to 10 mm facilitated H 2 O 2 -independent MnP reactions (41). We FIG. 5. Effect of ph on laccase-catalyzed formation of Mn 3 -oxalate ( ), -malonate ( ), and -pyrophosphate ( ) at 100 mm chelator, respectively (A); oxidation of 1 mm ABTS (ƒ) and 1 mm 2,6- DMP ( ) in 100 mm pyrophosphate (B); and production of H 2 O 2 at 1mMMn 2 and 100 mm oxalate (Œ) (C). In panels A and C, 100% corresponds (except for malonate where 100% refers to a Mn 3 - malonate concentration of 8.5 M, according to the slightly diverging ph optimum of 4.5) to the corresponding values shown in Fig. 4 and Table 1, respectively, and all other conditions were as described there. In panel B, 100% corresponds to specific activities of and U/mg of protein for ABTS and 2,6-DMP oxidation, respectively. Symbols represent means standard deviations for triplicate experiments. therefore employed high chelator concentrations of 100 mm to demonstrate the effect of laccase on MnP reactions unequivocally. In experiments containing laccase, MnP, Mn 2, and organic chelators, but no additional H 2 O 2, the Mn 3

5 3518 SCHLOSSER AND HÖFER APPL. ENVIRON. MICROBIOL. TABLE 1. Effect of Mn and chelators on H 2 O 2 production and TNM reduction in the presence and absence of laccase a Chelator and Mn addition H 2 O 2 production ( M) TNM reduction ( mol/min/liter) Laccase Laccase Laccase Laccase Oxalate Mn 3 added ND b,c ND b,c at 0.1 mm Mn 2 added b,c b,c at1mm Mn omitted Malonate Mn 3 added ND b ND b at 0.1 mm Mn 2 added b b at1mm Mn omitted Pyrophosphate Mn 3 added ND ND at 0.1 mm Mn 2 added at1mm Mn omitted a Laccase ( ) was employed at 0.2 U/ml, and chelators were always applied at 100 mm. Air-saturated reaction mixtures (ph 5.0) were incubated at 35 C in the dark. H 2 O 2 concentrations and TNM-reducing activities were determined after 2 (laccase assays) and 4 (assays without laccase) h of incubation. All values represent means standard deviations for triplicate experiments. ND, not done. b Significantly different (P 0.05) from corresponding values detected in the absence of Mn (laccase assays) or from corresponding values observed in the presence of Mn 2 as well as from those without Mn (assays omitting laccase), as obtained by Student s t test. c Significantly different (P 0.05) from the corresponding values obtained upon employment of malonate. complex formation was increasingly speeded up (traces 1 in Fig. 7A and B, respectively). After 90 min, approximately 22- and 2-fold-higher Mn 3 -oxalate and -malonate concentrations were observed, respectively, compared to those in assays omitting MnP (Fig. 3). This indicates increasingly stimulated MnP reactions. No Mn 3 complex formation was found in assays containing MnP, Mn 2, and organic chelators, but omitting laccase (traces 3 in Fig. 7A and B, respectively). Thus, laccase was essential to initiate MnP reactions. In laccase-driven MnP reactions, Mn 3 -oxalate formation occurred much faster than Mn 3 -malonate production, which resulted in an approximately 30-fold-higher Mn 3 - oxalate than Mn 3 -malonate concentration after 90 min. No MnP reaction was observed at 100 mm pyrophosphate, 0.2 U of laccase/ml, 0.2 U of MnP/ml, and 1 mm Mn 2, where Mn 3 -pyrophosphate formation was identical to that of assays containing laccase as the only enzyme (not shown in Fig. 7). According to the stoichiometry of reactions 3 and 4, SODcatalyzed dismutation of superoxide produces 50% of the H 2 O 2 that could be derived from superoxide reduction by Mn 2. Provided that in the absence of SOD H 2 O 2 is generated from reduction of superoxide by Mn 2 at concentrations ratelimiting to MnP reactions, SOD would thus delay MnP-catalyzed Mn 3 production. This is evident from traces 2 in Fig. 7A and B, respectively. SOD inhibited Mn 3 production by approximately 50% upon application of oxalate as well as malonate, indicating that superoxide reduction by Mn 2 is the key process for H 2 O 2 production in laccase-driven MnP reactions. DISCUSSION S. rugosoannulata produced large amounts of exclusively laccase when shaking-culture conditions together with 200 M 2,5-xylidine, 30 M Mn 2,50 M Cu 2, and 12 mm diammonium tartrate (providing 24 mm nitrogen) were applied. Copper, nitrogen, and 2,5-xylidine are known to enhance laccase gene transcription and extracellular enzyme titers in ligninolytic fungi (8). The presence of a chromatographic form with a molecular mass of 66 kda and the absorbance peak at nearly 610 nm caused by laccase type 1 copper reveal that S. rugosoannulata produces a typical blue laccase (7, 15, 27, 40). To date, no laccase has been described with a type 1 copper redox potential exceeding approximately 800 mv (versus a normal hydrogen electrode) (7, 45), whereas the potential of the aqueous Mn 2 /Mn 3 couple at ph 7 is 1,510 mv (7). The redox potential difference between type 1 copper and substrate as the thermodynamically driving force for the electron transfer from substrate to type 1 copper is considered to be a major parameter in controlling the rate of laccase substrate oxidation under steady-state conditions (45). Thermodynamically, oxidation of substrates with higher redox potential than that of type 1 copper may become possible if a primary oxidation product would efficiently be removed from the reaction equilibrium (23). Compounds such as oxalate, malonate, and pyrophosphate rapidly chelate both the Mn 2 and Mn 3 states and form mononuclear complexes with various ligand ratios, depending on the respective equilibrium constant and chelator concentration (1, 9, 22). Increasing concentrations of oxalate, malonate, and pyrophosphate increasingly favor Mn complex formation (2, 9, 22). Thus, it remains to be elucidated whether free (hexa-aquo) or complexed Mn 2 acts as a laccase substrate. Free Mn 2 was reported to be the substrate for MnP compound II re-reduction (4). Mn 3 arising from oxidation of Mn 2 by laccase could be removed from the reaction equilibrium upon chelation, thus driving the reaction forward. Also, the redox potential of the Mn 2 /Mn 3 couple commonly decreases on complexation (9), which could support FIG. 6. Dependence of Mn 3 -oxalate ( ) and H 2 O 2 (Œ) formation on the amount of laccase. Air-saturated reaction mixtures (ph 5.0) contained 1 mm Mn 2, 100 mm oxalate, and laccase as indicated. Mn 3 -oxalate and H 2 O 2 concentrations were determined after 2 h of incubation at 35 C in the dark. Symbols represent means standard deviations for triplicate experiments.

6 VOL. 68, 2002 H 2 O 2 PRODUCTION THROUGH Mn 2 OXIDATION BY LACCASE 3519 FIG. 7. Kinetics of Mn 3 -oxalate (A) and -malonate (B) formation in the presence of 0.2 U of laccase/ml, 0.2 U of MnP/ml, 1 mm Mn 2, and 100 mm chelator ( trace 1); 0.2 U of laccase/ml, 0.2 U of MnP/ml, 1,500 U of SOD/ml, 1 mm Mn 2, and 100 mm chelator (trace 2); and 0.2 U of MnP/ml, 1 mm Mn 2, and 100 mm chelator (trace 3) at ph 5.0. Mn 2 oxidation by laccase. Our observation that Mn 3 formation was chelator-specifically enhanced with increasing chelator concentrations (Fig. 4) favors such effects. The true Mn 2 -oxidizing activities of laccase in the presence of oxalate and malonate may well be higher than the observed ones, which obviously reflect mixed kinetics involving simultaneous Mn 3 complex formation and decay. Potentially, the ph optima obtained for Mn 3 -oxalate and -malonate formation (Fig. 5A) may have been faked by Mn 3 complex decomposition. Such decay processes lead to complex reaction equilibria, which are affected by several parameters, such as ph, Mn 3, Mn 2, and oxygen concentrations, as well as the kind and concentration of the respective chelator (9, 41). The net Mn 3 - oxalate and -malonate decay rates are known to increase with decreasing ph, decreasing chelator, and increasing Mn 3 concentrations (9, 41). In contrast, Mn 3 -pyrophosphate was reported to be stable for months at excess concentrations of pyrophosphate (1). The ph optimum of Mn 3 -pyrophoshate production by S. rugosoannulata laccase is identical to that of T. versicolor laccase (15). Nonagitated S. rugosoannulata cultures supplemented with 1.2 mm diammonium tartrate and 100 M Mn 2 predominantly produced MnP concomitant with low levels of laccase, similar to previously published results (38). In Phanerochaete chrysosporium, the highest MnP transcript levels were observed in Mn 2 -containing, nonagitated cultures upon nitrogen limitation (12), whereas MnP production shows a different response toward Mn 2 and nitrogen in other white rot fungi (24, 30). Since S. rugosoannulata MnP does not oxidize veratryl alcohol, ABTS, and 2,6-DMP in the absence of Mn 2,itresembles P. chrysosporium MnP in essentially strictly requiring Mn 2 as a substrate (29). Mn 2 -independent oxidation of veratryl alcohol, ABTS, and 2,6-DMP was shown for Mn 2 - oxidizing peroxidases from other white rot basidiomycetes (14, 24, 29). Oxalate concentrations of up to 27.8 and 47.5 mm were found in white and brown rot fungi, respectively (37). H 2 O 2 - independent Cerporiopsis subvermispora MnP reactions clearly were speeded up by addition of 10 M Mn 3 (41). Mn 3 - oxalate production by S. rugosoannulata laccase thus meets physiologically relevant conditions (Fig. 4). For MnP, H 2 O 2 K m values still below the H 2 O 2 concentrations derived from the laccase-mn 2 -oxalate system in this study (Table 1 and Fig. 6) were described (29). The ph profiles of laccase-catalyzed H 2 O 2 production in the presence of oxalate (Fig. 5C) and Mn 3 formation upon oxalate and malonate (Fig. 5A) fit the ph activity profile of MnP (20, 24, 40). At 50 to 100 mm, malonate laccase produces Mn 3 at concentrations (Fig. 4) within the same order of magnitude of those previously shown to initiate H 2 O 2 -independent MnP reactions at 50 mm malonate (16). Only 20 to 30 M malonate has been observed in ligninolytic cultures of P. chrysosporium (43). Laccase-driven MnP reactions were much faster in presence of 100 mm oxalate than malonate (Fig. 7), likely due to the higher H 2 O 2 concentration resulting from Mn 3 -oxalate decay (Table 1). Oxalate and malonate concentrations of 50 mm did not differentially affect MnP activities in a previous study (43), whereas MnP shows a different response at lower oxalate and malonate concentrations (20). Moreover, Mn 2 was found to enhance levels of laccase mrna and extracellular laccase activities in litter-decomposing and white rot fungi (32), indicating a regulatory role in laccase expression. Hence, our results favor a physiological function of Mn 2 oxidation by laccase and are in further support of the role of oxalate as a natural chelator (21, 40). In ligninolytic fungi, laccase and MnP titers can considerably vary in terms of culture conditions and time (35), as also shown here. Consequently, we propose a novel cooperation between laccase and MnP according to the scheme shown in Fig. 8. In the presence of Mn 2 and oxalate, laccase produces Mn 3 -oxalate. The latter initializes a set of follow-up reactions leading to H 2 O 2 formation, which may initiate or support peroxidase reactions. This does not rule out other ways to generate H 2 O 2 (13, 16, 19, 27, 31, 41, 42). Concerning biotechnological applications, laccase offers a simple and convenient alternative to supply peroxidases with H 2 O 2, because laccases will become available at an economically feasible scale. H 2 O 2 is also considered to be an oxidant in extracellular Fenton-type degradation mechanisms, implicated in white and brown rot basidiomycetes (3, 34, 44). Laccases were also described in brown rot fungi (10). We found a purified blue laccase from the brown rot basidiomycete Laetiporus sulfureus

7 3520 SCHLOSSER AND HÖFER APPL. ENVIRON. MICROBIOL. FIG. 8. Proposed scheme for the extracellular generation of H 2 O 2 as a result of laccase-catalyzed Mn 2 oxidation in the presence of oxalate and its impact on MnP. (The stoichiometry has not been balanced for oxalate, due to the possible involvement of complexes with various ligand ratios.) also active in Mn 2 oxidation (C. Höfer and D. Schlosser, unpublished data). Moreover, microbial Mn 2 oxidation is an important biogeochemical process at present mainly attributed to bacterial activities (28), in which multicopper oxidases have been implicated (6, 11). Besides basidiomycetes, environmentally ubiquitous organisms such as ascomycetes, imperfect fungi, and yeasts are known to produce laccases (39). A contribution of laccase activities of such organisms to microbial Mn 2 oxidation in different environmental compartments has not been considered as yet, but seems reasonable. ACKNOWLEDGMENTS This work was supported by UFZ Centre for Environmental Research Leipzig-Halle and Thüringer Ministerium für Wissenschaft, Forschung und Kultur (grant B ). We thank A. Orthaus (Jena) for excellent technical assistance. REFERENCES 1. Archibald, F., and B. Roy Production of manganic chelates by laccase from the lignin-degrading fungus Trametes (Coriolus) versicolor. Appl. Environ. Microbiol. 58: Archibald, F. S., and I. Fridovich The scavenging of superoxide radical by manganous complexes: in vitro. Arch. Biochem. Biophys. 214: Backa, S., J. Gierer, T. Reitberger, and T. Nilsson Hydroxyl radical activity associated with the growth of white-rot fungi. Holzforschung 47: Banci, L., I. Bertini, L. Dal Pozzo, R. Del Conte, and M. Tien Monitoring the role of oxalate in manganese peroxidase. Biochemistry 37: Bradford, M. M A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: Brouwers, G. J., E. Vijgenboom, P. L. Corstjens, J. P. de Vrind, and E. W. de Vrind-de Jong Bacterial Mn 2 oxidizing systems and multicopper oxidases: an overview of mechanisms and functions. Geomicrobiol. J. 17: Call, H. P., and I. Mücke History, overview and applications of mediated ligninolytic systems, especially laccase-mediator-systems (Lignozym-process). J. Biotechnol. 53: Collins, P. J., and A. D. W. Dobson Regulation of laccase gene transcription in Trametes versicolor. Appl. Environ. Microbiol. 63: Demmer, H., I. Hinz, H. Keller-Rudek, K. Koeber, H. Kottelwesch, and D. Schneider Manganese D 2, p In E. Schleitzer-Rust (ed.), Coordination compounds of manganese, 8th ed., vol. 56. Springer-Verlag, New York, N.Y. 10. D Souza, T. M., K. Boominathan, and C. A. Reddy Isolation of laccase gene-specific sequences from white rot and brown rot fungi by PCR. Appl. Environ. Microbiol. 62: Francis, C. A., and B. M. Tebo cuma multicopper oxidase genes from diverse Mn(II)-oxidizing and non-mn(ii)-oxidizing Pseudomonas strains. Appl. Environ. Microbiol. 67: Gettemy, J. M., B. Ma, M. Alic, and M. H. Gold Reverse transcription- PCR analysis of the regulation of the manganese peroxidase gene family. Appl. Environ. Microbiol. 64: Gómez-Toribio, V., A. T. Martínez, M. J. Martínez, and F. Guillén Oxidation of hydroquinones by the versatile ligninolytic peroxidase from Pleurotus eryngii. H 2 O 2 generation and the influence of Mn 2. Eur. J. Biochem. 268: Heinfling, A., F. J. Ruiz-Dueñas, M. J. Martínez, M. Bergbauer, U. Szewzyk, and A. T. Martínez A study on reducing substrates of manganeseoxidizing peroxidases from Pleurotus eryngii and Bjerkandera adusta. FEBS Lett. 428: Höfer, C., and D. Schlosser Novel enzymatic oxidation of Mn 2 to Mn 3 catalyzed by a fungal laccase. FEBS Lett. 451: Hofrichter, M., D. Ziegenhagen, T. Vares, M. Friedrich, M. G. Jäger, W. Fritsche, and A. Hatakka Oxidative decomposition of malonic acid as basis for the action of manganese peroxidase in the absence of hydrogen peroxide. FEBS Lett. 434: Hyslop, P. A., and L. A. Sklar A quantitative fluorimetric assay for the determination of oxidant production by polymorphonuclear leukocytes: its use in the simultaneous fluorimetric assay of cellular activation processes. Anal. Biochem. 141: Kenten, R. H., and P. J. G. Mann The oxidation of manganese by illuminated chloroplast preparations. Biochem. J. 61: Kersten, P. J., and T. K. Kirk Involvement of a new enzyme, glyoxal oxidase, in extracellular H 2 O 2 production by Phanerochaete chrysosporium.j. Bacteriol. 169: Kishi, K., H. Wariishi, L. Marquez, H. B. Dunford, and M. H. Gold Mechanism of manganese peroxidase compound II reduction. Effect of organic acid chelators and ph. Biochemistry 33: Kuan, I.-C., and M. Tien Stimulation of Mn peroxidase activity: a possible role for oxalate in lignin biodegradation. Proc. Natl. Acad. Sci. USA 90: Kuan, I.-C., K. A. Johnson, and M. Tien Kinetic analysis of manganese peroxidase. The reaction with manganese complexes. J. Biol. Chem. 268: Majcherczyk, A., C. Johannes, and A. Hüttermann Oxidation of aromatic alcohols by laccase from Trametes versicolor mediated by the 2,2 azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) cation radical and dication. Appl. Microbiol. Biotechnol. 51: Mester, T., and J. A. Field Characterization of a novel manganese peroxidase-lignin peroxidase hybrid isoenzyme produced by Bjerkandera species strain BOS55 in the absence of manganese. J. Biol. Chem. 273: Min, K.-L., Y.-H. Kim, Y. W. Kim, H. S. Jung, and Y. C. Hah Characterization of a novel laccase produced by the wood-rotting fungus Phellinus ribis. Arch. Biochem. Biophys. 392: Moreira, M. T., G. Feijoo, T. Mester, P. Mayorga, R. Sierra-Alvarez, and J. A. Field Role of organic acids in the manganese-independent biobleaching system of Bjerkandera sp. strain BOS55. Appl. Environ. Microbiol. 64: Muñoz, C., F. Guillén, A. T. Martínez, and M. J. Martínez Laccase isoenzymes of Pleurotus eryngii: characterization, catalytic properties, and participation in activation of molecular oxygen and Mn 2 oxidation. Appl. Environ. Microbiol. 63: Nealson, K. H., B. M. Tebo, and R. A. Rosson Occurrence and mechanisms of microbial oxidation of manganese. Adv. Appl. Microbiol. 33:

8 VOL. 68, 2002 H 2 O 2 PRODUCTION THROUGH Mn 2 OXIDATION BY LACCASE Palma, C., A. T. Martínez, J. M. Lema, and M. J. Martínez Different fungal manganese-oxidizing peroxidases: a comparison between Bjerkandera sp. and Phanerochaete chrysosporium. J. Biotechnol. 77: Ruiz-Dueñas, F. J., F. Guillén, S. Camarero, M. Pérez-Boada, M. J. Martínez, and A. T. Martínez Regulation of peroxidase transcript levels in liquid cultures of the ligninolytic fungus Pleurotus eryngii. Appl. Environ. Microbiol. 65: Sannia, G., P. Limongi, E. Cocca, F. Buonocore, G. Nitti, and P. Giardina Purification and characterization of a veratryl alcohol oxidase enzyme from the lignin degrading basidiomycete Pleurotus ostreatus. Biochim. Biophys. Acta 1073: Scheel, T., M. Höfer, S. Ludwig, and U. Hölker Differential expression of manganese peroxidase and laccase in white-rot fungi in the presence of manganese or aromatic compounds. Appl. Microbiol. Biotechnol. 54: Scheibner, K., M. Hofrichter, A. Herre, and W. Fritsche Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene. Appl. Microbiol. Biotechnol. 47: Schlosser, D., K. Fahr, W. Karl, and H.-G. Wetzstein Hydroxylated metabolites of 2,4-dichlorophenol imply a Fenton-type reaction in Gloeophyllum striatum. Appl. Environ. Microbiol. 66: Schlosser, D., R. Grey, and W. Fritsche Patterns of ligninolytic enzymes in Trametes versicolor. Distribution of extra- and intracellular enzyme activities during cultivation on glucose, wheat straw and beech wood. Appl. Microbiol. Biotechnol. 47: Schlosser, D., R. Grey, C. Höfer, and K. Fahr Degradation of chlorophenols by basidiomycetes, p In D. L. Wise, D. J. Trantolo, E. J. Cichon, H. I. Inyang, and U. Stottmeister (ed.), Bioremediation of contaminated soils. Marcel Dekker, New York, N.Y. 37. Shimada, M., Y. Akamatsu, T. Tokimatsu, K. Mii, and T. Hattori Possible biochemical roles of oxalic acid as a low-molecular weight compound involved in brown-rot and white-rot wood decays. J. Biotechnol. 53: Steffen, K. T., M. Hofrichter, and A. Hatakka Mineralisation of 14 C- labelled synthetic lignin and ligninolytic enzyme activities of litter-decomposing basidiomycetous fungi. Appl. Microbiol. Biotechnol. 54: Thurston, C. F The structure and function of fungal laccases. Microbiology 140: Timofeevski, S. L., and S. D. Aust Effects of Mn 2 and oxalate on the catalytic activity of manganese peroxidase. Biochem. Biophys. Res. Commun. 239: Urzúa, U., P. J. Kersten, and R. Vicuña Kinetics of Mn 3 -oxalate formation and decay in reactions catalyzed by manganese peroxidase of Ceriporiopsis subvermispora. Arch. Biochem. Biophys. 360: Urzúa, U., P. J. Kersten, and R. Vicuña Manganese peroxidasedependent oxidation of glyoxylic and oxalic acids synthesized by Ceriporiopsis subvermispora produces extracellular hydrogen peroxide. Appl. Environ. Microbiol. 64: Wariishi, H., K. Valli, and M. H. Gold Manganese(II) oxidation by manganese peroxidase from the basidiomycete Phanerochaete chrysosporium. Kinetic mechanism and role of chelators. J. Biol. Chem. 267: Wetzstein, H.-G., M. Stadler, H.-V. Tichy, A. Dalhoff, and W. Karl Degradation of ciprofloxacin by basidiomycetes and identification of metabolites generated by the brown rot fungus Gloeophyllum striatum. Appl. Environ. Microbiol. 65: Xu, F., J. J. Kulys, K. Duke, K. Li, K. Krikstopaitis, H.-J.W. Deussen, E. Abbate, V. Galinyte, and P. Schneider Redox chemistry in laccasecatalyzed oxidation of N-hydroxy compounds. Appl. Environ. Microbiol. 66: